Biochemical characteristics of point mutated Capra hircus lysosome α-mannosidase

J Vet Med Sci. 2023 Feb 21;85(2):244-251. doi: 10.1292/jvms.22-0222. Epub 2023 Jan 2.

Abstract

Locoweeds, a type of poisonous weedare, are widely distributed throughout the world and have a significant impact on the development of herbivore animal husbandry. Swainsonine (SW), the main toxin in locoweeds, can competitively inhibit lysosomes α-mannosidase (LAM) in animal cells, resulting in α-mannosidosis. However, the specifics of the interaction between SW and LAM are still unclear. Here, we used molecular docking to predicte the interaction points between SW and LAM, built mutated lysosomes α-mannosidase (LAMM), and analyzed its biochemical properties changes in presumption points. The Trp at the 28th position and the Tyr at the 599th position of the LAM were interaction point candidates, and the above two amino acids in Capra hircus LAM (chLAM), were successfully mutated to glycine by constructing recombinant yeast GS115/PIC9K- LAMM. The results showed that the sensitivity of Capra hircus LAMM (chLAMM), to SW decreased significantly compared with wild-type LAM, the enzyme activity of LAM decreased approximately threefold, the optimum temperature of LAMM decreased from 55°C to 50°C, the optimum pH value increased from 4.5 to 5.0, and the effects of Mn2+, Fe3+, Al3+, Co2+, Cr3+, and ethylenediaminetetraacetic acid (EDTA) on LAM enzyme activity before and after point mutation changed significantly. These findings help us better understanding the molecular mechanism of the interaction mechanism between SW and chLAM, and provide new reference for solving locoweeds poisoning.

Keywords: locoweed poisoning; lysosomal α-mannosidase; site-directed mutagenesis; swainsonine.

MeSH terms

  • Animals
  • Goats / metabolism
  • Lysosomes* / metabolism
  • Mannosidases / metabolism
  • Molecular Docking Simulation
  • Swainsonine*
  • alpha-Mannosidase / genetics

Substances

  • alpha-Mannosidase
  • Swainsonine
  • Mannosidases