Site-Directed Mutagenesis Of Capra Hircus Lysosomal α-Mannosidase, And The Sensitivity To SW Of Mutant Product

Lysosomal α-Mannosidase (LAM) belongs to the glycoside hydrolyzing enzymes family38and involved in the biosynthesis and turnover of N-linked glycoproteins process. In many regions of the world, Locoweeds which contains swainsonine (SW) are the main poisoning plants. Locoweed poisoning due to its major toxic components Swainsonine (SW) inhibition of lysosomal a-mannosidase (AMA) leaving the body mannose metabolism abnormality, a large accumulation in the cells and cause cell degeneration and ultimately leading to animal poisoning or even death. At present, there is no effective solution to it. Kong Xiangya et al cloned and expressed Capra hircus lysosomal AMA. By homology modeling, molecular docking and mutant analysis, they obtained the probable binding modes of SW at the allosteric sites of chLAM, and the potential mutant sites for the resistance to SW. In this study, according to the prediction, we used overlap extension PCR to site-directed mutant chLAM gene, to construct wild-type and mutant-type recombinant yeast expression plasmid, to purified the supernatant and study the enzymatic characterization after expression in Pichia pastoris GS115. The obtained results lead to a better understanding of not only interactions between substrate/SW and chLAM, but also a potential strategy for a novel pharmaceutical preparation for locoweed poisoning. Results are as follows:1. Three pair of site-directed mutant primers were designed according to the the published Capra hircus LAM sequence in the GenBank(JN602369). Using the technology of overlap extension PCR, the predicted A28W/G, D58Y/G mutant chLAMs were obtained.2. The target mutant gene was sub-cloned into pEASY-Blunt Simple vector, than be sent to sequence, Blast showed that chLAM gene was mutated successfully, there is no frame shift mutation in CDS sequence, cloning vector was constructed successfully.3. Taking the correct recombinant cloning vectors as a template, re-design primers, which was added restriction sites and protect bases. After digested by restricted enzyme, the target gene can be directly sub-cloned into yeast expression vector pPIC9K to construct recombinant yeast expression plasmid. Blast showed that wild type and mutant recombinant yeast expression plasmids were constructed successfully.4. Using restricted enzyme Pme I to linearize the construction the yeast expression vector of pPIC9K-chLAM and pPIC9K-chLAM-A28-D58, the electric them into the Pichia yeast GS115. After induced by1%methanol, the enzyme activity of the recombinant protein was determined, we detected that the recombinant wild-type chLAM activity of expression product was104U,while the mutant-type one was39U. The activity of both type expression product can be promoted by Zn+、Ca2+5. Compared to the wild-type recombinant protein, the sensitivity to SW of purified mutant-type recombinant protein was really declined.