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

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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 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.

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Acknowledgments

This work was supported by Shaanxi Province Program for Science and Technology Research Development Plan (2014k01-17-02) of China and the central university basic scientific research operation cost special fund subsidizes (ZD2012009).

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Authors and Affiliations

  1. College of Veterinary Medicine, Northwest A&F University, Xian, China

    Jianfei Li, Jiangye Zhang, Bi Lai, Ying Zhao & Qinfan Li

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  1. Jianfei Li
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  2. Jiangye Zhang
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  3. Bi Lai
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  4. Ying Zhao
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Correspondence to Qinfan Li.

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ESM 1

Intracellular and extracellular expression of chGMII. Lanes M, protein standard; 1–6, intracellular expression of chGMII in different pH; 7 and 14, intracellular and extracellular expression of negative control; 8–13, extracellular expression products in different pH. (GIF 1.95 mb)

High resolution image file (TIF 486 kb)

ESM 2

Raw real-time PCR data of Fig. 1a (XLS 115 kb)

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Li, J., Zhang, J., Lai, B. et al. Cloning, Expression, and Characterization of Capra hircus Golgi α-Mannosidase II. Appl Biochem Biotechnol 177, 1241–1251 (2015). https://doi.org/10.1007/s12010-015-1810-0

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