title: Molecular characterization of Capra hircus lysosomal α-mannosidase and potential mutant site for the therapy of locoweed poisoning.
abstract: 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 (chLAM). Expression of cDNA in Pichia pastoris resulted in the secretion of aLAM activity into the culture medium. The recombinant chLAM was activated 1.6 and 1.2-fold with Zn(2+) and Ca(2+), respectively. By homology modeling, molecular docking and mutant analysis, we obtained the probable binding modes of SW at the allosteric sites of chLAM, and the potential mutant sites for the resistance to SW. Prediction of SW sensitivity to A28 W/G, D58 Y/G mutant chLAM is lower than wild type chLAM. The obtained results lead to a better understanding of not only interactions between substrate/SW and chLAM, but also of a potential strategy for a novel therapy for locoweed poisoning.
authors.affiliation: College of Veterinary Medicine, Northwest A & F University, Yangling 712100, Shaanxi, China.
authors.forename: Kong
authors.initials: K
authors.lastname: Xiangya
authors.affiliation: College of Veterinary Medicine, Northwest A & F University, Yangling 712100, Shaanxi, China.
authors.forename: Zhang
authors.initials: Z
authors.lastname: Jiangye
authors.affiliation: College of Veterinary Medicine, Northwest A & F University, Yangling 712100, Shaanxi, China.
authors.forename: Wu
authors.initials: W
authors.lastname: Ying
authors.affiliation: College of Veterinary Medicine, Northwest A & F University, Yangling 712100, Shaanxi, China.
authors.forename: Li
authors.initials: L
authors.lastname: Jianfei
authors.affiliation: College of Veterinary Medicine, Northwest A & F University, Yangling 712100, Shaanxi, China.
authors.forename: Li
authors.initials: L
authors.lastname: Qinfan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics*; D006041:Goats / Q000235:genetics; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D009154:Mutation; D033021:Oxytropis / Q000235:genetics / Q000506:poisoning*; D010843:Pichia; D043323:alpha-Mannosidase / Q000737:chemistry / Q000235:genetics*
chemical_list: D006023:Glycoproteins; D043323:alpha-Mannosidase