voltaesequence, with the targeted gene sequence replaced by the purrcassette

voltaesequence, with the targeted gene sequence replaced by the purrcassette. deletion of two Ro 31-8220 mesylate putativeM. voltaeglycosyl transferase genes, designatedaglC(forarchaealglycosylation) andaglK, interfered with proper N-glycosylation. This resulted in flagellin and S-layer proteins with significantly reduced apparent molecular masses, loss of flagellar assembly, and absence of glycan attachment. Given previous knowledge of both the N-glycosylation pathway inM. voltaeand the general characteristics of N-glycosylation components, it appears that AglC and AglK are involved in the biosynthesis or transfer of diacetylated glucuronic acid within the glycan structure. In addition, a knockout of the putative flippase candidate gene (Mv891) had no effect on N-glycosylation but did result in the production of giant cells with diameters three to four times that of wild-type cells. It has become widely accepted that glycosylation is an important posttranslational protein modification within all three domains of life. Long recognized and studied in eukaryotes, glycosylation pathways in prokaryotes have received more attention in recent years. Several reviews summarizing the current state of knowledge of protein glycosylation inArchaea(2,11,42) and flagellar glycosylation inBacteriaandArchaea(25) attest to the progress that has been made in understanding this important process from a prokaryotic perspective. Specifically, significant advances in comprehending the process of N-linked glycosylation, which is the attachment of polysaccharide structures to specific Asn residues within a conserved Asn-Xaa-Ser/Thr motif (where Xaa is any amino acid except Pro) have occurred. Of note is the N-glycosylation system inCampylobacter jejuni, where the gene products from thepgllocus assemble a branched heptasaccharide on a membrane-bound lipid carrier and then translocate the glycan across the cytoplasmic membrane to facilitate transfer to the appropriate Asn residue of the target protein (24,34). The study of archaeal glycosylation in recent years has begun to yield an understanding of how these organisms modify proteins. The first requirement for assembling a glycan of any Rabbit polyclonal to Synaptotagmin.SYT2 May have a regulatory role in the membrane interactions during trafficking of synaptic vesicles at the active zone of the synapse. linkage type is a pool of nucleotide-activated monosaccharide precursors. Several enzymes have been identified fromMethanococcus maripaludisthat are required for UDP-acetamido sugar synthesis and are predicted to be precursors for flagellin and surface (S) layer protein modifications (29). With these Ro 31-8220 mesylate nucleotide-activated sugars, a set of genes known as thearchaealglycosylation (agl) genes then assemble and attach Ro 31-8220 mesylate the desired glycan to its target protein in a stepwise fashion. First, a set of glycosyl transferases construct the glycan on a dolichol phosphate anchor at the cytoplasmic face of the cytoplasmic membrane. In the obligate anaerobic methanogenMethanococcus voltae, at least three glycosyl transferases are predicted to be necessary to assemble the trisaccharide -ManpNAcA6Thr-(1-4)–GlcpNAc3NAcA-(1-3)– GlcpNAc, which has been characterized from both the S-layer protein and the flagellins (two major flagellins, FlaB1 and FlaB2, and two minor flagellins, FlaA and FlaB3) (39). In this system, AglH has been proposed as the glycosyl transferase responsible for the attachment of Ro 31-8220 mesylate the linkingN-acetylglucosamine (33), while AglA is responsible for the terminal sugar attachment (9). In the moderate halophileHaloferax volcanii, a pentasaccharide is found on the S-layer protein that requires the activities of at least five glycosyl transferases, with AglD, AglE, AglF, AglG, and AglI identified as involved so far (1,3,4,41,42). The next step in the process is to translocate the glycan from the cytoplasmic to the extracellular side of the membrane. No homologs of either the proposed eukaryotic flippase (Rft1; a unique transporter) (14) or the bacterial flippase (PglK; an ABC transporter) (21) have been detected in eitherM. voltaeorH. volcanii(1,9). Furthermore, a knockout of the most likely flippase candidate inM. voltae, based on very weak BLAST scores with Rft1 and PglK, had no detectable effect on the flagellin or S-layer glycan (9). This appears to indicate that the protein responsible for glycan translocation is unique in each domain of life and that the archaeal flippase may prove to be an archaeal-specific protein. The final step in the N-glycosylation pathway is the attachment of the completed glycan to its target protein via the action of an oligosaccharyl transferase. In bothM. voltaeandH. volcanii, this protein has been identified as AglB (1,9). Both of the identified proteins are homologs of the eukaryotic Stt3p and the bacterial PglB proteins. A key feature of these proteins is the highly conserved active site, WWDXG, responsible for its transferase activity (40). Interestingly, the soluble domain of AglB from the thermophilic archaeonPyrococcus furiosushas been crystallized (17) and appears to extend the conserved active site to include a DXXK motif located approximately 60 residues upstream of the WWDXG motif. Significantly, subsequent mutagenesis of the DXXK motif revealed its essential role in the catalytic activity of the yeast STT3 (16). The apparent universality of Stt3p/PglB/AglB to catalyze the N-glycosyl bond within all three domains of life, despite the presence of different linking sugars in different organisms, illustrates the conserved nature of this step in the N-glycosylation process. The currently proposed glycan assembly model forM. voltaeis summarized in Fig.1. This contribution reports the testing of three genes fromM. voltae,.