Substitution of the N-glycan function in glycosyltransferases by specific amino acids: ST3Gal-V as a model enzyme

Satoshi Uemura, Takahiro Kurose, Tomoko Suzuki, Sayaka Yoshida, Makoto Ito, Masaki Saito, Masataka Horiuchi, Fuyuhiko Inagaki, Yasuyuki Igarashi, Jin Ichi Inokuchi

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)258-270
Number of pages13
JournalGlycobiology
Volume16
Issue number3
DOIs
Publication statusPublished - Mar 1 2006

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Glycosyltransferases
Polysaccharides
Substitution reactions
Glycosylation
Amino Acids
beta-galactoside alpha-2,3-sialyltransferase
Enzymes
Enzyme activity
Sialyltransferases
Asparagine
N-Acetylneuraminic Acid
Oryzias
Glutamine
Escherichia coli
Zebrafish
Complementary DNA
Cricetulus
Cells
Ovary

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Uemura, S., Kurose, T., Suzuki, T., Yoshida, S., Ito, M., Saito, M., ... Inokuchi, J. I. (2006). Substitution of the N-glycan function in glycosyltransferases by specific amino acids: ST3Gal-V as a model enzyme. Glycobiology, 16(3), 258-270. https://doi.org/10.1093/glycob/cwj060

Substitution of the N-glycan function in glycosyltransferases by specific amino acids : ST3Gal-V as a model enzyme. / Uemura, Satoshi; Kurose, Takahiro; Suzuki, Tomoko; Yoshida, Sayaka; Ito, Makoto; Saito, Masaki; Horiuchi, Masataka; Inagaki, Fuyuhiko; Igarashi, Yasuyuki; Inokuchi, Jin Ichi.

In: Glycobiology, Vol. 16, No. 3, 01.03.2006, p. 258-270.

Research output: Contribution to journalArticle

Uemura, S, Kurose, T, Suzuki, T, Yoshida, S, Ito, M, Saito, M, Horiuchi, M, Inagaki, F, Igarashi, Y & Inokuchi, JI 2006, 'Substitution of the N-glycan function in glycosyltransferases by specific amino acids: ST3Gal-V as a model enzyme', Glycobiology, vol. 16, no. 3, pp. 258-270. https://doi.org/10.1093/glycob/cwj060
Uemura, Satoshi ; Kurose, Takahiro ; Suzuki, Tomoko ; Yoshida, Sayaka ; Ito, Makoto ; Saito, Masaki ; Horiuchi, Masataka ; Inagaki, Fuyuhiko ; Igarashi, Yasuyuki ; Inokuchi, Jin Ichi. / Substitution of the N-glycan function in glycosyltransferases by specific amino acids : ST3Gal-V as a model enzyme. In: Glycobiology. 2006 ; Vol. 16, No. 3. pp. 258-270.
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