**4. Comparison of the relative catalytic activity of Abs from different MS patients**

#### **4.1. Abzymes with DNase and RNase activities**

Anti-DNA Abs are detectable even in the sera of healthy mammals, but their titters vary sig‐ nificantly [59]. In comparison with healthy donors, concentrations of anti-DNA antibodies are higher in patients with rheumatoid arthritis (7% of patients) [59], myasthenia gravis (6%), Sjogren's syndrome (18%), multiple sclerosis (17–18%), primary Hashimoto's thyroidi‐ tis (23%), and systemic lupus erythematosus (SLE, 36%). Anti-DNA Abs are directed against histone-DNA nucleosomal complexes appearing in the circulation from internucleosomal cleavage during apoptosis in many SLE patients [60].

Healthy humans do not develop Abzs with detectable DNase activity, their levels being usually on the borderline of sensitivity of the detection methods [13–21]. The RAs of Abs from the sera of MS patients vary markedly from patient to patient [54]. **Figure 3A** illustrates cleavage of plasmid DNA by Abs from sera of ten MS patients after 2 h of incubation. During this time, some Abs cause only single breaks in one strand of plasmid supercoiled DNA converting it to the relaxed form (lanes 1–3), whereas others cause multiple breaks causing DNA linearization (lanes 4–6). The most active Abs hydrolyze DNA into short- and medium-length oligonucleo‐ tides (lanes 7–10). Relative activity (RA) of different MS IgG preparations was first estimated using arbitrary units (a/u) from 0 to 10 a/u (**Figure 3A**).

IgGs from 53 of 55 MS patients (~96%) demonstrated detectable RAs from 1 to 10 a/u; the average values of IgG DNase activity were 5 ± 4 a/u. MS Abzs hydrolyze single- and doublestranded DNAs of different sequences and length [52, 54–56]. The affinity of Abzs with DNase activity for DNA is usually high (1–10 nM) and corresponds to the typical affinity of Abs for nucleic acids and ~104- and 105-fold higher than that for canonical DNases [54–56].

Recently, we have shown, for the first time, that average concentration of total proteins (132 fold), total IgGs (194-fold), and anti-DNA antibodies (1986-fold; **Figure 3B**) in the sera (average value 437 ± 311 A450 units) is significantly higher than that in the CSF (average value 0.22 ± 0.008 A450 units, **Figure 3B**) of fifteen MS patients [52]. In the sera and CSFs, the relative activities of total protein varied remarkably from patient to patient. Specific DNase activity of the total protein of CSF reparations were surprising approximately 198-fold higher than the serum ones. We present first evidence showing that IgGs from CSF not only bind but efficiently hydrolyze DNA and that average specific DNase activity of homogeneous Autoimmune Processes in Multiple Sclerosis: Production of Harmful Catalytic Antibodies Associated with Significant Changes in the Hematopoietic Stem Cell Differentiation and Proliferation http://dx.doi.org/10.5772/63824 107

antibodies from CSF (543.3 ± 239.7 pmole DNA/1 mg of Ab/1 h) is unpredictably ~49-fold higher than that from the sera (average value 11.2±4.3 pmole DNA/1 mg of Ab/1 h) of the same MS patients (**Figure 3C**) [52].

**Figure 3.** DNase activities of catalytic IgGs from sera of ten different patients with MS in the cleavage of supercoiled (sc) and nicked (n) plasmid DNA (A). Lanes 1–10, IgGs from the sera of 10 different patients; C1, scDNA incubated alone; C2 and C3, scDNA incubated with Ab from the sera of two healthy donors [54, 55]. Relative content (A450 units) of anti-DNA IgGs determined by ELISA in serum (the right hand axis) and in CSF (the left hand axis) preparations of fifteen MS patients (B) and RAs of polyclonal IgGs purified from these fifteen serum and CSF preparations in the hy‐ drolysis of supercoiled DNA (C) [52].

#### **4.2. Abzymes with protease activity**

protease, and amylase Abzs from MS patients are located on the light chains of these Abs [48– 53, 54, 56–58]. Intact proteins usually interact with both light and heavy chains of Abzs, thus ensuring the specificity of the target protein recognition and its cleavage. Overall, it is obvious that nuclease, amylase, and MBP-hydrolyzing activities are intrinsic properties of Abzs from sera and CSF of MS patients and do not due to the admixture of any possible canonical

**4. Comparison of the relative catalytic activity of Abs from different MS**

Anti-DNA Abs are detectable even in the sera of healthy mammals, but their titters vary sig‐ nificantly [59]. In comparison with healthy donors, concentrations of anti-DNA antibodies are higher in patients with rheumatoid arthritis (7% of patients) [59], myasthenia gravis (6%), Sjogren's syndrome (18%), multiple sclerosis (17–18%), primary Hashimoto's thyroidi‐ tis (23%), and systemic lupus erythematosus (SLE, 36%). Anti-DNA Abs are directed against histone-DNA nucleosomal complexes appearing in the circulation from internucleosomal

Healthy humans do not develop Abzs with detectable DNase activity, their levels being usually on the borderline of sensitivity of the detection methods [13–21]. The RAs of Abs from the sera of MS patients vary markedly from patient to patient [54]. **Figure 3A** illustrates cleavage of plasmid DNA by Abs from sera of ten MS patients after 2 h of incubation. During this time, some Abs cause only single breaks in one strand of plasmid supercoiled DNA converting it to the relaxed form (lanes 1–3), whereas others cause multiple breaks causing DNA linearization (lanes 4–6). The most active Abs hydrolyze DNA into short- and medium-length oligonucleo‐ tides (lanes 7–10). Relative activity (RA) of different MS IgG preparations was first estimated

IgGs from 53 of 55 MS patients (~96%) demonstrated detectable RAs from 1 to 10 a/u; the average values of IgG DNase activity were 5 ± 4 a/u. MS Abzs hydrolyze single- and doublestranded DNAs of different sequences and length [52, 54–56]. The affinity of Abzs with DNase activity for DNA is usually high (1–10 nM) and corresponds to the typical affinity of Abs for

Recently, we have shown, for the first time, that average concentration of total proteins (132 fold), total IgGs (194-fold), and anti-DNA antibodies (1986-fold; **Figure 3B**) in the sera (average value 437 ± 311 A450 units) is significantly higher than that in the CSF (average value 0.22 ± 0.008 A450 units, **Figure 3B**) of fifteen MS patients [52]. In the sera and CSFs, the relative activities of total protein varied remarkably from patient to patient. Specific DNase activity of the total protein of CSF reparations were surprising approximately 198-fold higher than the serum ones. We present first evidence showing that IgGs from CSF not only bind but efficiently hydrolyze DNA and that average specific DNase activity of homogeneous

nucleic acids and ~104- and 105-fold higher than that for canonical DNases [54–56].

enzymes.

106 Trending Topics in Multiple Sclerosis

**patients**

**4.1. Abzymes with DNase and RNase activities**

cleavage during apoptosis in many SLE patients [60].

using arbitrary units (a/u) from 0 to 10 a/u (**Figure 3A**).

It was shown that IgGs from sera of healthy donors do not hydrolyze MBP [13–21]. We have compared the RAs of IgGs, IgAs, and IgMs from sera of 35 MS patients in the hydrolysis of MBP (**Figures 4A** and **B**) [48, 49]. Specific activities of IgGs from sera of any single patient were usually significantly lower than those of IgMs and sIgAs. Specific inhibitors of thiol and acidic proteases had a weak effect on protease activity of IgGs and IgMs. But, specific inhibitors of serine proteases (PMSF and AEBSF) significantly suppressed proteolytic activity of the Abzs. IgGs, IgMs, and IgAs hydrolyze specifically human MBP, but not many other tested proteins [48, 49].

The sera of healthy donors demonstrated ~four- and fivefold lower concentration of anti-MBP concentration (average value 0.08 ± 0.05 A450 units) than that for MS patients (average value 0.32 ± 0.08 A450 units) [43, 44]. We used ELISA to compare the relative levels of Abs against MBP in the sera and CSFs of 15 MS patients (**Figure 4C**) [53]. The average relative content of anti-MBP Abs in the sera of MS patients is approximately 230-fold higher than in the corre‐ sponding CSFs (**Figure 4C**). We present first evidence showing that IgGs from CSF efficiently hydrolyze MBP and that their average specific catalytic activity is unpredictably ~54-fold higher than that of Abs from sera of the same MS patients (**Figure 4D**) [53].

**Figure 4.** The relative activities (RA) of polyclonal IgGs and IgMs from the sera of 25 different MS patients (A) and of IgGs, IgMs, and IgAs from the sera of other 10 MS patients (B) in the hydrolysis of MBP. The RAs were determined from decrease of initial MBP; the relative intensity of the main bands of MBP incubated alone for 3 h was taken as 100% [49]. Relative content (A450 units) of anti-MBP IgGs determined by ELISA in serum (the right hand axis) and in CSF (the left hand axis) preparations of fifteen MS patients (C) and RAs of polyclonal IgGs purified from these fifteen preparations of biological fluids in the hydrolysis of supercoiled MBP (D) [53].

IgGs and sIgAs from breast milk demonstrated high ATPase activity [61]. It has recently been shown that during spontaneous development of a profound SLE-like pathology in MRLlpr/lpr mice leads to a production of DNase, protease, ATPase, and amylase Abzs [22–24]. Interestingly, individual IgGs from sera and CSF of MS patients did not possess detectable ATPase activity [62].

The sera of healthy donors demonstrated ~four- and fivefold lower concentration of anti-MBP concentration (average value 0.08 ± 0.05 A450 units) than that for MS patients (average value 0.32 ± 0.08 A450 units) [43, 44]. We used ELISA to compare the relative levels of Abs against MBP in the sera and CSFs of 15 MS patients (**Figure 4C**) [53]. The average relative content of anti-MBP Abs in the sera of MS patients is approximately 230-fold higher than in the corre‐ sponding CSFs (**Figure 4C**). We present first evidence showing that IgGs from CSF efficiently hydrolyze MBP and that their average specific catalytic activity is unpredictably ~54-fold

**Figure 4.** The relative activities (RA) of polyclonal IgGs and IgMs from the sera of 25 different MS patients (A) and of IgGs, IgMs, and IgAs from the sera of other 10 MS patients (B) in the hydrolysis of MBP. The RAs were determined from decrease of initial MBP; the relative intensity of the main bands of MBP incubated alone for 3 h was taken as 100% [49]. Relative content (A450 units) of anti-MBP IgGs determined by ELISA in serum (the right hand axis) and in CSF (the left hand axis) preparations of fifteen MS patients (C) and RAs of polyclonal IgGs purified from these fifteen

preparations of biological fluids in the hydrolysis of supercoiled MBP (D) [53].

higher than that of Abs from sera of the same MS patients (**Figure 4D**) [53].

108 Trending Topics in Multiple Sclerosis

IgGs and IgMs from sera of patients with several autoimmune diseases [57, 58, 63–66] and sIgAs from human breast milk [67] possess amylase activity; however, the maximal activity was observed for Abs from sera of patients with MS [57, 58, 63] and SLE [63, 65, 66]. Individual IgGs and IgMs isolated from patients with MS and SLE had approximately three orders of magnitude higher specific amylolytic activity than that for healthy donors [57, 58, 63–66].

The ability of the presence of MBP- and DNA-hydrolyzing antibodies in the cerebrospinal fluid of MS patients may be related to the fact that anti-MBP and anti-DNA Abs may play an important role in the pathogenesis of this disease. Anti-MBP Abzs can attack MBP of the myelin-proteolipid shell of axons [48–51]. In addition, DNase Abzs of MS patients [16, 21] similarly to SLE patients [68] are cytotoxic and induce cell apoptosis, which can play an important role in SLE and MS pathogenesis. At the same time, the involvement of antibodies with amylase activity in the pathogenesis of any autoimmune diseases has not yet been identified. We have recently shown the first unpredictable evidence showing that IgGs from CSF (average value 9.0 ± 4.9 μM/1 h/mg) possess amylase activity and efficiently hydrolyze maltoheptaose; their average specific Ab activity is ~30-fold higher than that of antibodies from sera (average value 0.30 ± 0.14 μM/1 h/mg) of the same MS patients [62]. Specific average RA for IgGs from healthy volunteers was approximately ~1000 lower than that for MS patients [57, 58]. It was shown that a relative RA of total proteins of CSF (including Abs) is ~15-fold lower than that for purified IgGs, while the RAs of the total sera protein is higher than that of sera IgGs by a factor of 1033. This result speaks in favor of the fact that amylolytic activity of CSF proteins is mainly caused by the activity of amylase Abzs. One cannot exclude that amylase Abzs of CSF can also play a, as yet unknown, role in the pathogenesis of MS.

As it was mentioned above, the correlation coefficients between titters of Abs to DNA and to MBP and 13 different standard clinical parameters, including Poser criteria in the case of 49 patients with MS, were very low [21, 44]. We have not revealed high correlation coefficients between different RAs characterizing IgGs of CSF and serum as well as RAs of these IgGs and total proteins corresponding CSF and serum. All CCs varied from −0.03 to +0.18 except unpredicted correlation of RAs for IgGs from serum and total protein corresponding to CSF (CC = 0.7). Similar situation was observed earlier for CCs between RAs of these IgGs and total proteins corresponding CSF and serum in the hydrolysis of DNA (CCs = −0.05 − +0.03) [52]. In addition, we calculated a possible correlation between RAs characterizing 15 IgGs in the hydrolysis of DNA [52], MBP [53], and oligosaccharide [62] (**Table 1**). All CCs were relatively low (−0.009 − +0.2) except some of them: IgG amylase activity of CSF correlates with MBPhydrolyzing activity of serum (CC = +0.41) and CSF (CC = +0.45); DNase activity of serum IgGs correlates with MBP-hydrolyzing activity of serum (CC = +0.44) and CSF (CC = +0.61), as well as MBP-hydrolyzing activity of serum and CSF (CC = +0.59). However, all these correlation coefficients were very low or relatively low. Thus, an additional question is why there is no good correlation between various indexes, characterizing different MS patients. As mentioned above, each patient can be characterized by an individual combination of genetic, environ‐ mental, chronic, inflammatory, autoimmune, demyelinating, neurodegenerative, and other factors [46, 47]. This can be the main reason for the lack of a good correlation between the different indicators of MS development by various patients. However, taking all data together, it is reasonable to propose that MBP-, DNA-, and polysaccharide-hydrolyzing Abzs can probably cooperatively promote important neuropathological mechanisms in MS.


\* Parameters 1 and 2 [62], 3 and 4 [52] and 5 and 6 [53]. Here the same 15 preparations of IgGs from the same MS patients were compared.

**Table 1.** Correlation coefficients between relative activities of 15 IgGs from sera and CSFs of MS patients in the hydrolysis of oligosaccharide, DNA, and MBP.

#### **4.3. Extreme diversity of MS Abzs**

The extreme diversity of RNase and DNase activity of IgG and/or IgM Abzs from the sera of autoimmune patients and also of MRL-lpr/lpr mice was observed [13–21, 69–76]. It was demonstrated that different patients (and animals) may have an extremely large a relatively small content of polyclonal nuclease Abzs containing different relative amounts of kappa and lambda light chains and demonstrating at various optimal pHs maximal activity, possessing a different net charge, dependent or not on different metal ions, and characterized by different substrate specificities.

Different auto-Abs, including Abs to DNA, and different DNA-dependent enzymes can interact with DNA [13–21, 77, 78]. DNase, RNase, and MBP-hydrolyzing IgGs from the sera of autoimmune patients are usually very heterogeneous in their affinity for these substrates and can be separated into many fractions by chromatography on DNA-cellulose and MBP-Sepharose ([13–21] and refs therein). MS Abs with DNase [54–56] and MBP-hydrolyzing [43] activities were distributed all over the profiles of chromatographies (e.g., **Figure 5A**). The affinity of abzyme fractions for these substrates (in terms of Km values) was increased gradually with the increase in eluting salts concentrations. In contrast to MS IgGs, Abzs from SLE patients are more sensitive to EDTA (**Figure 5B** and **C**) and less sensitive to specific inhibitors of serine-like proteases. Similar data of extreme diversity of DNase activity of IgGs were observed in the case of other autoimmune diseases and autoimmune mice [13–21, 75]. All these data are indicative of the extreme diversity of Abzs with different activities in their affinity for specific substrates and in their relative specific activities.

Autoimmune Processes in Multiple Sclerosis: Production of Harmful Catalytic Antibodies Associated with Significant Changes in the Hematopoietic Stem Cell Differentiation and Proliferation http://dx.doi.org/10.5772/63824 111

good correlation between various indexes, characterizing different MS patients. As mentioned above, each patient can be characterized by an individual combination of genetic, environ‐ mental, chronic, inflammatory, autoimmune, demyelinating, neurodegenerative, and other factors [46, 47]. This can be the main reason for the lack of a good correlation between the different indicators of MS development by various patients. However, taking all data together, it is reasonable to propose that MBP-, DNA-, and polysaccharide-hydrolyzing Abzs can

probably cooperatively promote important neuropathological mechanisms in MS.

**DNase activity of sera IgGs**

\* Parameters 1 and 2 [62], 3 and 4 [52] and 5 and 6 [53]. Here the same 15 preparations of IgGs from the same MS

**Table 1.** Correlation coefficients between relative activities of 15 IgGs from sera and CSFs of MS patients in the

The extreme diversity of RNase and DNase activity of IgG and/or IgM Abzs from the sera of autoimmune patients and also of MRL-lpr/lpr mice was observed [13–21, 69–76]. It was demonstrated that different patients (and animals) may have an extremely large a relatively small content of polyclonal nuclease Abzs containing different relative amounts of kappa and lambda light chains and demonstrating at various optimal pHs maximal activity, possessing a different net charge, dependent or not on different metal ions, and characterized by different

Different auto-Abs, including Abs to DNA, and different DNA-dependent enzymes can interact with DNA [13–21, 77, 78]. DNase, RNase, and MBP-hydrolyzing IgGs from the sera of autoimmune patients are usually very heterogeneous in their affinity for these substrates and can be separated into many fractions by chromatography on DNA-cellulose and MBP-Sepharose ([13–21] and refs therein). MS Abs with DNase [54–56] and MBP-hydrolyzing [43] activities were distributed all over the profiles of chromatographies (e.g., **Figure 5A**). The affinity of abzyme fractions for these substrates (in terms of Km values) was increased gradually with the increase in eluting salts concentrations. In contrast to MS IgGs, Abzs from SLE patients are more sensitive to EDTA (**Figure 5B** and **C**) and less sensitive to specific inhibitors of serine-like proteases. Similar data of extreme diversity of DNase activity of IgGs were observed in the case of other autoimmune diseases and autoimmune mice [13–21, 75]. All these data are indicative of the extreme diversity of Abzs with different activities in their

affinity for specific substrates and in their relative specific activities.

**Parameter number 1 2 3\* 4\* 5\* 6\*** Correlation coefficient 1/2: 0.22 1/3: 0.03 1/4: - 0.009 1/5: - 0.13 1/6: 0.1 2/3: 0.22

**DNase activity of CSF IgGs**

2/4: 0.03 2/5: 0.41 2/6: 0.45 3/4: 0.11 3/5: 0.44 3/6: 0.61 4/5: 0.34 4/6: 0.11 5/6: 0.59 − − −

**MBP-hydrolyzing activity of plasma**

**MBP-hydrolyzing activity of CSF**

**IgGs**

**IgGs**

**Amylase activity of CSF IgGs**

**Number of patient Amylase**

110 Trending Topics in Multiple Sclerosis

patients were compared.

substrate specificities.

hydrolysis of oligosaccharide, DNA, and MBP.

**4.3. Extreme diversity of MS Abzs**

**activity of sera IgGs**

**Figure 5.** Affinity chromatography of MS IgGs on DNA cellulose: (–), absorbance at 280 nm; columns, relative DNase activity (A) [54, 56]. Affinity chromatography of mixtures of IgGs from sera of 15 SLE (B) and 15 MS (C) patients on MBP-Sepharose [43]: (—), absorbance at 280 nm; the relative catalytic activities (RA) of IgGs of different fractions in the absence (□) and in the presence of EDTA (■) was measured. Depending on the RA, the reaction mixtures were incu‐ bated for 0.3–16 h in the presence of 0.01–0.1 mg/ml IgGs and then the RAs were normalized to the standard condi‐ tions: the complete transition oligopeptide corresponding to MBP and DNA to their products was taken for 100%.

Theoretically, the human immune system can produce up to 106 specificities of Abs against one antigen. Affinity chromatography data (**Figure 5**) do not give possibility to estimate a

possible number of monoclonal DNase antibodies that correspond to the polyclonal IgGs, eluting at various Ab peaks. For this evaluation, we have used the analysis of possible number of monoclonal Abs with DNase activity [79–81]. An immunoglobulin kappa light chain library from blood of SLE patients was cloned into a phagemid vector. Phage parti‐ cles displaying recombinant monoclonal light chains (MLChs) capable of binding DNA were isolated by affinity chromatography on DNA cellulose. The phage particles contain‐ ing MLChs bound with DNA-cellulose were distributed between eleven peaks (11 different fractions) eluted during chromatography, and all fractions obtained were active in the hy‐ drolysis of DNA. The fraction eluted by 0.5 M NaCl (peak 7) and an acidic buffer (pH 2.6; peak 11) were used for preparation of individual monoclonal light chains (MLChs, ~28 kDa). Then, 45 of 451 and 33 of 687 individual colonies corresponding respectively to peaks 7 and 11 were randomly chosen for study of MLCh DNase activity [79–81]. Fourteen of 45 and nineteen of 33 clones, respectively, contained MLChs with DNase activity. Totally, all 33 of 78 purified MLChs (42%) containing no canonical DNases demonstrated various en‐ zymatic properties including different kcat values, one or two pH optima, were inactive af‐ ter dialysis against EDTA, but could be activated by several externally added metal ions; the ratio of relative activity in the presence of Mn2+, Ca2+, Mg2+, Ni2+, Zn2+, Cu2+, and Co2+ was individual for each MLCh, with Mn2+, Mg2+, and Co2+ being among the best activators of their DNase activity [79–81]. In the presence of Mn2+ or Mg2+, the DNase activity of some MLChs did not require K+ or Na+ , while the others could be activated by KCl or NaCl at optimal concentrations from 1 to 100 mM. It should be mentioned that not only Abs from sera of patients with different ADs containing kappa, but also lambda light chains possess DNase activity [13–21]. In addition, we have analyzed MLChs corresponding only to two of eleven peaks obtained [79–81], while Abs of all 11 peaks were catalytically active. It means that the sera of SLE (and MS) patients in principle can contain some hundreds of catalytic DNase lambda- and kappa-IgGs with different catalytic properties.

In order to compare the substrate specificity of Abzs, the hydrolysis of various oligonucleo‐ tides by MS IgGs was analyzed [55]. The oligonucleotides (ODNs) cleavage patterns varied from patient to patient. Some Abs demonstrated sequence-independent hydrolysis of ODNs, while other produced in parallel both 5′-phosphate terminated products (similar to those of DNase I) or 3′-phosphate terminated ODNs, which are typical for DNase II. Several IgGs demonstrate sequence-dependent manner, while for other the products correspond to both 3′- and 5′-exonuclease activities. The hydrolysis of ODNs by different IgGs was also strongly dependent on the reaction conditions used including addition of MgCl2, EDTA, in‐ crease in NaCl or potassium phosphate concentration [55]. All the data obtained show that MS IgGs can demonstrate different combinations of endo- and exonuclease activities and properties of the MS DNase IgGs distinguished them from all known canonical DNases [55]. The extreme diversity of RNase and DNase IgG and/or IgM Abzs in their affinity to DNA (and in kcat values) was also revealed for several other autoimmune diseases [13–21, 55, 68, 69, 76].

Autoimmune Processes in Multiple Sclerosis: Production of Harmful Catalytic Antibodies Associated with Significant Changes in the Hematopoietic Stem Cell Differentiation and Proliferation http://dx.doi.org/10.5772/63824 113

possible number of monoclonal DNase antibodies that correspond to the polyclonal IgGs, eluting at various Ab peaks. For this evaluation, we have used the analysis of possible number of monoclonal Abs with DNase activity [79–81]. An immunoglobulin kappa light chain library from blood of SLE patients was cloned into a phagemid vector. Phage parti‐ cles displaying recombinant monoclonal light chains (MLChs) capable of binding DNA were isolated by affinity chromatography on DNA cellulose. The phage particles contain‐ ing MLChs bound with DNA-cellulose were distributed between eleven peaks (11 different fractions) eluted during chromatography, and all fractions obtained were active in the hy‐ drolysis of DNA. The fraction eluted by 0.5 M NaCl (peak 7) and an acidic buffer (pH 2.6; peak 11) were used for preparation of individual monoclonal light chains (MLChs, ~28 kDa). Then, 45 of 451 and 33 of 687 individual colonies corresponding respectively to peaks 7 and 11 were randomly chosen for study of MLCh DNase activity [79–81]. Fourteen of 45 and nineteen of 33 clones, respectively, contained MLChs with DNase activity. Totally, all 33 of 78 purified MLChs (42%) containing no canonical DNases demonstrated various en‐ zymatic properties including different kcat values, one or two pH optima, were inactive af‐ ter dialysis against EDTA, but could be activated by several externally added metal ions; the ratio of relative activity in the presence of Mn2+, Ca2+, Mg2+, Ni2+, Zn2+, Cu2+, and Co2+ was individual for each MLCh, with Mn2+, Mg2+, and Co2+ being among the best activators of their DNase activity [79–81]. In the presence of Mn2+ or Mg2+, the DNase activity of some

optimal concentrations from 1 to 100 mM. It should be mentioned that not only Abs from sera of patients with different ADs containing kappa, but also lambda light chains possess DNase activity [13–21]. In addition, we have analyzed MLChs corresponding only to two of eleven peaks obtained [79–81], while Abs of all 11 peaks were catalytically active. It means that the sera of SLE (and MS) patients in principle can contain some hundreds of

In order to compare the substrate specificity of Abzs, the hydrolysis of various oligonucleo‐ tides by MS IgGs was analyzed [55]. The oligonucleotides (ODNs) cleavage patterns varied from patient to patient. Some Abs demonstrated sequence-independent hydrolysis of ODNs, while other produced in parallel both 5′-phosphate terminated products (similar to those of DNase I) or 3′-phosphate terminated ODNs, which are typical for DNase II. Several IgGs demonstrate sequence-dependent manner, while for other the products correspond to both 3′- and 5′-exonuclease activities. The hydrolysis of ODNs by different IgGs was also strongly dependent on the reaction conditions used including addition of MgCl2, EDTA, in‐ crease in NaCl or potassium phosphate concentration [55]. All the data obtained show that MS IgGs can demonstrate different combinations of endo- and exonuclease activities and properties of the MS DNase IgGs distinguished them from all known canonical DNases [55]. The extreme diversity of RNase and DNase IgG and/or IgM Abzs in their affinity to DNA (and in kcat values) was also revealed for several other autoimmune diseases [13–21, 55, 68,

catalytic DNase lambda- and kappa-IgGs with different catalytic properties.

, while the others could be activated by KCl or NaCl at

MLChs did not require K+ or Na+

112 Trending Topics in Multiple Sclerosis

69, 76].

**Figure 6.** Complete sequence of human MBP (on the top); the positions of OP17, OP19, OP21, and OP25 sequences in the human MBP sequence are shown in bold. All sites of cleavage of X-OP17 (B–C) and X-OP19 (E–F) (X is fluorescent label of OPs), corresponding to detectable major and minor products of these OPs digestion after their mild (B and E) and deep (C and F) hydrolysis by equimolar mixture of 15 SLE IgGs are shown [80]. Panels A and D show the major cleavage sites, which were found previously in the case of hydrolysis of globular intact MBP by MS and SLE IgGs [43, 78]; trypsin hydrolyzes these OPs at the same sites. All sites of the OP21 (I) and OP25 (L) corresponding to major and moderate products of the cleavage are shown by long and short arrows respectively, while corresponding to minor ones by diamonds [81]. Panels G and J show all possible sites of these OPs cleavage by trypsin, while panels H and K demonstrate the major cleavage sites of MBP, which were found previously in the case of hydrolysis of globular intact MBP by MS and SLE IgGs [43, 78]. Clustered major and minor sites of cleavage are underlined.

Anti-MBP Abzs of MS patients' cleavage MBP at several sites corresponding to four known immunodominant regions of human MBP [82]. It was demonstrated that anti-MBP Abzs of SLE patients hydrolyze MBP at the same four immunodominant regions of MBP [43]. Four different encephalytogenic peptides corresponding to known antigenic determinants (AGDs) of MBP can play a negative role in the MS and SLE pathogenesis [82]. Therefore, it was interesting to study the specific sequences of MBP in more detail. Interestingly, MS and SLE anti-MBP IgGs hydrolyze nonspecific tri- and tetrapeptides with an extremely low efficiency and they cannot efficiently cleavage longer oligopeptides corresponding AGDs of HIV-1 integrase [83]. To identify all MBP cleavage sites corresponding to anti-MBP IgGs in the case of four AGDs of MBP, we have used a combination of MALDI mass spectrometry, reversephase chromatography, and thin layer chromatography for revealing of the cleavage products of four (17-, 19-, 21, and 25-mer) encephalytogenic oligopeptides corresponding to these AGDs [84, 85]. Several clustered major and minor sites of cleavage were revealed in the case of all oligopeptides (**Figure 6**). It was shown that the number of oligopeptide cleavage sites is greater than that of intact MBP. The active sites of anti-MBP Abzs are located on their light chains, while the heavy chains are responsible for Abs increased affinity for protein substrates. Interactions of both light and heavy chains of Abzs with intact globular proteins provide high affinity and specificity of intact MBP hydrolysis. The affinity of anti-MBP Abzs for oligopep‐ tides was ~103-fold lower than that for the intact MBP. These data indicate that a relatively short oligopeptides interact mainly with the light chain of different monoclonal Abzs of total pool of IgGs, which possesses lower affinity for substrates, and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific [84, 85].

IgGs and IgMs from the sera of patients with MS and SLE were found to possess amylolytic activity hydrolyzing α—(1-4)-glucosyl linkages of maltooligosaccharides, glycogen, and several synthetic substrates [57, 58, 63–67]. The specific amylolytic activity of individual IgGs and IgMs from MS patients was about three orders of magnitude higher than that of healthy donors [57, 58]. Fractions of auto-Abs from human milk [67] and from sera of patients with different autoimmune pathologies [57, 58 63–65] revealed different modes of action in the hydrolysis of maltooligosaccharides, p-nitrophenyl maltooligosaccharides, p-nitrophenyl, and α-D-glucopyranoside; several samples of MS Abzs demonstrated β-xylosidase activity, which is not observed in known mammalian polysaccharide-hydrolyzing enzymes.

Proteolytic Abs from the sera of patients with various autoimmune diseases [9, 86] caseinhydrolyzing Abzs from human milk [87], casein-, human serum albumin-, and HIV reverse transcriptase-hydrolyzing Abs from AIDS patients [88] are serine-like proteases, and their activity is most strongly reduced by specific serine protease inhibitors PMSF or AEBSF. Specific inhibitors of acidic and thiol proteases demonstrated a weak effect on proteolytic activity of MS polyclonal IgGs and IgMs [48–51]. However, specific inhibitors of serine proteases significantly inhibited proteolytic activity. In addition, MS polyclonal IgGs contained several chelated metals and the relative amount of which decreases in the order: Fe2+ > Ca2+ > Cu2+ > Zn2+ > Mg2+ > Mn2+ > Co2+ > Ni2+ [50]. After removing of Mg2+ ions by dialysis against EDTA, MS IgGs have not completely lost proteolytic activity, which was increased after addition of different external Me2+ ions. After chromatography of MS IgGs on Chelex-100, a minor metalAutoimmune Processes in Multiple Sclerosis: Production of Harmful Catalytic Antibodies Associated with Significant Changes in the Hematopoietic Stem Cell Differentiation and Proliferation http://dx.doi.org/10.5772/63824 115

dependent fraction did not cleavage MBP in the absence Mg2+ ions, but it was activated after addition of metal ions: Ca2+ < Cu2+ < Mn2+ < Mg2+ [50]. Protease activities of several MS IgGs were also activated by other metal ions (Ni2+, Fe2+, Co2+, Zn2+, Pb2+, and Co2+) and especially Ni2+. Observed properties of MS Abzs distinguish them from other known mammalian metalloproteases and demonstrate their pronounced catalytic diversity. Metal-dependent IgG from MS patients was the first example of Abzs with metal-dependent proteolytic activity [50]. Later, we have revealed an important metal-dependent casein-hydrolyzing sIgA from human milk [89] and MBP-hydrolyzing IgGs from SLE patients [43]. We have recently shown that anti-integrase IgGs and IgMs from AIDS patients can contain Abzs cleaving HIV integrase by small subfractions resembling thiol, serine, acidic, and metal-dependent proteases, the ratio of which may be individual for every HIV-infected patient [90, 91]. Since anti-MBP from sera of MS and SLE patients are very similar, we have analyzed in more details monoclonal Abzs from SLE patients [92, 93].

Anti-MBP Abzs of MS patients' cleavage MBP at several sites corresponding to four known immunodominant regions of human MBP [82]. It was demonstrated that anti-MBP Abzs of SLE patients hydrolyze MBP at the same four immunodominant regions of MBP [43]. Four different encephalytogenic peptides corresponding to known antigenic determinants (AGDs) of MBP can play a negative role in the MS and SLE pathogenesis [82]. Therefore, it was interesting to study the specific sequences of MBP in more detail. Interestingly, MS and SLE anti-MBP IgGs hydrolyze nonspecific tri- and tetrapeptides with an extremely low efficiency and they cannot efficiently cleavage longer oligopeptides corresponding AGDs of HIV-1 integrase [83]. To identify all MBP cleavage sites corresponding to anti-MBP IgGs in the case of four AGDs of MBP, we have used a combination of MALDI mass spectrometry, reversephase chromatography, and thin layer chromatography for revealing of the cleavage products of four (17-, 19-, 21, and 25-mer) encephalytogenic oligopeptides corresponding to these AGDs [84, 85]. Several clustered major and minor sites of cleavage were revealed in the case of all oligopeptides (**Figure 6**). It was shown that the number of oligopeptide cleavage sites is greater than that of intact MBP. The active sites of anti-MBP Abzs are located on their light chains, while the heavy chains are responsible for Abs increased affinity for protein substrates. Interactions of both light and heavy chains of Abzs with intact globular proteins provide high affinity and specificity of intact MBP hydrolysis. The affinity of anti-MBP Abzs for oligopep‐ tides was ~103-fold lower than that for the intact MBP. These data indicate that a relatively short oligopeptides interact mainly with the light chain of different monoclonal Abzs of total pool of IgGs, which possesses lower affinity for substrates, and therefore, depending on the

114 Trending Topics in Multiple Sclerosis

oligopeptide sequences, their hydrolysis may be less specific [84, 85].

IgGs and IgMs from the sera of patients with MS and SLE were found to possess amylolytic activity hydrolyzing α—(1-4)-glucosyl linkages of maltooligosaccharides, glycogen, and several synthetic substrates [57, 58, 63–67]. The specific amylolytic activity of individual IgGs and IgMs from MS patients was about three orders of magnitude higher than that of healthy donors [57, 58]. Fractions of auto-Abs from human milk [67] and from sera of patients with different autoimmune pathologies [57, 58 63–65] revealed different modes of action in the hydrolysis of maltooligosaccharides, p-nitrophenyl maltooligosaccharides, p-nitrophenyl, and α-D-glucopyranoside; several samples of MS Abzs demonstrated β-xylosidase activity,

which is not observed in known mammalian polysaccharide-hydrolyzing enzymes.

Proteolytic Abs from the sera of patients with various autoimmune diseases [9, 86] caseinhydrolyzing Abzs from human milk [87], casein-, human serum albumin-, and HIV reverse transcriptase-hydrolyzing Abs from AIDS patients [88] are serine-like proteases, and their activity is most strongly reduced by specific serine protease inhibitors PMSF or AEBSF. Specific inhibitors of acidic and thiol proteases demonstrated a weak effect on proteolytic activity of MS polyclonal IgGs and IgMs [48–51]. However, specific inhibitors of serine proteases significantly inhibited proteolytic activity. In addition, MS polyclonal IgGs contained several chelated metals and the relative amount of which decreases in the order: Fe2+ > Ca2+ > Cu2+ > Zn2+ > Mg2+ > Mn2+ > Co2+ > Ni2+ [50]. After removing of Mg2+ ions by dialysis against EDTA, MS IgGs have not completely lost proteolytic activity, which was increased after addition of different external Me2+ ions. After chromatography of MS IgGs on Chelex-100, a minor metal-

**Figure 7.** The relative MBP-hydrolyzing activity of 22 MLChs after their pre-incubation with specific inhibitors of dif‐ ferent type proteases [92]. Various MLChs (0.1 mg/ml) were pre-incubated in the absence of other components (black bars, control—C), in the presence of 50 mM EDTA (gray bars) or 1 mM PMSF (white bars) and then added to standard reaction mixture containing MBP substrate (A and B). Panel C shows several examples of the relative activity of MLChs with metalloprotease activity (numbers: 1, 5, 12, 15, and 21) and serine-like activity (numbers: 4 and 11), which do not change their activity after pre-incubation with iodoacetamide; three MLChs (numbers: 10, 14, and 18) demon‐ strating negative response to PMSF and EDTA as well as chimeric MLCh-22 after their pre-incubation with iodoaceta‐ mide leading to a significant decrease in the activity. White and grey bars correspond respectively to the activity before (control) and after these MLChs pre-incubation with iodoacetamide (Panel C). The relative activity of all MLChs before their pre-incubation with different inhibitors was taken for 100% [92].

An immunoglobulin light chain phagemid library corresponding to peripheral blood of SLE patients lymphocytes was used [92, 93]. Small pools of phage particles displaying light chains with different affinity for MBP were obtained using affinity chromatography on MBP-Sepharose. For preparation of the individual monoclonal light chains (MLChs, 27–28 kDa), the fraction eluted with 0.5 M NaCl was used. Seventy two of 440 individual colonies were randomly chosen, expressed in E. coli in a soluble form, and individual MLChs were purified by metal chelating chromatography. 22 of 72 MLChs efficiently cleaved MBP showing various pH optima in a 5.7–9.0 range and different substrate specificity in the hydrolysis of four various MBP oligopeptides. Four light chains demonstrated serine-like, three MLChs—thiol proteaselike activities, while eleven MLChs were metal-dependent (**Figure 7**). PMSF and EDTA inhibited by the activity of three chimeric MLChs, two other chains were supressed by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. In the presence of Ca2+, Mg2+, Mn2+, Ni2+, Zn2+, Cu2+, and Co2+, the ratio of relative activity was individual for each of 22 MLCh preparations [92, 93]. These observations suggest an extreme diversity of anti-MBP Abzs in SLE (and most probably MS) patients.

As it was mentioned above, iodoacetamide weakly (5–15%) suppress MBP-hydrolyzing activity of polyclonal Abs from sera of patients with MS and SLE [43, 48–51]. It was shown that the relative number of iodoacetamide-dependent MLChs is only approximately 27% of all MLChs analyzed, and several of them possess at the same time activity of serine- and metal‐ loproteases (**Figure 7**) [92, 93]. Thus, a relative contribution of thiol-like proteolytic activity to a total MBP-hydrolyzing activity of polyclonal Abzs pools in SLE and MS patients can be remarkably lower than that of antibodies with serine-like and metalloprotease activities and may be estimated for total IgGs depending on patients comparable to 5–15%, as found previously [43, 48–51]. Using different methods, it has been proven that recombinant mono‐ clonal kappa light chain NGTA2-Me-pro-ChTr possess two different activities—trypsin-like and metalloprotease activities [93], while other with trypsin-like, metalloprotease and DNase activities (Timofeeva AM, personal communication). Thus, it reasonable to believe that the immune system of SLE and MS patients can produce anti-MBP Abzs not only with one type but also with a combined structure of the active center, carrying amino acid residues typical for different proteases with metal-dependent, serine-, thiol-, and acidic-like activities.

Mammalian blood DNase II and DNase I demonstrate only one pronounced pH optimum in hydrolysis of scDNA (pH 5.2 and 7.0, respectively) [94–96]. In contrast to all human DNases, all polyclonal MS IgGs demonstrated high or detectable activity at a wide range of pH values between 5.0 and 9.5 [56]. The heights of the peaks corresponding to various optima were different, and the ratios of RAs at these pH values were individual for each polyclonal IgG preparation. **Figure 8A** demonstrates typical data for two individual MS patients. Similar results were obtained for DNase IgGs and/or IgMs from patients with other autoimmune diseases [13–21].

It is well known that mammalian, bacterial, and plant canonical proteases, depending on their biological function, can have optimal pHs ranging from acidic (2.0) to neutral and alkaline (8– 10) [97, 98]. We have measured the relative activity of polyclonal IgGs at pH from 2.6 to 10.5. In contrast to canonical proteases, the pH profile of each preparation of polyclonal IgGs from Autoimmune Processes in Multiple Sclerosis: Production of Harmful Catalytic Antibodies Associated with Significant Changes in the Hematopoietic Stem Cell Differentiation and Proliferation http://dx.doi.org/10.5772/63824 117

MS [51] and SLE [43] patients was unique (**Figures 8C** and **8D**). In contrast to all human proteases having one pronounced pH optimum, catalytic MS and SLE polyclonal IgGs demonstrated high specific MBP-hydrolyzing activity within a wide range of pH values (2.6– 10). Taking this into account, one cannot exclude that human immune system could, in principle, produce Abzs with very different proteolytic activities including similar to that of stomach acidic proteases. The above results clearly demonstrate that polyclonal IgGs from individual MS and SLE patients are very heterogeneous and can consist of different sets of catalytic IgG subfractions demonstrating quite distinct pH dependencies.

An immunoglobulin light chain phagemid library corresponding to peripheral blood of SLE patients lymphocytes was used [92, 93]. Small pools of phage particles displaying light chains with different affinity for MBP were obtained using affinity chromatography on MBP-Sepharose. For preparation of the individual monoclonal light chains (MLChs, 27–28 kDa), the fraction eluted with 0.5 M NaCl was used. Seventy two of 440 individual colonies were randomly chosen, expressed in E. coli in a soluble form, and individual MLChs were purified by metal chelating chromatography. 22 of 72 MLChs efficiently cleaved MBP showing various pH optima in a 5.7–9.0 range and different substrate specificity in the hydrolysis of four various MBP oligopeptides. Four light chains demonstrated serine-like, three MLChs—thiol proteaselike activities, while eleven MLChs were metal-dependent (**Figure 7**). PMSF and EDTA inhibited by the activity of three chimeric MLChs, two other chains were supressed by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. In the presence of Ca2+, Mg2+, Mn2+, Ni2+, Zn2+, Cu2+, and Co2+, the ratio of relative activity was individual for each of 22 MLCh preparations [92, 93]. These observations suggest an extreme diversity of anti-MBP Abzs

As it was mentioned above, iodoacetamide weakly (5–15%) suppress MBP-hydrolyzing activity of polyclonal Abs from sera of patients with MS and SLE [43, 48–51]. It was shown that the relative number of iodoacetamide-dependent MLChs is only approximately 27% of all MLChs analyzed, and several of them possess at the same time activity of serine- and metal‐ loproteases (**Figure 7**) [92, 93]. Thus, a relative contribution of thiol-like proteolytic activity to a total MBP-hydrolyzing activity of polyclonal Abzs pools in SLE and MS patients can be remarkably lower than that of antibodies with serine-like and metalloprotease activities and may be estimated for total IgGs depending on patients comparable to 5–15%, as found previously [43, 48–51]. Using different methods, it has been proven that recombinant mono‐ clonal kappa light chain NGTA2-Me-pro-ChTr possess two different activities—trypsin-like and metalloprotease activities [93], while other with trypsin-like, metalloprotease and DNase activities (Timofeeva AM, personal communication). Thus, it reasonable to believe that the immune system of SLE and MS patients can produce anti-MBP Abzs not only with one type but also with a combined structure of the active center, carrying amino acid residues typical

for different proteases with metal-dependent, serine-, thiol-, and acidic-like activities.

Mammalian blood DNase II and DNase I demonstrate only one pronounced pH optimum in hydrolysis of scDNA (pH 5.2 and 7.0, respectively) [94–96]. In contrast to all human DNases, all polyclonal MS IgGs demonstrated high or detectable activity at a wide range of pH values between 5.0 and 9.5 [56]. The heights of the peaks corresponding to various optima were different, and the ratios of RAs at these pH values were individual for each polyclonal IgG preparation. **Figure 8A** demonstrates typical data for two individual MS patients. Similar results were obtained for DNase IgGs and/or IgMs from patients with other autoimmune

It is well known that mammalian, bacterial, and plant canonical proteases, depending on their biological function, can have optimal pHs ranging from acidic (2.0) to neutral and alkaline (8– 10) [97, 98]. We have measured the relative activity of polyclonal IgGs at pH from 2.6 to 10.5. In contrast to canonical proteases, the pH profile of each preparation of polyclonal IgGs from

in SLE (and most probably MS) patients.

116 Trending Topics in Multiple Sclerosis

diseases [13–21].

**Figure 8.** pH dependences of the RAs in the hydrolysis of plasmid DNA by five individual MS polyclonal IgGs (IgG-1– IgG-5) (A) and by canonical DNase I and DNase II (B) [56] as well as pH dependences of the relative MBP-hydrolyzing activity of five individual IgGs from the sera of five different MS (C) and six various SLE (D) patients [43, 51]. Hydroly‐ sis of DNA incubated alone was used as control (A, "Con.") The relative DNase and protease activities corresponding to a complete transition of substrates to their shorter products as well as maximal activities of DNase I and DNase II were taken for 100%.

The next question concerning structural diversity of MS abzymes is related to a possibility of existence of catalytic Abs of different subclasses. It was interesting whether the cerebrospinal fluid similarly to serum of MS patients can contain IgG antibodies of all four subclasses. We have recently analyzed CSF and sera of MS patients, the average content of lambda- and kappa-IgGs as well as IgGs of four different subclasses (IgG1-IgG4) [53]. The average relative content of lambda-IgGs and kappa-IgGs in the case of CSFs (8.0 and 92.0%) and sera (12.3 and 87.7%) are comparable, while IgG1, IgG2, IgG3, and IgG4: CSF—40.4, 49.0, 8.2, and 2.5% of total IgGs, respectively, and the sera—53.6, 36.0, 5.6, and 4.8%, decreased in different order [53].

To analyze an "average" situation concerning a possible catalytic heterogeneity of MBP- and DNA-hydrolyzing IgGs from sera, we have prepared a mixture of equal amounts of IgGs from the sera of ten MS patients [51, 99]. The purity of IgGs of all types was confirmed by ELISA. It was shown that small fractions of IgGs of all four subclasses (IgG1–IgG4) are catalytically active in the DNA cleavage and their relative activity (nM supercoiled DNA/1 mg IgG/1 h) on average decreases in the order: IgG4 (4.1) < IgG3 (1.4) < IgG2 (0.94) < IgG1 (0.58), while their approxi‐ mate relative contribution to the total activity of Abzs (taking into account their relative content in the sera) decreases in the order: IgG4 (65.6%) > IgG2 (18.2%) > IgG3 (9.3%) > IgG1 (6.9% [99]. In the hydrolysis of DNA on average, k-IgGs are several folds more active than λ-IgGs. By different physico-chemical methods of Abs analysis, it was shown that the immune systems of MS patients can generate a variety of different type of anti-DNA Abzs and with various catalytic properties, which can play an important role in MS pathogenesis.

**Figure 9.** Affinity chromatography of polyclonal IgGs (mixture of 10 preparations from sera of MS patients) on anti-IgG1 (A), anti-IgG2 (B), anti-IgG3 (C), and anti-IgG4 (D) Sepharoses [84]: (—), absorbance at 280 nm, (□) and (■), rela‐ tive catalytic activities (RA) in the hydrolysis of MBP and OP-19 corresponding to one of immunodominant region of MBP, respectively. Depending on the RA, the reaction mixtures were incubated for 0.3–16 h in the presence of 10–100 μg/ml IgGs and then the RAs were normalized to the standard conditions: the complete transition of 0.19 mg/ml MBP and 0.33 mM OP-19 to their hydrolyzed forms in the presence of 0.1 mg/ml IgGs after 1 h of incubation was taken for 100%. The average error in the initial rate determination from two experiments in each case did not exceed 7–10%.

Autoimmune Processes in Multiple Sclerosis: Production of Harmful Catalytic Antibodies Associated with Significant Changes in the Hematopoietic Stem Cell Differentiation and Proliferation http://dx.doi.org/10.5772/63824 119

It was shown that IgGs from sera of MS patients containing k- and λ-types of light chains demonstrated comparable relative activities in the hydrolysis of MBP [51]. IgGs of all four subclasses (IgG1–IgG4) demonstrate catalytic activity (**Figure 9**), their contribution to the total activity of Abzs in the hydrolysis of MBP and its 19-mer oligopeptide increasing in the order: IgG1 (1.5–2.1%) < IgG2 (4.9–12.8%) < IgG3 (14.7–25.0%) < IgG4 (71–78%). Interestingly, the RAs of DNase polyclonal IgGs from MS patients increased in the same order (IgG1 < IgG2 < IgG3 < IgG4 [99]) as the RAs of MBP-hydrolyzing IgGs from SLE patients [43]. These data provide the evidence that all types of human MS and SLE IgGs, IgG1–IgG4 can possess various catalytic activities, but differ in their RAs and contribution into the total activity of MBP- and DNAhydrolyzing polyclonal Abzs.

#### **4.4. Relative catalytic activity of MS abzymes**

have recently analyzed CSF and sera of MS patients, the average content of lambda- and kappa-IgGs as well as IgGs of four different subclasses (IgG1-IgG4) [53]. The average relative content of lambda-IgGs and kappa-IgGs in the case of CSFs (8.0 and 92.0%) and sera (12.3 and 87.7%) are comparable, while IgG1, IgG2, IgG3, and IgG4: CSF—40.4, 49.0, 8.2, and 2.5% of total IgGs,

To analyze an "average" situation concerning a possible catalytic heterogeneity of MBP- and DNA-hydrolyzing IgGs from sera, we have prepared a mixture of equal amounts of IgGs from the sera of ten MS patients [51, 99]. The purity of IgGs of all types was confirmed by ELISA. It was shown that small fractions of IgGs of all four subclasses (IgG1–IgG4) are catalytically active in the DNA cleavage and their relative activity (nM supercoiled DNA/1 mg IgG/1 h) on average decreases in the order: IgG4 (4.1) < IgG3 (1.4) < IgG2 (0.94) < IgG1 (0.58), while their approxi‐ mate relative contribution to the total activity of Abzs (taking into account their relative content in the sera) decreases in the order: IgG4 (65.6%) > IgG2 (18.2%) > IgG3 (9.3%) > IgG1 (6.9% [99]. In the hydrolysis of DNA on average, k-IgGs are several folds more active than λ-IgGs. By different physico-chemical methods of Abs analysis, it was shown that the immune systems of MS patients can generate a variety of different type of anti-DNA Abzs and with various

**Figure 9.** Affinity chromatography of polyclonal IgGs (mixture of 10 preparations from sera of MS patients) on anti-IgG1 (A), anti-IgG2 (B), anti-IgG3 (C), and anti-IgG4 (D) Sepharoses [84]: (—), absorbance at 280 nm, (□) and (■), rela‐ tive catalytic activities (RA) in the hydrolysis of MBP and OP-19 corresponding to one of immunodominant region of MBP, respectively. Depending on the RA, the reaction mixtures were incubated for 0.3–16 h in the presence of 10–100 μg/ml IgGs and then the RAs were normalized to the standard conditions: the complete transition of 0.19 mg/ml MBP and 0.33 mM OP-19 to their hydrolyzed forms in the presence of 0.1 mg/ml IgGs after 1 h of incubation was taken for 100%. The average error in the initial rate determination from two experiments in each case did not exceed 7–10%.

respectively, and the sera—53.6, 36.0, 5.6, and 4.8%, decreased in different order [53].

118 Trending Topics in Multiple Sclerosis

catalytic properties, which can play an important role in MS pathogenesis.

The catalysis mediated by artificial Abzs is usually characterized by relatively low kcat values, which are 102- to 106-fold lower than those for canonical enzymes [1–8]. The known kcat values for natural Abzs from AI patients vary in the range of 0.0001–40 min−1 [8–21]. The kcat of polyclonal IgGs with various activities from sera of MS patients is in average varied in different ranges. Overall, the kcat values characterizing hydrolysis of DNA, RNA, MBP, oligosaccharides in the reactions catalyzed by non-separated polyclonal MS IgGs and their subfractions separated by affinity chromatographies are comparable with or even higher than those for other known Abzs [8–21]. In addition, the RAs of DNase non-separated polyclonal Abs from patients with different diseases increase approximately in the order: diabetes [100] ≤ viral hepatitis [101] ≈ thick bone encephalitis [102] < polyarthritis ≤ Hashimoto's thyroiditis [71, 76, 103] ≤ schizophrenia [104] < AIDS [105] ≤ MS [54–56, 99] < SLE [10, 68–70]. At first glance, the kcat values characterizing some polyclonal Abs may seem relatively low compared with those for canonical enzymes with the same enzymatic functions. At the same time, some DNAdependent enzymes with very important functions, for example, repair enzymes and EcoRI endonuclease demonstrate the kcat values comparable or even lower [106] than those for Abzs analyzed. In addition, the RAs of RNase IgGs and IgMs from the sera of patients with different autoimmune diseases including MS may be 10- to 1000-fold higher than those of DNase Abzs from the same patients. RNase activities of autoimmune Abzs were more often 0.1–20% of that of RNase A and of six known human sera RNases, while poly(A) was hydrolyzed by MS and other autoimmune Abs 2–10 times faster than by RNase A, one of the most active RNases known [11, 13–21, 54, 101]. The specific activity of IgGs of several MS patients was 40–400% of that of RNase A [101].

Notably, currently there are no methods that could efficiently separate Abzs from catalytically inactive antibodies against the same substrate (antigen). The specific activities were calculated using the total concentrations of polyclonal Abs, and affinity chromatography on DNAcellulose or MBP-Sepharose leads only to partial enrichment of individual fractions with proteolytic and DNase activities. Thus, the specific substrate-hydrolyzing activities of the individual monoclonal Ab subfractions in pool of polyclonal Abs may be significantly higher than those of the non-fractionated or partially fractionated polyclonal IgGs, IgAs, and IgMs. In addition, it is impossible not to take into account catalytic activities of Abs from patients with MS and other autoimmune diseases in the analysis of their possible role in the patho‐ genesis of these pathologies, since some of them may be very harmful for people health.
