Interaction Of Mannan-binding Lectin With MBL-associated Serine Proteases

The inbeing of innate immunity is to identify molecular-pattem possessed specially by microorganism and it's the first-line of host to defense infection. At present, it has been established Mannan-binding lectin (MBL) is a key molecule of innate immune system. MBL, the member of collectins which belongs to C-type lectin superfamily, is a serum protein secreted by liver cells. And MBL can through the pattern-recognition function to selectively recognize sugars presented on pathogens. On binding to pathogens, MBL may activate the complement cascade via lectin pathway, leads to the killing of microbes by exerting lysis effect and indirect opsonizing function. At the same time, MBL has a direct opsonization when binding to lectin receptors on the phagocytic cells, and it also can mediate the MBL dependent cell-mediated cytotoxicity reaction. MBL also play an important role on surfactant defenses of mucous membrane.The overall polypeptide structure of MBL from N-terminal domain to C-terminal domain includes four domains, a cysteine-rich N-terminal domain, a collagen-like region (CLR), a neck region and a C-terminal carbohydrate- recognition domain (CRD). Integrity MBL is a multimeric protein composed by three of homogeneity trimer subunits and as many as six subunits. Only the high oligomeric forms of MBL have the biologic activity and can fix complement. CRD is recognition function domain of MBL, can selectively recognize sugars presented on pathogens. And CLR is effect function domain. MBL activates the complement and bind to C 1 qR location on the domain. MBL can form compound with MBL-associated serine proteases (MASPs) through its CLR. After its CRD domain recognize and binding to sugars presented on pathogens, MBL can activity the zymogens of MASPs, so activate the complement cascade via lectin pathway to play the function of anti-infection by innate immunity.The peptide chain of MASPs are composed of six modules and a linker region: an N-terminal complement subcomponent C 1 r/C 1 s-like domain (CUB 1) followed by an epidermal growth factor (EGF)-like domain of the Ca -binding type, a second CUB domain (CUB2), two contiguous complement control protein modules (CCP 1 and CCP2), a short linker and finally a chymotrypsin-like serine protease (SP) domain. The present study indicates that the three domains of MASPs interacted with MBL CLR via the N-terminal three modules (CUB 1-EGF-CUB2) depend on the Ca2+. However the detail of this interaction is not clearly, especially the bind location is not clearly.The structure gene of MBL CLR has been found three point mutations in exon 1 indepednently. CGT52TGT, GGC54GAC and GGA57GAA mutations have the high frequency in mankind and lead the concentration of MBL in serum is low, the activity and the function of opsonophagocytosis also descendent. MBL deficiency had been reported to be an important element in an acute and chronicity recurrent infections, even with the infection of HBV and HIV. The mutations of structure gene of MBL also closely correlate with the autoimmune diseases, such as SLE, RA, IgA nephritis and dermatomyositis. But the mechanisms which the mutations of MBL gene cause immunodeficiency are not definite.In order to study the interaction of MBL and Masps, to revealed and elucidate the subtle structure and function of MBL-CLR, we use the prokaryotic expression system to express the proteins of MBL-CLR and MASPs, at the same time espression the MBL in mammalian cells and established a two proteins binding system. Then we synthesize a range of peptides to block up the two proteins' interaction to reveal the subtle structure of MBL CLR and from one aspect elucidations of the molecular mechanism of defense function by innate immunity. The human MBL collagen-like region (CLR) gene fragment was amplified by PCR from pGEM-MBL plasmid which contains human MBL cDNA, and then inserted into prokaryotic expression vector pET32a, which named pET32a-MBL-CLR. After identified by restriction mapping and sequencing, the recombinant plasmid pET32a/His MBL-CLR was transformed into E. coli BL21 (DE3) cells. The expressed product was purified by Immobilized Metal Affinity Chromatography (IMAC) and identified by SDS-PAGE, Western blot and indirect enzyme-linked immunosorbent assay (ELISA) using the antibody from Balb/C mice immunized with the recombinant human MBL protein. The cDNA fragment of 180 bp was amplified from pGEM-MBL plasmid and the recombinant expression vector pET32/His MBL-CLR was constructed. The recombinant plasmid was consistent with those expected by restriction maps and sequence. Three components of Mr 30 000, 60 000 and 120 000 in the purified recombinant product were detected by SDS-PAGE and all the components could be recognized by anti-6His antibody in Western blot assay. The three components were correspondingly with the band of the monomer and oligomer of the fusion protein. The purified recombinant product could react with the antibody against the recombinant human MBL protein in the indirect ELISA. So the prokaryotic expression strains that efficiently express recombinant human MBL-CLR and the recombinant human MBL-CLR-Trx fusion protein were obtained successfully, which will help the further structure function research of MBL molecule.Partâ…¡Prokaryotie expression of MASP1,2-N and MASP1,2-C proteins and identity the target proteinsUsing the same methods, the target sequences in pGEM-MASP1 and 2 plasmids that contain human MBL-MASP1, 2 cDNA were amplified by PCRs, then inserted into prokaryotic expression vector pGEX4T-1 and identified by restriction mapping and sequencing. The two recombinant expression vectors were transformed into E. coli BL21 (DE3) cells. The expressed products were purified by GSTrap Immobilized Metal Affinity Chromatography (IMAC) and identified by SDS-PAGE and Western blot assay. The DNA fragments of 860 and 840bp, which encode the N-terminal region of human MASP 1 and 2, were amplified from pGEM-MASP 1 and 2 plasmids and the recombinant expression vector, pGEX4T-MASP 1-N and pGEX4T-MASP2-N, were constructed, whose restriction maps and sequence were consistent with those expected. The components of Mr 60 000 in the purified recombinant products were found by SDS-PAGE and could be recognized by anti-GST antibody in Western blot assay. The prokaryotic cell strain that expresses efficiently recombinant human MASP1 and 2-N (rhMASP1 and 2-N) proteins and the purified rhMASP1 and 2-N proteins were successfully obtained, which provides the basis for further research of MASPs molecule. At the same time, we also constructed the pET17b-MASP1-C and pET17b-MASP2-C prokaryotic vectors, and purified rhMASP 1 and 2-C proteins.Partâ…¢Structure Location of MBL Binding with MASPsAt present, the relationship between the structural and function of the MBL-CLR is not clearly. In this study, we build an ELISA system of MASPs binding with wild type MBL and with MBL-CLR protein at first, then design and synthesize a range of peptides which imitation the CLR to block the bindings in order to location the binding site on MBL-CLR. The result of MBL-CLR inhibition the binding shows CLR can successfully block the MASP1, 2-N binding with MASPs, and the inhibiting concentration(IC50) are 0.3g/L and 0.15g/L when the inhibiting ratio are 50%; MBL-CLR also can binding with MASPs that prove MASPs are really interaction with MBL-CLR.The N-terminal portion of the MBP polypeptide consists of a collagen-like domain composed of 19 Gly-X-Y triplets with a single interruption that forms a bend in the domain and total is 59 amino acids.Based on the sequence and characteristic of MBL-CLR, we design and synthesize nine peptides to inhibit the interaction. Nos. 1-4 peptides has about 22 amino acids, from the begin to the end coverage the 59 amino acids and some parts are overlap. Nos. 5-8 peptides aim directly at the point mutations; No.5 is wild type peptide; No.6 is 52Cys mutation peptide; No.7 is 54Asp mutation peptide; No.8 is 57Glu mutation peptide. In the experiment of peptides inhibition of the binding with MASP1-N, MASP2-N of MBL-CLR, the result shows No.3 and No.5 peptides have some activity of inhibition, the inhibiting ratio are 25%-45%; No.2 peptides have the high activity of inhibition, the inhibiting ratio is 50%. No.9 peptide is designed basied on the result of the activity of inhibition of No.2, 3, 5 peptides and no activity of inhibition of No.1, 4, 6, 7, 8 peptides, also considered the single interruption of CLR and based on the reference, pyrrolidinecarboxylic acids(P) in the peptide are substituted by the hydroxyprolines(O). No.9 peptides have the high activity of inhibition, the inhibiting ratio is 70%.The sequence of No.9 peptide is GLRGLQGPOGKLGPOG, local on MBL from 45 to 60 amino acid site, total 16 amino acids just behind the interruption of the CLR. In mammal, the sequence of No.9 peptide on CLR is conserved. So perhaps the binding location of MBL with MASPs is same in other genera. This conclusion also needs to be proved. But the avidity of No.9 binding with MASPs is low and great research efforts have to be made to develop some effective peptides to inhibitor of MASPs.Also using the character of MBL-CLR binding with the ClqR of U937 cell, we build an cell ELISA system of U937 binding with MBL-CLR protein. The result shows CLR really can bind with the ClqR ofU937 cell, but no peptides can efficiently inhibit the binding. This proves that the location of CLR binding with the C 1 qR of U937 cell is not same as binding with the Masps, but the really binding location need to continue research.Partâ…£Interaction of MASPs with Mutation Proteins of MBLThe MASP1-N and MASP2-N can bind not only with wild type of MBL but also with the 32Cys, 34Asp, 37Glu mutation proteins of MBL. At the same coating concentration of MASPs, bonding force of the wild type MBL is higher than the others. At the same situation, the bonding force of the 32Cys mutation protein binding with the MASPs is the lowest, 34Asp mutation protein in the middle, and the 37Glu mutation protein at the top relative. In addition, the No.9 peptide can inhibit the MASP1 and 2-N binding with the mutation proteins of MBL. It shows the three point mutation sites is not location on the sites of MASPs binding with mutation protein of MBL-CLR. Although the bonding force has been influenced, we suppose the mutations of MBL lead to the low oligomerization and influence the interaction with the MASPs, not because of the MASPs directly binding on the mutation sites.