Systemic lupus erythematosus: molecular cloning and analysis of recombinant monoclonal kappa light chain NGTA2-Me-pro-ChTr possessing two different activities—trypsin-like and metalloprotease

Abstract

Polyclonal antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. The small pools of phage particles displaying light chains with different affinity for MBP were isolated by affinity chromatography on MBP-Sepharose. The fraction eluted with 0.5M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27kDa). The clones were expressed in Escherichia coli in a soluble form; MLChs were purified by metal-chelating chromatography followed by gel filtration. In mammalians, there are serine proteases and metalloproteases. These and many other enzymes usually have only one active site and catalyze only one chemical reaction. In contrast to canonical proteases, one MLCh (NGTA2-Me-pro-ChTr) efficiently hydrolyzed MBP (but not other proteins) and four different oligopeptides corresponding to four immunodominant sequences containing cleavage sites of MBP. The proteolytic activity of MLCh was efficiently inhibited only by specific inhibitors of serine-like (phenylmethanesulfonylfluoride, PMSF) and metalloproteases (EDTA). It was shown that MLCh possess independent serine-like and metal-dependent activities. The principal existence of monoclonal antibodies with two different proteolytic activities is unexpected but very important for the further understanding of at present unknown biological functions of human antibodies.

Catalytic Antibodies in Norm and Systemic Lupus Erythematosus

“…On the next step we analyzed in more detail three additional MLChs (numbers 23-25) corresponding to peak 7 eluted from MBP-Sepharose with 0.5 M NaCl ( Figure 18A) [119,120]. These three MLChs were purified and characterized in detail exactly similar to above described 22 preparations [118].…”

“…However, in the case of some patients, a possible number of abzymes can be very large. In our studies, we used a cDNA library only kappa light chains of Abs from three patients with SLE [111,112,[118][119][120]. We have analyzed only 45 of 451 single of colonies corresponding one peak eluted from DNA-cellulose with 0.5 M NaCl and 33 of 687 colonies of peak eluded with acidic buffer.…”

“…Addition of EDTA to NGTA2-Me-pro-Tr containing only intrinsically bound Me 2+ -ions results in a decrease in its activity for 58 ± 5% ( Figure 23A) [119]. Average serine-like activity of NGTA2-Me-pro-Tr containing only intrinsically bound Me 2+ ions was ~1.4-fold lower than its Me 2+ -dependent protease activity.…”

“…Figure 23(A) shows that NGTA2-Me-pro-Tr is not sensitive to pepstatin and iodoacetamide [119]. Preincubation of this MLCh with specific inhibitor of serine-like proteases results in a decrease of its activity for 42 ± 4%.…”

“…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).…”