AglP is a S‐adenosyl‐L‐methionine‐dependent methyltransferase that participates in the N‐glycosylation pathway of Haloferax volcanii

Abstract

SummaryWhile pathways for N-glycosylation in Eukarya and Bacteria have been solved, considerably less is known of this post-translational modification in Archaea. In the halophilic archaeon Haloferax volcanii, proteins encoded by the agl genes are involved in the assembly and attachment of a pentasaccharide to select asparagine residues of the S-layer glycoprotein. AglP, originally identified based on the proximity of its encoding gene to other agl genes whose products were shown to participate in N-glycosylation, was proposed, based on sequence homology, to serve as a methyltransferase. In the present report, gene deletion and mass spectrometry were employed to reveal that AglP is responsible for adding a 14 Da moiety to a hexuronic acid found at position four of the pentasaccharide decorating the Hfx. volcanii S-layer glycoprotein. Subsequent purification of a tagged version of AglP and development of an in vitro assay to test the function of the protein confirmed that AglP is a S-adenosyl-L-methioninedependent methyltransferase.

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

“…volcanii cells lacking AglP. In this way, it was confirmed that AglP acts as a SAM-dependent methyltransferase that modifies the HexA found at position 4 of the pentasaccharide N-linked to the S-layer glycoprotein (79).…”

“…As the 190-kDa pentasaccharide subunit was previously identified as a dimethylated Hex or a methyl ester of HexA (72), it was predicted that the 14-Da decrease in mass in cells lacking AglP likely reflected the loss of a methyl group. This assumption was confirmed in a methyl esterification assay involving an Asn-13-containing S-layer glycoprotein-derived peptide (79). This assay revealed that the 176-Da sugar detected at position 4 of the pentasaccharide in cells where AglP is absent is a HexA, meaning that the 190-Da sugar normally found at this position is a methyl ester of HexA and that AglP is a methyltransferase.…”

“…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.…”

“…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). The topologies of AglJ and AglD were identified through a combination of bioinformatics topology prediction software, assays designed to assess the accessibility of CBD-tagged versions of these proteins to proteinase K, and the use of cysteine-modifying reagents.…”

A Rhomboid Protease Gene Deletion Affects a Novel Oligosaccharide N-Linked to the S-layer Glycoprotein of Haloferax volcanii

“…O-Glycosylation of this protein occurs through binding of glucosyl (132) galactose disaccharides to a C-terminal threonine cluster (13,14). Of the seven putative N-glycosidic sequons found within the S-layer glycoprotein, it was determined that Asn-13 and Asn-83 are modified by a pentasaccharide comprising two hexoses, two hexuronic acids, and a methyl ester of hexuronic acid (15,16). It was also determined that the sequon at Asn-370 is not modified (15).…”