Identification of critical residues in Gap3 of <Emphasis Type="Italic">Streptococcus parasanguinis</Emphasis> involved in Fap1 glycosylation,fimbrial formation and <Emphasis Type="Italic">in vitro</Emphasis>adhesion

Background  

Streptococcus parasanguinis is a primary colonizer of human tooth surfaces and plays an important role in dental plaque formation. Bacterial adhesion and biofilm formation are mediated by long peritrichous fimbriae that are composed of a 200 kDa serine rich glycoprotein named Fap1 (fimbriae-associated protein). Glycosylation and biogenesis of Fap1 are modulated by a gene cluster downstream of the fap1 locus. A gene encoding a glycosylation-associated protein, Gap3, was found to be important for Fap1 glycosylation, long fimbrial formation and Fap1-mediated biofilm formation.     

Incorporating significant amino acid pairs to identify O-linked glycosylation sites on transmembrane proteins and non-transmembrane proteins

Background  

While occurring enzymatically in biological systems, O-linked glycosylation affects protein folding, localization and trafficking, protein solubility, antigenicity, biological activity, as well as cell-cell interactions on membrane proteins. Catalytic enzymes involve glycotransferases, sugar-transferring enzymes and glycosidases which trim specific monosaccharides from precursors to form intermediate structures. Due to the difficulty of experimental identification, several works have used computational methods to identify glycosylation sites.     

IgG glycosylation and DNA methylation are interconnected with smoking

Background

Glycosylation is one of the most common post-translation modifications with large influences on protein structure and function. The effector function of immunoglobulin G (IgG) alters between pro- and anti-inflammatory, based on its glycosylation. IgG glycan synthesis is highly complex and dynamic.

Characterization of Endogenous Human FcγRIII by Mass Spectrometry Reveals Site,Allele and Sequence Specific Glycosylation*

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Highlights

• •Glycosylation of endogenous FcγRIII from neutrophils and matched plasma from more than 40 donors characterized at two sites involved in IgG binding.
• •Glycosylation of soluble FcγRIII glycosylation at N45 can be used to assign FcγRIIIb alleles.
• •FcγRIIIb allele specific differences in glycosylation at N162 may influence differential activity observed for primary cells.

     

Glycosylation of Sodium/Iodide Symporter (NIS) Regulates Its Membrane Translocation and Radioiodine Uptake

Purpose

Human sodium/iodide symporter (hNIS) protein is a membrane glycoprotein that transports iodide ions into thyroid cells. The function of this membrane protein is closely regulated by post-translational glycosylation. In this study, we measured glycosylation-mediated changes in subcellular location of hNIS and its function of iodine uptake.

Methods

HeLa cells were stably transfected with hNIS/tdTomato fusion gene in order to monitor the expression of hNIS. Cellular localization of hNIS was visualized by confocal microscopy of the red fluorescence of tdTomato. The expression of hNIS was evaluated by RT-PCR and immunoblot analysis. Functional activity of hNIS was estimated by radioiodine uptake. Cyclic AMP (cAMP) and tunicamycin were used to stimulate and inhibit glycosylation, respectively. In vivo images were obtained using a Maestro fluorescence imaging system.

Results

cAMP-mediated Glycosylation of NIS resulted in increased expression of hNIS, stimulating membrane translocation, and enhanced radioiodine uptake. In contrast, inhibition of glycosylation by treatment with tunicamycin dramatically reduced membrane translocation of intracellular hNIS, resulting in reduced radioiodine uptake. In addition, our hNIS/tdTomato fusion reporter successfully visualized cAMP-induced hNIS expression in xenografted tumors from mouse model.

Conclusions

These findings clearly reveal that the membrane localization of hNIS and its function of iodine uptake are glycosylation-dependent, as our results highlight enhancement of NIS expression and glycosylation with subsequent membrane localization after cAMP treatment. Therefore, enhancing functional NIS by the increasing level of glycosylation may be suggested as a promising therapeutic strategy for cancer patients who show refractory response to conventional radioiodine treatment.     

Glycosylation Site Analysis of Human Platelets by Electrostatic Repulsion Hydrophilic Interaction Chromatography

Introduction

Glycosylations range among the most common posttranslational modifications with an estimated 50% of all proteins supposed to be glycosylated. These modifications are required for essential cellular processes including cell–cell recognition, protein structure and activity, e.g., of surface receptors, as well as subcellular localization of proteins. Beside the elucidation of the carbohydrate structures, the annotation of glycosylation sites is of primary interest as a basis for subsequent functional characterization. Although mass spectrometry is the method of choice for large-scale analysis of glycosylation sites, it requires initial enrichment of glycopeptides prior mass spectrometric detection in most cases.

Materials and Methods

In this paper, we present a novel approach for glycopeptide enrichment by electrostatic repulsion hydrophilic interaction chromatography (ERLIC). Glycopeptides were separated from the bulk of non-modified peptides and gradually eluted from the stationary phase with potential for isoform resolution. Applied to human platelets, 125 glycosylation sites on 66 proteins were identified including major platelet glycoproteins responsible for cellular function.

Conclusion

These sites add a major contribution to the now more than 250 glycosylation sites annotated for platelets, which enable the clinically relevant design of quantification assays for platelet glycoproteins.     

An automated workflow for enhancing microbial bioprocess optimization on a novel microbioreactor platform

Background

Although the occurrence, biosynthesis and possible functions of glycoproteins are increasingly documented for pathogens, glycoproteins are not yet widely described in probiotic bacteria. Nevertheless, knowledge of protein glycosylation holds important potential for better understanding specific glycan-mediated interactions of probiotics and for glycoengineering in food-grade microbes.

Results

Here, we provide evidence that the major secreted protein Msp1/p75 of the probiotic Lactobacillus rhamnosus GG is glycosylated. Msp1 was shown to stain positive with periodic-acid Schiff staining, to be susceptible to chemical deglycosylation, and to bind with the mannose-specific Concanavalin A (ConA) lectin. Recombinant expression in Escherichia coli resulted in a significant reduction in molecular mass, loss of ConA reactivity and increased sensitivity towards pronase E and proteinase K. Mass spectrometry showed that Msp1 is O-glycosylated and identified a glycopeptide TVETPSSA (amino acids 101-108) bearing hexoses presumably linked to the serine residues. Interestingly, these serine residues are not present in the homologous protein of several Lactobacillus casei strains tested, which also did not bind to ConA. The role of the glycan substitutions in known functions of Msp1 was also investigated. Glycosylation did not seem to impact significantly on the peptidoglycan hydrolase activity of Msp1. In addition, the glycan chain appeared not to be required for the activation of Akt signaling in intestinal epithelial cells by Msp1. On the other hand, examination of different cell extracts showed that Msp1 is a glycosylated protein in the supernatant, but not in the cell wall and cytosol fraction, suggesting a link between glycosylation and secretion of this protein.

Conclusions

In this study we have provided the first evidence of protein O-glycosylation in the probiotic L rhamnosus GG. The major secreted protein Msp1 is glycosylated with ConA reactive sugars at the serine residues at 106 and 107. Glycosylation is not required for the peptidoglycan hydrolase activity of Msp1 nor for Akt activation capacity in epithelial cells, but appears to be important for its stability and protection against proteases.     

Aberrant glycosylation associated with enzymes as cancer biomarkers

Background  

One of the new roles for enzymes in personalized medicine builds on a rational approach to cancer biomarker discovery using enzyme-associated aberrant glycosylation. A hallmark of cancer, aberrant glycosylation is associated with differential expressions of enzymes such as glycosyltransferase and glycosidases. The aberrant expressions of the enzymes in turn cause cancer cells to produce glycoproteins with specific cancer-associated aberrations in glycan structures.     

Chondroitin sulfate-cell membrane effectors as regulators of growth factor-mediated vascular and cancer cell migration

Background

Glycosylation is a multi-step post-translational enzymatic process which enhances the functional diversity of secreted or membrane proteins and is implicated in physiological and pathological conditions. Chondroitin sulfate (CS) chains are glycosaminoglycan chains, consisting of disaccharide units of glucuronic acid and N-acetylgalactosamine, attached to proteins as part of proteoglycans.

Scope of Review

The existing knowledge on glycosylation by CS (CS glycanation) of cell membrane proteins and receptors, such as syndecans, chondroitin sulfate proteoglycan 4, betaglycan, neuropilin-1, integrins and receptor protein tyrosine phosphatase β/ζ, is summarized and the importance of CS glycanation in growth factor-induced migration, angiogenesis and tumor growth and invasion is described.

Major Conclusions

Identification of glycosylation so far used to be a means of further characterizing and categorizing proteins and receptors. Although there is a significant amount of information regarding the interaction of growth factors with CS chains, very little information exists on the core proteins involved. It is now evident that there is more than meets the eye regarding the addition of glycans.

General Significance

Future effort should focus on characterizing CS glycanation of membrane proteins and receptors of interest in an attempt to elucidate its contribution in fine-tuning growth factor-induced signaling. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Detection of the Heterogeneous O-Glycosylation Profile of MT1-MMP Expressed in Cancer Cells by a Simple MALDI-MS Method

Background

Glycosylation is an important and universal post-translational modification for many proteins, and regulates protein functions. However, simple and rapid methods to analyze glycans on individual proteins have not been available until recently.

Methods/Principal Findings

A new technique to analyze glycopeptides in a highly sensitive manner by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using the liquid matrix 3AQ/CHCA was developed recently and we optimized this technique to analyze a small amount of transmembrane protein separated by SDS-PAGE. We used the MALDI-MS method to evaluate glycosylation status of membrane-type 1 matrix metalloproteinase (MT1-MMP). O-glycosylation of MT1-MMP is reported to modulate its protease activity and thereby to affect cancer cell invasion. MT1-MMP expressed in human fibrosarcoma HT1080 cells was immunoprecipitated and resolved by SDS-PAGE. After in-gel tryptic digestion of the protein, a single droplet of the digest was applied directly to the liquid matrix on a MALDI target plate. Concentration of hydrophilic glycopeptides within the central area occurred due to gradual evaporation of the sample solution, whereas nonglycosylated hydrophobic peptides remained at the periphery. This specific separation and concentration of the glycopeptides enabled comprehensive analysis of the MT1-MMP O-glycosylation.

Conclusions/Significance

We demonstrate, for the first time, heterogeneous O-glycosylation profile of a protein by a whole protein analysis using MALDI-MS. Since cancer cells are reported to have altered glycosylation of proteins, this easy-to-use method for glycopeptide analysis opens up the possibility to identify specific glycosylation patterns of proteins that can be used as new biomarkers for malignant tumors.     

Identification of N-Glycosylation in Hepatocellular Carcinoma Patients’ Serum with a Comparative Proteomic Approach

Aim

This study is to explore the different expressions of serum N-glycoproteins and glycosylation sites between hepatocellular carcinoma (HCC) patients and healthy controls.

Method

We combined high abundant proteins depletion and hydrophilic affinity method to enrich the glycoproteins. Through liquid chromatography-tandem mass spectrometry (LC-MS/MS), we extensively surveyed different expressions of glycosylation sites and glycoproteins between the two groups.

Result

This approach identified 152 glycosylation sites and 54 glycoproteins expressed differently between HCC patients and healthy controls. With the absolute values of Pearson coefficients of at least 0.8, eight proteins were identified significantly up or down regulated in HCC serum. Those proteins are supposed to be involved in several biological processes, cellular components and molecular functions of hepatocarcinogenesis. Several of them had been reported abnormally regulated in several kinds of malignant tumors, and may be promising biomarkers of HCC.

Conclusion

Our work provides a systematic and quantitative method of glycoproteomics and demonstrates some key changes in clinical HCC serum. These proteomic signatures may help to unveil the underlying mechanisms of hepatocarcinogenesis and may be useful for the exploration of candidate biomarkers.     

Linkage Specific Fucosylation of Alpha-1-Antitrypsin in Liver Cirrhosis and Cancer Patients: Implications for a Biomarker of Hepatocellular Carcinoma

Background

We previously reported increased levels of protein-linked fucosylation with the development of liver cancer and identified many of the proteins containing the altered glycan structures. One such protein is alpha-1-antitrypsin (A1AT). To advance these studies, we performed N-linked glycan analysis on the five major isoforms of A1AT and completed a comprehensive study of the glycosylation of A1AT found in healthy controls, patients with hepatitis C- (HCV) induced liver cirrhosis, and in patients infected with HCV with a diagnosis of hepatocellular carcinoma (HCC).

Methodology/Principal Findings

Patients with liver cirrhosis and liver cancer had increased levels of triantennary glycan-containing outer arm (α-1,3) fucosylation. Increases in core (α-1,6) fucosylation were observed only on A1AT from patients with cancer. We performed a lectin fluorophore-linked immunosorbent assay using Aleuria Aurantia lectin (AAL), specific for core and outer arm fucosylation in over 400 patients with liver disease. AAL-reactive A1AT was able to detect HCC with a sensitivity of 70% and a specificity of 86%, which was greater than that observed with the current marker of HCC, alpha-fetoprotein. Glycosylation analysis of the false positives was performed; results indicated that these patients had increases in outer arm fucosylation but not in core fucosylation, suggesting that core fucosylation is cancer specific.

Conclusions/Significance

This report details the stepwise change in the glycosylation of A1AT with the progression from liver cirrhosis to cancer and identifies core fucosylation on A1AT as an HCC specific modification.     

N-Glycosylation of the Drosophila neural protein Chaoptin is essential for its stability, cell surface transport and adhesive activity

Glycosylation of proteins can modulate their function in a striking variety of systems, including immune responses, neuronal activities and development. The Drosophila protein, Chaoptin (Chp), is essential for the development and maintenance of photoreceptor cells. This protein is heavily glycosylated, but the possible role of this glycosylation is not well-understood. Here we show that mutations introduced into about 1/3 of 16 potential N-linked glycosylation sites within Chp impaired its cell adhesive activities when expressed in Drosophila S2 cells. Mutation of 2/3 of the glycosylation sites resulted in a marked decrease in Chp protein abundance. These results suggest that N-linked glycosylation of Chp is essential for its stability and activity.     

Why Glycosylation Matters in Building a Better Flu Vaccine

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Highlights

• •Glycosylation is not currently considered in flu vaccine design.
• •Glycosylation influences on immunodominance are not well understood.
• •Identification of site-specific glycosylation using mass spectrometry has matured.
• •New methods are needed to quantify site-specific glycosylation for vaccine design.

     

Comparative analyses of the Conserved Oligomeric Golgi (COG) complex in vertebrates

Background  

The Conserved Oligomeric Golgi (COG) complex is an eight-subunit assembly that localizes peripherally to Golgi membranes and is involved in retrograde vesicular trafficking. COG subunits are organized in two heterotrimeric groups, Cog2, -3, -4 and Cog5, -6, -7, linked by a dimeric group formed by Cog1 and Cog8. Dysfunction of COG complex in humans has been associated with new forms of Congenital Disorders of Glycosylation (CDG), therefore highlighting its essential role. In the present study, we intended to gain further insights into the evolution of COG subunits in vertebrates, using comparative analyses of all eight COG proteins.     

Moisture-induced solid state instabilities in α-chymotrypsin and their reduction through chemical glycosylation

Background  

Protein instability remains the main factor limiting the development of protein therapeutics. The fragile nature (structurally and chemically) of proteins makes them susceptible to detrimental events during processing, storage, and delivery. To overcome this, proteins are often formulated in the solid-state which combines superior stability properties with reduced operational costs. Nevertheless, solid protein pharmaceuticals can also suffer from instability problems due to moisture sorption. Chemical protein glycosylation has evolved into an important tool to overcome several instability issues associated with proteins. Herein, we employed chemical glycosylation to stabilize a solid-state protein formulation against moisture-induced deterioration in the lyophilized state.     

Spatial partitioning of secretory cargo from Golgi resident proteins in live cells

Background  

To maintain organelle integrity, resident proteins must segregate from itinerant cargo during secretory transport. However, Golgi resident enzymes must have intimate access to secretory cargo in order to carry out glycosylation reactions. The amount of cargo and associated membrane may be significant compared to the amount of Golgi membrane and resident protein, but upon Golgi exit, cargo and resident are efficiently sorted. How this occurs in live cells is not known.     

The Compartmentalisation of Phosphorylated Free Oligosaccharides in Cells from a CDG Ig Patient Reveals a Novel ER-to-Cytosol Translocation Process

Background

Biosynthesis of the dolichol linked oligosaccharide (DLO) required for protein N-glycosylation starts on the cytoplasmic face of the ER to give Man₅GlcNAc₂-PP-dolichol, which then flips into the ER for further glycosylation yielding mature DLO (Glc₃Man₉GlcNAc₂-PP-dolichol). After transfer of Glc₃Man₉GlcNAc₂ onto protein, dolichol-PP is recycled to dolichol-P and reused for DLO biosynthesis. Because de novo dolichol synthesis is slow, dolichol recycling is rate limiting for protein glycosylation. Immature DLO intermediates may also be recycled by pyrophosphatase-mediated cleavage to yield dolichol-P and phosphorylated oligosaccharides (fOSGN2-P). Here, we examine fOSGN2-P generation in cells from patients with type I Congenital Disorders of Glycosylation (CDG I) in which defects in the dolichol cycle cause accumulation of immature DLO intermediates and protein hypoglycosylation.

Methods and Principal Findings

In EBV-transformed lymphoblastoid cells from CDG I patients and normal subjects a correlation exists between the quantities of metabolically radiolabeled fOSGN2-P and truncated DLO intermediates only when these two classes of compounds possess 7 or less hexose residues. Larger fOSGN2-P were difficult to detect despite an abundance of more fully mannosylated and glucosylated DLO. When CDG Ig cells, which accumulate Man₇GlcNAc₂-PP-dolichol, are permeabilised so that vesicular transport and protein synthesis are abolished, the DLO pool required for Man₇GlcNAc₂-P generation could be depleted by adding exogenous glycosylation acceptor peptide. Under conditions where a glycotripeptide and neutral free oligosaccharides remain predominantly in the lumen of the ER, Man₇GlcNAc₂-P appears in the cytosol without detectable generation of ER luminal Man₇GlcNAc₂-P.

Conclusions and Significance

The DLO pools required for N-glycosylation and fOSGN2-P generation are functionally linked and this substantiates the hypothesis that pyrophosphatase-mediated cleavage of DLO intermediates yields recyclable dolichol-P. The kinetics of cytosolic fOSGN2-P generation from a luminally-generated DLO intermediate demonstrate the presence of a previously undetected ER-to-cytosol translocation process for either fOSGN2-P or DLO.     

Expression in Pichia pastoris and characterization of two novel dirigent proteins for atropselective formation of gossypol

We established an efficient fed-batch fermentation process for two novel dirigent proteins from cotton plants, GbDIR2 from Gossypium barbadense and GhDIR3 from G. hirsutum, using the engineered Pichia pastoris GlycoSwitch® SuperMan₅ strain to prevent hyperglycosylation. The two (His)₆-tagged proteins were purified by metal-chelate affinity chromatography and obtained in quantities of 12 and 15 mg L⁻¹ of culture volume, respectively. Glycosylation sites were identified for the native and for the enzymatically deglycosylated proteins by mass spectrometry, confirming five to six of the seven predicted glycosylation sites in the NxS/T sequence context. The predominant glycan structure was Man₅GlcNAc₂ with, however, a significant contribution of Man_4–10GlcNAc₂. Both dirigent proteins (DIRs) mediated the formation of (+)-gossypol by atropselective coupling of hemigossypol radicals. Similar to previously characterized DIRs, GbDIR2 and GhDIR3 lacked oxidizing activity and depended on an oxidizing system (laccase/O₂) for the generation of substrate radicals. In contrast to DIRs involved in the biosynthesis of lignans, glycosylation was not essential for function. Quantitative enzymatic deglycosylation yielded active GbDIR2 and GhDIR3 in excellent purity. The described fermentation process in combination with enzymatic deglycosylation will pave the way for mechanistic and structural studies and, eventually, the application of cotton DIRs in a biomimetic approach towards atropselective biaryl synthesis.

     

COG5-CDG: expanding the clinical spectrum

Background

The Conserved Oligomeric Golgi (COG) complex is involved in the retrograde trafficking of Golgi components, thereby affecting the localization of Golgi glycosyltransferases. Deficiency of a COG-subunit leads to defective protein glycosylation, and thus Congenital Disorders of Glycosylation (CDG). Mutations in subunits 1, 4, 5, 6, 7 and 8 have been associated with CDG-II. The first patient with COG5-CDG was recently described (Paesold-Burda et al. Hum Mol Genet 2009; 18:4350–6). Contrary to most other COG-CDG cases, the patient presented a mild/moderate phenotype, i.e. moderate psychomotor retardation with language delay, truncal ataxia and slight hypotonia.

Methods

CDG-IIx patients from our database were screened for mutations in COG5. Clinical data were compared. Brefeldin A treatment of fibroblasts and immunoblotting experiments were performed to support the diagnosis.

Results and conclusion

We identified five new patients with proven COG5 deficiency. We conclude that the clinical picture is not always as mild as previously described. It rather comprises a broad spectrum with phenotypes ranging from mild to very severe. Interestingly, on a clinical basis some of the patients present a significant overlap with COG7-CDG, a finding which can probably be explained by subunit interactions at the protein level.