The second glutamic acid in the C-terminal CRD affects the carbohydrate-binding properties of recombinant galectins of Haemonchus contortus

Abstract

The effects of the second glutamic acid (E) in the C-terminal CRDs on the hemagglutination and lactose-binding characteristics of the recombinant galectins of nematode Haemonchus contortus were observed using two isoforms of recombinant galectins as models, and the sugar-binding abilities of the N-terminal and C-terminal CRDs of the galectins were also compared. 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) with a CRD of WGNEGR, 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. At the same time, the CRDs of the N-terminal (FNh,MNh) and C-terminal (FCh,MCh) of Hco–GAL-f, Hco–GAL-m were amplified by PCR. The abilities of carbohydrate binding and hemagglutination of the four galectins and the four CRDs were analysed, respectively, by α-lactose–agarose affinity chromatography and hemagglutination assay. The results showed that Hco–GAL-m and FE mutate bound effectively to α-lactose–agarose compared to Hco–GAL-f and MG mutate, which almost could not bind to the conjugate column. The hemagglutinating abilities of the Hco–GAL-m and FE mutate to human B type red blood cells were similar and were nearly two times higher than that of the Hco–GAL-f and MG mutate. The hemagglutinating ability of the MCh was five times to that of the MNh and FNh and almost two times to that of the FCh. 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 amino acid E in the C-ternminal CRD motif of H. contortus galectin was involved in carbohydrate binding and hemagglutination, and C-terminal CRDs had stronger carbohydrate ability than N-terminal CRDs.