The Direct Effects Of Human Mannan-binding Lectin Carbohydrate-recognition Domain On Pathogens

Mannan-binding lectin (MBL), the member of collectins which belongs to C-type lectin superfamily, is a serum protein secreted by liver cells. It is an important pattern-recognition receptor(PRR) in the innate immune system. The overall polypeptide structure of MBL includes a cysteine-rich N-terminal domain, a collagen-like region (CLR), a neck region and a C-terminal carbohydrate- recognition domain (CRD). MBL can selectively recognize sugars such as D-mannose, N-acetyl-glucosamine, N-acetyl-mannosamine, N-acetyl-galactosamine and fucose presented on pathogens via the CRD. It is clear that the CRD can recognize a wide range of microbes such as bacteria, viruses, yeast and Leishmania. On binding to pathogens, MBL may activate the complement cascade via lectin pathway, killing the pathogens or take parting in phagocytosis of them. In addition, MBL has a direct opsonization when binding to lectin receptors on the phagocytic cells.In the researches of MBL-CRD, the biochemists have being worked on the molecular mechanisms of carbohydrate-recognition property, while the immunologists have being interested in the microorganism-recognition pattern, regardless of other functions of CRD. Recent evidences suggest that MBL can inhibit influenza A virus infection independent of complement activation, while other groups showed that MBL can interfere with the viral attachment to host cells, thus affecting viral spread and release. But no study on the correlation between this function and thestructure of MBL has been done. We propose that MBL-CRD multimer would directly neutralize viruses and inhibit bacterial adhesion to host cells, just as that of the Fab of antibodies.To verify the hypothesis, we transformed the MBL-CRD gene into E.coli expression system and acquired the purified CRD protein, and then, carried out a preliminary study on the direct effects of MBL-CRD on pathogens.Part 1 Preparation of the recombination human MBL-CRD multimerA 447 bp gene fragment of the CRD and neck region was amplified by PCR from the plasmid pGEM-mbl that contains human MBL cDNA and inserted into the prokaryotic expression vector pGEX-4Tl. The recombinant expression vector pGEX-4T-CRD was transformed into E.coli and the expression was induced by IPTG. The inclusion body was dissolved in 8M urea and denatured via gradient dilution. The GST-CRD fusion protein was purified by GSTrap gel affinity chromatography and refolded in the presence of Ca2+. Purified CRD protein was acquired using thrombin, but the yield was low.Part 2 The ligand-binding activities of MBL-CRD fusion proteinThe ligand-binding activities of GST-CRD fusion protein were evaluated by ELISA and morphologic methods, in which six carbohydrates, mannose, mannan, mannosamine, galactosamine, glucosamine and GalNAc, and five pathogens, E. coli, S. flexneri, S. typhimurium, type I S. pneumoniae and influenza A virus, were used. GST-CRD fusion protein could bind to all of them and the interaction of the protein with E. coli could be inhibited by mannan. After S. typhimuria were incubated with FITC-CRD protein for 1 h at 37°C followed by washing, the fluorescence and aggregation of the bacteria were observed under fluorescent microscopy, in which FITC-CRD protein bound the bacteria in a calcium dependent manner. These results suggest that the GST-CRD fusion protein we prepared has ligand-binding activities.Part 3 The direct effects of human MBL-CRD fusion protein on pathogensA549 and Lovo cells were selected as target cells of type I Streptococcus pneumoniae (a gram-positive bacterium) and Shigella flexneri (a gram-negativebacterium) respectively in the studies of the direct inhibitory effects of CRD on bacterial adherence. FITC- labeled bacteria can specifically adhere to target cells. FITC-labeled bacteria were incubated with the GST-CRD fusion protein at various concentrations for 1 h, added to the target cells and incubated for 3 h at 37°C followed by washing. It was found that the fusion protein can effectively inhibit the adherence of pathogens to target cells, which was indicated by the fact that the number of bacteria adherent to target cells increased statistics-significantly (P<0.01) as the concentration of the CRD fusion protein decreased. So, it is clear that GST-CRD fusion protein may depress directly bacterial adhesion to target cells in a dose- dependent manner.Most pathogens invade the host cells in the premise of adherence. Our research suggested that MBL-CRD may directly suppress the adherence of pathogens to the target cells. This observation is helpful for the elucidations of the correlation of structure and functions of MBL molecule and may be of potential therapeutic benefit to the patients with infections.