Identification of AglE, a Second Glycosyltransferase Involved in N Glycosylation of the Haloferax volcanii S-Layer Glycoprotein

Abstract

Archaea, like Eukarya and Bacteria, are able to N glycosylate select protein targets. However, in contrast to relatively advanced understanding of the eukaryal N glycosylation process and the information being amassed on the bacterial process, little is known of this posttranslational modification in Archaea. Toward remedying this situation, the present report continues ongoing efforts to identify components involved in the N glycosylation of the Haloferax volcanii S-layer glycoprotein. By combining gene deletion together with mass spectrometry, AglE, originally identified as a homologue of murine Dpm1, was shown to play a role in the addition of the 190-Da sugar subunit of the novel pentasaccharide decorating the S-layer glycoprotein. Topological analysis of an AglE-based chimeric reporter assigns AglE as an integral membrane protein, with its N terminus and putative active site facing the cytoplasm. These finding, therefore, contribute to the developing picture of the N glycosylation pathway in Archaea.

N-Linked Glycosylation in Archaea: a Structural, Functional, and Genetic Analysis

“…Such analysis of the glycan-charged DolP pool from parent strain cells revealed peaks corresponding to C 55 and C 60 DolPs charged with only the first four subunits of the N-linked pentasaccharide, as well as by its precursors, but not by the complete glycan bound to S-layer glycoprotein Asn-13 and Asn-83 (86). When the same analysis was also performed on lipid extracts from cells lacking AglG, AglI, or AglE, namely, those GTs involved in adding the second, third, and fourth subunits to the S-layer glycoprotein, respectively (74,76,87), the same effects seen at the level of the N-linked glycan were observed at the DolP level (86).…”

“…In the unannotated and partially assembled version of the Hfx. volcanii genome available at that time, dpm1-B was detected eight open reading frames upstream of the aglB gene, on the opposite strand (74). Mass spectrometry analysis of an S-layer glycoprotein-derived tryptic peptide containing Asn-13 from a dpm1-B deletion mutant revealed peaks lacking the N-linked pentasaccharide and its tetrasaccharide precursor, suggesting that Dpm1-B participates in the addition of the 190-Da moiety found at pentasaccharide position 4.…”

“…Initially, the topology of AglE was determined by subcellular fractionation designed to define the position of the protein fused to a Clostridium thermocellum cellulose-binding domain (CBD) tag. Such efforts revealed AglE to be a transmembrane (TM) protein with the N-terminal end facing the cytoplasm (74). AglP was determined to be found in the cytoplasmic fraction by both bioinformatics and subcellular localization of a CBD-AglP fusion (79).…”

“…In this manner, both AglJ and AglD were demonstrated to be membrane-spanning proteins presenting two and six membranespanning domains, respectively, with the N-terminal portion of each protein facing the cytoplasm. Given the largely cytoplasmic orientation of AglJ, AglE, AglP, and AglD (74,79,81), it was concluded that the assembly of the N-linked pentasaccharide occurs on the cytoplasmic leaflet of the cytoplasmic membrane.…”

“…Mass spectrometry analysis of an S-layer glycoprotein-derived tryptic peptide containing Asn-13 from a dpm1-B deletion mutant revealed peaks lacking the N-linked pentasaccharide and its tetrasaccharide precursor, suggesting that Dpm1-B participates in the addition of the 190-Da moiety found at pentasaccharide position 4. Given its role in the N-glycosylation process, Dpm1-B was renamed AglE (74). In contrast to cells lacking either AglB or AglD, aglE deletion mutants showed no effects on cell growth or S-layer stability.…”