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.

Biochemical characteristics of point mutated <i>Capra hircus</i> lysosome α-mannosidase

“…The plasmid pMD19T-LAM containing the chLAM gene fragment has been constructed by our laboratory [43]; The pEASY-Blunt Simple vector is preserved in our laboratory; The yeast expression strain Pichia pastoris GS115 and the Pichia pastoris protein expression vector pPIC9K were presented by Mr. Zhang Yanming (Northwest A&F University, Yangling, China).…”

“…Therefore, an overall quality of modeled structure was good as checked by the program. The chLAM consists of 949 amino acid residues which can be divided into five peptide fractions: A (1-298 amino acid), B (299-382 amino acid), C (383-541 amino acid), D (542-823 amino acid) and E (824-949 amino acid) [43]. Before and after the mutation, SW and oligosaccharide D-Mannose 5 (D-Mannose 5, abbreviated as man5) were respectively molecularly docked with chLAM.…”

“…Before and after the mutation, SW and oligosaccharide D-Mannose 5 (D-Mannose 5, abbreviated as man5) were respectively molecularly docked with chLAM. According to Kong et al [43], the chLAM genes were docked and analyzed using the AutoDock v4.2.6(The Center for Computational Structural Biology, La Jolla, CA, USA). The corresponding amino acid position was denoted by the following: peptide + colon + single-letter amino acid symbol + amino acid sequence number (such as A: H23, which means A-peptide histidine acid residue 23).…”

“…The system and operation for determining the specific enzyme activity can be found in Kong's report [43]. The number of nanomoles of p-nitrophenol were produced by hydrolysis per liter of bacterial solution per second (nM L -1 sec -1 ) was α-mannosidase activity units (U).…”

Cloning, Expression, and Characterization of Capra hircus Golgi α-Mannosidase II

“…All reactions and controls were carried out in triplicate and the results averaged. One unit of enzyme activity was defined as the amount of enzyme releasing 1 nmol of p-nitrophenol per second at 37°C [25].…”