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http://dx.doi.org/10.5352/JLS.2016.26.3.275

Identification and Functional Analysis of Pig β-1,4-N-Acetylglucosaminyltransferase A (MGAT4A)  

Kim, Ji-Youn (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration)
Hwang, Hwan-Jin (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration)
Chung, Hak-Jae (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration)
Park, Mi-Ryung (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration)
Byun, Sung June (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration)
Kim, Kyung-Woon (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration)
Publication Information
Journal of Life Science / v.26, no.3, 2016 , pp. 275-281 More about this Journal
Abstract
Glycan modification is important in pharmaceutical industry. Especially, sialic acid affects the bioactivity and stability of medicine. Milk of pig has been used as bioreactor to produce various pharmaceutical proteins. Therefore, it is necessary to modify the glycan chain in pig mammary grand. β-1,4-N-Acetylglucosaminyltransferase A (pMGAT4A) is one of the essential enzymes for increase of sialic acid content, but pig MGAT4A is unclear. In this study, the pMGAT4A was identified and characterized. The pMGAT4A has 1638 nucleotides encoding 535 amino acids and type II membrane topology, which is one of the common features in many glycosyltransferases. The gene was strongly expressed in liver and mammary gland, whereas was weakly expressed in small intestine, stomach and bladder. For functional test, HA-tagged MGAT4A was over-expressed in porcine kidney (PK-15) cell line. Forced expression of pMGAT4A gene was identified by qPCR, and we identified that pMGAT4A is located in Golgi complex by co- staining with HA antibody and BODIPY TR ceramide. In addition, we identified the increase of mannose-β-1,4-N-acetylglucosamine structure by ELISA and immunofluorescence using Datura stramonium agglutinin (DSA), which recognizes mannose-β-1,4-Nacetylglucosamine. Through the specific activity analysis, we showed that pMGAT4A modified bi-antennary to tri-antennary. This event affects sialic acid content. Therefore, we thought that over-expression of pMGAT4A will be necessary in pig mammary grand for improved medicine.
Keywords
DSA; PK-15; Pig β -1; 4-N-Acetylglucosaminyltransferase A; sialic acid;
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1 Egrie, J. C., Dwyer, E., Browne, J. K., Hitz, A. and Lykos, M. A. 2003. Darbepoetin alfa has a longer circulating half-life and greater in vivo potency than recombinant human erythropoietin. Exp. Hematol. 31, 290-299.   DOI
2 Elliott, S., Egrie, J., Browne, J., Lorenzini, T., Busse, L., Rogers, N. and Ponting, I. 2004. Control of rHuEPO biological activity: the role of carbohydrate. Exp. Hematol. 32, 1146-1155.   DOI
3 Freitas, V. J., Serova, I. A., Andreeva, L. E., Dvoryanchikov, G. A., Lopes, E. S. Jr., Teixeira, D. I., Dias, L. P., Avelar, S. R., Moura, R. R., Melo, L. M., Pereira, A. F., Cajazeiras, J. B., Andrade, M. L., Almeida, K. C., Sousa, F. C., Carvalho, A. C. and Serov, O. L. 2007. Production of transgenic goat (Capra hircus) with human granulocyte colony stimulating factor (hG-CSF) gene in Brazil. An. Acad. Bras. Cienc. 79, 585-592.   DOI
4 Fukuta, K., Abe, R., Yokomatsu, T., Kono, N., Asanagi, M., Omae, F., Minowa, M. T., Takeuchi, M. and Makino, T. 2000. Remodeling of sugar chain structures of human interferon-gamma. Glycobiology 10, 421-430.   DOI
5 Fukuta, K., Yokomatsu, T., Abe, R., Asanagi, M. and Makino, T. 2000. Genetic engineering of CHO cells producing human interferon-gamma by transfection of sialyltransferases. Glycoconj. J. 17, 895-904.   DOI
6 Gil, G. C., Velander, W. H. and Van Cott, K. E. 2008. Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk. Glycobiology 18, 526-539.   DOI
7 Houdebine, L. M. 2009. Production of pharmaceutical proteins by transgenic animals. Comp. Immunol. Microbiol. Infect. Dis. 32, 107-121.   DOI
8 Jänne, J., Hyttinen, J. M., Peura, T., Tolvanen, M., Alhonen, L., Sinervirta, R. and Halmekytö, M. 1994. Transgenic bioreactors. Int. J. Biochem. 26, 859-870.   DOI
9 Ko, H. K., Song, K. H., Jin, U. H., Seong, H. H., Chang, Y. C., Kim, N. H., Kim, D. S., Lee, Y. C. and Kim, C. H. 2010. Molecular characterization of pig alpha2,3-Gal-beta1,3- GalNAc-alpha2,6-sialyltransferase (pST6GalNAc IV) gene specific for Neu5Acalpha2-3Galbeta1-3GalNAc trisaccharide structure. Glycoconj. J. 27, 367-374.   DOI
10 Koles, K., van Berkel, P. H., Mannesse, M. L., Zoetemelk, R., Vliegenthart, J. F. and Kamerling, J. P. 2004. Influence of lactation parameters on the N-glycosylation of recombinant human C1 inhibitor isolated from the milk of trans-genic rabbits. Glycobiology 14, 979-986.   DOI
11 Kyte, J. and Doolittle, R. F. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105-132.   DOI
12 Lee, H. G., Lee, H. C., Kim, S. W., Lee, P., Chung, H. J., Lee, Y. K., Han, J. H., Hwang, I. S., Yoo, J. I., Kim, Y. K., Kim, H. T., Lee, H. T., Chang, W. K. and Park, J. K. 2009. Production of recombinant human von Willebrand factor in the milk of transgenic pigs. J. Reprod. Dev. 55, 484-490.   DOI
13 Lee, P., Chung, H. K., Lee, H. G., Lee, H. C., Woo, J. S., Lee, S., Jo, S. J., Chang, W. K., Lee, H. T., Kwon, M. and Park, J. K. 2008. Cloning and characterization of 5'-untranslated region of porcine beta casein gene (CSN2). Domest. Anim. Endocrinol. 35, 245-253.   DOI
14 Schnieke, A. E., Kind, A. J., Ritchie, W. A., Mycock, K., Scott, A. R., Ritchie, M., Wilmut, I., Colman, A. and Campbell, K. H. 1997. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science. 278, 2130-2133.   DOI
15 Lindsay, M., Gil, G. C., Cadiz, A., Velander, W. H., Zhang, C. and Van Cott, K. E. 2004. Purification of recombinant DNA-derived factor IX produced in transgenic pig milk and fractionation of active and inactive subpopulations. J. Chromatogr. A. 1026, 149-157.   DOI
16 Montesino, R., Toledo, J. R., Sánchez, O., Sánchez, A., Harvey, D. J., Royle, L., Dwek, R. A., Rudd, P. M., Gerwig, G. J., Kamerling, J. P. and Cremata, J. A. 2008. Monosialylated biantennary N-glycoforms containing GalNAc-GlcNAc antennae predominate when human EPO is expressed in goat milk. Arch. Biochem. Biophys. 470, 163-175.   DOI
17 Park, J. K., Lee, Y. K., Lee, P., Chung, H. J., Kim, S., Lee, H. G., Seo, M. K., Han, J. H., Park, C. G., Kim, H. T., Kim, Y. K., Min, K. S., Kim, J. H., Lee, H. T. and Chang, W. K. 2006. Recombinant human erythropoietin produced in milk of transgenic pigs. J. Biotechnol. 122, 362-371.   DOI
18 Su, D., Zhao, H. and Xia, H. 2010. Glycosylation-modified erythropoietin with improved half-life and biological activity. Int. J. Hematol. 91, 238-244.   DOI
19 Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W. and Etzler, M. E. 2009. Essentials of Glycobiology, 2nd ed. Cold Spring Harbor Laboratory Press, NY.
20 Wu, Z. L., Ethen, C. M., Prather, B., Machacek, M. and Jiang, W. 2011. Universal phosphatase-coupled glycosyltransferase assay. Glycobiology 21, 727-733.   DOI
21 Yoshida, A., Minowa, M. T., Takamatsu, S., Hara, T., Oguri, S., Ikenaga, H. and Takeuchi, M. 1999. Tissue specific expression and chromosomal mapping of a human UDP-Nacetylglucosamine: alpha1,3-d-mannoside beta1, 4-N-acetylglucosaminyltransferase. Glycobiology 9, 303-310.   DOI