Enterococcus faecalis rnjB is required for pilin gene expression and biofilm formation.

Pili in Gram-positive bacteria play a major role in the colonization of host tissue and in the development of biofilms. They are promising candidates for vaccines or drug targets since they are highly immunogenic and share common structural and functional features among various Gram-positive pathogens. Numerous publications have helped build a detailed understanding of pilus surface assembly, yet regulation of pilin gene expression has not been well defined. Utilizing a monoclonal antibody developed against the Enterococcus faecalis major pilus protein EbpC, we identified mutants from a transposon (Tn) insertion library which lack surface-exposed Ebp pili. In addition to insertions in the ebp regulon, an insertion in ef1184 (dapA) significantly reduced levels of EbpC. Analysis of in-frame dapA deletion mutants and mutants with the downstream gene rnjB deleted further demonstrated that rnjB was responsible for the deficiency of EbpC. Sequence analysis revealed that rnjB encodes a putative RNase J2. Subsequent quantitative real-time PCR (qRT-PCR) and Northern blotting demonstrated that the ebpABC mRNA transcript level was significantly decreased in the rnjB deletion mutant. In addition, using a reporter gene assay, we confirmed that rnjB affects the expression of the ebpABC operon. Functionally, the rnjB deletion mutant was attenuated in its ability to produce biofilm, similar to that of an ebpABC deletion mutant which lacks Ebp pili. Together, these results demonstrate the involvement of rnjB in E. faecalis pilin gene expression and provide insight into a novel mechanism of regulation of pilus production in Gram-positive pathogens.

Evidence for VirB4-mediated dislocation of membrane-integrated VirB2 pilin during biogenesis of the Agrobacterium VirB/VirD4 type IV secretion system.

Agrobacterium VirB2 pilin is required for assembly of the VirB/VirD4 type IV secretion system (T4SS). The propilin is processed by signal sequence cleavage and covalent linkage of the N and C termini, and the cyclized pilin integrates into the inner membrane (IM) as a pool for assembly of the secretion channel and T pilus. Here, by use of the substituted cysteine accessibility method (SCAM), we defined the VirB2 IM topology and then identified distinct contributions of the T4SS ATPase subunits to the pilin structural organization. Labeling patterns of Cys-substituted pilins exposed to the membrane-impermeative, thiol-reactive reagent 3-(N-maleimidopropionyl)biocytin (MPB) supported a topology model in which two hydrophobic stretches comprise transmembrane domains, an intervening hydrophilic loop (residues 90 to 94) is cytoplasmic, and the hydrophilic N and C termini joined at residues 48 and 121 form a periplasmic loop. Interestingly, the VirB4 ATPase, but not a Walker A nucleoside triphosphate (NTP) binding motif mutant, induced (i) MPB labeling of Cys94, a residue that in the absence of the ATPase is located in the cytoplasmic loop, and (ii) release of pilin from the IM upon osmotic shock. These findings, coupled with evidence for VirB2-VirB4 complex formation by coimmunoprecipitation, support a model in which VirB4 functions as a dislocation motor to extract pilins from the IM during T4SS biogenesis. The VirB11 ATPase functioned together with VirB4 to induce a structural change in the pilin that was detectable by MPB labeling, suggestive of a role for VirB11 as a modulator of VirB4 dislocase activity.

Cancer intelligence acquired (CIA): tumor glycosylation and sialylation codes dismantling antitumor defense

Aberrant glycosylation is a key feature of malignant transformation and reflects epigenetic and genetic anomalies among the multitude of molecules involved in glycan biosynthesis. Although glycan biosynthesis is not template bound, altered tumor glycosylation is not random, but associated with common glycosylation patterns. Evidence suggests that acquisition of distinct glycosylation patterns evolves from a ‘microevolutionary’ process conferring advantages in terms of tumor growth, tumor dissemination, and immune escape. Such glycosylation modifications also involve xeno- and hypersialylation. Xeno-autoantigens such as Neu5Gc-gangliosides provide potential targets for immunotherapy. Hypersialylation may display ‘enhanced self’ to escape immunosurveillance and involves several not mutually exclusive inhibitory pathways that all rely on protein–glycan interactions. A better understanding of tumor ‘glycan codes’ as deciphered by lectins, such as siglecs, selectins, C-type lectins and galectins, may lead to novel treatment strategies, not only in cancer, but also in autoimmune disease or transplantation.

Glycosylation of TRPM4 and TRPM5 channels: molecular determinants and functional aspects

The transient receptor potential channel, TRPM4, and its closest homolog, TRPM5, are non-selective cation channels that are activated by an increase in intracellular calcium. They are expressed in many cell types, including neurons and myocytes. Although the electrophysiological and pharmacological properties of these two channels have been previously studied, less is known about their regulation, in particular their post-translational modifications. We, and others, have reported that wild-type (WT) TRPM4 channels expressed in HEK293 cells, migrated on SDS-PAGE gel as doublets, similar to other ion channels and membrane proteins. In the present study, we provide evidence that TRPM4 and TRPM5 are each N-linked glycosylated at a unique residue, Asn(992) and Asn(932), respectively. N-linked glycosylated TRPM4 is also found in native cardiac cells. Biochemical experiments using HEK293 cells over-expressing WT TRPM4/5 or N992Q/N932Q mutants demonstrated that the abolishment of N-linked glycosylation did not alter the number of channels at the plasma membrane. In parallel, electrophysiological experiments demonstrated a decrease in the current density of both mutant channels, as compared to their respective controls, either due to the Asn to Gln mutations themselves or abolition of glycosylation. To discriminate between these possibilities, HEK293 cells expressing TRPM4 WT were treated with tunicamycin, an inhibitor of glycosylation. In contrast to N-glycosylation signal abolishment by mutagenesis, tunicamycin treatment led to an increase in the TRPM4-mediated current. Altogether, these results demonstrate that TRPM4 and TRPM5 are both N-linked glycosylated at a unique site and also suggest that TRPM4/5 glycosylation seems not to be involved in channel trafficking, but mainly in their functional regulation.

A glycosylation mutant of Trypanosoma brucei links social motility defects in vitro to impaired colonisation of tsetse in vivo

Transmission of African trypanosomes by tsetse flies requires that the parasites migrate out of the midgut lumen and colonise the ectoperitrophic space. Early procyclic culture forms correspond to trypanosomes in the lumen; on agarose plates they exhibit social motility, migrating en masse as radial projections from an inoculation site. We show that an Rft1-/- mutant needs to reach a greater threshold number before migration begins, and that it forms fewer projections than its wild-type parent. The mutant is also up to 4 times less efficient at establishing midgut infections. Ectopic expression of Rft1 rescues social motility defects and restores the ability to colonise the fly. These results are consistent with social motility reflecting movement to the ectoperitrophic space, implicate N-glycans in the signalling cascades for migration in vivo and in vitro, and provide the first evidence that parasite-parasite interactions determine the success of transmission by the insect host.

Enriched mannose glycosylation contributes to Act d 2 allergenicity.

Allergens are responsible for the Th2 response in patients as part of complex mixtures of proteins, fatty acids and other molecules. Plant allergens have hitherto been included in several protein families that share no common biochemical features. Their physical, biochemical and immunological characteristics have been widely studied, but no definite conclusion has been reached about what makes a protein an allergen. N-glycosylation is characteristic of plant allergen sources but is not present in mammals.

Glycosylation differences between the normal and pathogenic prion protein isoforms

Prion protein consists of an ensemble of glycosylated variants or glycoforms. The enzymes that direct oligosaccharide processing, and hence control the glycan profile for any given glycoprotein, are often exquisitely sensitive to other events taking place within the cell in which the glycoprotein is expressed. Alterations in the populations of sugars attached to proteins can reflect changes caused, for example, by developmental processes or by disease. Here we report that normal (PrPC) and pathogenic (PrPSc) prion proteins (PrP) from Syrian hamsters contain the same set of at least 52 bi-, tri-, and tetraantennary N-linked oligosaccharides, although the relative proportions of individual glycans differ. This conservation of structure suggests that the conversion of PrPC into PrPSc is not confined to a subset of PrPs that contain specific sugars. Compared with PrPC, PrPSc contains decreased levels of glycans with bisecting GlcNAc residues and increased levels of tri- and tetraantennary sugars. This change is consistent with a decrease in the activity of N-acetylglucosaminyltransferase III (GnTIII) toward PrPC in cells where PrPSc is formed and argues that, in at least some cells forming PrPSc, the glycosylation machinery has been perturbed. The reduction in GnTIII activity is intriguing both with respect to the pathogenesis of the prion disease and the replication pathway for prions.

Conformational influences of glycosylation of a peptide: A possible model for the effect of glycosylation on the rate of protein folding

Improved strategies for synthesis make it possible to expand the range of glycopeptides available for detailed conformational studies. The glycopeptide 1 was synthesized using a new solid phase synthesis of carbohydrates and a convergent coupling to peptide followed by deprotection. Its conformational properties were subjected to NMR analysis and compared with a control peptide 2 prepared by conventional solid phase methods. Whereas peptide 2 fails to manifest any appreciable secondary structure, the glycopeptide 1 does show considerable conformational bias suggestive of an equilibrium between an ordered and a random state. The implications of this ordering effect for the larger issue of protein folding are considered.

High-affinity binding of bioactive glycosylation-inhibiting factor to antigen-primed T cells and natural killer cells

High-affinity binding was demonstrated between suppressor-T-cell-derived bioactive glycosylation-inhibiting factor (GIF) and helper T hybridomas and natural killer cell line cells. Inactive GIF present in cytosol of suppressor T cells and Escherichia coli-derived recombinant human GIF (rhGIF) failed to bind to these cells. However, affinity of rhGIF for the target cells was generated by replacement of Cys-57 in the sequence with Ala or of Asn-106 with Ser or binding of 5-thio-2-nitrobenzoic acid to Cys-60 in the molecule. Such mutations and the chemical modification of rhGIF synergistically increased the affinity of GIF molecules for the target cells. The results indicated that receptors on the target cells recognize conformational structures of bioactive GIF. Equilibrium dissociation constant (Kd) of the specific binding between bioactive rGIF derivatives and high-affinity receptors was 10–100 pM. Receptors for bioactive GIF derivatives were detected on Th1 and Th2 T helper clones and natural killer NK1.1+ cells in normal spleen but not on naive T or B cells. Neither the inactive rGIF nor bioactive rGIF derivatives bound to macrophage and monocyte lines or induced macrophages for tumor necrosis factor α production.

Synthetic genes for glycoprotein design and the elucidation of hydroxyproline-O-glycosylation codes

Design of hydroxyproline (Hyp)-rich glycoproteins (HRGPs) offers an approach for the structural and functional analysis of these wall components, which are broadly implicated in plant growth and development. HRGPs consist of multiple small repetitive “glycomodules” extensively O-glycosylated through the Hyp residues. The patterns of Hyp-O-glycosylation are putatively coded by the primary sequence as described by the Hyp contiguity hypothesis, which predicts contiguous Hyp residues to be attachment sites of small arabinooligosaccharides (1–5 Ara residues/Hyp); while clustered, noncontiguous Hyp residues are sites of arabinogalactan polysaccharide attachment. As a test, we designed two simple HRGPs as fusion proteins with green fluorescent protein. The first was a repetitive Ser-Hyp motif that encoded only clustered noncontiguous Hyp residues, predicted polysaccharide addition sites. The resulting glycoprotein had arabinogalactan polysaccharide O-linked to all Hyp residues. The second construct, based on the consensus sequence of a gum arabic HRGP, contained both arabinogalactan and arabinooligosaccharide addition sites and, as predicted, gave a product that contained both saccharide types. These results identify an O-glycosylation code of plants.

Enhanced Glycosylation and Sulfation of Secretory Proteoglycans Is Coupled to the Expression of a Basic Secretory Protein

We have used coexpression of a salivary basic proline-rich protein (PRP) along with a proline-rich proteoglycan (PRPg) in pituitary AtT-20 cells to examine the regulation of glycosaminoglycan (GAG) biosynthesis and the storage of these secretory products for regulated secretion. The basic PRP caused a dose-dependent increase in sulfation of PRPg and also increased the extent to which PRPg polypeptide backbones are modified by a GAG chain. The sulfation of an endogenous proteoglycan was similarly increased in the presence of basic PRP; however, other sulfated secretory products of AtT-20 cells were unaffected. These results imply that enzymes functioning in elongation and sulfation of proteoglycans are coordinately regulated and that their activities respond to a change in the milieu of the intracellular transport pathway. Analysis of the regulated secretion of both the basic PRP and PRPg has indicated that while the presence of the GAG chain improves the storage of PRPg, the presence of PRPg does not increase the storage of basic PRP. Therefore, sulfation of GAGs does not appear to be a primary factor in regulated secretory sorting.

The medial-Golgi Ion Pump Pmr1 Supplies the Yeast Secretory Pathway with Ca2+ and Mn2+ Required for Glycosylation, Sorting, and Endoplasmic Reticulum-Associated Protein Degradation

The yeast Ca2+ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca2+ and Mn2+ ions. We show here that addition of Mn2+ greatly alleviates defects of pmr1 mutants in N-linked and O-linked protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca2+. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum proteins. Growth inhibition of pmr1 mutants on Ca2+-deficient media is overcome by expression of other Ca2+ pumps, including a SERCA-type Ca2+ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca2+ and Mn2+ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes.

Apical sorting of a voltage- and Ca2+-activated K+ channel α-subunit in Madin-Darby canine kidney cells is independent of N-glycosylation

The voltage- and Ca2+-activated K+ (KV,Ca) channel is expressed in a variety of polarized epithelial cells seemingly displaying a tissue-dependent apical-to-basolateral regionalization, as revealed by electrophysiology. Using domain-specific biotinylation and immunofluorescence we show that the human channel KV,Ca α-subunit (human Slowpoke channel, hSlo) is predominantly found in the apical plasma membrane domain of permanently transfected Madin-Darby canine kidney cells. Both the wild-type and a mutant hSlo protein lacking its only potential N-glycosylation site were efficiently transported to the cell surface and concentrated in the apical domain even when they were overexpressed to levels 200- to 300-fold higher than the density of intrinsic Slo channels. Furthermore, tunicamycin treatment did not prevent apical segregation of hSlo, indicating that endogenous glycosylated proteins (e.g., KV,Ca β-subunits) were not required. hSlo seems to display properties for lipid-raft targeting, as judged by its buoyant distribution in sucrose gradients after extraction with either detergent or sodium carbonate. The evidence indicates that the hSlo protein possesses intrinsic information for transport to the apical cell surface through a mechanism that may involve association with lipid rafts and that is independent of glycosylation of the channel itself or an associated protein. Thus, this particular polytopic model protein shows that glycosylation-independent apical pathways exist for endogenous membrane proteins in Madin-Darby canine kidney cells.

Posttranslational modification of the glycosylation inhibiting factor (GIF) gene product generates bioactive GIF

Glycosylation inhibiting factor (GIF) and macrophage migration inhibitory factor (MIF) share an identical structure gene. Here we unravel two steps of posttranslational modifications in GIF/MIF molecules in human suppressor T (Ts) cell hybridomas. Peptide mapping and MS analysis of the affinity-purified GIF from the Ts cells revealed that one modification is cysteinylation at Cys-60, and the other is phosphorylation at Ser-91. Cysteinylated GIF, but not the wild-type GIF/MIF, possessed immunosuppressive effects on the in vitro IgE antibody response and had high affinity for GIF receptors on the T helper hybridoma cells. In vitro treatment of wild-type recombinant human GIF/MIF with cystine resulted in preferential cysteinylation of Cys-60 in the molecules. The cysteinylated recombinant human GIF and the Ts hybridoma-derived cysteinylated GIF were comparable both in the affinity for the receptors and in the immunosuppressive activity. Polyclonal antibodies specific for a stretch of the amino acid sequence in α2-helix of GIF bound bioactive cysteinylated GIF but failed to bind wild-type GIF/MIF. These results strongly suggest that cysteinylation of Cys-60 and consequent conformational changes in the GIF/MIF molecules are responsible for the generation of GIF bioactivity.

Arabidopsis cyt1 mutants are deficient in a mannose-1-phosphate guanylyltransferase and point to a requirement of N-linked glycosylation for cellulose biosynthesis

Arabidopsis cyt1 mutants have a complex phenotype indicative of a severe defect in cell wall biogenesis. Mutant embryos arrest as wide, heart-shaped structures characterized by ectopic accumulation of callose and the occurrence of incomplete cell walls. Texture and thickness of the cell walls are irregular, and unesterified pectins show an abnormally diffuse distribution. To determine the molecular basis of these defects, we have cloned the CYT1 gene by a map-based approach and found that it encodes mannose-1-phosphate guanylyltransferase. A weak mutation in the same gene, called vtc1, has previously been identified on the basis of ozone sensitivity due to reduced levels of ascorbic acid. Mutant cyt1 embryos are deficient in N-glycosylation and have an altered composition of cell wall polysaccharides. Most notably, they show a 5-fold decrease in cellulose content. Characteristic aspects of the cyt1 phenotype, including radial swelling and accumulation of callose, can be mimicked with the inhibitor of N-glycosylation, tunicamycin. Our results suggest that N-glycosylation is required for cellulose biosynthesis and that a deficiency in this process can account for most phenotypic features of cyt1 embryos.