Functional analysis of recombinant human and Yarrowia lipolytica O-GlcNAc transferases expressed in Saccharomyces cerevisiae.
O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important post-translational modification in many cellular processes. It is mediated by O-GlcNAc transferases (OGTs), which catalyze the addition of O-GlcNAc to serine or threonine residues of the target proteins. In this study, we expressed a putative Yarrowia lipolytica OGT (YlOGT), the only homolog identified in the subphylum Saccharomycotina through bioinformatics analysis, and the human OGT (hOGT) as recombinant proteins in Saccharomyces cerevisiae, and performed their functional characterization. Immunoblotting assays using antibody against O-GlcNAc revealed that recombinant hOGT (rhOGT), but not the recombinant YlOGT (rYlOGT), undergoes auto-O-GlcNAcylation in the heterologous host S. cerevisiae. Moreover, the rhOGT expressed in S. cerevisiae showed a catalytic activity during in vitro assays using casein kinase II substrates, whereas no such activity was obtained in rYlOGT. However, the chimeric human-Y. lipolytica OGT, carrying the human tetratricopeptide repeat (TPR) domain along with the Y. lipolytica catalytic domain (CTD), mediated the transfer of O-GlcNAc moiety during the in vitro assays. Although the overexpression of full-length OGTs inhibited the growth of S. cerevisiae, no such inhibition was obtained upon overexpression of only the CTD fragment, indicating the role of TPR domain in growth inhibition. This is the first report on the functional analysis of the fungal OGT, indicating that the Y. lipolytica OGT retains its catalytic activity, although the physiological role and substrates of YlOGT remain to be elucidated.
Department of Life Science, College of Natural Science, Seoul, 06974, Republic of Korea.
Hye Ji
HJ
Oh
Department of Life Science, College of Natural Science, Seoul, 06974, Republic of Korea.
Hye Yun
HY
Moon
Department of Life Science, College of Natural Science, Seoul, 06974, Republic of Korea.
Seon Ah
SA
Cheon
Department of Life Science, College of Natural Science, Seoul, 06974, Republic of Korea. hahny@cau.ac.kr.|Research Center for Biomolecules and Biosystems, Chung-Ang University, Seoul, 06974, Republic of Korea. hahny@cau.ac.kr.
Yoonsoo
Y
Hahn
Department of Life Science, College of Natural Science, Seoul, 06974, Republic of Korea.|Research Center for Biomolecules and Biosystems, Chung-Ang University, Seoul, 06974, Republic of Korea.
Hyun Ah
HA
Kang
D000215:Acylation; D047390:Casein Kinase II / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D019295:Computational Biology; D006031:Glycosylation; D006801:Humans; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D013379:Substrate Specificity; D025062:Yarrowia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; D047390:Casein Kinase II
O-GlcNAc glycosylation; O-GlcNAc transferase (OGT); Yarrowia lipolytica
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