High prevalence of anti-alpha-crystallin antibodies in multiple sclerosis: correlation with severity and activity of disease.

INTRODUCTION: The presence of T-cell reactivity to alphaB-crystallin in patients with multiple sclerosis (MS) has suggested that this small molecular weight heat shock protein (Hsp) may be an autoantigen in MS. MATERIAL AND METHODS: We have tested the serum of patients with clinically definite MS (n=30), other inflammatory neurological disease (n=22), non-inflammatory neurological disease (n=42) and healthy individuals (n=23) for systemic humoral responses to bovine alphaB-crystallin, to the homologous chaperone protein, alphaA-crystallin, and to another small Hsp, Hsp 27. RESULTS: Sixty-three percent of MS patients exhibited immunoreactivity to alpha-crystallin and this was present in all 4 of 4 non-ambulatory patients with MS. In contrast, serum concentrations in MS patients of antibodies to the small Hsp, Hsp27, and to myelin basic protein were negligible (P<0.001). Serum anti-alpha-crystallin immune responses were detected in significantly lower percentages of patients with other inflammatory neurological diseases (32%, P<0.025), and with non-inflammatory neurological diseases (12%, P<0.001). None of the healthy control individuals showed anti-alpha-crystallin reactivity. The concentration of anti-alpha-crystallin antibodies in patients with MS correlated with severe disease (P<0.05) and with active disease (P<0.025). CONCLUSION: Our observations support the notion that anti-alpha-crystallin autoimmune responses may contribute to pathogenicity in MS and may represent a mechanism of how recurrent attacks of MS develop subsequent to an isolated demyelinating episode.

Differential surface accessibility of alpha(187-199) in the Torpedo acetylcholine receptor alpha subunits.

We have probed the surface accessibility of residues alpha187 to alpha199 of the Torpedo acetylcholine receptor with monoclonal antibody 383C, which binds uniquely to these residues. However, 383C binds to only one of the two alpha subunits in the membrane-bound receptor, neither of the two subunits in carbamylcholine-desensitized receptor, and to both alpha subunits in Triton X-100 solubilized receptor. The kinetics of association and dissoci-ation of 383C with the peptide alpha(183-199) compared to those with the membrane-bound receptor suggest that all but a single hydrogen bond of affinity derives from contacts between this peptide and the monoclonal antibody paratope. Inhibition of 383C binding by alpha-bungarotoxin selectively directed to the alpha subunit correlated with the high-affinity d-tubocurarine binding site, along with a lack of inhibition by alpha-bungarotoxin directed to the alpha subunit correlated with the low-affinity d-tubocurarine binding site, suggests that the 383C epitope on the membrane-bound receptor resides on the alpha subunit associated with the high-affinity d-tubocurarine binding site. The results presented here suggest a structural basis for the differences between the two receptor acetylcholine binding sites.

Mapping the mAb 383C epitope to alpha 2(187-199) of the Torpedo acetylcholine receptor on the three-dimensional model.

Monoclonal antibody 383C is an anti-acetylcholine receptor antibody whose binding to the receptor is blocked by alpha-bungarotoxin and by carbamylcholine. Monoclonal antibody 383C binds to the alpha subunit of the Torpedo acetylcholine (ACh) receptor as well as to its V8-protease 20 kDa fragment that possesses the affinity alkylatable Cys192/193. In an epitope scanning experiment spanning the N-terminal 211 amino acid residues of the alpha subunit, 383C binds uniquely to three overlapping peptides; alpha(184-196), alpha(187-199) and alpha(190-202). These peptides span a cluster of amino acid residues implicated in the binding of acetylcholine, including Cys192/193. To map the location of these residues on the three-dimensional model of the ACh receptor, we have employed a combination of X-ray diffraction from oriented complexes of 383C with ACh receptor-enriched membrane vesicles and electron microscopy of negatively stained tubular arrays of 383C/receptor complexes. The X-ray diffraction study finds extra electron density in the presence of 383C centered 35 A above the synaptic side phosphate head groups. The electron micrographic images display extra stain exclusion from the antibody at a site adjacent to the alpha2 subunit on the periphery of the rosette clockwise to the alpha2 vertex. This mapping localizes several residues of the ACh receptor alpha subunit involved in the binding of acetylcholine. Despite these residues being present in both alpha subunits, only the alpha2 subunit is decorated with this monoclonal antibody.

Entrapment of enzymes using organo-functionalized polysiloxane copolymers.

The present study expands previous work [(1984) Biochim. Biophys. Acta. 797, 343-347] by showing that organo-functionalized polysiloxane copolymers could entrap two of the most frequently immobilized enzymes, i.e. urease and invertase with retention of biological activity. Urease was solidly entrapped in the polymer formed from a 1:3 mixture of 3-aminopropyltriethoxysilane and tetraethylorthosilicate. The entrapment yield and the activity of the entrapped enzyme are significantly greater than with other techniques reported to date. Significantly, the entrapped enzyme possessed greater activity than its solution counterpart (36% at higher amounts of enzyme entrapped). The entrapment process also rendered the enzyme more stable toward pH and temperature, and less susceptible toward the action of urea at high concentrations. In addition, the entrapment process significantly increased the stability, both operational and storage, of the urease enzyme. When invertase was entrapped in the same copolymer, it retained two thirds of its solution activity, but the entrapment yield was lower than that of urease. Results obtained during this study also suggested that the protein may be influencing polymer development in these systems and that the resultant polymer in turn may be affecting the enzyme's activity (see following paper for further discussion).

Influence of protein on polysiloxane polymer formation: evidence for induction of complementary protein-polymer interactions.

Results presented in the companion paper suggested that the protein itself might be actively involved in the polymerization process while being entrapped in polysiloxane polymers. It was speculated that the organo-functional side chains on the silanol monomers (or small oligomers) tended to associate with complementary residues on the protein surface during the polymerization process. This phenomenon might lead to complementary binding pockets for the protein on the polymer. To investigate this possibility, polysiloxane polymers were prepared from 3-aminopropyltriethoxysilane and tetraethylorthosilicate (1:3) in the presence of two proteins: urease and BSA. The entrapped proteins were removed by pronase digestion and washing and the resulting polymers evaluated for their ability to again bind the two proteins. It was found that urease preferentially bound to the polymer made in the presence of urease, and BSA preferentially bound to the polymer made in the presence of BSA. The absolute preferential binding excess was greater (30%) for urease binding relative to that observed for BSA (3%). However, in both cases the same relative binding ratio of 1.5 or 50% excess was found. A similar study using the closely related hemoglobin and myoglobin proteins failed to show comparable excess binding in the presence of the predetermined protein. In the latter case, it was demonstrated that the rebound proteins did not equilibrate with labeled solution proteins, indicating a very tight association with the polymer surface possibly masking any specificity which existed. However, it was possible to show that urea release of rebound hemoglobin from the polymer made in the presence of hemoglobin was less than for myoglobin bound to the same polymer and visa versa, again suggesting induced properties unique to the polymer prepared with the predetermined protein. To the extent that this notion of induced complementary order is correct, it may have implications in the development of protein specific adsorbants and in our understanding of polymer surface adhesion and the molding of template fine structure.