Proteins harboring intrinsically disordered regions (IDRs) lacking stable secondary or tertiary structures are abundant across the three domains of life. These regions have not been systematically studied in prokaryotes. Our genome-wide analysis identifies extracytoplasmic serine/threonine-rich IDRs in several biologically important membrane proteins in streptococci.
View Article and Find Full Text PDFThe cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate.
View Article and Find Full Text PDFIn ovoid-shaped, Gram-positive bacteria, MapZ guides FtsZ-ring positioning at cell equators. The cell wall of the ovococcus Streptococcus mutans contains peptidoglycan decorated with serotype c carbohydrates (SCCs). In the present study, we identify the major cell separation autolysin AtlA as an SCC-binding protein.
View Article and Find Full Text PDFCell wall glycopolymers on the surface of Gram-positive bacteria are fundamental to bacterial physiology and infection biology. Here we identify gacH, a gene in the Streptococcus pyogenes group A carbohydrate (GAC) biosynthetic cluster, in two independent transposon library screens for its ability to confer resistance to zinc and susceptibility to the bactericidal enzyme human group IIA-secreted phospholipase A. Subsequent structural and phylogenetic analysis of the GacH extracellular domain revealed that GacH represents an alternative class of glycerol phosphate transferase.
View Article and Find Full Text PDFThe human pathogen encodes a proteasome that carries out regulated degradation of bacterial proteins. It has been proposed that the proteasome contributes to nitrogen metabolism in , although this hypothesis had not been tested. Upon assessing growth in several nitrogen sources, we found that a mutant strain lacking the proteasomal activator Mpa was unable to use nitrate as a sole nitrogen source due to a specific failure in the pathway of nitrate reduction to ammonium.
View Article and Find Full Text PDFIn many Lactobacillales species ( lactic acid bacteria), peptidoglycan is decorated by polyrhamnose polysaccharides that are critical for cell envelope integrity and cell shape and also represent key antigenic determinants. Despite the biological importance of these polysaccharides, their biosynthetic pathways have received limited attention. The important human pathogen, , synthesizes a key antigenic surface polymer, the Lancefield group A carbohydrate (GAC).
View Article and Find Full Text PDFFront Cell Infect Microbiol
September 2017
(Group A or GAS) is a hemolytic human pathogen associated with a wide variety of infections ranging from minor skin and throat infections to life-threatening invasive diseases. The cell wall of GAS consists of peptidoglycan sacculus decorated with a carbohydrate comprising a polyrhamnose backbone with immunodominant N-acetylglucosamine side-chains. All GAS genomes contain the operon, which encodes a 35-amino-acid membrane protein SpyB, and a membrane-bound C3-like ADP-ribosyltransferase SpyA.
View Article and Find Full Text PDFCongenital disorders of glycosylation (CDG) arise from pathogenic mutations in over 100 genes leading to impaired protein or lipid glycosylation. ALG1 encodes a β1,4 mannosyltransferase that catalyzes the addition of the first of nine mannose moieties to form a dolichol-lipid linked oligosaccharide intermediate required for proper N-linked glycosylation. ALG1 mutations cause a rare autosomal recessive disorder termed ALG1-CDG.
View Article and Find Full Text PDFLipid Insights
February 2016
Glycosylation is essential to the synthesis, folding, and function of glycoproteins in eukaryotes. Proteins are co- and posttranslationally modified by a variety of glycans in the endoplasmic reticulum (ER); modifications include C- and O-mannosylation, N-glycosylation, and the addition of glycosylphosphatidylinositol membrane anchors. Protein glycosylation in the ER of eukaryotes involves enzymatic steps on both the cytosolic and lumenal surfaces of the ER membrane.
View Article and Find Full Text PDFBackground: Dolichyl phosphate-linked mono- and oligosaccharides (DLO) are essential intermediates in protein N-glycosylation, C- and O-mannosylation and GPI anchor biosynthesis. While many membrane proteins in the endoplasmic reticulum (ER) involved in the assembly of DLOs are known, essential proteins believed to be required for the transbilayer movement (flip-flopping) and proteins potentially involved in the regulation of DLO synthesis remain to be identified.
Methods: The synthesis of a series of Dol-P derivatives composed of citronellyl-based photoprobes with benzophenone groups equipped with alkyne moieties for Huisgen "click" chemistry is now described to utilize as tools for identifying ER proteins involved in regulating the biosynthesis and transbilayer movement of lipid intermediates enzymatic assays were used to establish that the photoprobes contain the critical structural features recognized by pertinent enzymes in the dolichol pathway.
Congenital disorders of glycosylation are a group of metabolic disorders with an expansive and highly variable clinical presentation caused by abnormal glycosylation of proteins and lipids. Dolichol kinase (DOLK) catalyzes the final step in biosynthesis of dolichol phosphate (Dol-P), which is the oligosaccharide carrier required for protein N-glycosylation. Human DOLK deficiency, also known as DOLK-CDG or CDG-Im, results in a syndrome that has been reported to manifest with dilated cardiomyopathy of variable severity.
View Article and Find Full Text PDFDolichol monophosphate (Dol-P) functions as an obligate glycosyl carrier lipid in protein glycosylation reactions. Dol-P is synthesized by the successive condensation of isopentenyl diphosphate (IPP), with farnesyl diphosphate catalysed by a cis-isoprenyltransferase (cis-IPTase) activity. Despite the recognition of cis-IPTase activity 40 years ago and the molecular cloning of the human cDNA encoding the mammalian enzyme, the molecular machinery responsible for regulating this activity remains incompletely understood.
View Article and Find Full Text PDFDuring evolution the average chain length of polyisoprenoid glycosyl carrier lipids increased from C55 (prokaryotes) to C75 (yeast) to C95 (mammalian cells). In this study, the ability of the E. coli enzyme, undecaprenyl pyrophosphate synthase (UPPS), to complement the loss of the yeast cis-isoprenyltransferase in the rer2Δ mutant was tested to determine if (55)dolichyl phosphate (Dol-P) could functionally substitute in the protein N-glycosylation pathway for (75)Dol-P, the normal isoprenologue synthesized in S.
View Article and Find Full Text PDFEscherichia coli strain O157 produces an O-antigen with the repeating tetrasaccharide unit alpha-D-PerNAc-alpha-l-Fuc-beta-D-Glc-alpha-D-GalNAc, preassembled on undecaprenyl pyrophosphate (Und-P-P). These studies were conducted to determine whether the biosynthesis of the lipid-linked repeating tetrasaccharide was initiated by the formation of GalNAc-P-P-Und by WecA. When membrane fractions from E.
View Article and Find Full Text PDFTo further evaluate the role of Rft1 in the transbilayer movement of Man(5)GlcNAc(2)-P-P-dolichol (M5-DLO), a series of experiments was conducted with intact cells and sealed microsomal vesicles. First, an unexpectedly large accumulation (37-fold) of M5-DLO was observed in Rft1-depleted cells (YG1137) relative to Glc(3)Man(9)GlcNAc(2)-P-P-Dol in wild type (SS328) cells when glycolipid levels were compared by fluorophore-assisted carbohydrate electrophoresis analysis. When sealed microsomes from wild type cells and cells depleted of Rft1 were incubated with GDP-[(3)H]mannose or UDP-[(3)H]GlcNAc in the presence of unlabeled GDP-Man, no difference was observed in the rate of synthesis of [(3)H]Man(9)GlcNAc(2)-P-P-dolichol or Man(9)[(3)H]GlcNAc(2)-P-P-dolichol, respectively.
View Article and Find Full Text PDFProtein N-glycosylation requires flipping of the glycolipid Man(5)GlcNAc(2)-diphosphate dolichol (Man(5)GlcNAc(2)-PP-Dol) across the endoplasmic reticulum (ER). Helenius et al. report genetic evidence suggesting that Rft1, an essential ER membrane protein in yeast, is required directly to translocate Man(5)GlcNAc(2)-PP-Dol.
View Article and Find Full Text PDFDuring protein N-glycosylation, dolichyl pyrophosphate (Dol-P-P) is discharged in the lumenal monolayer of the endoplasmic reticulum (ER). Dol-P-P is then cleaved to Dol-P by Dol-P-P phosphatase (DPPase). Studies with the yeast mutant cwh8Delta, lacking DPPase activity, indicate that recycling of Dol-P produced by DPPase contributes significantly to the pool of Dol-P utilized for lipid intermediate biosynthesis on the cytoplasmic leaflet.
View Article and Find Full Text PDFIn glycosyltransferase-catalyzed reactions a new carbohydrate-carbohydrate bond is formed between a carbohydrate acceptor and the carbohydrate moiety of either a sugar nucleotide donor or lipid-linked saccharide donor. It is currently believed that most glycosyltransferase-catalyzed reactions occur via an electrophilic activation mechanism with the formation of an oxocarbenium ion-like transition state, a hypothesis that makes clear predictions regarding the charge development on the donor (strong positive charge) and acceptor (minimal negative charge) substrates. To better understand the mechanism of these enzyme-catalyzed reactions, we have introduced a strongly electron-withdrawing group (fluorine) at C-5 of both donor and acceptor substrates in order to explore its effect on catalysis.
View Article and Find Full Text PDFMembrane fractions from Micrococcus luteus catalyze the transfer of mannose from GDP-mannose to mono- and dimannosyldiacylglycerol, mannosylphosphorylundecaprenol (Man-P-Undec), and a membrane-associated lipomannan. This chapter describes the detergent solubilization, partial purification, and properties of Man-P-Undec synthase. The mobility of the mannosyltransferase activity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is a polypeptide with a molecular weight of approx 30.
View Article and Find Full Text PDFFlippases are a class of membrane proteins that are proposed to facilitate the transbilayer movement of amphipathic polar lipids that are required for membrane biogenesis and the assembly of many diverse complex glycoconjugates in eukaryotic and prokaryotic cells. Despite their crucial roles in membrane biology, very little is known about their structures and the precise mechanism(s) by which they overcome the biophysical barriers of the hydrophobic core, and allow polar head groups to traverse membrane bilayers. This chapter presents methods based on the transport of water-soluble analogues that can be applied to investigate membrane proteins mediating the transverse diffusion of polyisoprenoid-linked glycolipid intermediates involved in the biosynthesis of N-linked glycoproteins, glycosylphosphatidylinositol anchors and bacterial polysaccharides.
View Article and Find Full Text PDFMannosylphosphoryldolichol (Man-P-Dol) is synthesized on the cytosolic leaflet of the rough endoplasmic reticulum (ER), and functions as a mannosyl donor in the biosynthesis of Glc(3)Man(9)GlcNAc(2)-P-P-Dol after being translocated to the lumenal leaflet. An assay, based on the transport of Man-P-citronellol (Man-P-Dol(10)), a water-soluble analogue of Man-P-Dol(95), into sealed microsomal vesicles, has been devised to identify protein(s) (flippases) that could mediate the thermodynamically unfavorable movement of Man-P-Dol between the two leaflets of the ER. To develop a defined system for the systematic investigation of the properties of the Man-P-Dol(10) transporter, and as an initial step toward purification of the protein(s) involved in the transport of Man-P-Dol(10), the activity has been solubilized from rat liver microsomes with n-octyl-beta-D-glucoside and reconstituted into proteoliposomes (approximately 0.
View Article and Find Full Text PDFA long-chain cis-isoprenyltransferase (cis-IPTase) located in the endoplasmic reticulum (ER) catalyzes the chain elongation stage in the pathway for the de novo biosynthesis of dolichyl monophosphate (Dol-P) in eukaryotic cells. In Saccharomyces cerevisiae, the ER-associated cis-IPTase is encoded by the RER2 gene. Mutations in the RER2 gene result in defects in growth and protein N-glycosylation.
View Article and Find Full Text PDFDefects in the assembly of dolichol-linked oligosaccharide or its transfer to proteins result in severe, multi-system human diseases called Type I congenital disorders of glycosylation. We have identified a novel CDG type, CDG-Ij, resulting from deficiency in UDP-GlcNAc: dolichol phosphate N-acetyl-glucosamine-1 phosphate transferase (GPT) activity encoded by DPAGT1. The patient presents with severe hypotonia, medically intractable seizures, mental retardation, microcephaly, and exotropia.
View Article and Find Full Text PDFThe assembly of many bacterial cell surface polysaccharides requires the transbilayer movement of polyisoprenoid-linked saccharide intermediates across the cytoplasmic membrane. It is generally believed that transverse diffusion of glycolipid intermediates is mediated by integral membrane proteins called translocases or "flippases." The bacterial genes proposed to encode these translocases have been collectively designated wzx genes.
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