The Mutation Of The Diversity Amino Acid In The C Terminal CRD Of Galectins Of Haemonchus Contortus Affects The Bioactivities Of The Recombinant Proteins

The galectins of Haemonchus contortus contained two tandemly repeated carbohydrate-recognition domain (CRD). The motifs of C-temminal CRD of recombinant female and male galectins of adult H. contortus were WGNEGR and WGNEER respectively, varied by only one amino acid. The two galectins presented different capabilities in hemagglutination, lactose-binding, decreasing cytokine mRNA transcription and inducing apoptosis of peripheral blood lymphocytes (PBLCs) of goats in vitro.Based on the previous researchs, we carried on the following researchs:1. Mutagenesis of the diversity amino acid in the C terminal CRD of recombinant galectins and the clone of the CRDs of Haemonchus contortusSite-dirceted mutagenesis was performed to substitute the different amino acid in the C terminal CRD of Hco-gal-m and Hco-gal-f. The second E in the CRD, WGNEER, of Hco-gal-m was mutated to glycine acid (G) and resulted in a recombinant galectin (MG mutate), identical to that of Hco-gal-f. The G in Hco-gal-f CRD, WGNEGR, was mutated to E and produced a recombinant galectin (FE mutate) equal to that of Hco-gal-m. DNA sequence analysis proved that the sequence of the two mutants were the same with the designed sequences. At the same time, the 340bp CRDs of the N-terminal (FNh, MNh), and the 540bp CRDs of the C-terminal (FCh,MCh) of Hco-gal-f and Hco-gal-m were amplified by PCR. DNA sequence analysis confirmed the correctness.2. Prokaryotic expression of the recombinant proteins and the refolding of expressional products.Recombinant MG mutate, FE mutate, MNh, MCh, FNh and FCh were expressed using temperature sensitive plamid pBV220 in E. coll. SDS-PAGE gel showed that the molecular weight of recombinant MG mutate and FE mutate were 32.5kDa; MNh and FNh were 14.4kDa; MCh and FCh were 20.0kDa. The six recombinant galectins formed insouble inclusion bodies. The yield of recombinant galectins was higher utilizing 8mol/L urea to solubilize inclusion bodies and then dialyzed in TE buffer containing degressive concentration of urea step by step.3. The mutated amino acid in the C terminal CRD affects the hemagglutination characteristics of galectins of Haemonchus contortusThe effects of the second glutamic acid (E) in the C-terminal CRDs on the hemagglutination characteristics of the recombinant galectins of nematode Haemonchus contortus were observed by comparing the hemagglutinating ability of HCO-GAL-f, Hco-GAL-m and their mutants MG mutate, FE mutate. Hemagglutinating assay indicated that the mutation of the second E in the C-terminal CRD reversed the abilities of the recombinant galectins binding to type B human erythrocytes. The agglutination titers of purified recombinant Hco-GAL-m and HCO-GAL-f were 2^-8 and 2^-6, respectively. Their mutants, MG mutate and FE mutate, presented titers of 2^-5 and 2^-9. These results suggested that the second E in WGxExR motif played an important role in the hemagglutination and sugar-binding of the galectin. The hemagglutinating ability of the N-terminal and C-terminal CRDs of the galectins were also compared. The titers of MCh and FCh were2^-5 and 2^-4, respectively, compared to 2^-1 and 2^-1 of MNh and FNh. The results showed that the C-terminal CRDs had higher hemagglutinating capabilities to type B human erythrocytes than the N-terminal CRDs. Sugar inhibition assay suggested that lactose and D-galactose were both effective inhibitors of the agglutination of human type B erythrocytes by the 8 recombinant galectins. The minimal inhibitory concentration of lactose was lower than that of D-galactose.4. The mutated amino acid in the C terminal CRD affects the carbohydrate-binding ability of galectins of Haemonchus contortusThe effects of the second glutamic acid (E) in the C-terminal CRDs on the lactose-binding characteristics of the recombinant galectins of nematode Haemonchus contortus and the sugar-binding abilities of CRDs were observed by comparing the lactose-binding ability of recombinant Hco-GAL-f, HCO-GAL-m, MG mutate, FE mutate, MCh, FCh, MNh, FNh. The results showed that Hco-GAL-m and FE mutate (the mutant of Hco-GAL-f) bound effectively to a-lactose-agarose compared to Hco-GAL-f and MG mutate (the mutant of Hco-GAL-m), which almost could not bind to the conjugate column. The binding ability of the MCh and FCh were significantly reduced compared to that of the Hco-GAL-m and FE mutate ,but still remained. As for the MNh and FNh, no elution peak was observed in the lactose-agarose affinity chromatography. These results suggested that the second E in WGxExR motif played an important role in sugar-binding of the galectin, and C-terminal CRDs had stronger carbohydrate-binding ability than N-terminal CRDs.5. The research on the specific active site of recombinant galectins of Hemonchus contortus to induce apoptosis of goat peripheral blood lymphocytesThe specific active site of recombinant galectins of male and female Hemonchus contortus (Hco-GAL-m/f) to induce apoptosis of goat peripheral blood lymphocytes were examined by DNA agarose gel electrophoresis and flow cytometry analysis. The results showed that all of the 8 recombinant galectins (Hco-GAL-m, Hco-GAL-f, MG mutate, FE mutate and the four CRDs) could induce apoptosis of goat peripheral blood lymphocytes. Typical DNA ladders were showed in the DNA agarose electrophoresis gels. Quantitative analysis of the cells by flow cytometry indicated that Hco-GAL-m and FE mutate could induce more lymphocytes apoptosis than Hco-GAL-f and MG mutate. The apoptosis cells induced by MNh, MCh, FNh and FCh were much less than that of the Hco-GAL-m, FE mutate, Hco-GAL-f and MG mutate. The specific active site to induce apoptosis of goat peripheral blood lymphocytes could not be confirmed in each of the CRDs.