Global Profiling of N-Glycoproteins and N-Glycans in the Diatom Phaeodactylum tricornutum

N-glycosylation is an important posttranslational modification in all eukaryotes, but little is known about the N-glycoproteins and N-glycans in microalgae. Here, N-glycoproteomic and N-glycomic approaches were used to unveil the N-glycoproteins and N-glycans in the model diatom Phaeodactylum tricornutum. In total, 863 different N-glycopeptides corresponding to 639 N-glycoproteins were identified from P. tricornutum. These N-glycoproteins participated in a variety of important metabolic pathways in P. tricornutum. Twelve proteins participating in the N-glycosylation pathway were identified as N-glycoproteins, indicating that the N-glycosylation of these proteins might be important for the protein N-glycosylation pathway. Subsequently, 69 N-glycans corresponding to 59 N-glycoproteins were identified and classified into high mannose and hybrid type N-glycans. High mannose type N-glycans contained four different classes, such as Man-5, Man-7, Man-9, and Man-10 with a terminal glucose residue. Hybrid type N-glycan harbored Man-4 with a terminal GlcNAc residue. The identification of N-glycosylation on nascent proteins expanded our understanding of this modification at a N-glycoproteomic scale, the analysis of N-glycan structures updated the N-glycan database in microalgae. The results obtained from this study facilitate the elucidation of the precise function of these N-glycoproteins and are beneficial for future designing the microalga to produce the functional humanized biopharmaceutical N-glycoproteins for the clinical therapeutics.

N-acetylglucosaminyltransferase II Is Involved in Plant Growth and Development Under Stress Conditions

Alpha-1,6-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase [EC 2.4.1.143, N-acetylglucosaminyltransferase II (GnTII)] catalyzes the transfer of N-acetylglucosamine (GlcNAc) residue from the nucleotide sugar donor UDP-GlcNAc to the α1,6-mannose residue of the di-antennary N-glycan acceptor GlcNAc(Xyl)Man3(Fuc)GlcNAc2 in the Golgi apparatus. Although the formation of the GlcNAc2(Xyl)Man3(Fuc)GlcNAc2 N-glycan is known to be associated with GnTII activity in Arabidopsis thaliana, its physiological significance is still not fully understood in plants. To address the physiological importance of the GlcNAc2(Xyl)Man3(Fuc)GlcNAc2 N-glycan, we examined the phenotypic effects of loss-of-function mutations in GnTII in the presence and absence of stress, and responsiveness to phytohormones. Prolonged stress induced by tunicamycin (TM) or sodium chloride (NaCl) treatment increased GnTII expression in wild-type Arabidopsis (ecotype Col-0) but caused severe developmental damage in GnTII loss-of-function mutants (gnt2-1 and gnt2-2). The absence of the 6-arm GlcNAc residue in the N-glycans in gnt2-1 facilitated the TM-induced unfolded protein response, accelerated dark-induced leaf senescence, and reduced cytokinin signaling, as well as susceptibility to cytokinin-induced root growth inhibition. Furthermore, gnt2-1 and gnt2-2 seedlings exhibited enhanced N-1-naphthylphthalamic acid-induced inhibition of tropic growth and development. Thus, GnTII’s promotion of the 6-arm GlcNAc addition to N-glycans is important for plant growth and development under stress conditions, possibly via affecting glycoprotein folding and/or distribution.

Bombyx mori β1,4-N-acetylgalactosaminyltransferase possesses relaxed donor substrate specificity in N-glycan synthesis

N-Glycosylation is one of the most important post-translational protein modifications in eukaryotic cells. Although more than 200 N-glycogenes contributing to N-glycan biosynthesis have been identified and characterized, the information on insect N-glycosylation is still limited. Here, focusing on insect N-glycosylation, we characterized Bombyx mori N-acetylgalactosaminyltransferase (BmGalNAcT) participating in complex N-glycan biosynthesis in mammals. BmGalNAcT localized at the Golgi and was ubiquitously expressed in every organ and in the developmental stage of the middle silk gland of fifth instar larvae. Analysis of recombinant BmGalNAcT expressed in Sf9 cells showed that BmGalNAcT transferred GalNAc to non-reducing terminals of GlcNAcβ1,2-R with β1,4-linkage. In addition, BmGalNAcT mediated transfer of galactose and N-acetylglucosamine residues but not transfer of either glucose or glucuronic acid from the UDP-sugar donor substrate to the N-glycan. Despite this tri-functional sugar transfer activity, however, most of the endogenous glycoproteins of insect cells were present without GalNAc, Gal, or GlcNAc residues at the non-reducing terminal of β1,2-GlcNAc residue(s). Moreover, overexpression of BmGalNAcT in insect cells had no effect on N-acetylgalactosaminylation, galactosylation, or N-acetylglucosaminylation of the major N-glycan during biosynthesis. These results suggested that B. mori has a novel multifunctional glycosyltransferase, but the N-glycosylation is highly and strictly regulated by the endogenous N-glycosylation machineries.

The wclY gene of Escherichia coli serotype O117 encodes an α1,4-glucosyltransferase with strict acceptor specificity but broad donor specificity

The O antigen of enterotoxigenic Escherichia coli serotype O117 consists of repeating units with the structure [-D-GalNAcβ1-3-L-Rhaα1-4-D-Glcα1-4-D-Galβ1-3-D-GalNAcα1-4]n. A related structure is found in E. coli O107 where Glc is replaced by a GlcNAc residue. The O117 and O107 antigen biosynthesis gene clusters are homologous and reveal the presence of four putative glycosyltransferase (GT) genes, wclW, wclX, wclY and wclZ, but the enzymes have not yet been biochemically characterized. We show here that the His6-tagged WclY protein expressed in E. coli Lemo21(DE3) cells is an α1,4-Glc-transferase that transfers Glc to the Gal moiety of Galβ1-3GalNAcα-OPO3-PO3-phenoxyundecyl as a specific acceptor and that the diphosphate moiety of this acceptor is required. WclY utilized UDP-Glc, TDP-Glc, ADP-Glc, as well as UDP-GlcNAc, UDP-Gal or UDP-GalNAc as donor substrates, suggesting an unusual broad donor specificity. Activity using GDP-Man suggested the presence of a novel Man-transferase in Lemo21(DE3) cells. Mutations of WclY revealed that both Glu residues of the Ex7E motif within the predicted GT domain are essential for activity. High GlcNAc-transferase (GlcNAc-T) activities of WclY were created by mutating Arg194 to Cys. A triple mutant identical to WclY in E. coli O107 was identified as an α1,4 GlcNAc-T. The characterization of WclY opens the door for the development of antibacterial approaches.

Synthesis of 3-Azidopropyl-Functionalized GalNAc-β-(1→4)-Gal Natural Disaccharide

Oligosaccharides such as the GalNAc-β-(1→4)-Gal disaccharide are involved in host-pathogen interactions and their synthesis is a continuing challenge for organic chemists. Only a few reports have discussed the synthesis of functionalized GalNAc-β-(1→4)-Gal for its further conjugation and applications in glycobiology. The synthetic route described here is taking advantage of (1) a simple and affordable GlcNAc donor which is epimerized to the more expensive GalNAc donor and (2) a 1,6-anhydro-galactose acceptor exalting the reactivity at the 4-position of galactose. The allyloxycarbonyl (Alloc) protecting group used at the 2-position of the GalNAc residue was important (1) for a successful epimerization of the GlcNAc residue into the corresponding GalNAc donor but also (2) for the stereoselective β-glycosylation through anchimeric assistance. The key disaccharide intermediate was further transformed to a trichloroacetimidate donor which could then be glycosylated with any alcohol. The example chosen here is the 3-azidopropyl aglycon for the design of multivalent glycoclusters.<br>

Synthesis of 3-Azidopropyl-Functionalized GalNAc-β-(1→4)-Gal Natural Disaccharide

Oligosaccharides such as the GalNAc-β-(1→4)-Gal disaccharide are involved in host-pathogen interactions and their synthesis is a continuing challenge for organic chemists. Only a few reports have discussed the synthesis of functionalized GalNAc-β-(1→4)-Gal for its further conjugation and applications in glycobiology. The synthetic route described here is taking advantage of (1) a simple and affordable GlcNAc donor which is epimerized to the more expensive GalNAc donor and (2) a 1,6-anhydro-galactose acceptor exalting the reactivity at the 4-position of galactose. The allyloxycarbonyl (Alloc) protecting group used at the 2-position of the GalNAc residue was important (1) for a successful epimerization of the GlcNAc residue into the corresponding GalNAc donor but also (2) for the stereoselective β-glycosylation through anchimeric assistance. The key disaccharide intermediate was further transformed to a trichloroacetimidate donor which could then be glycosylated with any alcohol. The example chosen here is the 3-azidopropyl aglycon for the design of multivalent glycoclusters.<br>

Analysis host-recognition mechanism of staphylococcal kayvirus ɸSA039 reveals a novel strategy that protects Staphylococcus aureus against infection by Staphylococcus pseudintermedius Siphoviridae phages

Following the emergence of antibiotic resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP), phage therapy has attracted significant attention as an alternative to antibiotic treatment. Bacteriophages belonging to kayvirus (previously known as Twort-like phages) have broad host range and are strictly lytic in Staphylococcus spp. Previous work revealed that kayvirus ɸSA039 has a host-recognition mechanism distinct from those of other known kayviruses: most of kayviruses use the backbone of wall teichoic acid (WTA) as their receptor; by contrast, ɸSA039 uses the β-N-acetylglucosamine (β-GlcNAc) residue in WTA. In this study, we found that ɸSA039 could switch its receptor to be able to infect S. aureus lacking the β-GlcNAc residue by acquiring a spontaneous mutation in open reading frame (ORF) 100 and ORF102. Moreover, ɸSA039 could infect S. pseudintermedius, which has a different WTA structure than S. aureus. By comparison with newly isolated S. pseudintermedius–specific phage (SP phages), we determined that glycosylation in WTA of S. pseudintermedius is essential for adsorption of SP phages, but not ɸSA039. Finally, we describe a novel strategy of S. aureus which protects the bacteria from infection of SP phages. Notably, glycosylation of ribitol phosphate (RboP) WTA by TarM or/and TarS prevents infection of S. aureus by SP phages. These findings could help to establish a new strategy for treatment of S. aureus and S. pseudintermedius infection, as well as provide valuable insights into the biology of phage–host interactions.

Endohexosaminidase-catalyzed synthesis of glycopeptides and proteins

The synthetic application of endohexosaminidase enzymes (e.g., Endo A, Endo M, Endo D) promises to allow ready access to a wide variety of defined homogeneous glycoproteins and glycopeptides. The use ofN-glycan oligosaccharides that are activated at the reducing terminus as oxazolines allows their high-yielding attachment to almost any amino acid, peptide, or protein that contains a GlcNAc residue as an acceptor. A wide variety of oxazoline donors are readily available, either by total synthesis or by isolation of the corresponding oligosaccharide from natural sources and then conversion to the oxazoline in water. The synthetic potential of the enzymes is particularly augmented by the production of mutant glycosynthases, the use of which allows the synthesis of a wide variety of glycopeptides and glycoproteins bearing defined homogeneousN-glycan structures.