Identification of a novel glycosyltransferase involved in LOS biosynthesis of Moraxella catarrhalis

Moraxella catarrhalis is an important human mucosal pathogen that contributes to otitis media in infants and exacerbates conditions such as chronic obstructive pulmonary disease in the elderly. This study describes the identification of a novel gene, lgt5 that encodes a glycosyltransferase involved in the LOS biosynthesis of M. catarrhalis. Analysis of NMR data of LOS-derived oligosaccharide from a Serotype A lgt5 mutant strain of M. catarrhalis indicate that lgt5 encodes an alpha-(1-->4)-galactosyltransferase.     

A Putative Polypeptide N-Acetylgalactosaminyltransferase/Williams-Beuren Syndrome Chromosome Region 17 (WBSCR17) Regulates Lamellipodium Formation and Macropinocytosis

We previously identified a novel polypeptide N-acetylgalactosaminyltransferase (GalNAc-T) gene, which is designated Williams-Beuren syndrome chromosome region 17 (WBSCR17) because it is located in the chromosomal flanking region of the Williams-Beuren syndrome deletion. Recent genome-scale analysis of HEK293T cells treated with a high concentration of N-acetylglucosamine (GlcNAc) demonstrated that WBSCR17 was one of the up-regulated genes possibly involved in endocytosis (Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteomics 8, 3294-3302). To assess its roles, we first expressed recombinant WBSCR17 in COS7 cells and demonstrated that it was N-glycosylated and localized mainly in the Golgi apparatus, as is the case for the other GalNAc-Ts. Assay of recombinant WBSCR17 expressed in insect cells showed very low activity toward typical mucin peptide substrates. We then suppressed the expression of endogenous WBSCR17 in HEK293T cells using siRNAs and observed phenotypic changes of the knockdown cells with reduced lamellipodium formation, altered O-glycan profiles, and unusual accumulation of glycoconjugates in the late endosomes/lysosomes. Analyses of endocytic pathways revealed that macropinocytosis, but neither clathrin- nor caveolin-dependent endocytosis, was elevated in the knockdown cells. This was further supported by the findings that the overexpression of recombinant WBSCR17 stimulated lamellipodium formation, altered O-glycosylation, and inhibited macropinocytosis. WBSCR17 therefore plays important roles in lamellipodium formation and the regulation of macropinocytosis as well as lysosomes. Our study suggests that a subset of O-glycosylation produced by WBSCR17 controls dynamic membrane trafficking, probably between the cell surface and the late endosomes through macropinocytosis, in response to the nutrient concentration as exemplified by environmental GlcNAc.     

A systematic study of site-specific GalNAc-type O-glycosylation modulating proprotein convertase processing

Site-specific GalNAc-type O-glycosylation is emerging as an important co-regulator of proprotein convertase (PC) processing of proteins. PC processing is crucial in regulating many fundamental biological pathways and O-glycans in or immediately adjacent to processing sites may affect recognition and function of PCs. Thus, we previously demonstrated that deficiency in site-specific O-glycosylation in a PC site of the fibroblast growth factor, FGF23, resulted in marked reduction in secretion of active unprocessed FGF23, which cause familial tumoral calcinosis and hyperostosis hyperphosphatemia. GalNAc-type O-glycosylation is found on serine and threonine amino acids and up to 20 distinct polypeptide GalNAc transferases catalyze the first addition of GalNAc to proteins making this step the most complex and differentially regulated steps in protein glycosylation. There is no reliable prediction model for O-glycosylation especially of isolated sites, but serine and to a lesser extent threonine residues are frequently found adjacent to PC processing sites. In the present study we used in vitro enzyme assays and ex vivo cell models to systematically address the boundaries of the region within site-specific O-glycosylation affect PC processing. The results demonstrate that O-glycans within at least ±3 residues of the RXXR furin cleavage site may affect PC processing suggesting that site-specific O-glycosylation is a major co-regulator of PC processing.     

Exploiting bacterial glycosylation machineries for the synthesis of a Lewis antigen-containing glycoprotein

Glycoproteins constitute a class of compounds of increasing importance for pharmaceutical applications. The manipulation of bacterial protein glycosylation systems from Gram-negative bacteria for the synthesis of recombinant glycoproteins is a promising alternative to the current production methods. Proteins carrying Lewis antigens have been shown to have potential applications for the treatment of diverse autoimmune diseases. In this work, we developed a mixed approach consisting of in vivo and in vitro steps for the synthesis of glycoproteins containing the Lewis x antigen. Using glycosyltransferases from Haemophilus influenzae, we engineered Escherichia coli to assemble a tetrasaccharide on the lipid carrier undecaprenylphosphate. This glycan was transferred in vivo from the lipid to a carrier protein by the Campylobacter jejuni oligosaccharyltransferase PglB. The glycoprotein was then fucosylated in vitro by a truncated fucosyltransferase from Helicobacter pylori. Diverse mass spectrometry techniques were used to confirm the structure of the glycan. The strategy presented here could be adapted in the future for the synthesis of diverse glycoproteins. Our experiments demonstrate that bacterial enzymes can be exploited for the production of glycoproteins carrying glycans present in human cells for potential therapeutic applications.     

Trans-activity of Plasma Membrane-associated Ganglioside Sialyltransferase in Mammalian Cells

Gangliosides are acidic glycosphingolipids that contain sialic acid residues and are expressed in nearly all vertebrate cells. They are synthesized at the Golgi complex by a combination of glycosyltransferase activities followed by vesicular delivery to the plasma membrane, where they participate in a variety of physiological as well as pathological processes. Recently, a number of enzymes of ganglioside anabolism and catabolism have been shown to be associated with the plasma membrane. In particular, it was observed that CMP-NeuAc:GM3 sialyltransferase (Sial-T2) is able to sialylate GM3 at the plasma membrane (cis-catalytic activity). In this work, we demonstrated that plasma membrane-integrated ecto-Sial-T2 also displays a trans-catalytic activity at the cell surface of epithelial and melanoma cells. By using a highly sensitive enzyme-linked immunosorbent assay combined with confocal fluorescence microscopy, we observed that ecto-Sial-T2 was able to sialylate hydrophobically or covalently immobilized GM3 onto a solid surface. More interestingly, we observed that ecto-Sial-T2 was able to sialylate GM3 exposed on the membrane of neighboring cells by using both the exogenous and endogenous donor substrate (CMP-N-acetylneuraminic acid) available at the extracellular milieu. In addition, the trans-activity of ecto-Sial-T2 was considerably reduced when the expression of the acceptor substrate was inhibited by using a specific inhibitor of biosynthesis of glycolipids, indicating the lipidic nature of the acceptor. Our findings provide the first direct evidence that an ecto-sialyltransferase is able to trans-sialylate substrates exposed in the plasma membrane from mammalian cells, which represents a novel insight into the molecular events that regulate the local glycosphingolipid composition.     

A network-based approach to identify substrate classes of bacterial glycosyltransferases

Background

Bacterial interactions with the environment- and/or host largely depend on the bacterial glycome. The specificities of a bacterial glycome are largely determined by glycosyltransferases (GTs), the enzymes involved in transferring sugar moieties from an activated donor to a specific substrate. Of these GTs their coding regions, but mainly also their substrate specificity are still largely unannotated as most sequence-based annotation flows suffer from the lack of characterized sequence motifs that can aid in the prediction of the substrate specificity.

Results

In this work, we developed an analysis flow that uses sequence-based strategies to predict novel GTs, but also exploits a network-based approach to infer the putative substrate classes of these predicted GTs. Our analysis flow was benchmarked with the well-documented GT-repertoire of Campylobacter jejuni NCTC 11168 and applied to the probiotic model Lactobacillus rhamnosus GG to expand our insights in the glycosylation potential of this bacterium. In L. rhamnosus GG we could predict 48 GTs of which eight were not previously reported. For at least 20 of these GTs a substrate relation was inferred.

Conclusions

We confirmed through experimental validation our prediction of WelI acting upstream of WelE in the biosynthesis of exopolysaccharides. We further hypothesize to have identified in L. rhamnosus GG the yet undiscovered genes involved in the biosynthesis of glucose-rich glycans and novel GTs involved in the glycosylation of proteins. Interestingly, we also predict GTs with well-known functions in peptidoglycan synthesis to also play a role in protein glycosylation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-349) contains supplementary material, which is available to authorized users.     

Protein O-Mannosyltransferases Associate with the Translocon to Modify Translocating Polypeptide Chains

O-Mannosylation and N-glycosylation are essential protein modifications that are initiated in the endoplasmic reticulum (ER). Protein translocation across the ER membrane and N-glycosylation are highly coordinated processes that take place at the translocon-oligosaccharyltransferase (OST) complex. In analogy, it was assumed that protein O-mannosyltransferases (PMTs) also act at the translocon, however, in recent years it turned out that prolonged ER residence allows O-mannosylation of un-/misfolded proteins or slow folding intermediates by Pmt1-Pmt2 complexes. Here, we reinvestigate protein O-mannosylation in the context of protein translocation. We demonstrate the association of Pmt1-Pmt2 with the OST, the trimeric Sec61, and the tetrameric Sec63 complex in vivo by co-immunoprecipitation. The coordinated interplay between PMTs and OST in vivo is further shown by a comprehensive mass spectrometry-based analysis of N-glycosylation site occupancy in pmtΔ mutants. In addition, we established a microsomal translation/translocation/O-mannosylation system. Using the serine/threonine-rich cell wall protein Ccw5 as a model, we show that PMTs efficiently mannosylate proteins during their translocation into microsomes. This in vitro system will help to unravel mechanistic differences between co- and post-translocational O-mannosylation.