Biochemical Characteristics of Point Mutated Goat Lysosome Α- Mannosidase

Abstract

Background Locoweeds are widely distributed all over the world. Goats and other herbivores often get poisoning if they eat a large number of locoweeds by mistake. Swainsonine (SW), the main toxin in locoweeds, can inhibit lysosomes α-Mannosidase (LAM) competitively in animal cells. The insufficient activity of lysosomes α-Mannosidase can lead to abnormal metabolism of animals, forming alpha mannosidosis. However，the details of interaction between SW and LAM are not clear yet. Methods In this study, molecular docking was used to predict the interaction points between SW and LAM. The effect of putative points was investigated by constructing mutated LAM (LAMM) and analyzing its biochemical characteristics. Results The results showed that the Trp at the 28th position and Tyr at the 599th position of the LAM were the candidates of interaction points. Both tryptophan at position 28 of A peptide residue and tyrosine at position 599 of D peptide residue of goat LAM were mutated into glycine, and goat lysosomes were obtained α- The mutant sequence of mannosidase; The recombinant yeast GS115 / pPIC9K LAMM was successfully constructed; After SW induction, the sensitivity of goat LAMM to SW decreased significantly, and the enzyme activity decreased about 3 times compared with wild-type LAM; The optimum temperature of LAMM decreased from 55 °C to 50 °C, and the optimum pH value increased from 4.5 to 5.0; All the trivalent metal ions (Fe3+, Al3+, Cr3+) had significant activation (P < 0.05), and EDTA had inhibitory effect on goat LAM before and after mutation. Conclusions These results indicated that the sensitivity of goat LAMM to SW was significantly reduced, but other characteristics changed little, which was consistent with the molecular docking results of goat LAMM. This study provides help for the study of interaction mechanism between SW and goat LAM, and also provides a new idea for the development of animal locoweed disease control technology.