Cloning, Expression, and Characterization of Capra hircus Golgi α-Mannosidase II

• Published in: Applied biochemistry and biotechnology 2015-11, Vol.177 (6), p.1241-1251
• Main Authors: Li, Jianfei, Zhang, Jiangye, Lai, Bi, Zhao, Ying, Li, Qinfan
• Format: Article
• Language: English
• Subjects: alpha-mannosidase; amino acids; Animals; Biochemistry; Biotechnology; calcium; Capra hircus; Chemistry; Chemistry and Materials Science; Cloning, Molecular; cobalt; complementary DNA; copper; EDTA (chelating agent); Gene Expression; Gene Expression Regulation, Enzymologic - physiology; glycosylation; Goats - genetics; Goats - metabolism; iron; mammals; manganese; Mannosidases - biosynthesis; Mannosidases - genetics; molecular models; molecular weight; open reading frames; Organ Specificity - physiology; Pichia pastoris; polypeptides; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; spleen; Spleen - enzymology; therapeutics; zinc
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-015-1810-0

Golgi α-mannosidase II (GMII), a key glycosyl hydrolase in the N-linked glycosylation pathway, has been demonstrated to be closely associated with the genesis and development of cancer. In this study, we cloned cDNA-encoding Capra hircus GMII (chGMII) and expressed it in Pichia pastoris expression system. The chGMII cDNA contains an open reading frame of 3432 bp encoding a polypeptide of 1144 amino acids. The deduced molecular mass and pI of chGMII was 130.5 kDa and 8.04, respectively. The gene expression profile analysis showed GMII was the highest expressed gene in the spleen. The recombinant chGMII showed maximum activity at pH 5.4 and 42 °C and was activated by Fe²⁺, Zn²⁺, Ca²⁺, and Mn²⁺ and strongly inhibited by Co²⁺, Cu²⁺, and EDTA. By homology modeling and molecular docking, we obtained the predicted 3D structure of chGMII and the probable binding modes of chGMII-GnMan₅Gn, chGMII-SW. A small cavity containing Tyr355 and zinc ion fixed by residues Asp290, His176, Asp178, and His570 was identified as the active center of chGMII. These results not only provide a clue for clarifying the catalytic mechanism of chGMII but also lay a theoretical foundation for subsequent investigations in the field of anticancer therapy for mammals.