Expression Of Human CGT52TGT, GGC54GAC And GGA57GAA MBL Mutants In Mammal Cells And Analysis Of The Expression Products

Mannan-binding lectin (MBL), a member of the collectin superfamily (C-type lectin with a collagen-like domain), is a plasma glycoprotein mainly secreted by liver cells. As an important molecule of the innate immune system, MBL triggers the lectin pathway of complement activation. Recently, more and more data indicate that MBL insufficiency is closely associated with recurrent infection of a wide range pathogen.Human MBL gene locates at chromosome 10q11.2-q21. Three different genetic polymorphisms in exon 1 independently lead to MBL insufficiency and futher to opsonization deficiency. CGT52TGT, GGC54GAC and GGA57GAA mutations result in Arg→Cys, Gly→Asp and Gly→Glu substitutions, respectively. These mutant genes are codominant inheritance on euchromosome. The frequencies of these point mutations vary significantly among ethnics but are very high overall. For instance, the frequency of mutation of codon 54 in exon 1 of MBL gene in Eurasian populations is higher (0.11~0.14), that of codon 57 in sub-Saharan African very high (0.23~0.29), but that of mutation of codon 52 relatively low (<0.1) in all tested populations. Obviously, MBL insufficiency is one of the most common immunodeficiencies. Therefore, it's of great significance to illustrate the mechanisms by which the mutations of MBL gene cause immunodeficiency.However, it is still not clear what directly causes MBL protein level reduction inserum——whether is it due to low secretion or unstable structure of MBL proteincaused by the mutations of MBL gene? To better understand the molecular mechanism how the mutations of MBL gene lead to immunodeficiency and to further clarify the struction-function relationship of MBL molecule, we set three variantMBL genes, CGT52TGT, GGC54GAC and GGA57GAA mutants, as our research objects. The transient and stable expressions of them in mammalian cells and the expression products were analyzed.1. Preparation and identification of MBL specific-epitope antibodyBased on the prediction of the dominant epitopes for B cell's recognition in natural MBL molecule, the 15mer peptide, NH2-EPGQGLRGLQGPPGK-C00H, was synthesized. A polyclonal antibody against human MBL specific-epitope was produced by immunizing rabbits with the immunogen that was prepared by conjugating the peptide with carrier protein KLH (peptide-KLH). The antiserum titer was 1:128 000 by ELISA using peptide-OVA as the coated antigen. The polyclonal antibody was purified by antigenic peptides affinity columns. It was showed that the product of purification, named MBL specific-epitope antibody (MSA), can detect 5(i,g/ml peptide-OVA at the level of 3.32 ng/ml by ELISA and react with recombinant wild type MBL by Western blotting.2. Transient expression of wild type and variant MBL genes in COS7 cellsPreviously, our research team constructed the plasmids pMBLm52, pMBLm54 and pMBLm57 that contain respevtively three MBL mutant genes. Using Primer Premier 5.0, we designed and synthesized a pair of special primmer. The MBL genes containing CGT52TGT, GGC54GAC or GGA57GAA point mutations were amplified from the plasmid pMBLm52, pMBLm54 and pMBLm57 by PCR and then were inserted into the eukaryotic expression vector pcDNA4/HisMax C. It was showed three recombinant expression vectors, pcDNA4/HisMax C-MBLm52, pcDNA4/ HisMax C-MBLm54 and pcDNA4/HisMax C-MBLm57, are successfully consturcted by DNA sequencing.The recombinant expression vectors, pcDNA4/HisMax C-MBLm52, pcDNA4/ HisMax C-MBLm54, pcDNA4/HisMax C-MBLm57 and pcDNA4/HisMax C-MBLw, were transfected into COS7 cells by Iipofectamine? Reagent, respectively. In each transfection, the mass of the expression vectors and the number of COS7 cells were the same. 72 hours later, the supematants were collected and the expression product in each supernatant was quantitated by ELISA. It was found that there are no obvious differencies among the levels of four forms of MBL proteins secreted by COS7 cells.So, we infer that the point mutations in the exon 1 of MBL gene might not interrupt the synthesis and secretion of MBL protein.3. Expression of MBL mutants in CHO cells and the analysis of the productsThe three recombinant expression vectors, pcDNA4/HisMax C-MBLm52, pcDNA4/HisMax C-MBLm54 and pcDNA4/HisMax C-MBLm57, were transfected into Chinese-hamster ovary (CHO) cells by electroporation, respectively. Zeocin of 800 mg/L has been used for 30 days to select electroporated CHO cells, and then 200 mg/L for another 30 days to obtain transfected stably cells (CHO-MBLm52, CHO-MBLm54 and CHO-MBLm57). The expression of mRNA was analyzed by RT-PCR; the recombinant protein was purified from the culture supernatant by Ni2+-NTA agarose chromatography and analyzed by SDS-PAGE under nonreducing conditions and Western blot. It was found that there are no evident differencies among the transcription levels of the wild type gene and the three mutant genes and all the purified recombinant proteins, named 52Cys, 54Asp and 57Glu mutant MBL proteins respectively, can react with the MBL specific-epitope antibody, anti-His antibody or anti-wild type MBL antibody, which were identified by Western blot or ELISA. It was showed by SDS-PAGE that the mutant proteins appear mainly at the site of Mr 60 000 but wild type protein appears mainly at Mr 120 000 or above, which indicates that mutant proteins have a much lower oligomerization level than those of the recombinant human wild MBL and human plasma-derived MBL proteins. It was found by the ligand-binding assay that only protein forms with high oligomerization level, such as wild type or plasma-derived natual MBL, can bind with mannan, but three mutant MBL proteins lose this function due to the low oligomerization.We concluded that CGT52TGT, GGC54GAC and GGA57GAA point mutations in MBL gene do not affect the synthesis and secretion of their products, but Arg-?Cys, Gly->Asp or Gly-?Glu substitution in MBL peptide may disrupt the structure of MBL molecule, as well as its function.