PUBLICATION
Substitution of the N-glycan Function in Glycosyltransferases by Specific Amino Acids (SUNGA): ST3Gal-V as a model enzyme
- Authors
- Uemura, S., Kurose, T., Suzuki, T., Yoshida, S., Ito, M., Saito, M., Horiuchi, M., Inagaki, F., Igarashi, Y., and Inokuchi, J.I.
- ID
- ZDB-PUB-051128-9
- Date
- 2006
- Source
- Glycobiology 16(3): 258-270 (Journal)
- Registered Authors
- Keywords
- ST3Gal-V, N-glycan, glycosyltransferase, sialyltransferase, glycotechnology
- MeSH Terms
-
- Escherichia coli/genetics
- Escherichia coli/metabolism
- Molecular Sequence Data
- Sialyltransferases/chemistry*
- Sialyltransferases/genetics
- PubMed
- 16306051 Full text @ Glycobiology
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 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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping