title: Cloning, Expression, and Characterization of Capra hircus Golgi α-Mannosidase II.
abstract: 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 Fe(2+), Zn(2+), Ca(2+), and Mn(2+) and strongly inhibited by Co(2+), Cu(2+), 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.
authors: College of Veterinary Medicine, Northwest A&F University, Xian, China.
authors: Jianfei
authors: J
authors: Li
authors: College of Veterinary Medicine, Northwest A&F University, Xian, China.
authors: Jiangye
authors: J
authors: Zhang
authors: College of Veterinary Medicine, Northwest A&F University, Xian, China.
authors: Bi
authors: B
authors: Lai
authors: College of Veterinary Medicine, Northwest A&F University, Xian, China.
authors: Ying
authors: Y
authors: Zhao
authors: College of Veterinary Medicine, Northwest A&F University, Xian, China. liqinfan@nwsuaf.edu.cn.
authors: Qinfan
authors: Q
authors: Li
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D015870:Gene Expression; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006041:Goats / Q000235:genetics / Q000378:metabolism; D008361:Mannosidases / Q000096:biosynthesis / Q000235:genetics; D009928:Organ Specificity / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013154:Spleen / Q000201:enzymology*
chemical_list: D011994:Recombinant Proteins; D008361:Mannosidases; C038829:mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase
keywords: Active site; Anticancer; Capra hircus; Golgi α-mannosidase II; Pichia pastoris expression