title: Systemic lupus erythematosus: molecular cloning and analysis of recombinant monoclonal kappa light chain NGTA1-Me-pro with two metalloprotease active centers.
abstract: It was shown previously that approximately 30% ± 5% of antibodies against myelin basic protein (MBP) and the DNA of patients with systemic lupus erythematosus (SLE) and multiple sclerosis (MS) possess catalytic activities that play an important negative role in the pathogenesis of MS and 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, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27 kDa). The clones were expressed in E. coli in a soluble form. MLChs were purified by metal chelating chromatography followed by FPLC-gel filtration. The activity of one MLCh (NGTA1-Me-pro) was inhibited only by EDTA, and it efficiently hydrolyzed MBP (but not other proteins) and four different oligopeptides corresponding to four known immunodominant sequences containing cleavage sites of MBP only in the presence of several different metal ions. An unexpected result was obtained: NGTA1-Me-pro demonstrated two pH optima, two optimal concentrations of Me2+ions, and two Kmvalues for MBP. The protein sequence of NGTA1-Me-pro, having two metalloprotease active centers, has homology with several mammalian metalloproteases. Recently, it was shown that one other MLCh possesses serine-like and metalloprotease activity. The principal possibility of the existence of MLChs with several different active centers is unexpected, but very important for the further understanding of unknown possibilities for immune systems and the biological functions of antibodies.
authors: SB RAS Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, 8 Lavrentiev Ave., Novosibirsk 630090, Russia. nevinsky@niboch.nsc.ru.
authors: Anna M
authors: AM
authors: Timofeeva
authors: SB RAS Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, 8 Lavrentiev Ave., Novosibirsk 630090, Russia. nevinsky@niboch.nsc.ru.
authors: Valentina N
authors: VN
authors: Buneva
authors: SB RAS Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, 8 Lavrentiev Ave., Novosibirsk 630090, Russia. nevinsky@niboch.nsc.ru.
authors: Georgy A
authors: GA
authors: Nevinsky
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics*; D003001:Cloning, Molecular; D006801:Humans; D006868:Hydrolysis; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D008180:Lupus Erythematosus, Systemic / Q000235:genetics* / Q000276:immunology; D045726:Metalloproteases / Q000737:chemistry / Q000235:genetics*; D008670:Metals / Q000737:chemistry; D004676:Myelin Basic Protein / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins
chemical_list: D007145:Immunoglobulin kappa-Chains; D008670:Metals; D004676:Myelin Basic Protein; D011993:Recombinant Fusion Proteins; D045726:Metalloproteases