Glycobiology Advance Access originally published online on November 22, 2005
Glycobiology 2006 16(3):258-270; doi:10.1093/glycob/cwj060
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Substitution of the N-glycan function in glycosyltransferases by specific amino acids: ST3Gal-V as a model enzyme
2 Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Science, Frontier Research Center for Post-Genomic Science and Technology; 3 Core Research for Evaluational Science and Technology Program (CREST), Japan Science and Technology Agency (JST), Graduate School of Pharmaceutical Science, Frontier Research Center for Post-Genomic Science and Technology; and 4 Department of Structural Biology, Graduate School of Pharmaceutical Science, Hokkaido University, Kita 21-jo, Nishi 11-choume, Kita-ku, Sapporo 001-0021, Japan; 5 Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8581, Japan; and 6 Pharmacodynamics, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan
1 To whom correspondence should be addressed; Tohoku Pharmaceutical University, 4-4-1, Komatsujima, Aoba-Ku, Sendai, 981-8558, Miyagi, Japan; e-mail: inokuchi{at}kinou02.pharm.hokudai.ac.jp
Received on October 12, 2005; revised on November 8, 2005; accepted on November 9, 2005
The sialyltranferase ST3Gal-V transfers a sialic acid to lactosylceramide. We investigated the role of each of the N-glycans modifying mouse ST3Gal-V (mST3Gal-V) by measuring the in vitro enzyme activity of Chinese hamster ovary (CHO) cells transfected with ST3Gal-V cDNA or its mutants. By examining mutants of mST3Gal-V, in which each asparagine was replaced with glutamine (N180Q, N224Q, N334Q), we determined that all three sites are N-glycosylated and that each N-glycan is required for enzyme activity. Despite their importance, N-glycosylation sites in ST3Gal-V are not conserved among species. Therefore, we considered whether the function in the activity that is performed in mST3Gal-V by the N-glycan could be substituted for by specific amino acid residues selected from the ST3Gal-V of other species or from related sialyltransferases (ST3Gal-I, -II, -III, and -IV), placed at or near the glycosylation sites. To this end, we constructed a series of interspecies mutants for mST3Gal-V, specifically, mST3Gal-V-H177D-N180S (medaka or tetraodon type), mST3Gal-V-N224K (human type), and mST3Gal-V-T336Q (zebrafish type). The ST3Gal-V activity of these mutants was quite similar to that of the wild-type enzyme. Thus, we have demonstrated here that the N-glycans on mST3Gal-V are required for activity but can be substituted for specific amino acid residues placed at or near the glycosylation sites. We named this method SUNGA (substitution of N-glycan functions in glycosyltransferases by specific amino acids). Furthermore, we verified that the ST3Gal-V mutant created using the SUNGA method maintains its high activity when expressed in Escherichia coli thereby establishing the usefulness of the SUNGA method in exploring the function of N-glycans in vivo.
Key words: ST3Gal-V / N-glycan / glycosyltransferase / sialyltransferase / glycotechnology
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