Cloninq Expression, Purification, And Characterization Of Goat Lysosomal Acid α-Mannosidase

-Mannosidase (AMA) belongs to the glycoside hydrolyzing enzymes family. VariousAMA widely distributed in animals, plants and microorganisms are involved in thebiosynthesis and turnover of N-linked glycoproteins process. Locoweeds are main poisoningplants on the western lands in China. In addition to the destruction of grassland ecosystem, ithas causing goats, horses, and sheep death and large economical loss every year. The plantscontaining the toxic indolizidine alkaloid swainsonine (SW) that inhibits AMA, and therebyresulting in animal poisoning, eventually death. At present, it has been reported a variety ofanimal’ AMA gene sequences. However, there is no detailed research about the characteristicof locoweeds sensitivity animal Capra hircas lysosomal AMA (chLAM). Many domestic andforeign scholars have made great progress on prevention and treatment of locoweed poisoning,but has not yet been reveal the mechanism about SW inhabits chLAM. In this work, the geneof goat lysosomal α-AMA will be cloned, expressed, and the characterization of α-AMA willbe studied. In addition, homology modeling and molecular docking methods will also beperformed to further predict the structure properties, the probable binding modes of SW at theallosteric sites of α-AMA, and the potential mutant sites for the resistance to SW. Results areas follows：1. Three primers were designed according to the the published cattle LAM sequence inthe GenBank and the whole open reading frame of the chLAM cDNA sequence were obtainedby RT-PCR technology and has been submitted to the GenBank (JN602369). Throughbioinformatic analysis, chLAM protein signal peptide sequence should be set at1aa~50aa.The molecular formula, pI, and weight of the removal signal peptide chLAM protein isC4825H7440N1350O1392S29,8.01, and107.56KD, respectively.2. Using the technology of fluorescence quantitative PCR, chLAM gene expressionprofiling was obtained. The results showed that the chLAM gene was expressed in heart, liver,spleen, lung, kidney, brain, cerebellum, muscle, and ovarian tissue, but of different expressionlevel. In the lungs and liver, it is significantly higher than that in other tissues (P <0.05), andcardiac and muscle tissue showed the lowest.3. Using the SWISS-MODEL homology modeling tools and procheck software analysis, three-dimensional structure of remove signal peptide chLAM protein were obtained.4. Through molecular docking and site-directed mutagenesis, we confirmed that theactivity site of chLAM was formed by9amino acids of A, C, D chain. SW, KIF, and dMNJcould be competitive inhibit chLAM’ activity. A28SW sensitivity to Trp Gly, D58Tyr Glymutant chLAM is lower than wild type, which can be applied to the prevention and treatmentof locoweed poisoning.5. By construction the yeast expression vector of pPICZα A-chLAM, linearized, electricinto the Pichia yeast X-33,1%methanol induced expression, and determination of activity,we detected the chLAM activity of expression product of was55U.