Glycobiology Advance Access published online on November 22, 2005
Glycobiology, doi:10.1093/glycob/cwj060
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1 Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Science, Frontier Research Center for Post-Genomic Science and Technology, Hokkaido University, Kita 21-jo, Nishi 11-choume, Kita-ku, Sapporo 001-0021, Japan; Core Research for Evaluational Science and Technology Program (CREST), Japan Science and Technology Corporation (JST), Graduate School of Pharmaceutical Science, Frontier Research Center for Post-Genomic Science and Technology, Hokkaido University, Kita 21-jo, Nishi 11-choume, Kita-ku, Sapporo 001-0021, Japan
* To whom correspondence should be addressed. 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 CHO cells transfected with ST3Gal-V cDNA or its mutants. By examining mutants of mST3Gal-V, in which each asparagines 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 inter-species 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 E. coli. thereby establishing the usefulness of the SUNGA method in exploring the function of N-glycans in vivo.
Received October 12, 2005
Revised November 8, 2005
Accepted November 9, 2005
Article
Substitution of the N-glycan Function in Glycosyltransferases by Specific Amino Acids (SUNGA): ST3Gal-V as a model enzyme
Satoshi Uemura 1,
Takahiro Kurose 2,
Tomoko Suzuki 3,
Sayaka Yoshida 3,
Makoto Ito 4,
Masaki Saito 5,
Masataka Horiuchi 2,
Fuyuhiko Inagaki 2,
Yasuyuki Igarashi 3,
and
Jin-ichi Inokuchi 3 *
2 Department of Structural Biology, Graduate School of Pharmaceutical Science, Hokkaido University, Kita 21-jo, Nishi 11-choume, Kita-ku, Sapporo 001-0021, Japan
3 Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Science, Frontier Research Center for Post-Genomic Science and Technology, Hokkaido University, Kita 21-jo, Nishi 11-choume, Kita-ku, Sapporo 001-0021, Japan
4 Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8581, Japan
5 Pharmacodynamics, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan
Jin-ichi Inokuchi, E-mail: inokuchi{at}kinou02.pharm.hokudai.ac.jp
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