Molecular characterization of Capra hircus lysosomal α-mannosidase and potential mutant site for the therapy of locoweed poisoning

摘要

Lysosomal α-Mannosidase (LAM) belongs to the glycoside hydrolyzing enzymes family 38 and is involved in the biosynthesis and turnover of N-linked glycoproteins process. Locoweeds, which contain swainsonine (SW) that inhibits LAM, are the main poisoning plants in many regions of the world, and thereby resulting in animal poisoning or even death. Based on regions of protein sequence conservation between LAM from Bos taurus and Homo sapiens , we cloned cDNA encoding Capra hircus LAM ( ch LAM). Expression of cDNA in Pichia pastoris resulted in the secretion of aLAM activity into the culture medium. The recombinant ch LAM was activated 1.6 and 1.2-fold with Zn²⁺ and Ca²⁺, respectively. By homology modeling, molecular docking and mutant analysis, we obtained the probable binding modes of SW at the allosteric sites of ch LAM, and the potential mutant sites for the resistance to SW. Prediction of SW sensitivity to A28 W/G, D58 Y/G mutant ch LAM is lower than wild type ch LAM. The obtained results lead to a better understanding of not only interactions between substrate/SW and ch LAM, but also of a potential strategy for a novel therapy for locoweed poisoning.