Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein

Abstract

Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP-Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27 kDa). Seventy-two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty-two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7-9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease-like and three thiol protease-like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca(2+), Mg(2+), Mn(2+), Ni(2+), Zn(2+), Cu(2+), and Co(2+) was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti-MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti-MBP abzymes, which can attack MBP of myelin-proteolipid sheath of axons and play an important role in MS and SLE pathogenesis.

Catalytic Antibodies in Norm and Systemic Lupus Erythematosus

“…Seven different metal ions did not effect on the activity of MLChs with serine-like (9, 6, and 4) and thiol-dependent (18, 14, and 10) All 22 MLCh preparations hydrolyzed efficiently MBP within a wide range of pHs (5.0-10), but in contrast to polyclonal SLE IgGs, they show mainly only one pH optimum [118]. Only the pH profile for preparation 4 demonstrates optimal pH at 5.7-5.9 and pronounced shoulder at pHs 6.5-7.5 ( Figure 21C).…”

“…For analysis of MBP-hydrolyzing activity of Abs, we have used the same phagemid library of kappa light chains [118][119][120] as for analysis of MLChs with DNase activity [111,112]. The phage particles containing MLChs with different for MBP were separated by affinity chromatography on MBP-Sepharose (Figure 18A).…”

“…Similar distribution all over the chromatography profiles was Figure 18. Affinity chromatography on MBP-Sepharose of phage particles: (− −) and (-) absorbance at 280 nm of particles corresponding plasmid respectively without and with kappa light chains cDNA (A) [118]. Relative titres of phage particle and NaCl concentrations corresponding to various peaks are shown on panel A.…”

“…The effect of PMSF and EDTA on the RAs of 22 different MLChs was analyzed [118]. Four MLChs (4,6,9, and 11) demonstrate serine-like proteolytic activity; PMSF suppressed their activity, but there was no noticeable effect of EDTA ( Figure 20B).…”

“…A very intriguing situation was observed for three MLChs (18, 14, and 10); EDTA and PMSF do not remarkably decreased their proteolytic activity ( Figure 20B). No significant suppression (5-15%) of MS and SLE polyclonal MBP-hydrolyzing abzymes by specific [118]. Several examples (C) of the RAs of MLChs having metal-dependent activity (1, 5, 12, 15, and 21) and serine-protease-like activity (4 and 11), demonstrating no iodoacetamide-dependent activity; three MLChs (10, 14, and 18) showing negative response to EDTA and PMSF.…”