Assembling Glycan-Charged Dolichol Phosphates: Chemoenzymatic Synthesis of a Haloferax volcanii N-Glycosylation Pathway Intermediate

5 years ago

Assembling Glycan-Charged Dolichol Phosphates: Chemoenzymatic Synthesis of a Haloferax volcanii N-Glycosylation Pathway Intermediate

Jerry Eichler, Ziqiang Guan, Yifat Elharar, Suvarn S. Kulkarni, Ananda Rao Podilapu

N-glycosylation, the covalent attachment of glycans to select protein target Asn residues, is a post-translational modification performed by all three domains of life. In the halophilic archaea Haloferax volcanii, in which understanding of this universal protein-processing event is relatively well-advanced, genes encoding the components of the archaeal glycosylation (Agl) pathway responsible for the assembly and attachment of an N-linked pentasaccharide have been identified. As elsewhere, the N-linked glycan is assembled on phosphodolichol carriers before transfer to target Asn residues. However, as little is presently known of the Hfx. volcanii Agl pathway at the protein level, the seemingly unique ability of Archaea to use dolichol phosphate (DolP) as the glycan lipid carrier, rather than dolichol pyrophosphate used by eukaryotes, remains poorly understood. With this in mind, a chemoenzymatic approach was taken to biochemically study AglG, one of the five glycosyltransferases of the pathway. Accordingly, a novel regio- and stereoselective reduction of naturally isolated polyprenol gave facile access to S-dolichol via asymmetric transfer hydrogenation under very mild conditions. This compound was used to generate glucose-charged DolP, a precursor of the N-linked pentasaccharide, as well as DolP–glucose–glucuronic acid and DolP–glucuronic acid. AglG, purified from Hfx. volcanii membranes in hypersaline conditions, like those encountered in situ, was subsequently combined with uridine diphosphate (UDP)–glucuronic acid and DolP–glucose to yield DolP–glucose–glucuronic acid. The in vitro system for the study of AglG activity developed here represents the first such tool for studying halophilic glycosyltransferases and will allow for a detailed understanding of archaeal N-glycosylation.

Publisher URL: http://dx.doi.org/10.1021/acs.bioconjchem.7b00436

DOI: 10.1021/acs.bioconjchem.7b00436