Cloning, Expression And Characterization Of Capra Hircus Golgi α-mannosidase Ⅱ

Golgi α-mannosidase Ⅱ(GMⅡ), a key glycosyl hydrolase in the N-linked glycosylation pathway, regulates cell recognition by modifying the glycoprotein on the cell surface. It has been reported that the inhibition of GMⅡ impede the growth and metastasis of cancer cells. For example, swainsonine(SW), a natural inhibitor of GMⅡ, has been shown to reduce certain tumors and hematological dysfunctions. Unfortunately, this potent inhibitor often co-inhibits lysosomal α-mannosidases, inducing symptoms similar to lysosomal storage disease α-mannosidosis. Hence, it is indispensable for anticancer treatment to find specific inhibitors of GMⅡ activity. Understanding the structural basis for the catalytic and inhibition process of GMⅡ might provide unique opportunities for designing the selective inhibitors. However, the crystal structure was only isolated from Drosophila melanogaster. The GMⅡ c DNA and gene have been isolated from Drosophila melanogaster, murine, human, Arabidopsis thaliana and Spodoptera frugiperda, and successfully expressed in cells. But, there is no detailed report on GMⅡ of any mammalian species, which narrowed the research scope of anticancer therapy. This study focused on a common mammal, goat. The chGMⅡ c DNA was cloned by RT-PCR, and expressed by Pichia pastoris expression system. The 3D structure and the binding sites of chGMⅡ-substrate and chGMⅡ-intibitor were predicted with the help of homology modeling and molecular docking to find out the catalytic mechanism, laying the theoretical foundation for subsequent anticancer therapy of mammals. Results are as follows:1. The 3552 bp chGMⅡ cDNA sequence was obtained by RT-PCR, and has been deposited into GenBank data base under accession number KF717587. Bioinformatic analysis showed that, the ORF of 3432 bp encodes a polypeptide of 1144 amino acids with a calculated molecular weight of 130.5 kDa and theoretical isoelectric point(pI) of 8.04. No signal peptide and just one transmembrane domain were detected in the amino acid sequence. The predicted secondary structure of chGMⅡ comprise of 23.5% of α-helix, 22.1% of β-strand and 54.4% of loop.2. The relative transcript level of chGMⅡ gene in different tissues was detected by qPCR. The results showed chGMⅡ were expressed in all examined tissues with different levels. The highest expression was detected in spleen, followed by liver, lung and ovary. In contrast, the heart and kidney exhibited very low expression levels of GMⅡ.3. 3D structure of chGMⅡ was created by SWISS-MODEL Repository, the quality was assessed by Procheck. The representative substrate and inhibitor of GMⅡ, Gn Man5Gn2 and SW, were respectively docked with the modeled enzyme. Result showed that the T5-square-based pyramidal geometry, involving a zinc ion and residues Asp178, His176, Asp290 and His570, was the active site of chGMⅡ.4. The recombinant expression plasmid pPICZαA-chGMⅡ was constructed and introduced into X-33 for methanol induction. The expression products were identified by SDS-PAGE and Western-blot. Result indicated the recombinant chGMⅡ with molecular weight 130 kDa was successfully expressed, and its intracellular expression was more abundant than extracellular; it was appropriate for alkalinity and abundant expressed in pH 8.5. After purified by Ni-NTA Resin, target protein was collected for enzyme assay; the enzyme activity of chGMⅡ was computative 149.38 U. The recombinant chGMⅡ showed optimum activity at pH 5.4 and temperature around 42 ℃ for substrate pNP-α-Man. chGMⅡ was activated by Fe2+, Zn2+, Ca2+ and Mn2+, and strongly inhibited by Co2+, Cu2+ and EDTA.