**Identification and Characterization of Feruloyl Esterases Produced by Probiotic Bacteria**

Kin-Kwan Lai, Clara Vu, Ricardo B. Valladares, Anastasia H. Potts and Claudio F. Gonzalez *University of Florida, USA* 

#### **1. Introduction**

150 Protein Purification

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Bacteriological Analytical Manual (8th ed.). FDA/Center for Food Safety &

A variety of phenolic compounds are naturally available, and contain one or more phenolic rings with or without substituents such as hydroxyl or methoxy groups. The term phytophenol, or phytochemical, is also used due to the widespread distribution of these chemicals throughout the plant kingdom (Huang *et al.*, 2007). Phytophenols are secondary metabolites of plants, which are primarily used in defense against ultraviolet radiation and pathogens (Beckman, 2000). These chemicals also participate in the formation of macromolecular structures in plant cell walls, and are naturally present in the form of monophenols or polyphenols with ester linkages. The presence of phenolic ester linkages limits the hydrolytic activity of enzymes such as xylanases, cellulases, and pectinases, by shielding the site of hydrolysis on plant cell walls from these enzymes. Hydrolyzing the ester linkages within the phytophenols releases the phenolic acids and relaxes the structure of the plant cell wall, aiding in the degradation and maximizing the nutritional value of dietary fiber. Phenolic acids such as ferulic acid, caffeic acid, chlorogenic acid, and rosmarinic acid are studied extensively due to their anti-oxidative, anti-inflammatory, and other health related properties which have been demonstrated both *in vitro* and *in vivo* (Srinivasan *et al.*, 2007). Even though phenolic acids can be easily found in dietary fiber, the ester linkages prevent their absorption in the human intestine. It has been demonstrated that only small monophenolic acids, but not esterified phenolic acids, can be absorbed efficiently by the monocarboxylic acid transporter (Konishi *et al.*, 2005). Thus, an enzymatic step is required to convert the esterified phenolic acids into monophenolic acids prior to absorption. In the presence of water, a specific type of enzyme, feruloyl esterases (FAEs), is able to hydrolyze the phenolic compounds into respective alcohols and phenolic acids. Thus, FAEs become one of the target fields of study to improve the bioavailiability and assimilation of phenolic acids in the human diet.

Feruloyl esterases (EC 3.1.1.73) are classified as a subclass of carboxylic acid esterases (EC 3.1.1.1). Alternate names such as ferulic acid esterases, cinnamoyl ester hydrolases, cinnamoyl esterases, and hydroxycinnamoyl esterases are generally used in the literature. They are also termed hemicellulase accessory enzymes because they can act synergistically with xylanases, cellulases, and pectinases to degrade the hemicellulose of plant cell walls. High substrate preference of FAEs is achieved when the carboxylic ester is in the phenolic/aromatic form, such that an aromatic hydrocarbon is attached to the carbon atom of the carbonyl group of the ester. FAEs are important enzymes in the rumen ecosystem due to their ability to increase the absorption of plant-based energy sources in ruminant animals. In recent years, several FAEs from fungi were partially characterized, but little is known about bacterial or plant FAEs. For both medicinal and industrial applications, there is an increasing amount of research focused on FAEs that are capable of releasing monophenols from plant biomass. To the best of today's knowledge, humans do not synthesize FAEs. However, FAE activity is found in total human gut microbiota (Kroon *et al.*, 1997, Gonthier *et al.*, 2006), indicating that FAEs are present in the intestine and may contribute to the release of phytophenols from dietary fiber. Currently, most characterized FAEs have been identified from fungi, and the lack of FAEs identified in other organisms, particularly intestinal bacteria, has limited their application.

Tertiary structure information on FAEs is scarce, while primary and secondary structure is poorly conserved between fungi and bacteria. Identifying and characterizing FAEs in bacteria is an important challenge. This chapter tells the story of the identification, purification, characterization, and crystallization of FAEs from probiotic bacteria. The potential FAEs were identified based on the enzymatic activity displayed by the bacterial strains as well as bioinformatics analysis. The work discussed herein will provide insight for further exploration of FAEs in other species, enhancing the path for medicinal and industrial application of these enzymes.

#### **2. Identification of feruloyl esterases from bacteria**

Our understanding of the relevance of the commensal microbiota in relation to the healthy status of the host is rapidly expanding. However, the mechanisms by which these microorganisms interact with the host are still unclear. Important technological advances such as rapid sequencing methods, bioinformatics, and species identification using 16S rDNA are valuable tools to describe the variability and composition of these small ecosystems. One of the most interesting applications of the study of commensal microbiota is the identification of species potentially responsible for, or correlated with, specific host diseases. For example, there are noticeable changes in the composition of the gut microbial ecosystem of diabetes patients compared to healthy individuals (Vaarala *et al.*, 2008). Some studies indicate that there is a predominant presence of probiotic bacteria such as *Lactobacillus johnsonii*, *Lactobacillus reuteri*, and *Bifidobacterium* species in healthy individuals (Roesch *et al.*, 2009).

Lactic acid bacteria (Lactobacilli) are well known bacteria present in the human intestine and used in probiotic supplements. There are a variety of explanations in the literature as to the mechanisms responsible for the probiotic effects of these bacteria. These mechanisms include competitive exclusion of pathogens, secretion of bioactive compounds, immune system alteration, and host metabolism modification. However, there is no general consensus as to the mechanism of probiotics action, and studies of mechanism typically differ depending on the species or strain of bacteria. For example, a feeding study using the biobreeding diabetes-prone (BB-DP) rat model for type 1 diabetes with the intestinal bacteria *L. johnsonii* showed that oral administration of the probiotic bacterium *L. johnsonii* decreases the incidence of type 1 diabetes, possibly by decreasing the intestinal oxidative stress response (Valladares *et al.*, 2010). The decreased oxidative stress at the intestinal level

phenolic/aromatic form, such that an aromatic hydrocarbon is attached to the carbon atom of the carbonyl group of the ester. FAEs are important enzymes in the rumen ecosystem due to their ability to increase the absorption of plant-based energy sources in ruminant animals. In recent years, several FAEs from fungi were partially characterized, but little is known about bacterial or plant FAEs. For both medicinal and industrial applications, there is an increasing amount of research focused on FAEs that are capable of releasing monophenols from plant biomass. To the best of today's knowledge, humans do not synthesize FAEs. However, FAE activity is found in total human gut microbiota (Kroon *et al.*, 1997, Gonthier *et al.*, 2006), indicating that FAEs are present in the intestine and may contribute to the release of phytophenols from dietary fiber. Currently, most characterized FAEs have been identified from fungi, and the lack of FAEs identified in other organisms, particularly

Tertiary structure information on FAEs is scarce, while primary and secondary structure is poorly conserved between fungi and bacteria. Identifying and characterizing FAEs in bacteria is an important challenge. This chapter tells the story of the identification, purification, characterization, and crystallization of FAEs from probiotic bacteria. The potential FAEs were identified based on the enzymatic activity displayed by the bacterial strains as well as bioinformatics analysis. The work discussed herein will provide insight for further exploration of FAEs in other species, enhancing the path for medicinal and industrial

Our understanding of the relevance of the commensal microbiota in relation to the healthy status of the host is rapidly expanding. However, the mechanisms by which these microorganisms interact with the host are still unclear. Important technological advances such as rapid sequencing methods, bioinformatics, and species identification using 16S rDNA are valuable tools to describe the variability and composition of these small ecosystems. One of the most interesting applications of the study of commensal microbiota is the identification of species potentially responsible for, or correlated with, specific host diseases. For example, there are noticeable changes in the composition of the gut microbial ecosystem of diabetes patients compared to healthy individuals (Vaarala *et al.*, 2008). Some studies indicate that there is a predominant presence of probiotic bacteria such as *Lactobacillus johnsonii*, *Lactobacillus reuteri*, and *Bifidobacterium* species in healthy individuals

Lactic acid bacteria (Lactobacilli) are well known bacteria present in the human intestine and used in probiotic supplements. There are a variety of explanations in the literature as to the mechanisms responsible for the probiotic effects of these bacteria. These mechanisms include competitive exclusion of pathogens, secretion of bioactive compounds, immune system alteration, and host metabolism modification. However, there is no general consensus as to the mechanism of probiotics action, and studies of mechanism typically differ depending on the species or strain of bacteria. For example, a feeding study using the biobreeding diabetes-prone (BB-DP) rat model for type 1 diabetes with the intestinal bacteria *L. johnsonii* showed that oral administration of the probiotic bacterium *L. johnsonii* decreases the incidence of type 1 diabetes, possibly by decreasing the intestinal oxidative stress response (Valladares *et al.*, 2010). The decreased oxidative stress at the intestinal level

intestinal bacteria, has limited their application.

**2. Identification of feruloyl esterases from bacteria** 

application of these enzymes.

(Roesch *et al.*, 2009).

could be a consequence of multiple factors. For example, the rat chow is formulated with many ingredients containing 6% to 8% (weight to weight) of fiber in the form of sugar beet pulp. The sugar beet pulp is an important source of ferulic acid, a phytophenol with antioxidative and anti-inflammatory effects (Couteau *et al.*, 1998). It has been demonstrated that low dosage of cinnamic acids (especially ferulic acid) has been related with the stimulation of insulin secretion (Balasubashini *et al.*, 2003, Adisakwattana *et al.*, 2008), prevention of oxidative stress and lipid peroxidation (Balasubashini *et al.*, 2004, Srinivasan *et al.*, 2007), and inhibition of diabetic nephropathy progression (Fujita *et al.*, 2008). The interaction of select bacteria in the host intestines with dietary fiber, and the possible release of phenolic acids, is an interesting process to be analyzed in order to generate a rationale understanding of the problem.

Despite the fact that total human gut microbiota displays FAE activity, specific bacterial species producing FAEs have not been investigated in depth. FAE activity was identified in several lactobacilli, including *L. fermentum*, *L. reuteri*, *L. leichmanni*, and *L. farciminis*, however the genes encoding the FAEs were not identified (Donaghy *et al.*, 1998). It is hypothesized that probiotic bacteria could enhance the release of bioactive phenolic acids from dietary fiber by producing the necessary FAE activity and aiding in the assimilation of phenolic acids. It is necessary to identify and characterize the FAEs from probiotic bacteria to further investigate this hypothesis.

#### **2.1 Identification of FAE-producing bacterial strains**

A screening assay for detection of FAE activity from *Lactobacillus* strains was first described by Donaghy *et al*. (Donaghy *et al.*, 1998). A model substrate for FAEs, ethyl ferulate, was embedded in de Man Rogosa Sharpe (MRS) plates. The presence of ethyl ferulate created a turbid appearance in the MRS agar due to the semi-soluble ethyl ferulate at 0.1% (weight to volume) final concentration. Ferulate assay (MRS-EF) plates were inoculated with cells obtained from individual overnight MRS cultures. The plates were incubated at 37°C in a gas pack system for a maximum of 3 days. The formation of halo (clear area) around the colonies due to the hydrolysis of ethyl ferulate indicated the presence of FAE activity. This technique was sucessfully applied by Lai *et al*. (Lai *et al.*, 2009), which identified that *L. johnsonii*, *L. reutri*, and *L. helveticus* are able to produce FAEs (Fig. 1).

Fig. 1. Halo zone created by *L. johnsonii* colony on MRS-EF screening plate.

#### **2.2 Selection of potential FAEs using bioinformatics analysis**

Since FAEs are classified as carboxylic esterases, they display the characteristics of serine esterases (Brenner, 1988, Cygler *et al.*, 1993). These enyzmes have a classically conserved pentapeptide esterase motif with a consensus sequence glycine-X-serine-X-glycine (GlyXSerXGly), where X represents any amino acid. Public databases such as Comprehensive Microbial Resource (CMR) Database (Davidsen *et al.*, 2010) can be used to predict potential FAEs from the genomic sequences of FAE producing strains. Since there are only a handful of bacterial FAEs currently identified, most of the potential FAEs are annotated as hypothetical hydrolases or esterases. Two FAEs, LJ0536 and LJ1228 were previously identified in a probiotic bacterium *L. johnsonii* (Lai *et al.*, 2009). The amino acid sequences of both FAEs can also be used to identify potential FAE homologs using a BLASTP search in NCBI database (Altschul *et al.*, 1997).

#### **2.3 Purification and characterization of FAEs**

Apart from fungal FAEs, most of the bacterial FAEs described in the literature are purified directly from the growth media of bacterial strains, without previous knowledge of their coding gene or protein amino acid sequence. In order to obtain a large amount of enzyme to carry out thorough biochemical characterization, it is necessary to express and purify the target FAEs as recombinant proteins.

#### **2.3.1 Cloning and purification of FAEs**

After selection of potential FAEs from genomic analysis of FAE-producing strains, the genes of interest can be cloned into an expression vector. pET vectors are one of the most common expression vectors used for the cloning and expression of recombinant proteins in *Escherichia coli*. The pET System is driven by the T7 promoter, so target genes are regulated by the strong bacteriophage T7 transcription and translation signals. In this system, gene expression is effectively induced by the presence of isopropyl β-D-1-thiogalactopyranoside (IPTG). The host cells, such as *E. coli* BL21 DE3, provide T7 RNA polymerase during protein expression. The proteins are expressed with specific tags such as Histidine Tag or S epitope tag, depending on the vector used.

Using pET15 vector as an example, the expression of His6-tagged proteins is carried out in *E. coli* BL21 using IPTG (1 mM) to induce gene transcription on the recombinant vector p15TV-L. The cells are collected by centrifugation at 8000 RPM (JLA8.1000 rotor, Beckman Coulter) for 25 min. The collected cell mass is resuspended in 25 mL binding buffer (5 mM imidazole, 500 mM NaCl, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5) and then disrupted by French press (20000 psi). The cell free extract is collected by centrifugation at 4°C, 17500 RPM (JA25.50 rotor, Beckman Coulter) for 25 min. The soluble His6-tagged proteins are purified by affinity chromatography. All solutions pass through the nickel-nitriloacetic acid (Ni-NTA) column by gravity flow. The Ni-NTA column is first washed with 30 mL of ultra-pure water to wash out unbound nickel ions. It is then preequilibrated with 30 mL binding buffer. The cell free extract is then applied to the Ni-NTA column. During this step, the His6-tagged proteins bind to nickel ions that are immobilized by NTA. The resin is washed with 30 mL of binding buffer to wash out any unbound proteins. 200 mL of wash buffer (20 mM imidazole, 500 mM NaCl, 20 mM HEPES pH 7.5), which contains a higher concentration of imidazole, is used to remove unspecific proteins that are bound to the resin. Imidazole is a competitive molecule that displaces the nickel ions bound to His6-tagged protein. The His6-tagged proteins are eluted using 20 mL elution buffer (250 mM imidazole, 500 mM NaCl, 20 mM HEPES pH 7.5). The purified proteins are dialyzed at 4°C for 16 hours. The dialysis buffer is composed of 50 mM HEPES buffer pH 7.5, 500 mM sodium chloride (NaCl), and 1 mM dithiothreitol (DTT). After dialysis, the samples are flash frozen and preserved at -80°C in 200 μL aliquots until needed. The His6 tag can be removed by treatment with tobacco etch virus (TEV) protease (60 ug TEV protease per 1 mg of target protein) at 4°C for 16 hours. The sample is applied to the nickel affinity chromatography column to eliminate the released His6-tag. Collected proteins are dialyzed at 4°C against dialysis buffer for 16 hours. The purified proteins without His6-tag are flash-frozen and preserved in small aliquots at -80°C until needed.

A rapid method to evaluate the FAE activity can be used immediately after purification. 3 µl of the purified proteins (3-5 µl equivalent to 0.1 µg total protein) are dropped on the surface of the MRS-EF screening plate. The formation of halo zones indicates the presence of FAE activity. This system was used to purify the recombinant proteins LJ0536 and LJ1228 (Lai *et al.*, 2009). By using the same strategy, a hypothetical protein LREU1684 was purified from *L. reuteri* and identified as a FAE.

#### **2.3.2 Enzymatic substrate profile analysis**

154 Protein Purification

Since FAEs are classified as carboxylic esterases, they display the characteristics of serine esterases (Brenner, 1988, Cygler *et al.*, 1993). These enyzmes have a classically conserved pentapeptide esterase motif with a consensus sequence glycine-X-serine-X-glycine (GlyXSerXGly), where X represents any amino acid. Public databases such as Comprehensive Microbial Resource (CMR) Database (Davidsen *et al.*, 2010) can be used to predict potential FAEs from the genomic sequences of FAE producing strains. Since there are only a handful of bacterial FAEs currently identified, most of the potential FAEs are annotated as hypothetical hydrolases or esterases. Two FAEs, LJ0536 and LJ1228 were previously identified in a probiotic bacterium *L. johnsonii* (Lai *et al.*, 2009). The amino acid sequences of both FAEs can also be used to identify potential FAE homologs using a

Apart from fungal FAEs, most of the bacterial FAEs described in the literature are purified directly from the growth media of bacterial strains, without previous knowledge of their coding gene or protein amino acid sequence. In order to obtain a large amount of enzyme to carry out thorough biochemical characterization, it is necessary to express and purify the

After selection of potential FAEs from genomic analysis of FAE-producing strains, the genes of interest can be cloned into an expression vector. pET vectors are one of the most common expression vectors used for the cloning and expression of recombinant proteins in *Escherichia coli*. The pET System is driven by the T7 promoter, so target genes are regulated by the strong bacteriophage T7 transcription and translation signals. In this system, gene expression is effectively induced by the presence of isopropyl β-D-1-thiogalactopyranoside (IPTG). The host cells, such as *E. coli* BL21 DE3, provide T7 RNA polymerase during protein expression. The proteins are expressed with specific tags such as Histidine Tag or S epitope

Using pET15 vector as an example, the expression of His6-tagged proteins is carried out in *E. coli* BL21 using IPTG (1 mM) to induce gene transcription on the recombinant vector p15TV-L. The cells are collected by centrifugation at 8000 RPM (JLA8.1000 rotor, Beckman Coulter) for 25 min. The collected cell mass is resuspended in 25 mL binding buffer (5 mM imidazole, 500 mM NaCl, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5) and then disrupted by French press (20000 psi). The cell free extract is collected by centrifugation at 4°C, 17500 RPM (JA25.50 rotor, Beckman Coulter) for 25 min. The soluble His6-tagged proteins are purified by affinity chromatography. All solutions pass through the nickel-nitriloacetic acid (Ni-NTA) column by gravity flow. The Ni-NTA column is first washed with 30 mL of ultra-pure water to wash out unbound nickel ions. It is then preequilibrated with 30 mL binding buffer. The cell free extract is then applied to the Ni-NTA column. During this step, the His6-tagged proteins bind to nickel ions that are immobilized by NTA. The resin is washed with 30 mL of binding buffer to wash out any unbound proteins. 200 mL of wash buffer (20 mM imidazole, 500 mM NaCl, 20 mM HEPES pH 7.5),

**2.2 Selection of potential FAEs using bioinformatics analysis** 

BLASTP search in NCBI database (Altschul *et al.*, 1997).

**2.3 Purification and characterization of FAEs** 

target FAEs as recombinant proteins.

tag, depending on the vector used.

**2.3.1 Cloning and purification of FAEs** 

The change in pH that occurs during ester hydrolysis can be used to screen for substrate preference of FAEs. A pH indicator such as 4-nitrophenol (Janes *et al.*, 1998) can be used to detect the change of pH during a reaction by monitoring the absorbance with a spectrophotometer. From the information in absorbance change, an estimate of enzymatic activity on different ester substrates can be determined. Enzymatic substrate profiles are determined at 25°C using an ester library composed of a variety of aliphatic and phenolic ester substrates (Liu *et al.*, 2001). The purified enzymes are first thawed from -80°C and redialyzed against 5 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer pH 7.2. The reactions are carried out in 96 well plates. Each enzymatic reaction contains 1 mM ester substrate, 0.44 mM 4-nitrophenol (proton acceptor), 4.39 mM BES pH 7.2, 7.1% (volume to volume) acetonitrile, and 30 - 35 µg . mL-1 of enzyme in a total volume of 105 μL reaction mixture. The 96 well plates are incubated at 25°C using Synergy™ HT Multi-Detection Microplate Reader (Biotek). The reactions are continuously monitored for 30 min at 404 nm. FAEs such as LJ0536 and LJ1228 display high activity towards phenolic esters (Lai *et al.*, 2009).

This techique is used only to demonstrate enzyme substrate preferences since it allows the use of several substrates in parallel. It utilizes specific conditions to detect the release of hydrogen ion (proton) during hydrolysis. The buffer (BES buffer) and the pH indicator (4 nitrophenol) to be used in this type of assay must have similar affinity (BES buffer pKa = 7.09; 4-nitrophenol pKa = 7.15) for the release of protons. In this way, the ratio of protonated buffer and the protonated indicator remains constant. The pH shifts by the proton release during the enzymatic reaction and it is detected as a change in the yellow color of the indicator present in the mixture. Thus, this technique is not flexible enough to adjust to the best conditions (pH, type of buffers, ions, etc.) that many enzymes require to function at their maximum activity. The specific enzyme activity determined using this method does not reflect the true specific enzyme activity. In addition, the stability of several enzymes could be affected by exhaustive dialysis in the reaction buffer. The dialysis was performed using 120 to 150 times the volume of the original enzyme suspension. Consequently, the technique is valid only to demonstrate substrate preferences. An alternate method involving the commerical equipment HydroPlate® instead of the tranditional 4-nitrophenol pH indicator can be used to monitor the pH during ester hydrolysis. The HydroPlate® is a 96 well plate containing immobilized pH sensor layers in each well (PreSens). The sensor contains a stable reference dye and one sensitive to oxygen. Thus, the measured fluorescence used to determine pH are internally referenced for precision across the plate.

#### **2.3.3 Biochemical properties of FAEs**

As enzyme reactions are saturable, the biochemical parameters such as Km (Michaelis constant: amount of substrate required to reach half of Vmax), Vmax (maximum rate of reaction or maximum enzyme specific activity, μmol . mg-1 . min-1), Kcat (catalytic rate constant, s-1), and Kcat / Km (catalytic efficiency, M-1 . s-1) can be determined by measuring the initial rate of the reaction over a range of substrate concentrations. The optimal conditions such as pH and temperature should be determined with model substrates before attempting to determine these biochemical parameters. Naphthyl esters and 4-nitrophenyl esters are common model substrates used to determine the biochemical parameters of esterases using saturation kinetics. Hydrolysis of naphthyl esters or 4-nitrophenyl esters generates napthol or 4-nitrophenol respectively, resulting in a specific absorbance at 412 nm. In similar ferulic acid esterase screenings, phenolic esters such as ethyl ferulate and chlorogenic acid should be included to compare the differences in biochemical parameters between aliphatic and phenolic esters. The hydrolysis of these phenolic esters can be monitored at 324 nm (Lai *et al.*, 2009). FAEs have higher catalytic efficiency and affinity towards phenolic esters. However, phenolic esters have higher values of absorbance and are less stable compared to the model substrates such as 4-nitrophenyl esters. The concentration of phenolic esters used to obtain the initial rate of the reaction is usually limited to 0.1 mM due to the upper limit of plate reader absorbance. Using absorbance to obtain enzyme kinetic parameters from phenolic ester hydrolysis can be difficult. An alternative method such as high performance liquid chromatography could be used to estimate the enzymatic activity by monitoring the release of products during phenolic ester hydrolysis (Mastihuba *et al.*, 2002).

#### **3. Structural analysis of FAEs**

FAEs belong to a structural group described as α / β fold hydrolases (Ollis *et al.*, 1992). The secondary structure of this group is composed of a minimum of eight β-strands in the center core surrounded by α-helices. The term α / β barrel is also used to describe the structure. The β-strands in the central core and α-helices are mostly parallel. The α-helices and βstrands tend to alternate along the chain of the polypeptide. There are only few structures of FAEs deposited in public databases (PDB) (Berman *et al.*, 2000). All the structures (apoenzymes or co-crystallized with a substrate) deposited in the PDB belong to two enzymes purified from only two species, a fungus *Aspergillus niger* and a bacterium *Butyrivibrio*  *proteoclasticus*. Five additional structures related to the probiotic bacterium *L. johnsonii* FAE LJ0536 were recently released in the PDB. The recently available structures of bacterial FAEs will allow researchers to identify new FAEs based on structural alignments and conserved structural features.

#### **3.1 GlyXSerXGly is the classical esterase motif**

156 Protein Purification

method does not reflect the true specific enzyme activity. In addition, the stability of several enzymes could be affected by exhaustive dialysis in the reaction buffer. The dialysis was performed using 120 to 150 times the volume of the original enzyme suspension. Consequently, the technique is valid only to demonstrate substrate preferences. An alternate method involving the commerical equipment HydroPlate® instead of the tranditional 4-nitrophenol pH indicator can be used to monitor the pH during ester hydrolysis. The HydroPlate® is a 96 well plate containing immobilized pH sensor layers in each well (PreSens). The sensor contains a stable reference dye and one sensitive to oxygen. Thus, the measured fluorescence used to determine pH are internally

As enzyme reactions are saturable, the biochemical parameters such as Km (Michaelis constant: amount of substrate required to reach half of Vmax), Vmax (maximum rate of

the initial rate of the reaction over a range of substrate concentrations. The optimal conditions such as pH and temperature should be determined with model substrates before attempting to determine these biochemical parameters. Naphthyl esters and 4-nitrophenyl esters are common model substrates used to determine the biochemical parameters of esterases using saturation kinetics. Hydrolysis of naphthyl esters or 4-nitrophenyl esters generates napthol or 4-nitrophenol respectively, resulting in a specific absorbance at 412 nm. In similar ferulic acid esterase screenings, phenolic esters such as ethyl ferulate and chlorogenic acid should be included to compare the differences in biochemical parameters between aliphatic and phenolic esters. The hydrolysis of these phenolic esters can be monitored at 324 nm (Lai *et al.*, 2009). FAEs have higher catalytic efficiency and affinity towards phenolic esters. However, phenolic esters have higher values of absorbance and are less stable compared to the model substrates such as 4-nitrophenyl esters. The concentration of phenolic esters used to obtain the initial rate of the reaction is usually limited to 0.1 mM due to the upper limit of plate reader absorbance. Using absorbance to obtain enzyme kinetic parameters from phenolic ester hydrolysis can be difficult. An alternative method such as high performance liquid chromatography could be used to estimate the enzymatic activity by monitoring the release of products during phenolic ester hydrolysis (Mastihuba

FAEs belong to a structural group described as α / β fold hydrolases (Ollis *et al.*, 1992). The secondary structure of this group is composed of a minimum of eight β-strands in the center core surrounded by α-helices. The term α / β barrel is also used to describe the structure. The β-strands in the central core and α-helices are mostly parallel. The α-helices and βstrands tend to alternate along the chain of the polypeptide. There are only few structures of FAEs deposited in public databases (PDB) (Berman *et al.*, 2000). All the structures (apoenzymes or co-crystallized with a substrate) deposited in the PDB belong to two enzymes purified from only two species, a fungus *Aspergillus niger* and a bacterium *Butyrivibrio* 

mg-1 .

min-1), Kcat (catalytic rate

s-1) can be determined by measuring

referenced for precision across the plate.

**2.3.3 Biochemical properties of FAEs** 

*et al.*, 2002).

**3. Structural analysis of FAEs** 

reaction or maximum enzyme specific activity, μmol .

constant, s-1), and Kcat / Km (catalytic efficiency, M-1 .

In general, carboxylic acid esterases have one classical esterase motif composed of a consensus sequence GlyXSerXGly. Analysis of the LJ0536 amino acid sequence showed that LJ0536 has two GlyXSerXGly motifs, an exception to the general one motif rule for carboxylesterases. Mutiple sequence alignments indicated that LJ0536 and its homologs, including LREU1684, all have two GlyXSerXGly motifs (Fig. 2). The reason for the presence of two GlyXSerXGly motifs in enzymes has not been addressed clearly in the literature. A recent structural study on LJ0536 (Lai *et al*., 2011) shows that LJ0536 displays a typical α / β hydrolase fold, which is composed of twelve β-strands and nine α–helices. Only one GlyXSerXGly motif (Gly104-X-Ser106-X-Gly108) of LJ0536 is catalytically active, while the other (Gly66-X-Ser68-X-Gly70) maintains the folding of the protein by hydrogen bonds. The newly identified FAE LREU1684 shares 47% amino acid sequence identity with LJ0536.

Fig. 2. Multiple sequence alignment of LJ0536 with its respresentative homologs. LJ0536: *L. johnsonii* N6.2, cinnamoyl esterase, GI# 289594369; LJ1228: *L. johnsonii* N6.2, cinnamoyl esterase, GI# 289594371; LREU1684: *L. reuteri* DSM 20016, alpha / beta fold family hydrolase-like protein, GI# 148544890; LGAS1762: *L. gasseri* ATCC 33323, alpha / beta fold family hydrolase, GI# 116630316; LHV1882: *L. helveticus* DPC 4571, alpha / beta fold family hydrolase, GI# 161508065; LAF1318: *L. fermentum* IFO 3956, hypothetical protein, GI# 184155794; PBR1030: *Prevotella bryantii* B14, hydrolase of alpha-beta family, GI# 299776930; HMPREF9071: *Capnocytophaga sp*. oral taxon 338 str. F0234, hydrolase of alpha-beta family protein, GI# 325692879. Two GlyXSerXGly motifs are located and boxed in the sequences. Amino acids are color coded.

#### **3.2 Identification of critical amino acids involved in substrate hydrolysis**

Ferulic acid esterase features such as the catalytic triad and the oxyanion hole are usually maintained by several amino acid residues that are highly conserved among homologs. Other critical amino acids involved in substrate recognition and binding are also conserved among closely related homologs, but not necessarily with less related homologs. A technique called alanine scanning, or site-directed mutagenesis, is helpful to determine the conserved amino acids critical for catalysis in proteins with unknown structure. The target amino acids selected for modification are replaced by alanine. Alanine is chosen because the inert alanine methyl functional group generally does not interact with other residues or alter the overall protein structure. To introduce the alanine mutation, 39-nucleotide long complementary primers containing the desired amino acid replacement are used to introduce individual mutations. The protein variants are then constructed by Polymerase Chain Reaction using Finnymes PhusionTM high fidelity DNA polymerase. This approach was used to identify the critical amino acids of LJ0536 (Lai *et al*., 2011).

The enzymatic activities of alanine variants are impared when the mutated amino acids are critical to function of the proteins. However, the results obtained from alanine scanning may not be useful in distinguishing the specific function of the amino acids, such as the involvement of amino acids in the formation of catalytic triad, oxyanion hole, tertiary structure of the protein, or substrate recognition and binding. The amino acids involved in substrate recognition and binding can be determined by measuring the enzymatic activity of the protein variants with different substrate types. For example, mutation of the amino acids that are only necessary for phenolic ester binding would not impair the enzymatic activity when aliphatic esters are used as substrates (Lai *et al*., 2011). Ultimately, the tertiary structure of the proteins are still necessary to conclude the findings from alanine scanning.

#### **3.2.1 Catalytic triad of FAEs is composed of serine, histidine, and aspartic acid**

Two basic steps are involved during ester hydrolysis: acylation and deacylation (Ding *et al*., 1994). During acylation, the hydroxyl oxygen of the catalytic serine carries out a nucleophilic attack on the carbonyl carbon of the ester substrate. After the attack, a general base (the histidine of the catalytic triad) deprotonates the catalytic serine and the first tetrahedral intermediate is formed. The hydrogen bonding of the third member of the triad, aspartic acid, plays a critical role in the stabilization of the protonated histidine. The oxyanion of the resulting tetrahedral intermediate is positioned towards the oxyanion hole. The oxyanion hole is created by hydrogen bonding between the substrate carbonyl oxygen anion and the backbone of two nitrogen atoms from other residues of the catalytic pocket. The general base, histidine, transfers the proton to the leaving group. The deprotonation of histidine leads to the protonation of an ester oxygen to release the first product (for example: methanol with methyl ferulate as substrate). As a consequence, the tetrahedral intermediate collapses and the characteristic acylenzyme intermediate is formed. Thus, the residual half of the substrate remains attached to the catalytic serine.

The second step of the reaction, deacylation, takes place in the presence of water. A molecule of water performs a nucleophilic attack on the carbonyl carbon of the remaining substrate in the acylenzyme intermediate. The general base (histidine) immediately

Ferulic acid esterase features such as the catalytic triad and the oxyanion hole are usually maintained by several amino acid residues that are highly conserved among homologs. Other critical amino acids involved in substrate recognition and binding are also conserved among closely related homologs, but not necessarily with less related homologs. A technique called alanine scanning, or site-directed mutagenesis, is helpful to determine the conserved amino acids critical for catalysis in proteins with unknown structure. The target amino acids selected for modification are replaced by alanine. Alanine is chosen because the inert alanine methyl functional group generally does not interact with other residues or alter the overall protein structure. To introduce the alanine mutation, 39-nucleotide long complementary primers containing the desired amino acid replacement are used to introduce individual mutations. The protein variants are then constructed by Polymerase Chain Reaction using Finnymes PhusionTM high fidelity DNA polymerase. This approach

The enzymatic activities of alanine variants are impared when the mutated amino acids are critical to function of the proteins. However, the results obtained from alanine scanning may not be useful in distinguishing the specific function of the amino acids, such as the involvement of amino acids in the formation of catalytic triad, oxyanion hole, tertiary structure of the protein, or substrate recognition and binding. The amino acids involved in substrate recognition and binding can be determined by measuring the enzymatic activity of the protein variants with different substrate types. For example, mutation of the amino acids that are only necessary for phenolic ester binding would not impair the enzymatic activity when aliphatic esters are used as substrates (Lai *et al*., 2011). Ultimately, the tertiary structure of the proteins are still necessary to conclude the

**3.2.1 Catalytic triad of FAEs is composed of serine, histidine, and aspartic acid** 

Two basic steps are involved during ester hydrolysis: acylation and deacylation (Ding *et al*., 1994). During acylation, the hydroxyl oxygen of the catalytic serine carries out a nucleophilic attack on the carbonyl carbon of the ester substrate. After the attack, a general base (the histidine of the catalytic triad) deprotonates the catalytic serine and the first tetrahedral intermediate is formed. The hydrogen bonding of the third member of the triad, aspartic acid, plays a critical role in the stabilization of the protonated histidine. The oxyanion of the resulting tetrahedral intermediate is positioned towards the oxyanion hole. The oxyanion hole is created by hydrogen bonding between the substrate carbonyl oxygen anion and the backbone of two nitrogen atoms from other residues of the catalytic pocket. The general base, histidine, transfers the proton to the leaving group. The deprotonation of histidine leads to the protonation of an ester oxygen to release the first product (for example: methanol with methyl ferulate as substrate). As a consequence, the tetrahedral intermediate collapses and the characteristic acylenzyme intermediate is formed. Thus, the residual half

The second step of the reaction, deacylation, takes place in the presence of water. A molecule of water performs a nucleophilic attack on the carbonyl carbon of the remaining substrate in the acylenzyme intermediate. The general base (histidine) immediately

**3.2 Identification of critical amino acids involved in substrate hydrolysis** 

was used to identify the critical amino acids of LJ0536 (Lai *et al*., 2011).

findings from alanine scanning.

of the substrate remains attached to the catalytic serine.

deprotonates a molecule of water, leading to the formation of a second tetrahedral intermediate. The catalysis follows a similar pattern described for the acylation. The second tetrahedral intermediate is stabilized by the formation of the oxyanion hole. The proton of the general base moves to the nucleophilic serine. Consequently, the ester oxygen is protonated and the tetrahedral intermediate collapses. The protonation of ester oxygen releases the final product (for example: ferulic acid with methyl ferulate as substrate), and reconstitutes the native serine residue and the original state of the enzyme.

The catalytic center of esterases always consists of a triad composed of a nucleophile (serine or cysteine), a fully conserved histidine, and an acidic residue (aspartic acid). In order for the catalytic triad residues to carry out their roles during hydrolysis as described above, the histidine must be located next to the catalytic serine, while the aspartic acid must be located next to the histidine. The catalytic triad of LJ0536 is composed of serine, histidine, and

Fig. 3. Three dimensional structures of LJ0536 and LREU1684. (A) Ribbon and (B) surface representation of LJ0536. The catalytic triad of LJ0536 is colored orange. (C) Ribbon and (D) surface representation of LREU1684. The catalytic triad of LREU1684 is colored yellow.

aspartic acid (Ser106, His225, Asp197). Due to the high amino acid sequence identity between LJ0536 and LREU1684, it is expected that both enzymes could have similar tertiary structures. Hypothetical tertiary structure of LREU1684 is predicted using SWISS-MODEL (Arnold *et al.*, 2006). SWISS MODEL is a structure homology-modeling server, which allows users to predict the structure of a protein with a simple input of the peptide sequence. The modeling is generated based on the existing protein structures. The results indicate that the folding of LREU1684 is highly similar to LJ0536 (PDB: 3PF8). The catalytic triad of LREU1684 is arranged in an identical orientation as in LJ0536. It is composed of Ser109, His228, and Asp200 (Fig. 3). The catalytic serine residue (Ser109) is located on top of the sharp turn of an α-helix (nucleophilic elbow). The catalytic triad arrangement of both LJ0536 and LREU1684 follows the general rule of ester hydrolysis.

#### **3.2.2 Classical oxyanion hole aids in substrate binding**

Co-crystallization assays of the LJ0536 catalytic serine deficient mutant Ser106Ala (LJ0536- S106A) with various ligands identifies the classical oxyanion hole of LJ0536 (Lai *et al*., 2011). LJ0536-S106A was co-crystallized with ethyl ferulate (PDB: 3QM1), ferulic acid (PDB: 3PFC), and caffeic acid (PDB: 3S2Z). All these structures show that the oxyanion hole of LJ0536 is formed by the backbone nitrogen atoms of phenylalanine and glutamine (Phe34 and Gln107). The oxyanion hole is an important structural feature, which stabilizes the tetrahedral intermediates during hydrolysis. Structural superimposition of LREU1684 and LJ0536 shows that the oxyanion hole of LREU1684 is formed by the backbone nitrogen atoms of Phe34 and Gln110 (Fig. 4).

Fig. 4. Binding cavities of LJ0536-S106A co-crystallized with ethyl ferulate and LREU1684. (A) The oxyanion hole of LJ0536 is formed by Phe34 and Gln107 (palecyan). The catalytic triad residues are colored orange. Ethyl ferulate (EF) is colored pink. Dashed lines represent hydrogen bonds. (B) The oxyanion hole of LREU1684 is formed by Phe34 and Gln110 (cyans). The catalytic triad residues are colored yellow.

#### **3.2.3 Specific inserted domain contributes to substrate binding**

160 Protein Purification

aspartic acid (Ser106, His225, Asp197). Due to the high amino acid sequence identity between LJ0536 and LREU1684, it is expected that both enzymes could have similar tertiary structures. Hypothetical tertiary structure of LREU1684 is predicted using SWISS-MODEL (Arnold *et al.*, 2006). SWISS MODEL is a structure homology-modeling server, which allows users to predict the structure of a protein with a simple input of the peptide sequence. The modeling is generated based on the existing protein structures. The results indicate that the folding of LREU1684 is highly similar to LJ0536 (PDB: 3PF8). The catalytic triad of LREU1684 is arranged in an identical orientation as in LJ0536. It is composed of Ser109, His228, and Asp200 (Fig. 3). The catalytic serine residue (Ser109) is located on top of the sharp turn of an α-helix (nucleophilic elbow). The catalytic triad arrangement of both LJ0536

Co-crystallization assays of the LJ0536 catalytic serine deficient mutant Ser106Ala (LJ0536- S106A) with various ligands identifies the classical oxyanion hole of LJ0536 (Lai *et al*., 2011). LJ0536-S106A was co-crystallized with ethyl ferulate (PDB: 3QM1), ferulic acid (PDB: 3PFC), and caffeic acid (PDB: 3S2Z). All these structures show that the oxyanion hole of LJ0536 is formed by the backbone nitrogen atoms of phenylalanine and glutamine (Phe34 and Gln107). The oxyanion hole is an important structural feature, which stabilizes the tetrahedral intermediates during hydrolysis. Structural superimposition of LREU1684 and LJ0536 shows that the oxyanion hole of LREU1684 is formed by the backbone nitrogen

Fig. 4. Binding cavities of LJ0536-S106A co-crystallized with ethyl ferulate and LREU1684. (A) The oxyanion hole of LJ0536 is formed by Phe34 and Gln107 (palecyan). The catalytic triad residues are colored orange. Ethyl ferulate (EF) is colored pink. Dashed lines represent hydrogen bonds. (B) The oxyanion hole of LREU1684 is formed by Phe34 and Gln110

(cyans). The catalytic triad residues are colored yellow.

and LREU1684 follows the general rule of ester hydrolysis.

**3.2.2 Classical oxyanion hole aids in substrate binding** 

atoms of Phe34 and Gln110 (Fig. 4).

The study of the LJ0536 structure indicated that a specific α / β inserted domain is critical for substrate binding (Lai *et al*., 2011). The inserted domain of LJ0536 is formed by a sequence of 54 amino acids from proline to glutamine (Pro131 to Qln184), and is located on top of the binding cavity. The two protruding hairpins from the inserted domain decorate the entrance and form the roof of the catalytic compartment. The phenolic ring of the ester substrate binds in the deepest part of the binding cavity, towards the inserted domain. In addition, three amino acid residues of the inserted domain, Asp138, Tyr169, and Gln145, contribute to the specific phenolic ester binding. The 4-hydroxyl group (ethyl ferulate, ferulic acid, and caffeic acid) and 3-hydroxyl group (caffeic acid) of the phenolic ring of the substrates are hydrogen bonded to Asp138 and Tyr169, respectively. Gln145 creates a bridge-like structure on top of the binding cavity, serving as a physical clamp holding the substrate inside the binding cavity. It also orients a water molecule in the binding cavity, which is important for activating the catalytic serine residue during hydrolysis.

Similar to LJ0536, LREU1684 has an α / β inserted domain formed by a sequence of 53 amino acids from Pro134 to Qln185 (Fig. 5). Asp141, Gln148, and Tyr172 of LREU1684 correspond to Asp138, Gln145, and Tyr169 of LJ0536, respectively. They adopted the same orientations as the residues in LJ0536 (Fig. 6). Thus, it is highly possible that Asp141, Gln148, and Tyr172 of LREU1684 also adopt the functional roles of Asp138, Gln145, and Tyr169 of LJ0536. LREU1684 and LJ0536 have both high amino acid sequence identity and high structural conservation.

Fig. 5. (A) Ribbon and (B) surface representation of the LREU1684 α / β inserted domain. It is composed of two short β-hairpins and three α–helices. The domain is colored dark blue. The catalytic triad residues are colored yellow. The binding cavity is circled with dashed lines.

Fig. 6. Substrate binding mechanism of LJ0536 and LREU1684. (A) Binding cavity of LJ0536- S106A co-crystallized with caffeic acid. The phenolic ring of the ester is stabilized in the binding cavity by Asp138, Gln145, and Tyr169. The inserted domain is colored dark green. The catalytic triad residues are colored orange. Caffeic acid (CA) is colored grey. Dashed lines represent hydrogen bonds. (B) Binding cavity of LREU1684. Critical amino acid residues for phenolic ester binding are identified as Asp141, Gln148, and Tyr172. The inserted domain is colored dark blue. The catalytic triad residues are colored yellow.

#### **3.3 Folding of LJ0536 is conserved among homologs**

Since LREU1684 is predicted to have tertiary structure and binding mechanism that are similar to LJ0536, it is hypothesized that the other LJ0536 homologs should also contain the structural features of LJ0536. To test this hypothesis, the models of LJ0536 homologs are predicted using SWISS MODEL. The quality of the modeling is estimated by the E-value, QMEAN Z-Score, and QMEANscore4 (Benkert *et al.*, 2011). The E-value is a parameter that describes the number of hits that you expect to find a protein by chance when searching a database. The lower the E-value, the more structurally significant the hit is. The Q-MEAN Z-Score measures the absolute quality of a model. A strongly negative value indicates a model of low quality. The QMEANscore4 represents the probability that the input protein matches the predicted model. The value ranges between 0 and 1. The results obtained using an automatic template search are summarized in Table 1.

All predictions provided good quality models except for the modeling of EVE, a hypothetical protein from *Eubacterium ventriosum* ATCC 27560. EVE has an E-Value of 1.40E-28, a QMEANscore4 of 0.477, and a QMEAN Z-Score of -4.276. BFI-1, a cinnamoyl ester hydrolase from *Butyrivibrio fibrisolvens* E14, has the best quality of model with an E-Value of 1.61E-91, a QMEANscore4 of 0.82, and a QMEAN Z-Score of 0.425. Among all 12 homologs, 10 were predicted to have similar folding to Est1E (Goldstone *et al*., 2010), a feruloyl esterase from *Butyrivibrio proteoclasticus* (PDB: 2wtmC and 2wtnA). The structures of LJ0536 and Est1E are highly similar as previously studied (Lai *et al*., 2011). The predictions were validated by including the sequences of LJ0536 in the analysis. The homologs, LBA-1 and BFI-2, do not have a similar Est1E folding. LBA-1 is annotated as α / β superfamily hydrolase in *L. acidophilus* NCFM. It was predicted to be similar to lipase

Fig. 6. Substrate binding mechanism of LJ0536 and LREU1684. (A) Binding cavity of LJ0536- S106A co-crystallized with caffeic acid. The phenolic ring of the ester is stabilized in the binding cavity by Asp138, Gln145, and Tyr169. The inserted domain is colored dark green. The catalytic triad residues are colored orange. Caffeic acid (CA) is colored grey. Dashed lines represent hydrogen bonds. (B) Binding cavity of LREU1684. Critical amino acid residues for phenolic ester binding are identified as Asp141, Gln148, and Tyr172. The inserted domain is colored dark blue. The catalytic triad residues are colored yellow.

Since LREU1684 is predicted to have tertiary structure and binding mechanism that are similar to LJ0536, it is hypothesized that the other LJ0536 homologs should also contain the structural features of LJ0536. To test this hypothesis, the models of LJ0536 homologs are predicted using SWISS MODEL. The quality of the modeling is estimated by the E-value, QMEAN Z-Score, and QMEANscore4 (Benkert *et al.*, 2011). The E-value is a parameter that describes the number of hits that you expect to find a protein by chance when searching a database. The lower the E-value, the more structurally significant the hit is. The Q-MEAN Z-Score measures the absolute quality of a model. A strongly negative value indicates a model of low quality. The QMEANscore4 represents the probability that the input protein matches the predicted model. The value ranges between 0 and 1. The results obtained using an

All predictions provided good quality models except for the modeling of EVE, a hypothetical protein from *Eubacterium ventriosum* ATCC 27560. EVE has an E-Value of 1.40E-28, a QMEANscore4 of 0.477, and a QMEAN Z-Score of -4.276. BFI-1, a cinnamoyl ester hydrolase from *Butyrivibrio fibrisolvens* E14, has the best quality of model with an E-Value of 1.61E-91, a QMEANscore4 of 0.82, and a QMEAN Z-Score of 0.425. Among all 12 homologs, 10 were predicted to have similar folding to Est1E (Goldstone *et al*., 2010), a feruloyl esterase from *Butyrivibrio proteoclasticus* (PDB: 2wtmC and 2wtnA). The structures of LJ0536 and Est1E are highly similar as previously studied (Lai *et al*., 2011). The predictions were validated by including the sequences of LJ0536 in the analysis. The homologs, LBA-1 and BFI-2, do not have a similar Est1E folding. LBA-1 is annotated as α / β superfamily hydrolase in *L. acidophilus* NCFM. It was predicted to be similar to lipase

**3.3 Folding of LJ0536 is conserved among homologs** 

automatic template search are summarized in Table 1.


in *Burkholderia cepacia* (PDB: 1YS1). BFI-2 is annotated as a cinnamoyl ester hydrolase in *B. fibrisolvens* E14. It was predicted to be similar to acetyl xylan esterase in *Bacillus pumilus* (PDB: 3FVR).

Table 1. LJ0536 homologs model automatic prediction using SWISS MODEL. *L. johnsonii* N6.2 cinnamoyl esterase LJ0536 (LJO-1), GI# 289594369. *L. johnsonii* N6.2 cinnamoyl esterase LJ1228 (LJO-2), GI# 289594371. *L. gasseri* ATCC 33323 alpha/beta fold family hydrolase LGAS1762 (LGA), GI# 116630316. *L. acidophilus* NCFM alpha/beta superfamily hydrolase LBA1350 (LBA-1), GI# 58337623. *L. acidophilus* NCFM, alpha/beta superfamily hydrolase LBA1842 (LBA-2), GI# 58338090. *L. helveticus* DPC 4571 alpha / beta fold family hydrolase LHV1882 (LHV), GI#161508065. *L. plantarum* WCSF1 putative esterase LP2953 (LPL), GI# 28379396. *L. fermentum* IFO 3956 hypothetical protein LAF1318 (LAF), GI# 184155794. *L. reuteri* DSM 20016 alpha/beta fold family hydrolase-like protein LREU1684 (LRE), GI# 148544890. *Butyrivibrio fibrisolvens* E14 cinnamoyl ester hydrolase CinI (BFI-1), GI# 1622732. *B. fibrisolvens* E14 cinnamoyl ester hydrolase CinII (BFI-2), GI# 1765979. *Treponema denticola* ATCC 35405 cinnamoyl ester hydrolase TDE0358 (TDE), GI# 41815924. *Eubacterium ventriosum* ATCC 27560 hypothetical protein EUBVEN\_01801 (EVE), GI# 154484090. Numbers in parentheses indicate X-ray resolution.

In order to prove that the folding of LJ0536 is conserved in LBA-1 and BFI-2, a second prediction was preformed using Est1E or LJ0536 as the template structure. When Est1E was used as the template, the E-value of LBA-1 improved from 2.40E-08 to 2.70E-32. QMEAN Z-Score and QMEANscre4 decreased from -2.414 to -3.495 and from 0.556 to 0.527, respectively. When the prediction was done using LJ0536 as a template, the E-value improved to 1.2E-32, the QMEAN Z-Score decreased to -2.533, and the QMEANscre4 improved to 0.598. A similar scenario was observed when the protein BFI-2 was analyzed. The results indicated that the folding of LJ0536 is conserved in LBA-1 and BFI-2. The overall structure of LJ0536 is conserved among all homologs studied.

#### **4. Conclusion**

FAE application is one of the major fields of study for improving the bioavailability of phenolic acids in food components (phytophenols). After FAE activity on phytophenols in the intestinal tract, released phenolic acids become bioavailable and are absorbed in the intestines and can provide beneficial effects to the host. The identification and crystallization of the first intestinal probiotic bacterium FAE, LJ0536 identified from *L. johnsonii*, provides the fundamental knowledge (protein sequence and structural features) required to further identify FAEs from other species. Using a hypothetical protein LREU1684 as an example, this chapter provides a basic approach on how to identify, purify, and characterize FAEs, predict the model structure, and compare the model with known FAE structures. Further FAE crystallization is required to prove that the structure of LJ0536 is conserved among all homologs.

#### **5. References**


FAE application is one of the major fields of study for improving the bioavailability of phenolic acids in food components (phytophenols). After FAE activity on phytophenols in the intestinal tract, released phenolic acids become bioavailable and are absorbed in the intestines and can provide beneficial effects to the host. The identification and crystallization of the first intestinal probiotic bacterium FAE, LJ0536 identified from *L. johnsonii*, provides the fundamental knowledge (protein sequence and structural features) required to further identify FAEs from other species. Using a hypothetical protein LREU1684 as an example, this chapter provides a basic approach on how to identify, purify, and characterize FAEs, predict the model structure, and compare the model with known FAE structures. Further FAE crystallization is required to prove that the structure of LJ0536 is conserved among all

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**4. Conclusion** 

homologs.

**5. References** 

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Valladares, R., Sankar, D., Li, N., Williams, E., Lai, K., Abdelgeliel, A.*, et al.* (2010). *Lactobacillus johnsonii* N6.2 mitigates the development of type 1 diabetes in BB-DP rats. *PLoS One* 5, e10507.

## **Lectins: To Combat Infections**

Barira Islam and Asad U. Khan

*Interdisciplinary Biotechnology Unit, Aligarh Muslim University Aligarh, India* 

#### **1. Introduction**

166 Protein Purification

Valladares, R., Sankar, D., Li, N., Williams, E., Lai, K., Abdelgeliel, A.*, et al.* (2010).

rats. *PLoS One* 5, e10507.

*Lactobacillus johnsonii* N6.2 mitigates the development of type 1 diabetes in BB-DP

The term "lectin" was coined by William Boyd in 1954 from the Greek word "legere" which means "to select" or "to bind". Lectins and hemagglutinins are proteins/glycoproteins of non-immune origin, which have at least one non-catalytic domain that exhibits reversible binding to specific monosaccharides or oligosaccharides (Lis and Sharon, 1986). The lectinmonosaccharide interactions are relatively very weak and the dissociation constants lie in millimolar range. However, in nature for the multimeric sugars the dissociation constants are several folds higher indicating that multiple protein-carbohyrate interactions are involved in the recognition and binding events (Ambrosi et al., 2005). Thus, lectins are multivalent in nature and can bind to the carbohydrate moieties on the surface of erythrocytes and agglutinate the erythrocytes, without altering the properties of the carbohydrates (Lam and Ng, 2011). Lectins are ubiquitous and are extensively distributed in nature. Many hundreds of these lectins have been isolated from varied sources like plants, viruses, bacteria, invertebrates and vertebrates but in all, lectins from different sources show little similarity. Lectins are invaluable tools for the detection, isolation, and characterization of glycoconjugates, primarily of glycoproteins, for histochemistry of cells and tissues and for the examination of changes that occur on cell surfaces during physiological and pathological processes, from cell differentiation to cancer (Sharon and Lis, 2004).

Cell identification and separation Detection, isolation, and structural studies of glycoproteins Investigation of carbohydrates on cells and subcellular organelles; histochemistry and cytochemistry Mapping of neuronal pathways Mitogenic stimulation of lymphocytesb Purging of bone marrow for transplantationb Selection of lectin-resistant mutants Studies of glycoprotein biosynthesis

a Lectins from sources other than plants are rarely in use. b In clinical use.

Source: Sharon and Lis, 2004

Table 1. Major applications of lectinsa


Source: Van Damme and Peumans, 1996.

Table 2. Classification of lectins


Source: Ambrosi et al., 2005

Table 3. Examples of lectins, the families to which they belong and their glycan ligand specificities

domain. They are small, single-polypeptide proteins that are incapable of agglutinating cells because of their monovalent nature.

binding domains and an unrelated domain with a well-defined biological activity. Based on the number of sugar-binding sites, chimerolectins behave as either merolectins or homolectins.

(T-antigen)

GalNAc

galacturonic acid

GalNAca3GalNAc,

C-type Gal

P-type Man6P

Pentraxins Gal, Gal6P,

Merolectins Proteins that consist exclusively of a single carbohydrate-binding

Hololectins These are composed exclusively of carbohydrate binding domains,

Chimerolectins These are fusion proteins composed of one or more carbohydrate-

**Lectin name Family Glycan ligands** 

Wheat germ agglutinin (WGA; wheat) Gramineae (GlcNAc)1–3, Neu5Ac

Concanavalin A (Con A; jack bean) Leguminosae Man/Glc

*Phaseolus vulgaris* (PHA; French bean) Leguminosae None known Peanut agglutinin (PNA; peanut) Leguminosae Gal, Galb3GalNAca

Soybean agglutinin (SBA; soybean) Leguminosae Gal/GalNAc *Pisum sativum* (PSA; pea) Leguminosae Man/Glc *Lens culinaris* (LCA; lentil) Leguminosae Man/Glc *Galanthus nivalus* (GNA; snowdrop) Amaryllidaceae Man

*Solanum tuberosum* (STA; potato) (GlcNAc)n

Table 3. Examples of lectins, the families to which they belong and their glycan ligand

Galectin-3 galectins Gal Sialoadhesin I-type Neu5Ac

Ricin (castor bean) Euphorbiaceae Gal

but contain at least two such domains.

**Lectin Category Definition** 

Source: Van Damme and Peumans, 1996.

*Dolichos bifloris* (DBA; horse gram)

Asialoglycoprotein receptor

Cation-dependent mannose-6 phosphate receptor (CD-MPR)

Leguminosae

**Animal lectins** 

C-reactive protein

Source: Ambrosi et al., 2005

(CRP)

specificities

(ASGPR) H1

Plant lectins

Table 2. Classification of lectins

Lectins are often classified based on saccharide-specificity. Though this conventional method is familiar and practically useful, it is not necessarily relevant for refined specificity. Lectins in the same category (e.g., galactose-specific lectins) show considerably different sugar-binding preferences. Moreover, an increasing number of lectins which never show high affinity to simple saccharides have been found. They can also be categorized according to the overall structures into merolectins, hololectins, chimerolectins and superlectins, or be grouped into different families (legume lectins, type II ribosome-inactivating proteins, monocot mannose-binding lectins, and other lectins).

The first protein showing inhibition of microorganisms was isolated from wheat flour in 1942 (Balls et al., 1942). However, it was found as late as 1980 by Duguid that *E. coli* possesses the ability to agglutinate erythrocytes and this ability is inhibited by mannose and methyl a-mannoside. Erzler in 1986 reported that lectins in higher plants defend against pathogenic bacteria and fungi by recognizing and immobilizing the infecting microorganisms via binding, thereby preventing their subsequent growth and multiplication. The role of lectins as those of the herbaceous Amaranthus in inhibiting bacteria and fungi has long been known (Bolle et al., 1996). Microbes have lectins that help in recognition and the blocking of these can prevent the infection has been established in mouse models. However, success of such treatments in humans has not been achieved yet (Sharon, 2006). Lately, with the emerging problem of multiple drug resistance, research in characterization of newer lectins to combat infections is gaining momentum.

#### **2. Lectins as tools in cell recognition**

Lectins agglutinate cells and react with the glycoconjugates present on their surface. Cells at different stages of growth and differentiation express different glycoconjugates on their surface and this made lectins an important tool to investigate the cell pathology and physiology. James Sumner in 1919 isolated concanavalin A in crystalline form and in 1936, together with Howell, reported that it agglutinates cells such as erythrocytes and yeasts and that this agglutination is inhibited by sucrose, thus demonstrating for the first time the sugar specificity of lectins. Walter Morgan and Winifred Watkins in the early 1950s used blood type-specific hemagglutinins to show that the blood type A immunodeterminant is α-linked N-acetylgalactosamine and that the H(O) determinant is α-L-fucose. This was the first demonstration that cell surface carbohydrates can serve as carriers of biological information.

Several basic features of membranes were revealed, or their existence confirmed, with the aid of lectins. Singer and Nicolson using ferritin-conjugated concanavalin A and ricin as an electron microscopic probe found that the lectin derivatives bind specifically to the outer surface of the human and rabbit erythrocyte membrane and concluded that the oligosaccharides of the plasma membrane of eukaryotic cells are asymmetrically distributed (1971). Vincent Marchesi used ferritin-labeled phytohemagglutinin (PHA) and showed that glycophorin is oriented in such a way that its carbohydrate-carrying segment is exposed to the external medium, whereas the other segments of the same molecule are embedded in the lipid bilayer or protrude into the cytoplasm (Sharon, 2007).

It was later found that lectin-induced clustering and patching of the corresponding membrane receptors on lymphocytes and other kinds of cell, as illustrated for example by the treatment with fluorescein-labeled concanavalin A of rat or mouse lymphocytes (Inbar and Sachs, 1973). Reorganization of cell surface carbohydrates was later shown to be required for various activities of lectins on cells such as mitogenic stimulation and induction of apoptosis. There is increasing evidence that changes in cellular glycosylation attend alterations in cell behaviour in both normal and in pathological processes, and that this may be of particular interest in malignancy. The malignant cells differ from the normal cells in the distribution of carbohydrates on the outer surface; many of these have an affinity for lectins (Brooks et al., 2001, Kannan et al., 2003). The reports of Inbar and Sachs also proved that lectins agglutinated malignantly transformed cells but not their normal parental cells (1973). They provided compelling evidence that cancer might be associated with a change in cell surface sugars, an idea that only a few years before had been considered completely unfounded. It was also found that SBA (specific for galactose and N-acetylgalactosamine) also possesses the remarkable ability to distinguish between normal and malignant cells (Sharon, 2007). Numerous subsequent studies have demonstrated that high susceptibility to agglutination by lectins is a property shared by many, albeit not all, malignant cells. The herbal *Viscum album* (Mistletoe) lectin (ML-1), has been shown to have antitumoral activity because of its ability to modulate and activate natural killer cells (Joshi, S, 1993). ML-1 also induces apoptosis in myelomonocytic leukemia (Joshi, S et al., 1994). Another herbal medicine *Agaricus bisporus* lectin (ABL), has also been shown to reverse the proliferation of colorectal and breast cancer cells in humans (Yu L et al., 1983).

Bacterial lectins are typically elongated submicroscopic multi-protein appendages, known as fimbriae (or pili) which mediate their adhesion to glycocalyx. Adhesion appears to prevent bacterium removal by intestinal peristalsis, facilitating colonization of the small intestine. Lectins from human pathogens like *E.coli*, *Actinomyces naeslundii*, *C. jejuni*, *E. cloacae*, *H. influenzae*, *H. pylori*, *K. pneumoniae*, *N. gonorrhoea*, *N. meningitides*, *S. mutans* and *P. aeruginosa* with diverse specificities have been isolated and characterized. An individual bacterium may co-express more than one lectin, e.g., certain strains of *E. coli* are both mannose and galabiose specific and those of *H. pylori* recognize simultaneously the tri- and tetrasaccharide. The major function of the enterobacterial surface lectins, as that of similar lectins of other microorganisms, is to mediate the adhesion of the organisms to host cells, an initial stage of infection. This has been extensively demonstrated both in vitro, in studies with isolated cells and cell cultures, and in vivo in experimental animals, and is supported in some cases also by clinical data. It is best documented for *E. coli* type 1 and P fimbriae (Bergsten et al., 2005). P-fimbriae have been shown to enhance the early establishment of *E. coli* in the human urinary tract, and a strong association has been found between the presence of P-fimbriae with disease severity, suggesting that adherence mediated by these organelles has a direct effect on mucosal inflammation in vivo (Sauer et al., 2000). Concerning the type 1 fimbriae, it has been reported that mutants of *E. coli* deficient in Fim H, the carbohydrate-binding subunit of the fimbriae, are unable to cause cystitis in monkeys (Sauer et al., 2000). Attachment of a pathogen to a tissue does not of itself initiate disease. It must be coupled to specific responses that lead to infection. Adherence of P-fimbriated *E. coli* or of the isolated P fimbriae to galabiose of uroepithelial cells induces a two-way flow of biological crosstalk via the lectin bridge, affecting both partners. Following adherence, the target cells are activated, with resultant production of cytokines that engender acute

the treatment with fluorescein-labeled concanavalin A of rat or mouse lymphocytes (Inbar and Sachs, 1973). Reorganization of cell surface carbohydrates was later shown to be required for various activities of lectins on cells such as mitogenic stimulation and induction of apoptosis. There is increasing evidence that changes in cellular glycosylation attend alterations in cell behaviour in both normal and in pathological processes, and that this may be of particular interest in malignancy. The malignant cells differ from the normal cells in the distribution of carbohydrates on the outer surface; many of these have an affinity for lectins (Brooks et al., 2001, Kannan et al., 2003). The reports of Inbar and Sachs also proved that lectins agglutinated malignantly transformed cells but not their normal parental cells (1973). They provided compelling evidence that cancer might be associated with a change in cell surface sugars, an idea that only a few years before had been considered completely unfounded. It was also found that SBA (specific for galactose and N-acetylgalactosamine) also possesses the remarkable ability to distinguish between normal and malignant cells (Sharon, 2007). Numerous subsequent studies have demonstrated that high susceptibility to agglutination by lectins is a property shared by many, albeit not all, malignant cells. The herbal *Viscum album* (Mistletoe) lectin (ML-1), has been shown to have antitumoral activity because of its ability to modulate and activate natural killer cells (Joshi, S, 1993). ML-1 also induces apoptosis in myelomonocytic leukemia (Joshi, S et al., 1994). Another herbal medicine *Agaricus bisporus* lectin (ABL), has also been shown to reverse the proliferation of

Bacterial lectins are typically elongated submicroscopic multi-protein appendages, known as fimbriae (or pili) which mediate their adhesion to glycocalyx. Adhesion appears to prevent bacterium removal by intestinal peristalsis, facilitating colonization of the small intestine. Lectins from human pathogens like *E.coli*, *Actinomyces naeslundii*, *C. jejuni*, *E. cloacae*, *H. influenzae*, *H. pylori*, *K. pneumoniae*, *N. gonorrhoea*, *N. meningitides*, *S. mutans* and *P. aeruginosa* with diverse specificities have been isolated and characterized. An individual bacterium may co-express more than one lectin, e.g., certain strains of *E. coli* are both mannose and galabiose specific and those of *H. pylori* recognize simultaneously the tri- and tetrasaccharide. The major function of the enterobacterial surface lectins, as that of similar lectins of other microorganisms, is to mediate the adhesion of the organisms to host cells, an initial stage of infection. This has been extensively demonstrated both in vitro, in studies with isolated cells and cell cultures, and in vivo in experimental animals, and is supported in some cases also by clinical data. It is best documented for *E. coli* type 1 and P fimbriae (Bergsten et al., 2005). P-fimbriae have been shown to enhance the early establishment of *E. coli* in the human urinary tract, and a strong association has been found between the presence of P-fimbriae with disease severity, suggesting that adherence mediated by these organelles has a direct effect on mucosal inflammation in vivo (Sauer et al., 2000). Concerning the type 1 fimbriae, it has been reported that mutants of *E. coli* deficient in Fim H, the carbohydrate-binding subunit of the fimbriae, are unable to cause cystitis in monkeys (Sauer et al., 2000). Attachment of a pathogen to a tissue does not of itself initiate disease. It must be coupled to specific responses that lead to infection. Adherence of P-fimbriated *E. coli* or of the isolated P fimbriae to galabiose of uroepithelial cells induces a two-way flow of biological crosstalk via the lectin bridge, affecting both partners. Following adherence, the target cells are activated, with resultant production of cytokines that engender acute

colorectal and breast cancer cells in humans (Yu L et al., 1983).

inflammation and other symptoms of disease, while in the bacteria the interaction leads to up-regulation of signal transduction systems that allow responses to the changing environment (Sharon, 2006).

The FimH subunits of both *E. coli* and *K. pneumoniae* are 88% homologous, yet they have different specificities (Gupta et al., 2009). They mediate not only bacterial adhesion, but also invasion of human bladder and intestinal, respectively. In contrast, adhesion mediated by PapG, the lectin subunit of P fimbriae, did not initiate bacterial internalization. *E. coli* strains that cause urinary tract infections are not strictly extracellular pathogens and FimH can directly trigger host cell signalling cascades that lead to bacterial internalization. Type 1 fimbriae are instrumental also in the attachment of *E. coli* to human polymorphonuclear cells and human and mouse macrophages, in the absence of opsonins. This is often followed by the ingestion and killing of the bacteria, a phenomenon named "lectinophagocytosis**"** (Ofek and Sharon, 2000), an early example of innate immunity; it may function in vivo, for example in sites poor in opsonins, and in the peritoneal cavity. Indeed, injection of type 1 fimbriated *E. coli* into the peritoneal cavity of mice led to the activation of the peritoneal macrophages; no activation was observed in the presence of methyl α-mannoside or when non-fimbriated bacteria were used (Bernhard et al., 1992). Enterobacteria can attach by their surface lectins to mast cells as well, with resultant activation of the target cells and production of high levels of certain cytokines, in particular TNF-α (Malviya and Abraham, 2001). Activation of mast cells can also be induced by purified type 1 fimbriae, and by FimH. The cytokines released by the activated mast cells cause rapid recruitment of neutrophils into the site of infection, resulting in early clearance of the bacteria. As expected, mice lacking mast cells were significantly less efficient in clearing intranasal or intraperitoneal infection caused by *K. pneumoniae.*

Specific binding of lectin to Chlamydial cell wall structures is demonstrated by the binding of Galanthus nivalis lectin (GNA). Binding of sialic acid residues to peanut agglutinin (PNA), and jackfruit lectin (JFL), were also found in two Chlamydial glycopeptides (Siridewa, et al., 1993). The study suggests that lectins may be of use as therapeutic agents to keep Chlamydial organisms from entering human cells, thus rendering them more susceptible to immune system elimination.

#### **3. Antibacterial effect of lectins**

#### **3.1 Direct inhibition of lectin**

Quite recently novel lectins are usually tested for any potential antimicrobial activity. A novel galactoside binding lectin from *Bothrops leucurus* snake venom was purified and it exhibited antibacterial effect against the human pathogenic Gram positive bacteria *Staphylococcus aureus*, *Enterococcus faecalis* and *Bacillus subtilis* (Nunes et al., 2011). *Archidendron jiringa* seed lectin showed inhibitory activity against *B. subtilis* and *S. aureus* but did not show any activity against *E.coli* and *P. aeruginosa* (Charungchitrak et al., 2010). Lectins have been islated from serum, plasma, skin mucus and egg of fishes (Jensen et al., 1997;, Ottinger et al., 1999; Dong et al., 2004; Tasumi et al., 2004). A galactose binding lectin has been isolated from Indian catfish *Clarias batrachus*. The lectin agglutinated *E.coli*, *P aeruginosa* and *Klebsiella* strains (Dutta et al., 2005).


Table 4. Lectins with antibacterial activity, Source: Paiva et al., 2010.

#### **3.2 Lectins against the virulence properties of pathogenic bacteria**

The use of lectins in antiadhesion therapy has already been proposed in the literature (Ofek et al., 2003; Mody et al., 2005).This may be of particular importance for controlling diseases where opportunistic pathogens are involved and bacterial adhesion is critical, followed by attainment of sedentary mode of bacterial lifestyle (biofilms) like in oral infections. The acquired enamel pellicle is an organic and acellular film formed by selective adsorption of salivary molecules to the teeth (Yin et al., 2005). Oral bacteria adhere to this pellicle during the initial events of dental plaque formation (Saxton 1973; Yao et al., 2001), a crucial event to dental caries, pathology that represents a health expenditure of several billion dollars per year in the United States alone (Global Oral Health 2006). As bacterial adhesion to the acquired pellicle is one of the primary stages of plaque formation which may lead to caries (Scheie, 1994), it is reasonable to suppose that avoiding adhesion could be a good method to prevent this disease at early stages. Lectins may be good candidates to carry out this approach, as the adherence of bacteria to host cells is, in many cases, mediated by lectin-like adhesins on the bacterial surface that recognize carbohydrate receptors (Ofek and Sharon 1990; Hytonen et al., 2000). Marine algal lectins are especially interesting for biological applications because they have generally lower molecular masses as compared with most land plant lectins. An additional benefit might be that small algal lectin molecules may be expected to be less antigenic than the larger land plant lectins (Rogers and Hori, 1993). Further, they possess great stability on account of their several disulfide bridges and present high specificity for complex carbohydrates and glycoconjugates, especially for mucins (Ainouz et al., 1995; Sampaio et al., 1998; Nagano et al., 2005). The ability of two algal lectins BSL and BTL to bind to the SHA beads and their effectiveness in decreasing the adhesion of streptococci to the pellicle: BSL showed statistically significant results (<0·01), especially for *S. mutans*, whose adhesion was decreased almost totally; while BTL achieved this type of result for only two strains (*S. sobrinus* and *S. mitis*) (Teixeira et al., 2007)

The streptococcal cell wall contains four major antigenic polymers: peptidoglycan, group and type-specific polysaccharides, proteins and the glycerol form of teichoic and lipoteichoic acids. We studied lectins from edible sources and different specificities to the different

**Antibacterial activity** 

*Escherichia coli, Klebsiella sp., Pseudomonas aeruginosa,* 

GlcNAc *B. subtilis, Corynebacterium callunae, E. coli,* 

*Staphylococcus epidermidis,* 

*Streptococcus faecalis.* 

Fru-1,6-P2 *B. subtilis, K. pneumoniae,* 

*S. faecalis*

The use of lectins in antiadhesion therapy has already been proposed in the literature (Ofek et al., 2003; Mody et al., 2005).This may be of particular importance for controlling diseases where opportunistic pathogens are involved and bacterial adhesion is critical, followed by attainment of sedentary mode of bacterial lifestyle (biofilms) like in oral infections. The acquired enamel pellicle is an organic and acellular film formed by selective adsorption of salivary molecules to the teeth (Yin et al., 2005). Oral bacteria adhere to this pellicle during the initial events of dental plaque formation (Saxton 1973; Yao et al., 2001), a crucial event to dental caries, pathology that represents a health expenditure of several billion dollars per year in the United States alone (Global Oral Health 2006). As bacterial adhesion to the acquired pellicle is one of the primary stages of plaque formation which may lead to caries (Scheie, 1994), it is reasonable to suppose that avoiding adhesion could be a good method to prevent this disease at early stages. Lectins may be good candidates to carry out this approach, as the adherence of bacteria to host cells is, in many cases, mediated by lectin-like adhesins on the bacterial surface that recognize carbohydrate receptors (Ofek and Sharon 1990; Hytonen et al., 2000). Marine algal lectins are especially interesting for biological applications because they have generally lower molecular masses as compared with most land plant lectins. An additional benefit might be that small algal lectin molecules may be expected to be less antigenic than the larger land plant lectins (Rogers and Hori, 1993). Further, they possess great stability on account of their several disulfide bridges and present high specificity for complex carbohydrates and glycoconjugates, especially for mucins (Ainouz et al., 1995; Sampaio et al., 1998; Nagano et al., 2005). The ability of two algal lectins BSL and BTL to bind to the SHA beads and their effectiveness in decreasing the adhesion of streptococci to the pellicle: BSL showed statistically significant results (<0·01), especially for *S. mutans*, whose adhesion was decreased almost totally; while BTL achieved this type of

*Bacillus subtilis, Corynebacterium bovis,* 

*Streptococcus sp., Staphylococcus aureus*

*Klebsiella pneumoniae, P. aeruginosa, S. aureus,* 

**Plant (tissue) Lectin** 

*Eugenia uniflora*

*Myracrodruon urundeuva* (heartwood)

*Phthirusa pyrifolia*

(seeds)

(leaf)

**specificity** 

Carbohydrate complex

Table 4. Lectins with antibacterial activity, Source: Paiva et al., 2010.

**3.2 Lectins against the virulence properties of pathogenic bacteria** 

result for only two strains (*S. sobrinus* and *S. mitis*) (Teixeira et al., 2007)

The streptococcal cell wall contains four major antigenic polymers: peptidoglycan, group and type-specific polysaccharides, proteins and the glycerol form of teichoic and lipoteichoic acids. We studied lectins from edible sources and different specificities to the different components of the cell wall of oral pathogen, *Streptococcus mutans*. Lectins from *Canavalia ensiformis* (ConA), *Trigonella foenumgraecum* (TFA), *Triticum aestivum* (WGA), *Arachis hypogaea* (PNA), *Cajanus cajan* (CCL), *Phaseolus vulgaris* (PHA) and *Pisum sativum* (PSA) were tested against the growth and biofilm formation of *S*. *mutans* on saliva coated surface. None of these lectins inhibit the bacterial growth even up to a concentration of 1000mg/ml. However, all the lectins inhibited the biofilm formation by *S.mutans in-vitro*. Amongst these, lectins with Mannose/Glucose (ConA, TFA, CCL and PSA) specificity showed the highest inhibitory effect on the biofilm formation while lectins with N-acetylglucosamine specificity (WGA and PHA) and N-acetylgalactosamine specificity (PNA) also showed inhibition, albeit to a lesser degree (Islam et al., 2009).


Source: Gupta et al., 2009.

Table 5. Carbohydrates as attachment sites for bacterial pathogens on animal tissuesa

A surface glycoprotein of *S.mutans* of 60 kDa (with mannose and N-acetylgalactosamine) has been known to involve in saliva and bacterial interaction. The lesser adherence in the presence of glucose/mannose and galactosamine specific lectins could be because of the interaction with this protein. The PHA and WGA lectin binds to a constituent of the peptidoglycan of the cell wall (Sharon and Lis 2003). The attachment of bacteria is mediated by glucan binding lectin (GBL) and the presence of lectin in the growth media perhaps leads to competition between GBL of bacteria and plant lectins for the attachment sites on salivecoated plates resulting in less binding of the cells. With regard to bacterial surface lectins that often play a role in the initial step of adherence, plant lectins by interfering in this process show a promising future as anti-adherence agents (Islam et al., 2009). A schematic description of how lectins might inhibit attachment of bacteria to the host tissue is shown in Figure 1 (Ghazarian et al., 2011).

Use of bacterial lectin inhibitors such as mannose to prevent the adhesion of *Eschericia coli* to bladder epithelial cells has been employed in clinical practice for some time. Other bioglycans, such as that from *Crenomytalus grayanus* (mussels), has been found to considerably decrease the adhesion of the bacteria *Eschericia coli*, *Staphylococcus aureus* and P*seudomonas aeruginosa* (Zaporozhets et al., 1994). Plant lectins such as those from *Datura stramonium*, *Robinia pseudoacacia* and *Dolichos biflorus* agglutinated Streptococcal Group C bacterial cells (Kellens et al., 1994) which prevents them from adhering to human cell surfaces.

#### **4. Antiviral effect of lectin**

The surfaces of retroviruses such as human immunodeficiency virus (HIV) and many other enveloped viruses are covered by virally-encoded glycoproteins. Glycoproteins gp120 and gp41 present on the HIV envelope are heavily glycosylated, with glycans estimated to contribute almost 50% of the molecular weight of gp120 (Mizuochi et al., 1988; Ji et al., 2006). The antiviral activity of lectins appears to depend on their ability to bind mannosecontaining oligosaccharides present on the surface of viral envelope glycoproteins. Agents that specifically and strongly interact with the glycans may disturb interactions between the proteins of the viral envelope and the cells of the host (Botos & Wlodawer, 2005; Balzarini, 2006). Sugar-binding proteins can crosslink glycans on the viral surface (Sacchettini et al., 2001; Shenoy et al., 2002) and prevent further interactions with the co-receptors. Unlike the majority of current antiviral therapeutics that act through inhibition of the viral life cycle, lectins can prevent penetration of the host cells by the viruses. Antiviral lectins are best suited to topical applications and can exhibit lower toxicity than many currently used antiviral therapeutics. Additionally, these proteins are often resistant to high temperatures and low pH, as well as being odorless, which are favorable properties for potential microbicide drugs. Antiviral activity of a number of lectins that bind high-mannose carbohydrates has been described in the past. Examples of such lectins include jacalin (O'Keefe et al., 1997), concanavalin A (Hansen et al., 1989), Urtica diocia agglutinin (Balzarini et al., 1992), Myrianthus holstii lectin (Charan et al., 2000), and Narcissus pseudonarcissus lectin (Balzarini et al., 1991). However, lectins derived from marine organisms, a rich source of natural antiviral products (Tziveleka et al., 2003), such as CV-N (Boyd et al., 1997), SVN (Bokesch et al., 2003), MVL (Bewley et al., 2004) and GRFT (Mori et al., 2005), exhibit the highest activity among the lectins that have been investigated so far (Ziółkowska NE and Wlodawer A 2006). Some lectins found in algae, such as cyanovirin-N (CV-N) (Boyd et al., 1997; Esser et al., 1999; Barrientos et al., 2003; O'Keefe et al., 2003; Helleet al., 2006); scytovirin (SVN) (Bokesch et al., 2003), Microcystis viridis lectin (MVL) (Bewley et al., 2004), and griffithsin (GRFT) (Mori et al., 2005; Ziółkowska et al., 2006) exhibit significant activity against human immunodeficiency virus (HIV) and other enveloped viruses, which makes them particularly promising targets for the development as novel antiviral drugs (De Clercq, 2005; Reeves & Piefer, 2005)

that often play a role in the initial step of adherence, plant lectins by interfering in this process show a promising future as anti-adherence agents (Islam et al., 2009). A schematic description of how lectins might inhibit attachment of bacteria to the host tissue is shown in

Use of bacterial lectin inhibitors such as mannose to prevent the adhesion of *Eschericia coli* to bladder epithelial cells has been employed in clinical practice for some time. Other bioglycans, such as that from *Crenomytalus grayanus* (mussels), has been found to considerably decrease the adhesion of the bacteria *Eschericia coli*, *Staphylococcus aureus* and P*seudomonas aeruginosa* (Zaporozhets et al., 1994). Plant lectins such as those from *Datura stramonium*, *Robinia pseudoacacia* and *Dolichos biflorus* agglutinated Streptococcal Group C bacterial cells (Kellens et al., 1994) which prevents them from adhering to human cell

The surfaces of retroviruses such as human immunodeficiency virus (HIV) and many other enveloped viruses are covered by virally-encoded glycoproteins. Glycoproteins gp120 and gp41 present on the HIV envelope are heavily glycosylated, with glycans estimated to contribute almost 50% of the molecular weight of gp120 (Mizuochi et al., 1988; Ji et al., 2006). The antiviral activity of lectins appears to depend on their ability to bind mannosecontaining oligosaccharides present on the surface of viral envelope glycoproteins. Agents that specifically and strongly interact with the glycans may disturb interactions between the proteins of the viral envelope and the cells of the host (Botos & Wlodawer, 2005; Balzarini, 2006). Sugar-binding proteins can crosslink glycans on the viral surface (Sacchettini et al., 2001; Shenoy et al., 2002) and prevent further interactions with the co-receptors. Unlike the majority of current antiviral therapeutics that act through inhibition of the viral life cycle, lectins can prevent penetration of the host cells by the viruses. Antiviral lectins are best suited to topical applications and can exhibit lower toxicity than many currently used antiviral therapeutics. Additionally, these proteins are often resistant to high temperatures and low pH, as well as being odorless, which are favorable properties for potential microbicide drugs. Antiviral activity of a number of lectins that bind high-mannose carbohydrates has been described in the past. Examples of such lectins include jacalin (O'Keefe et al., 1997), concanavalin A (Hansen et al., 1989), Urtica diocia agglutinin (Balzarini et al., 1992), Myrianthus holstii lectin (Charan et al., 2000), and Narcissus pseudonarcissus lectin (Balzarini et al., 1991). However, lectins derived from marine organisms, a rich source of natural antiviral products (Tziveleka et al., 2003), such as CV-N (Boyd et al., 1997), SVN (Bokesch et al., 2003), MVL (Bewley et al., 2004) and GRFT (Mori et al., 2005), exhibit the highest activity among the lectins that have been investigated so far (Ziółkowska NE and Wlodawer A 2006). Some lectins found in algae, such as cyanovirin-N (CV-N) (Boyd et al., 1997; Esser et al., 1999; Barrientos et al., 2003; O'Keefe et al., 2003; Helleet al., 2006); scytovirin (SVN) (Bokesch et al., 2003), Microcystis viridis lectin (MVL) (Bewley et al., 2004), and griffithsin (GRFT) (Mori et al., 2005; Ziółkowska et al., 2006) exhibit significant activity against human immunodeficiency virus (HIV) and other enveloped viruses, which makes them particularly promising targets for the development as novel

Figure 1 (Ghazarian et al., 2011).

**4. Antiviral effect of lectin** 

antiviral drugs (De Clercq, 2005; Reeves & Piefer, 2005)

surfaces.

Fig. 1. Representation of bacterial lectins binding to the host cell (left) and specific lectins, used as drug interfering with this bacteria-host interaction (right)

Keyaerts et al., (2007) described the antiviral activity of plant lectins with specificity for different glycan structures against the severe acute respiratory syndrome coronavirus (SARS-CoV) and the feline infectious peritonitis virus (FIPV) in vitro. The SARS-CoV emerged in 2002 as an important cause of severe lower respiratory tract infection in humans, and FIPV infection causes a chronic and often fatal peritonitis in cats. A unique collection of 33 plant lectins with different specificities were evaluated. The plant lectins possessed marked antiviral properties against both coronaviruses with EC50 values in the lower microgram/ml range (middle nanomolar range), being non-toxic (CC50) at 50–100 μg/ml. The strongest anti-coronavirus activity was found predominantly among the mannosebinding lectins. In addition, a number of galactose-, N-acetylgalactosamine-, glucose-, and N-acetylglucosamine-specific plant agglutinines exhibited anti-coronaviral activity. A significant correlation (with an r-value of 0.70) between the EC50 values of the 10 mannosespecific plant lectins effective against the two coronaviruses was found. In contrast, little correlation was seen between the activities of other types of lectins. Two targets of possible antiviral intervention were identified in the replication cycle of SARS-CoV. The first target is located early in the replication cycle, most probably viral attachment, and the second target is located at the end of the infectious virus cycle (Keyaerts et al., 2007).

The carbohydrate binding profile of the red algal lectin KAA-2 from *Kappaphycus alvarezii* was studied by Sato et al (2011). They tested the anti-influenza virus activity of KAA-2 against various strains including the recent pandemic H1N1-2009 influenza virus. KAA-2 inhibited infection of various influenza strains with EC50s of low nanomolar levels. Immunofluorescence microscopy using an anti-influenza antibody demonstrated that the antiviral activity of KAA-2 was exerted by interference with virus entry into host cells. This mechanism was further confirmed by evidence of direct binding of KAA-2 to a viral envelope protein, hemagglutinin (HA), using an ELISA assay. These results indicate that this lectin could be a useful antiviral agent (Sato Y et al., 2011).

#### **5. Antifungal effects of lectins**

Despite the large numbers of lectins and hemagglutinins that have been purified, only a few of them manifested antifungal activity (Table 5). The expression of *Gastrodia elata* lectins in the vascular cells of roots and stems was strongly induced by the fungus *Trichoderma viride*, indicating that lectin is an important defense protein in plants (Sá et al., 2009). Following insertion of the precursor gene of stinging nettle isolectin I into tobacco, the germination of spores of *Botrytis cinerea*, *Colletotrichum lindemuthianum*, and *T. viride* was significantly reduced (Does et al., 1999). Thus, lectins may be introduced into plants to protect them from fungal attack.

Plant lectins can neither bind to glycoconjugates on the fungal membranes nor penetrate the cytoplasm owing to the cell wall barrier. It is not likely that lectins directly inhibit fungal growth by modifying fungal membrane structure and/or permeability. However, there may be indirect effects produced by the binding of lectins to carbohydrates on the fungal cell wall surface. Chitinase-free chitin-binding stinging nettle (*Urtica dioica* lectin) impeded fungal growth. Cell wall synthesis was interrupted because of attenuated chitin synthesis and/or deposition (Van Parijs et al., 1991). The effects of nettle lectin on fungal cell wall and hyphal morphology suggest that the nettle lectin regulates endomycorrhizal colonization of the rhizomes. Severa1 other plant lectins inhibit fungal growth. The first group includes small chitin-binding merolectins with one chitin-binding domain, e.g., hevein from rubber tree latex (Van Parijs et al., 1991) and chitin-binding polypeptide from *Amaranthus caudatus* seeds (Broekaert et al., 1992). The only plant lectins that can be considered as fungicidal proteins are the chimerolectins belonging to the class I chitinases. However, the antifungal activity of these proteins is ascribed to their catalytic domain.

#### **6. Lectins and the immune system**

To initiate immune responses against infection, the surface receptors on antigen presenting cells must recognise the corresponding molecules on infectious agents. Pattern recognition receptors (PRR) which include C-type lectin like receptor (CLR) recognise and interact with carbohydrate moieties of many pathogens. Despite the presence of a highly conserved domain, C-type lectins are functionally diverse and have been implicated in various processes including cell adhesion, tissue integration and remodelling, platelet activation, complement activation, pathogen recognition, endocytosis, and phagocytosis.

The carbohydrate binding profile of the red algal lectin KAA-2 from *Kappaphycus alvarezii* was studied by Sato et al (2011). They tested the anti-influenza virus activity of KAA-2 against various strains including the recent pandemic H1N1-2009 influenza virus. KAA-2 inhibited infection of various influenza strains with EC50s of low nanomolar levels. Immunofluorescence microscopy using an anti-influenza antibody demonstrated that the antiviral activity of KAA-2 was exerted by interference with virus entry into host cells. This mechanism was further confirmed by evidence of direct binding of KAA-2 to a viral envelope protein, hemagglutinin (HA), using an ELISA assay. These results indicate that

Despite the large numbers of lectins and hemagglutinins that have been purified, only a few of them manifested antifungal activity (Table 5). The expression of *Gastrodia elata* lectins in the vascular cells of roots and stems was strongly induced by the fungus *Trichoderma viride*, indicating that lectin is an important defense protein in plants (Sá et al., 2009). Following insertion of the precursor gene of stinging nettle isolectin I into tobacco, the germination of spores of *Botrytis cinerea*, *Colletotrichum lindemuthianum*, and *T. viride* was significantly reduced (Does et al., 1999). Thus, lectins may be introduced into plants to protect them from

Plant lectins can neither bind to glycoconjugates on the fungal membranes nor penetrate the cytoplasm owing to the cell wall barrier. It is not likely that lectins directly inhibit fungal growth by modifying fungal membrane structure and/or permeability. However, there may be indirect effects produced by the binding of lectins to carbohydrates on the fungal cell wall surface. Chitinase-free chitin-binding stinging nettle (*Urtica dioica* lectin) impeded fungal growth. Cell wall synthesis was interrupted because of attenuated chitin synthesis and/or deposition (Van Parijs et al., 1991). The effects of nettle lectin on fungal cell wall and hyphal morphology suggest that the nettle lectin regulates endomycorrhizal colonization of the rhizomes. Severa1 other plant lectins inhibit fungal growth. The first group includes small chitin-binding merolectins with one chitin-binding domain, e.g., hevein from rubber tree latex (Van Parijs et al., 1991) and chitin-binding polypeptide from *Amaranthus caudatus* seeds (Broekaert et al., 1992). The only plant lectins that can be considered as fungicidal proteins are the chimerolectins belonging to the class I chitinases. However, the antifungal

To initiate immune responses against infection, the surface receptors on antigen presenting cells must recognise the corresponding molecules on infectious agents. Pattern recognition receptors (PRR) which include C-type lectin like receptor (CLR) recognise and interact with carbohydrate moieties of many pathogens. Despite the presence of a highly conserved domain, C-type lectins are functionally diverse and have been implicated in various processes including cell adhesion, tissue integration and remodelling, platelet activation, complement activation, pathogen recognition,

this lectin could be a useful antiviral agent (Sato Y et al., 2011).

activity of these proteins is ascribed to their catalytic domain.

**6. Lectins and the immune system** 

endocytosis, and phagocytosis.

**5. Antifungal effects of lectins** 

fungal attack.



Table 6. Examples of lectins with antifungal activity, Source: Lam and Ng, 2011

#### **6.1 Mannose Receptor**

The MR binds a broad array of microorganisms, including *Candida albicans, Pneumocystis carinii*, *Leishmania donovani*, *Mycobacterium tuberculosis*, and capsular polysaccharides of *Klebisella pneumoniae* and *Streptococcus pneumonia* (Chakraborty et al., 2001; Ezekowitz et al., 1991; Marodi et al., 1991; O'Riordan et al., 1995; Schlesinger, 1993; Zamze et al., 2002). The receptor recognises mannose, fucose or N-acetylglucosamine sugar residues on the surfaces of these microorganisms (Largent et al., 1984) and carbohydrate recognition is mediated by CTLDs 4–8 (Taylor et al., 1992). The MR has been implicated in the phagocytic uptake of pathogens, but there are limited examples actually demonstrating MR-dependent phagocytosis.

#### **6.2 Dectin-1**

Dectin-1 is a type II transmembrane protein that is classified as a Group V non-classical Ctype lectin and lacks the conserved residues involved in the ligation of calcium that are usually required to co-ordinate carbohydrate binding. Dectin-1 was initially identified as a dendritic cell specific receptor that modulates T cell function through recognition of an unidentified ligand (Ariizumi et al., 2000; Grunebach et al., 2002). It was subsequently reidentified as a receptor for β-glucans, which are carbohydrate polymers found primarily in the cell walls of fungi, but also in plants and some bacteria (Brown and Gordon, 2001, 2003). Dectin-1 can recognise a number of fungal species, including *C. albicans*, *P. carinii*, *Saccharomyces cerevisiae*, *Coccidioides posadasii* and *Aspergillus fumigatus* (Brown et al., 2003; Gersuk et al., 2006; Saijo et al., 2007; Steele et al., 2003, 2005; Taylor et al., 2007; Viriyakosol et al., 2005).The ligation of Dectin-1 also triggers intracellular signalling resulting in a variety of cellular responses, including phagocytosis.

#### **6.3 DC-SIGN (CD209)**

DC-SIGN is a type II transmembrane protein that is classified as a Group II C-type lectin. DC-SIGN was originally identified as a receptor for intercellular adhesion molecule-3 (ICAM-3) that facilitates DC-mediated T-cell proliferation and binds HIV-1 (Geijtenbeek et al., 2000a, b). It has since been reported that the receptor interacts with a range of pathogens, including *M. tuberculosis*, *C. albicans*, *Helicobacter pylori*, *Schistosoma mansoni* and *A. fumigatus* (Appelmelk et al., 2003; Cambi et al., 2008; Geijtenbeek et al., 2000b, 2003; Serrano-Gomez et al., 2004; Tailleux et al., 2003; van Die et al., 2003). There have been no reports of a

**Natural source of lectin Fungal species inhibited Sugar specificity Reference** 

Not found Ghosh

2009

al.,2009

*Fusarium moniliforme, Macrophomina phaseolina*

*Zea mays* (maize) endosperm *Aspergillus flavus* D(+)galactose Baker et

The MR binds a broad array of microorganisms, including *Candida albicans, Pneumocystis carinii*, *Leishmania donovani*, *Mycobacterium tuberculosis*, and capsular polysaccharides of *Klebisella pneumoniae* and *Streptococcus pneumonia* (Chakraborty et al., 2001; Ezekowitz et al., 1991; Marodi et al., 1991; O'Riordan et al., 1995; Schlesinger, 1993; Zamze et al., 2002). The receptor recognises mannose, fucose or N-acetylglucosamine sugar residues on the surfaces of these microorganisms (Largent et al., 1984) and carbohydrate recognition is mediated by CTLDs 4–8 (Taylor et al., 1992). The MR has been implicated in the phagocytic uptake of pathogens, but there are limited examples actually demonstrating MR-dependent

Dectin-1 is a type II transmembrane protein that is classified as a Group V non-classical Ctype lectin and lacks the conserved residues involved in the ligation of calcium that are usually required to co-ordinate carbohydrate binding. Dectin-1 was initially identified as a dendritic cell specific receptor that modulates T cell function through recognition of an unidentified ligand (Ariizumi et al., 2000; Grunebach et al., 2002). It was subsequently reidentified as a receptor for β-glucans, which are carbohydrate polymers found primarily in the cell walls of fungi, but also in plants and some bacteria (Brown and Gordon, 2001, 2003). Dectin-1 can recognise a number of fungal species, including *C. albicans*, *P. carinii*, *Saccharomyces cerevisiae*, *Coccidioides posadasii* and *Aspergillus fumigatus* (Brown et al., 2003; Gersuk et al., 2006; Saijo et al., 2007; Steele et al., 2003, 2005; Taylor et al., 2007; Viriyakosol et al., 2005).The ligation of Dectin-1 also triggers intracellular signalling resulting in a variety

DC-SIGN is a type II transmembrane protein that is classified as a Group II C-type lectin. DC-SIGN was originally identified as a receptor for intercellular adhesion molecule-3 (ICAM-3) that facilitates DC-mediated T-cell proliferation and binds HIV-1 (Geijtenbeek et al., 2000a, b). It has since been reported that the receptor interacts with a range of pathogens, including *M. tuberculosis*, *C. albicans*, *Helicobacter pylori*, *Schistosoma mansoni* and *A. fumigatus* (Appelmelk et al., 2003; Cambi et al., 2008; Geijtenbeek et al., 2000b, 2003; Serrano-Gomez et al., 2004; Tailleux et al., 2003; van Die et al., 2003). There have been no reports of a

Table 6. Examples of lectins with antifungal activity, Source: Lam and Ng, 2011

*Withania somnifera*  (Ashwagandha/Indian ginseng/Winter

cherry/Ajagandha/Kanaje Hindi/Amukkuram) leaves

**6.1 Mannose Receptor** 

phagocytosis.

**6.2 Dectin-1** 

of cellular responses, including phagocytosis.

**6.3 DC-SIGN (CD209)** 

mechanism for DC-SIGN mediated phagocytosis. However, activation of DC-SIGN triggers Rho-GTPase (Hodges et al., 2007) making it conceivable that Rho could be involved in phagocytosis mediated by this receptor.

#### **6.4 Mannose-binding lectin (MBL)**

Mannose-binding lectin (MBL) is a Group III C-type lectin belonging to the collectins (Holmskov et al., 2003), which are a group of soluble oligomeric proteins containing collagenous regions and CTLDs. MBL is secreted into the blood stream as a large multimeric complex and is primarily produced by the liver, although other sites of production, such as the intestine, have been proposed (Uemura et al., 2002). It recognises carbohydrates such as mannose, glucose, l-fucose, N-acetyl-mannosamine (ManNAc), and N-acetyl-glucosamine (GlcNAc). Oligomerisation of MBL enables high avidity binding to repetitive carbohydrate ligands, such as those present on a variety of microbial surfaces, including *E. coli*, *Klebisella aerogenes*, *Neisseria meningitides*, *Staphylococcus aureus*, *S. pneumoniae*, *A. fumigatus* and *C. albicans* (Davies et al., 2000; Neth et al., 2000; Schelenz et al., 1995; Tabona et al., 1995; van Emmerik et al., 1994).MBL has also been proposed to function directly as an opsonin by binding to carbohydrates on pathogens and then interacting with MBL receptors on phagocytic cells, promoting microbial uptake and stimulating immune responses (Kuhlman et al., 1989). It was shown in a recent study that MBL modifies cytokine responses through a novel cooperation with TLR2/6 in the phagosome (Ip et al., 2008).

#### **7. Lectins and drug delivery**

The concept of lectin-mediated specic drug delivery was proposed by Woodley and Naisbett in 1988 (Bies et al., 2004). Delivery of targeted therapeutics via direct and reverse drug delivery systems (DDS) to specic sites provides numerous advantages over traditional non-targeted therapeutics (Rek et al., 2009). Targeted drug delivery increases the efcacy of treatment by enhancing drug exposure to targeted sites while limiting side effects of drugs on normal and healthy tissues (Rek et al., 2009). Furthermore, specic drug delivery increases the uptake and internalization of therapeutics that have reduced cellular permeability (Rek et al., 2009). Lectin based drug-targeting can be done in two ways. In the first approach, carbohydrate moieties form a part of DDS. The carbohydrate tag drives the drug to the endogenous lectins present on the cell surface. In the second approach, lectins are present on the drug surface and it interacts with the glycosylated surfaces of the cells (Gabor et al., 2004). Considering the fact that epithelial cells contain a thin layer of mucus which has mucins that are highly glycosylated proteins, the lectin-encapsulated drug strategy offers great potential. As non-specific interactions are susceptible to changes in pH and to interactions with food digesta, which probably reduce the mucoadhesive effect, specific mucoadhesiva of the second generation seem to be preferable. The second target is the glycocalyx of the absorptive epithelium. In case of identical oligosaccharide structures of the mucin and the glycocalyx, partitioning of the formulation to the cell surface is facilitated due to full reversibility of the mucin–lectin interaction. In case of lectin-matching carbohydrates only at the glycocalyx, the formulation has to penetrate the mucuos layer. Both pathways result in fixation of the drug delivery system closer to the site of absorption. That way cytoadhesion will increase the concentration gradient between the extracellular and intracellular compartment, which facilitates at least passive diffusion of the drug into the cell. The third target is represented by glycosylated receptors at the cell membrane. The binding of some lectins, such as WGA to the EGF-receptor, induces active receptor mediated endocytosis, which can improve cytoinvasion of prodrugs as well as nanoscaled carrier systems (Gabor, 2004).

In an approach towards pulmonary delivery, lectinised liposomes (130–170 nm in diameter) were screened for binding to alveolar type II epithelial cells (Bruck et al., 2001). As compared to plain liposomes, the binding to A549 cells increased 6–11-fold upon surface modification with wheat germ agglutinin (WGA), Concanavalin A (ConA) or soybean agglutinin. The binding was not affected by a synthetic lung surfactant and no cytotoxic effect of the free lectins or the lectinised liposomes was observed. Upon incubation with primary cultured human alveolar epithelial cells, which exhibit barrier functions, the WGAliposomes were not only bound but also taken up into the cells. In search for non-viral vectors for gene therapy of cystic fibrosis and as a basis for lectin-mediated gene transfer, 32 lectins were screened for binding and uptake into living human airway epithelium (Yi et al., 2001). Whereas ConA was internalised within 1 h, the lectins from *Erythrina cristagalli* and *Glycine max*, peanut lectin, and Jacalin were taken up into the epithelium within 4 h. The endocytosis of WGA was minimal even after 4 h. Irrespective of the specificity of the lectin– carbohydrate interaction; the internalised lectins exhibited a non-selective binding pattern on the epithelium. Only peanut lectin bound to subpopulations of ciliated and non-ciliated cells.

Owing to their remarkable specificities, plant lectins with affinities for the carbohydrates on microbial cell surface are already well characterised. Given the potential of porphyrins to act as antimicrobials it is pertinent to ask whether lectins could be used *in vivo* to specifically deliver porphyrins into pathogenic microbial cells, thereby improving the efficacy of the treatment, reducing the concentration of the drug required to be introduced into the system and thereby reducing the possible side-effects. In particular, lectins could be successful oral and mucosal drug delivery agents. Not only are a large number of lectins part of our everyday diet, but also several of them are known to survive the harsh conditions of human gastro-intestinal tract. Similarly, attempts have been made to use lectins in ocular drug delivery. Specific hydrophobic binding sites on lectins provide the ideal opportunity to expand the use of these molecules in targeted therapy (Komath et al., 2006).

#### **8. Conclusions**

Lectins are ubiquitous in nature and have garnered much attention due to specificity of its interaction with the carbohydrates. Glycosylation is a key step in many cellular processes and with more reports about the change in cell-surface carbohydrates in different pathological conditions, research about exploiting lectins as a therapeutic tool is now at the forefront. Lectins are now routinely used in the identification and purification of glycoproteins. Their use in blood typing as well as in clinical diagnostics is well established. Many lectins show antibacterial, antiviral or antifungal activities in-vitro. However, clinical trials need to be done for establishing their therapeutic effect and optimising their dosage delivery. As microbes use their surface lectins for attachment to the host tissue, dietary/therapeutic lectins may interfere in this interaction. Thus lectins can be used antiadhesion agents and prevent the colonization of the microbe and hence the establishment of the infection. In the immune system, endogenous lectins play a role in ligand recognition and hence are an important component of the host's defense against microbes. Given their

the cell. The third target is represented by glycosylated receptors at the cell membrane. The binding of some lectins, such as WGA to the EGF-receptor, induces active receptor mediated endocytosis, which can improve cytoinvasion of prodrugs as well as nanoscaled carrier

In an approach towards pulmonary delivery, lectinised liposomes (130–170 nm in diameter) were screened for binding to alveolar type II epithelial cells (Bruck et al., 2001). As compared to plain liposomes, the binding to A549 cells increased 6–11-fold upon surface modification with wheat germ agglutinin (WGA), Concanavalin A (ConA) or soybean agglutinin. The binding was not affected by a synthetic lung surfactant and no cytotoxic effect of the free lectins or the lectinised liposomes was observed. Upon incubation with primary cultured human alveolar epithelial cells, which exhibit barrier functions, the WGAliposomes were not only bound but also taken up into the cells. In search for non-viral vectors for gene therapy of cystic fibrosis and as a basis for lectin-mediated gene transfer, 32 lectins were screened for binding and uptake into living human airway epithelium (Yi et al., 2001). Whereas ConA was internalised within 1 h, the lectins from *Erythrina cristagalli* and *Glycine max*, peanut lectin, and Jacalin were taken up into the epithelium within 4 h. The endocytosis of WGA was minimal even after 4 h. Irrespective of the specificity of the lectin– carbohydrate interaction; the internalised lectins exhibited a non-selective binding pattern on the epithelium. Only peanut lectin bound to subpopulations of ciliated and non-ciliated cells. Owing to their remarkable specificities, plant lectins with affinities for the carbohydrates on microbial cell surface are already well characterised. Given the potential of porphyrins to act as antimicrobials it is pertinent to ask whether lectins could be used *in vivo* to specifically deliver porphyrins into pathogenic microbial cells, thereby improving the efficacy of the treatment, reducing the concentration of the drug required to be introduced into the system and thereby reducing the possible side-effects. In particular, lectins could be successful oral and mucosal drug delivery agents. Not only are a large number of lectins part of our everyday diet, but also several of them are known to survive the harsh conditions of human gastro-intestinal tract. Similarly, attempts have been made to use lectins in ocular drug delivery. Specific hydrophobic binding sites on lectins provide the ideal opportunity to

expand the use of these molecules in targeted therapy (Komath et al., 2006).

Lectins are ubiquitous in nature and have garnered much attention due to specificity of its interaction with the carbohydrates. Glycosylation is a key step in many cellular processes and with more reports about the change in cell-surface carbohydrates in different pathological conditions, research about exploiting lectins as a therapeutic tool is now at the forefront. Lectins are now routinely used in the identification and purification of glycoproteins. Their use in blood typing as well as in clinical diagnostics is well established. Many lectins show antibacterial, antiviral or antifungal activities in-vitro. However, clinical trials need to be done for establishing their therapeutic effect and optimising their dosage delivery. As microbes use their surface lectins for attachment to the host tissue, dietary/therapeutic lectins may interfere in this interaction. Thus lectins can be used antiadhesion agents and prevent the colonization of the microbe and hence the establishment of the infection. In the immune system, endogenous lectins play a role in ligand recognition and hence are an important component of the host's defense against microbes. Given their

systems (Gabor, 2004).

**8. Conclusions** 

ability to specifically target different cell types, they have always been looked upon as useful candidates for targeted drug delivery. Research utilizing lectins as carriers of monoclonal antibodies or specific chemotherapeutic agents has been conducted. Alongwith the beneficial effect, lectins have been reported to have caused severe allergic reactions. Most of the information on the acute toxicity of lectins in humans has been derived from observations of incidences of accidental poisoning. Since no experimental data is available to show the possible adverse effects of lectins on humans but can be inferred from experiments with laboratory animals. Although results obtained with mice, rats or pigs cannot simply be extrapolated to humans, the observed effects on the gut and other organs of these animals demonstrate the possible toxicity of the lectins. Thus lectin-based therapeutics for combating infections is very promising owing to its highly selective nature, provided the dosage is well below the toxic limits.

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