**3.1 Myelin basic protein**

480 Autoimmune Disorders – Current Concepts and Advances from Bedside to Mechanistic Insights

with or without anti-MBP antibodies, in patients presenting with an initial clinical event suggestive of central nervous system demyelination and evidence of multifocal lesions on MRI studies, is predictive of subsequent clinical events that establish the diagnosis of clinically definitive MS. However, the presence of anti-MOG antibodies in patients with nondemyelinating diseases of the CNS, as well as in a substantial number of healthy individuals, has raised important questions about the role of these antibodies in MS, and

The neurodegenerative model of MS is based on the proposition of a primary lesion in myelin followed by myelin breakdown and the release of myelin-compartmentalized proteins, particularly MBP. This is followed by the generation of MBP-derived peptides that become the main sensitizers of T cells. (Hafler, 2004) The hypothesis that interactions between MBP and T cells must occur at sites other than the myelin membrane has been challenged by recent physicochemical studies demonstrating that myelin can become structurally unstable secondary to specific posttranslational modifications of MBP structure. This was shown to result in the increased surface exposure and susceptibility to proteolysis of MBP 83–92. (Husted, 2006) The close association between the proteolytic sensitivity of MBP and the pattern of posttranslational modifications of the molecule may represent one of the key regulatory mechanisms in epitope generation (Lolli et al., 2005). Proteolysis may proceed by any of four distinct pathways that may exert a concerted attack on the MBP molecule, although the mechanisms responsible for the activation and regulation of these

potential activities are not known. Thus, epitope generation may occur via: 1. autocatalytic cleavage of the MBP molecule (D'Souza et al., 2005);

3. autoantibody-mediated site-specific cleavage (Ponomarenko et al., 2006);

The last mechanism, which has been demonstrated in a number of autoimmune pathologies, remains unproven for MS (Wentworth et al., 2001). These pathways are characterized by dramatic differences in reaction velocities and in cleavage site specificity. Obviously the rates of enzymatic reactions are several times higher than those for autocatalysis and Abenzymes (abzymes), perhaps making this pathway the major player in epitope generation. In the case of abzymes, the large excess of the biocatalyst, its high specificity, and its close compartmentalization with the MBP substrate shown in demyelinating lesions are suggestive of its likely effectiveness in vivo. (Ponomarenko et al., 2006; Genain et al.,

Today, the advent of antibody catalysis has demonstrated that antibodies can be programmed to perform complex cell biochemistry, and thus a logical question arises: does the original (nature-gifted) potential for catalysis relate to antibody function? In the sense of the question, it is very interesting to consider the evolution of antibodies, while generally accepting that natural enzymes are primitive molecules compared with antibodies, and that

A great deal of evidence has been also adduced to support the medical concept of the *living soul* of abzymes and their significance for utilizing broader autoantibodies properties in the formation of pathogenic patterns and clinical settings at different autoimmune and other conditions. Moreover, the medical concept of abzymes was tested in two ways, clinically and experimentally, i.e., on human and animal models. The progress achieved earlier in

4. Abdependent oxidative pathway (Wentworth et al., 2001).

these need to be addressed in future studies.

2. protease digestion (D'Souza et al., 2006);

antibodies arose just after the birth of enzymes.

**2.1 Catalysis** 

1999)

Myelin basic protein is by far the best studied myelin component in MS. It is the second most abundant myelin protein (approximately 30-40%) after PLP. There are five MBP isoforms with 14-21.5 kDa molecular weights in mammals that result from differential splicing of 11 axons within the Golli-MBP locus. The highly basic MBP is positioned at the intracellular surface of myelin membranes and via interactions with acidic lipid moieties is involved in maintaining the structure of compact myelin. The most abundant 18.5 kDa isoform (170 amino acid length) has been used in most immunological studies.

Different from MOG, and PLP (proteolipid protein), MBP is found in both central and peripheral myelin, and MBP transcripts have also been demonstrated in peripheral lymphoid organs such as lymph nodes and thymus.

Fig. 1. MBP sites of proteolysis by different enzymes-proteases and Abs-proteases and as a result of spontaneous autocatalytic hydrolysis. The sequence of the encephalitogenic peptide 81–103 is shown in red. Abbrevations: GelB (Gelatinase B), MMP-3 (matrix mettaloproteinase-3), CatD (Cathepsin D), ACat (Autocatalytic cleavage), Abz (Abzyme), LP (Lysosomal Proteases), Tryp4 (Trypsin 4).

Antibody-Proteases in the Pathogenesis of Autoimmune

induction, etc.

Demyelination and Monitoring Patients with Multiple Sclerosis 483

abzymes are closely related. This allows to assume on a hypothesis on a new mechanism of the contribution of autoAbs into the pathogenesis of autoimmune disorders. Such mechanism acts independently of complement and cytotoxic T cells; it requires a catalytically active Fab fragment but ignores Fc fragment whose structure is deprived of

Two different mechanisms of DNA-abzymes cytotoxic potential utilization are establishedby means of direct cytotoxic effect on a target cell involving the catalytically active Fab fragment and by means of apoptosis due to high affinity of DNA-binding autoAbs for membrane receptors providing the cell with features of possible target. Cross-reactive with such such cells, DNA-abzymes can provoke their degradation resulting in the development of different syndromal manifestations: lupus nephritis in glomerule endothelium crossreactivity, articular syndrome in synovial cross-reactivity, extracardial manifestations and MCS progression in CM cross-reactivity. (Rekvig et al., February 2004; Raz et al., 1993) Cross-reactivity of DNA-binding autoAbs with glutamate receptors accumulating at neural cells is of a special interest. Such autoAbs with catalytic and cytotoxic activities can initiate neural cells apoptosis providing the possibility for the development of CNS autoimmune degenerative disorders (Kotzin et al., 2001). The penetration of DNA-binding autoAbs (and DNA-abzymes) into a cell results in activation of cytotoxicity mechanisms, apoptosis

**4.2 Molecular mechanisms of the involvement of DNA-abzymes in the development of different autoimmunity conditions and pathogenesis of autoimmune disorders** 

One antigen may generate up to 102-104 different antibody molecules, a number that may further increase by somatic mutagenesis. Therefore, it seems feasible that different DNAbinding and other non-catalytic antibodies, as well as antibodies with catalytic activities can be synthesized in the course of immune response, either directed against the substrate or as antiidiotypic antibodies to enzymes hydrolyzing nucleic substrates. It is certainly difficult to predict the clinical significance of these catalytic activities, but it is likely that they modify

The interest to catalytic (i.e., DNA-hydrolyzing activity) of the autoantibodies is kept on growing up and is strongly supported by the new data including those illustrating cytotoxic activities of the biocatalysts and evidence that abzyme-mediated cytotoxic effects observed in human SLE (system lupus erythematosis) and mouse SLE-like syndromes are caspase-

The use of assays for Abs to MBP and MOG for diagnostic and prognostic purposes in patients with a clinically isolated syndrome (CIS), a frequent precursor to clinically definite MS (CDMS), has yielded conflicting results. One study showed that the presence of myelin Abs in the sera of CIS patients was predictive of a shorter time course to the development of CDMS. (Tomassini et al., 2007) Others, however, indicated that positive tests had no

Symbols indicate OD450 values for Abs from individual subjects within each EDSS category for specific MBP peptides or intact MBP and MOG proteins. Lines indicate the relation between EDSS value and the binding activity of specific peptides/proteins: MBP 81-103 (R2 0.332; p 0.005), MBP 130-156 (R2 0.381; p 0.011), MBP 146-170 (R2 0.310; p 0.033), intact MBP

the pathogenesis or clinical process of these autoimmune diseases.

prognostic value for progression to CDMS. (Kuhle et al., 2007)

dependent and thus apoptosis-related.

**5. Prediction of MS** 

even buried resources providing direct cytotoxic effect. (Ponomarenko et al., 2000)

## **3.2 Proteolipid protein**

Proteolipid protein (PLP) is the most abundant protein in CNS compact myelin (about 50%), highly hydrophobic and evolutionarily conserved across species. There are two main transcripts, the full-length 276 amino acid isoform and DM-20, an isoform that lacks 35 amino acids and is mainly expressed in brain and spinal cord prior to myelination, but also in peripheral lymphoid organs such as the thymus, where full-length PLP is barely found. Interestingly, the major encephalittogenic and immunodominant PLP peptide (139-154) is contained in full-length PLP but not in DM-20. This observation is thought to account for the encephalittogenicity and immunodominance of the PLP (139-154) peptide, since it is essentially not available for thymic negative selection and consequently a high precursor frequency of PLP (139-154)-specific T cells has been observed even in naive unprimed animals.

### **3.3 Myelin oligodendrocyte glycoprotein**

Myelin oligodendrocyte glycoprotein, a 218 amino acid trancemembrane glycoprotein of the IG superfamily, is much less abundant (0.01 - 0.05%) than MBP and PLP, and also different from the two major myelin proteins in being located not in compact myelin but exposed on the outermost surface of the oligodendrocyte membrane. Because of this "strategic" location, it is directly accessible to antibodies and believed to be particularly relevant as a target for both cellular and humoral immune responses in MS. MOG, is expressed relatively late during myelination and is only found in the brain/spinal cord and the retina but not in peripheral nerve. Furthermore, MOG expression is either completely or almost completely lacking in peripheral lymphoid tissues, although MOG transcripts have been seen in nonhuman primate peripheral nerve and a few samples of human tonsils and thymus.
