Background

N-glycosylation is initiated from the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER), which is catalyzed by a series of Alg (asparagine-linked glycosylation) proteins.

Scope of review

This review summarizes our recent studies on the enzymology of Alg mannosyltransferases (MTases). We also discuss the membrane topology and physiological importance of several ER cytosolic Alg proteins.

Major conclusions

Utilizing an efficient prokaryotic protein expression system and a new LC–MS quantitative activity assay, we overexpressed all Alg MTases and performed enzymology studies. Moreover, by reconstituting the LLO pathway, the high-yield chemoenzymatic synthesis of high-mannose-type N-glycans was accomplished using recombinant Alg MTases.

General significance

The analysis of the enzymology and topology of Alg MTases has provided valuable biochemical information in the LLO biosynthesis pathway. In addition, an efficient chemoenzymatic strategy that could prepare various oligomannose-type N-glycans in sufficient amounts was established for further biological assays.