Faculty Opinions recommendation of An engineered eukaryotic protein glycosylation pathway in Escherichia coli.

Multiple sclerosis (MS) is an inflammatory demyelinating and neurodegenerative disease initiated by autoreactive T cells. Mgat5, a gene in the Asn (N-) linked protein glycosylation pathway, associates with MS severity and negatively regulates experimental autoimmune encephalomyelitis (EAE) and spontaneous inflammatory demyelination in mice. N-glycan branching by Mgat5 regulates interaction of surface glycoproteins with galectins, forming a molecular lattice that differentially controls the concentration of surface glycoproteins. T-cell receptor signaling, T-cell proliferation, TH1 differentiation, and CTLA-4 endocytosis are inhibited by Mgat5 branching. Non-T cells also contribute to MS pathogenesis and express abundant Mgat5 branched N-glycans. Here we explore whether Mgat5 deficiency in myelin-reactive T cells is sufficient to promote demyelinating disease. Adoptive transfer of myelin-reactive Mgat5−/− T cells into Mgat5+/+ versus Mgat5−/− recipients revealed more severe EAE in the latter, suggesting that Mgat5 branching deficiency in recipient naive T cells and/or non-T cells contribute to disease pathogenesis.

Download Full-text

Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0500044102 ◽

2005 ◽

Vol 102 (8) ◽

pp. 3016-3021 ◽

Cited By ~ 276

Author(s):

M. F. Feldman ◽

M. Wacker ◽

M. Hernandez ◽

P. G. Hitchen ◽

C. L. Marolda ◽

...

Keyword(s):

Escherichia Coli ◽

Protein Glycosylation ◽

O Antigen

Download Full-text

Hot and sweet: protein glycosylation in Crenarchaeota

Biochemical Society Transactions ◽

10.1042/bst20120296 ◽

2013 ◽

Vol 41 (1) ◽

pp. 384-392 ◽

Cited By ~ 33

Author(s):

Benjamin H. Meyer ◽

Sonja-Verena Albers

Keyword(s):

Living Cell ◽

Protein Glycosylation ◽

Haloferax Volcanii ◽

Present Review ◽

Sulfolobus Acidocaldarius ◽

Glycosylphosphatidylinositol Anchor ◽

Sweet Protein ◽

Domains Of Life ◽

Glycosylation Pathway ◽

Sugar Chains

Every living cell is covered with a dense and complex array of covalently attached sugars or sugar chains. The majority of these glycans are linked to proteins via the so-called glycosylation process. Protein glycosylation is found in all three domains of life: Eukarya, Bacteria and Archaea. However, on the basis of the limit in analytic tools for glycobiology and genetics in Archaea, only in the last few years has research on archaeal glycosylation pathways started mainly in the Euryarchaeota Haloferax volcanii, Methanocaldococcus maripaludis and Methanococcus voltae. Recently, major steps of the crenarchaeal glycosylation process of the thermoacidophilic archaeon Sulfolobus acidocaldarius have been described. The present review summarizes the proposed N-glycosylation pathway of S. acidocaldarius, describing the phenotypes of the mutants disrupted in N-glycan biosynthesis as well as giving insights into the archaeal O-linked and glycosylphosphatidylinositol anchor glycosylation process.

Download Full-text

Identification of the UDP-N-Acetylglucosamine 4-Epimerase Involved in Exosporium Protein Glycosylation in Bacillus anthracis

Journal of Bacteriology ◽

10.1128/jb.01050-09 ◽

2009 ◽

Vol 191 (22) ◽

pp. 7094-7101 ◽

Cited By ~ 10

Author(s):

Shengli Dong ◽

Olga N. Chesnokova ◽

Charles L. Turnbough ◽

David G. Pritchard

Keyword(s):

Escherichia Coli ◽

Bacillus Anthracis ◽

Basal Layer ◽

Amino Sugar ◽

Protein Glycosylation ◽

Side Chains ◽

Protein Backbone ◽

Gene Encoding ◽

Multiple Copies ◽

Late Stages

ABSTRACT Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a loosely fitting exosporium composed of a basal layer and an external hair-like nap. The filaments of the nap are formed by trimers of the collagen-like glycoprotein BclA. The side chains of BclA include multiple copies of two linear rhamnose-containing oligosaccharides, a trisaccharide and a pentasaccharide. The pentasaccharide terminates with the unusual deoxyamino sugar anthrose. Both oligosaccharide side chains are linked to the BclA protein backbone through an N-acetylgalactosamine (GalNAc) residue. To identify the gene encoding the epimerase required to produce GalNAc for BclA oligosaccharide biosynthesis, three annotated UDP-glucose 4-epimerase genes of B. anthracis were cloned and expressed in Escherichia coli. The candidate proteins were purified, and their enzymatic activities were assessed. Only two proteins, encoded by the BAS5114 and BAS5304 genes (B. anthracis Sterne designations), exhibited epimerase activity. Both proteins were able to convert UDP-glucose (Glc) to UDP-Gal, but only the BAS5304-encoded protein could convert UDP-GlcNAc to UDP-GalNAc, indicating that BAS5304 was the gene sought. Surprisingly, spores produced by a mutant strain lacking the BAS5304-encoded enzyme still contained normal levels of BclA-attached oligosaccharides. However, monosaccharide analysis of the oligosaccharides revealed that GlcNAc had replaced GalNAc. Thus, while GalNAc appears to be the preferred amino sugar for the linkage of oligosaccharides to the BclA protein backbone, in its absence, GlcNAc can serve as a substitute linker. Finally, we demonstrated that the expression of the BAS5304 gene occurred in a biphasic manner during both the early and late stages of sporulation.

Download Full-text

Cj1496c Encodes a Campylobacter jejuni Glycoprotein That Influences Invasion of Human Epithelial Cells and Colonization of the Chick Gastrointestinal Tract

Infection and Immunity ◽

10.1128/iai.00033-06 ◽

2006 ◽

Vol 74 (8) ◽

pp. 4715-4723 ◽

Cited By ~ 50

Author(s):

Tsutomu Kakuda ◽

Victor J. DiRita

Keyword(s):

Campylobacter Jejuni ◽

Cell Invasion ◽

Protein Glycosylation ◽

Epithelial Cell Line ◽

Intestinal Epithelial ◽

Human Intestinal Epithelial Cell ◽

Site Specific ◽

Glycosylation Pathway ◽

Gastrointestinal Colonization ◽

Insertion Mutants

ABSTRACT Campylobacter jejuni has an N-linked protein glycosylation pathway that is required for efficient cell invasion and chick gastrointestinal colonization by the microbe. In this study, we constructed insertion mutants of 22 putative glycoprotein genes and examined the ability of each to invade the human intestinal epithelial cell line INT-407. Among the mutants tested, one carrying an insertion in Cj1496c was defective for invasion into INT-407 cells; this defect was also observed in an in-frame deletion mutant of Cj1496c (ΔCj1496c). The ΔCj1496c mutant C. jejuni also showed a reduced ability to colonize chick ceca. Site-specific mutagenesis combined with Western blot analysis suggested that the Cj1496c protein is glycosylated at N73 and N169. However, the ΔCj1496c mutant expressing a nonglycosylated form of Cj1496c exhibited levels of invasion and colonization equivalent to those of the parent strain, suggesting that glycans are not directly involved in the function of Cj1496c.

Download Full-text

Cj1123c (PglD), a multifaceted acetyltransferase from Campylobacter jejuni

Biochemistry and Cell Biology ◽

10.1139/o09-002 ◽

2009 ◽

Vol 87 (3) ◽

pp. 469-483 ◽

Cited By ~ 9

Author(s):

Melinda Demendi ◽

Carole Creuzenet

Keyword(s):

Campylobacter Jejuni ◽

Structural Information ◽

Protein Glycosylation ◽

Acyl Donor ◽

Acetyl Coa ◽

Molecular Determinants ◽

Glycosylation Pathway ◽

Opening Up ◽

Potential Use

Campylobacter jejuni produces both N- and O-glycosylated proteins. Because protein glycosylation contributes to bacterial virulence, a thorough characterization of the enzymes involved in protein glycosylation is warranted to assess their potential use as therapeutic targets and as glyco-engineering tools. We performed a detailed biochemical analysis of the molecular determinants of the substrate and acyl-donor specificities of Cj1123c (also known as PglD), an acetyltransferase of the HexAT superfamily involved in N-glycosylation of proteins. We show that Cj1123c has acetyl-CoA-dependent N-acetyltransferase activity not only on the UDP-4-amino-4,6-dideoxy-GlcNAc intermediate of the N-glycosylation pathway but also on the UDP-4-amino-4,6-dideoxy-AltNAc intermediate of the O-glycosylation pathway, implying functional redundancy between both pathways. We further demonstrate that, despite its somewhat relaxed substrate specificity for N-acetylation, Cj1123c cannot acetylate aminoglycosides, indicating a preference for sugar-nucleotide substrates. In addition, we show that Cj1123c can O-acetylate UDP-GlcNAc and that Cj1123c is very versatile in terms of acyl-CoA donors as it can use propionyl- and butyryl-CoA instead of acetyl-CoA. Finally, using structural information available for Cj1123c and related enzymes, we identify three residues (H125, G143, and G173) involved in catalysis and (or) acyl-donor specificity, opening up possibilities of tailoring the specificity of Cj1123c for the synthesis of novel sugars.

Download Full-text