**5. Structure of a sialo-oligosaccharide from carp RBC membranes**

We examined the amino acid composition of carp GP followed by the kind of sialic acid. While the amino acid composition was not strikingly different compared to that of human GP A, with the exception of valine, lysine and arginine, only the *N*-glycolylneuraminic acid (NeuGc) form of sialic acid was detected in the carp GP by TLC and a colorimetric method [36].

There are several reports on the sialic acid component of mammals sources of GP. In humans, GP contains *N*-acetylneuraminic acid (NeuAc), whereas the presence of NeuGc has been reported in GPs of horses [37], bovine [38], pig [39], and monkeys (*Macaca fuscata*) [19] and others. However, to date, little is known about sialic acid in teleost GPs. Compared to the sialic acids from nonhuman sources, this result suggested that teleosts contained the NeuGc, not as NeuAc. Interestingly, NeuGc has also been reported in the eggs of rainbow trout, chum salmon and land-locked cherry salmon [40].

The carbohydrate fraction of carp GP was separated into two components (P-1 and P-2) using a Glyco-Pak DEAE column with a continuous linear gradient of 0–100 mM NaCl. This fraction contained at least two kinds of O-linked oligosaccharides. Based on the chromatogram obtained using a NeuAc oligomer (α,2→8), the electro-negativity suggested that P-1 contained one sialic acid residue, whereas P-2 contained two residues [36].

We obtained *ca.* 190 μg P-1 and *ca.* 7.0 μg P-2 (as total carbohydrate) by HPLC from carp GP (*ca.* 4.0 mg protein). Using the graphite carbon column with ammonium bicarbonate in acetonitrile solution as an eluent, the yield of desalted oligosaccharide was satisfactory (P-1: *ca.* 90% and P-2: *ca.* 100%) [36].

These O-linked oligosaccharides (P-1 and P-2) were composed of glucose, galactose, fucose, *N*-acetylgalactosamine (GalNAc) and NeuGc. Using NMR and GC–MS, we determined that the structures of P-1 and P-2 were NeuGcα2→6(Fucα1→4) (Glcα1→3) Galβ1→4GalNAc-ol [41] and NeuGcα2→6(Fucα1→4)(Glcα1→3) (NeuGcα2→2) Galβ1→4GalNAc-ol, respectively (**Figure 5**). These O-linked oligosaccharides were unique to vertebrates with respect to the hexosamine and hexose linkages and their nonchain structure.

Human GPs contain O-linked sialo-oligosaccharides, and the structure of these oligosaccharides has been analysed [42]. The most commonly elucidated GP oligosaccharides from mammals sources are reported as below: tetra-saccharide core, NeuAcα2→3Galβ1→3(NeuAcα2→6)GalNAc-ol; tri-saccharide cores, Galβ1→3(NeuAcα2→6)GalNAc-ol or NeuAcα2→3Galβ1→3GalNAc-ol (**Figure 5**). *Behaviour of a Sialo-Oligosaccharide from Glycophorin in Teleost Red Blood Cell Membranes DOI: http://dx.doi.org/10.5772/intechopen.107234*

O-linked oligosaccharides containing NeuGc have also been reported among horse, pig, and rabbit GPs, and the most commonly reported structure is a trisaccharide, Galβ1→3(NeuGcα2→6) GalNAc-ol [20]. Other derivatives are synthesized by attaching NeuGc and Gal residues to the trisaccharide core [28].

Although Glc residue in O-linked oligosaccharides has not been reported to detect in mammalian [20] and chicken GPs [43], Guérardel et al. [44] reported that *O*-glycans synthesized by nematodes contained the Glc residue, whereas the Fuc residue was detected in the O-linked oligosaccharides of human GP A [32].

From the NMR spectra obtained using the asialo P-1 fraction, the characterized proton signals revealed an overall downfield shift in the resonance of αGlc and αFuc, except for the H-1 signals [41]. This O-linked oligosaccharide indicates a globule form rather than chain-like structure. Furthermore, the linkage between Gal and GalNAc-ol is 1→4, unlike the 1→3 standard linkage for O-linked oligosaccharides. The 1→4 linkage of GalNAc is unique compared with other O-linked oligosaccharides of mammals sources of GP. Interestingly, the glycosidic linkage of xylan from the seaweed cell wall is the β1→3 unlike the standard β1→4 linkage of xylan from land plants [45]. It seems possible to detect the β1→4 linkage of GalNAc in marine organisms.

### **6. Physiological activity of GPs from carp, yellow tail and red sea bream**

We performed a sensitivity test using GP preparations from the carp RBC membranes. The sensitivity test for the growth of test bacteria was performed using the disc-plate method [36, 46]. All of the test bacteria (gram-positive bacteria: *Micrococcus luteus* and *Bacillus subtilis*, gram-negative bacteria: *Aeromonas hydrophila*, *Vibrio anguillarum*, *Pseudomonas fluorescens*, *Edwardsiella tarda* and *Escherichia coli*) formed inhibition zones around the paper disc containing the GP fraction (**Figure 6**). For *E. tarda*, the inhibition zones were observed over a light box to discern the production of FeS from SS agar medium (**Figure 6**f). *M. luteus and E. coli* produced yellow pigments and white pigments, respectively (**Figure 6**d and g). While the outer zone of *M. luteus and E. coli* did not produce pigments, the inner zone represented growth inhibition. In contrast, the inhibition zone was not formed around the paper discs containing PBS (**Figure 6**g).

To clarify the physiological activity of teleost fish GPs other than those from carp, we performed a sensitivity test for the growth of *M. luteus* using GP preparations from the RBC membranes of yellow tail and red sea bream (**Figure 7**) [47]. These results showed that not only carp GP preparations but also yellow tail and red sea bream GPs had antibiotic activities*.*

Compared with the profile of forming an inhibition zone, these results also suggested that the yellow tail or red sea bream GPs have a broad-spectrum antibiotic activity similar to that of carp GP*.* While carp are freshwater fish, yellow tail and red sea bream are marine red-flesh fish and white-flesh fish, respectively. Thus, it is assumed that the antimicrobial activity of sialo-origosaccharide from GP is not confined to these teleost species but can be found in all fish.

Then, we examined which GP fraction demonstrates bacteriostatic activity by using a sensitivity test [36]. The carp RBC membrane preparation, GP preparation, carbohydrate and P-1 fractions also exhibited bacteriostatic activity (**Figure 8**a–f). The P-2 fraction exhibited bacteriostatic activity within the area of the disc paper (**Figure 8**e and f). In contrast, the inhibition zones were not observed using the GP

#### **Figure 6.**

*Sensitivity test of the carp GP fraction for the growth of several bacteria by the disc-plate method. (a) Carp GP fraction (ca. 15 μg·protein/disc) to B. subtilis; (b) GP fraction (ca. 15 μg·protein/disc) to A. hydrophila; (c) GP fraction (ca. 15 μg·protein/disc) to V. anguillarum; (d) GP fraction (ca. 15 μg·protein/disc) to M. luteus; (e) GP fraction (ca. 15 μg·protein/disc) to P. fluorescens; (e) GP fraction (ca. 15 μg·protein/disc) to P. fluorescens; (f) GP fraction (ca. 15 μg·protein/disc) to E. tarda; (g) GP fraction (ca. 15 μg·protein/disc) to E. coli (left disc; GP fraction, right disc; PBS). A paper disc (8 mm diameter) containing each fraction was placed on the medium and incubated at 20°C. After 24–48 h, the inhibition zone was observed on each plate.*

fraction that lacked sialic acid or the human GP. These results suggest that the test bacteria are sensitive to monosialyl-oligosaccharides from teleost GPs.

Based on electron microscope observations [36], the carp GP molecules attach to the flagellum of *V. anguillarum* rather than the cell itself (**Figure 9**a). Conversely, the GP molecules attach to the cell surface (contained cleavage line) on *M. luteus* (**Figure 9**b). Carp GP exists with the size of various molecules and has a diameter of 40–220 nm from TEM images (**Figure 9**c-3). It seems that the smallest GP molecules selectively possessed bacteriostatic activity.

*Behaviour of a Sialo-Oligosaccharide from Glycophorin in Teleost Red Blood Cell Membranes DOI: http://dx.doi.org/10.5772/intechopen.107234*

#### **Figure 7.**

*Sensitivity test for M. luteus by the disc-plate method. (a) GP preparation from carp (ca. 15 μg protein/disc). GP, carp GP; control, PBS; (b) GP preparation from yellow tail (ca. 10 μg protein/disc). GP, yellow tail GP; control, PBS; (c) GP preparation from red sea bream (ca. 10 μg protein/disc). GP, red sea bream GP; control, PBS.*

#### **Figure 8.**

*Sensitivity test for the growth of E. tarda and M. luteus. (a) Carp RBC membranes (ca. 5 mg·protein/disc); (b) carp GP fraction without streptomycin treatment (ca. 17 μg·protein/disc); (c) carp GP fraction (ca. 15 μg·protein/ disc); (d) carbohydrate fraction from carp GP (ca. 4 μg/disc); (e, f) P-1 and P-2 fractions (ca. 8 μg/disc each); upper left disc, P-1; upper right disc, P-2; lower disc, PBS. (a–e) Plates containing E. tarda; (f) plate containing M. luteus.*

#### **Figure 9.**

*Electron microscope images of the bacteria and carp GP. (a) V. anguillarum and carp GP. 1, V. anguillarum without carp GP; 2, V. anguillarum with carp GP. An equal volume of glycophorin solution (ca. 0.4 μg·protein/20 μL) was added to the cell suspension (ca. 3 × 106 cfu/20 μL) at 25°C. (b) M. luteus and carp GP. 1, M. luteus without carp GP; 2, M. luteus with carp GP. An equal volume of glycophorin solution (ca. 0.4 μg·protein/20 μL) was added to the cell suspension (ca. 3 × 106 cfu/20 μL) at 25°C. (c) Carp GP. 1, SEM image under the same conditions of V. anguillarum with carp GP; 2, SEM image under the same conditions of M. luteus with carp GP; 3, TEM image.*

### **7. Behaviour of a sialo-oligosaccharide from GP in RBC membranes**

These bacteriostatic activities of teleost GP are caused by the contained monosialyl-oligosaccharide and are attributed to the property of the lectin receptor. It is supposed that some lectin-like proteins exist on the surface of gram-positive bacteria or the component of flagellum of gram-negative bacteria. Based on the obtained observations, (1) the teleost GPs are released from RBC membranes and aggregated with each other by hydrophobic areas within the protein moiety of GP. (2) The sialooligosaccharides are exposed on the outer layer of the aggregated GP molecules. (3) Aggregated GP molecules are adsorbed onto the surface or the flagellum of invading bacteria in the blood plasma. (4) The bacteria attached to GP molecules will be led to a bacteriostatic state (**Figure 10**) [28].

The bacteriostatic activity of sialo-oligosaccharides from carp GP is attributed to pentose formation. This may be related to the bacteriostatic activity caused by the penta- or hexa-saccharides obtained from chitin [48]. In the bacteriostatic reaction by teleost GP, it is supposed that the size of the oligosaccharide corresponds to that of the cleft occurs in the lectin-like protein and also might contribute to the negative charge of sialic acid. In teleost blood, IgG does not exist unlike human blood, and other antibodies exist at low levels [49]. It is suggested that GP may exist as a substitute for antibodies such as IgG in teleost blood on the immune system. Although the physiological function of human GP has not yet been clarified, the structure of human GP's O-linked tetra-oligosaccharide is a simpler form than that of carp's pentose. And NeuAc in human GP is also simpler than carp's NeuGc. IgG is considered a major component in the human immune system, and the bacteriostatic activity of human GPs has been lost in the process of evolution.

*Behaviour of a Sialo-Oligosaccharide from Glycophorin in Teleost Red Blood Cell Membranes DOI: http://dx.doi.org/10.5772/intechopen.107234*

#### **Figure 10.**

*Schematic representation of the teleost GP interaction with invading bacteria in fish blood.*
