Glycosylation Of Glycoproteins Research Articles

Identification of a glycosylphosphatidylinositol anchor-modifying β1-3 galactosyltransferase in Trypanosoma brucei.

Trypanosoma brucei is the causative agent of human African sleeping sickness and the cattle disease nagana. Trypanosoma brucei is dependent on glycoproteins for its survival and infectivity throughout its life cycle. Here we report the functional characterization of TbGT3, a glycosyltransferase expressed in the bloodstream and procyclic form of the parasite. Bloodstream and procyclic form TbGT3 conditional null mutants were created and both exhibited normal growth under permissive and nonpermissive conditions. Under nonpermissive conditions, the normal glycosylation of the major glycoprotein of bloodstream form T. brucei, the variant surface glycoprotein and the absence of major alterations in lectin binding to other glycoproteins suggested that the major function of TbGT3 occurs in the procyclic form of the parasite. Consistent with this, the major surface glycoprotein of the procyclic form, procyclin, exhibited a marked reduction in molecular weight due to changes in glycosylphosphatidylinositol (GPI) anchor side chains. Structural analysis of the mutant procyclin GPI anchors indicated that TbGT3 encodes a UDP-Gal: β-GlcNAc-GPI β1-3 Gal transferase. Despite the alterations in GPI anchor side chains, TbGT3 conditional null mutants remained infectious to tsetse flies under nonpermissive conditions.

Mutations altering the gammaretrovirus endoproteolytic motif affect glycosylation of the envelope glycoprotein and early events of the virus life cycle

Previously, we found that mutation of glutamine to proline in the endoproteolytic cleavage signal of the PERV-C envelope (RQKK to RPKK) resulted in non-infectious vectors. Here, we show that RPKK results in a non-infectious vector when placed in not only a PERV envelope, but also the envelope of a related gammaretrovirus, FeLV-B. The amino acid substitutions do not prevent envelope precursor cleavage, viral core and genome assembly, or receptor binding. Rather, the mutations result in the formation of hyperglycosylated glycoprotein and a reduction in the reverse transcribed minus strand synthesis and undetectable 2-LTR circular DNA in cells exposed to vectors with these mutated envelopes. Our findings suggest novel functions associated with the cleavage signal sequence that may affect trafficking through the glycosylation machinery of the cell. Further, the glycosylation status of the envelope appears to impact post-binding events of the viral life cycle, either membrane fusion, internalization, or reverse transcription.

Confident assignment of site-specific glycosylation in complex glycoproteins in a single step.

A glycoprotein may contain several sites of glycosylation, each of which is heterogeneous. As a consequence of glycoform diversity and signal suppression from nonglycosylated peptides that ionize more efficiently, typical reversed-phase LC–MS and bottom–up proteomics database searching workflows do not perform well for identification of site-specific glycosylation for complex glycoproteins. We present an LC–MS system for enrichment, separation, and analysis of glycopeptides from complex glycoproteins (>4 N-glycosylation sequons) in a single step. This system uses an online HILIC enrichment trap prior to reversed-phase C18-MS analysis. We demonstrated the effectiveness of the system using a set of glycoproteins including human transferrin (2 sequons), human alpha-1-acid glycoprotein (5 sequons), and influenza A virus hemagglutinin (9 sequons). The online enrichment renders glycopeptides the most abundant ions detected, thereby facilitating the generation of high-quality data-dependent tandem mass spectra. The tandem mass spectra exhibited product ions from both glycan and peptide backbone dissociation for a majority of the glycopeptides tested using collisionally activated dissociation that served to confidently assign site-specific glycosylation. We demonstrated the value of our system to define site-specific glycosylation using a hemagglutinin containing 9 N-glycosylation sequons from a single HILIC-C18-MS acquisition.

Glycan structure and site of glycosylation in the ER-resident glycoprotein, uridine 5′-diphosphate-glucose: glycoprotein glucosyltransferases 1 from rat, porcine, bovine, and human

Here we report glycan structures and their position of attachment to a carrier protein, uridine 5′-diphosphate-glucose: glycoprotein glucosyltransferase (UGGT1), as detected using tandem mass spectrometry. UGGT1 acts as a folding sensor of newly synthesized glycosylated polypeptides in the endoplasmic reticulum, and the transferase itself is known to be glycosylated. The structure of glycan attached to UGGT1, however, has not been investigated. In this study, we reveal the site of glycosylation (N269) and the glycan structures (Hex5–8HexNAc2) in UGGT1 obtained from rat (Rattus norvegicus), pig (Sus scrofa), cow (Bos taurus), and human (Homo sapiens).

Label-free glycopeptide quantification for biomarker discovery in human sera.

Glycan moieties of glycoproteins modulate many biological processes in mammals, such as immune response, inflammation, and cell signaling. Numerous studies show that many human diseases are correlated with quantitative alteration of protein glycosylation. In some cases, these changes can occur for certain types of glycans over specific sites in a glycoprotein rather than on the global abundance of the glycoprotein. Conventional analytical techniques that analyze the abundance of glycans cleaved from glycoproteins cannot reveal these subtle effects. Here we present a novel statistical method to quantify the site-specific glycosylation of glycoproteins in complex samples using label-free mass spectrometric techniques. Abundance variations between sites of a glycoprotein as well as different glycoforms, that is, glycopeptides with different glycans attached to the same site, can be detected using these techniques. We applied our method to an esophageal cancer study based on blood serum samples from cancer patients in an attempt to detect potential biomarkers of site-specific N-linked glycosylation. A few glycoproteins, including vitronectin, showed significantly different site-specific glycosylations within cancer/control samples, indicating that our method is ready to be used for the discovery of glycosylated biomarkers.

Differential proteomic approach for identification and verification of aberrantly glycosylated proteins in adenocarcinoma lung cancer (ADLC) plasmas by lectin-capturing and targeted mass spectrometry

To investigate quantitative differences in aberrant glycosylation of target glycoproteins between noncancerous group and patient group with adenocarcinoma lung cancer (ADLC), differential proteomic approach was developed by cooperatively using comparative lectin-capturing, targeted mass spectrometry (MRM MS), and antibody/lectin sandwich ELISA. Plasma samples comparatively prepared from 3 ADLC patients and 3 controls, with and without lectin-fractionation using fucose-specific Aleuria aurantia lectin (AAL), were trypsin-digested and analyzed for target glycoproteins, alpha-1-acid glycoprotein (AGP) and ceruloplasmin (CP), by MRM MS. From the MRM MS data the abundance levels of AAL-captured glycoforms of both targets were significantly higher in ADLC cases compared to controls, although the levels in total protein abundance were comparable between ADLC and control groups. This difference between ADLC and control groups in the fucosylated glycoform levels was originated mainly from aberrant fucosylation on the targets in ADLC plasmas rather than change in total protein abundance of the targets, and also confirmed by sandwich ELISA. AGP and CP were further verified to be biomarker candidates by MRM-based analysis of AAL-captured plasmas (30 ADLC cases, 30 controls), with AUROC 0.758 and 0.847 respectively. This differential proteomic approach can be useful for identifying and verifying biomarker candidate involved in aberrant protein glycosylation. The present paper introduces an efficient differential proteomic method to investigate quantitative differences in aberrant protein glycosylation of serological glycoproteins between noncancerous group and lung cancer patient group. This differential proteomic approach consisting of the targeted MRM MS of comparatively lectin-captured plasma fractions and the antibody/lectin sandwich ELISA-based assay was evaluated to be useful for identification of aberrantly fucosylated glycoproteins AGP and CP in lung cancer plasmas. In addition, we have demonstrated that the MRM MS-based differential proteomic approach is also useful for high-throughput verification of the aberrantly fucosylated glycoproteins AGP and CP using the large number of individual plasmas. Therefore, the present MRM MS-based differential proteomic strategy with lectin-capturing can be a powerful tool for high-throughput verification of aberrantly glycosylated biomarker candidates, identified preliminary by mass profiling experiments in proteomic fields but requiring further validation using a large number of cohorts.

Importance of N-glycosylation on CD147 for its biological functions.

Glycosylation of glycoproteins is one of many molecular changes that accompany malignant transformation. Post-translational modifications of proteins are closely associated with the adhesion, invasion, and metastasis of tumor cells. CD147, a tumor-associated antigen that is highly expressed on the cell surface of various tumors, is a potential target for cancer diagnosis and therapy. A significant biochemical property of CD147 is its high level of glycosylation. Studies on the structure and function of CD147 glycosylation provide valuable clues to the development of targeted therapies for cancer. Here, we review current understanding of the glycosylation characteristics of CD147 and the glycosyltransferases involved in the biosynthesis of CD147 N-glycans. Finally, we discuss proteins regulating CD147 glycosylation and the biological functions of CD147 glycosylation.

A Rhomboid Protease Gene Deletion Affects a Novel Oligosaccharide N-Linked to the S-layer Glycoprotein of Haloferax volcanii

Rhomboid proteases occur in all domains of life; however, their physiological role is not completely understood, and nothing is known of the biology of these enzymes in Archaea. One of the two rhomboid homologs of Haloferax volcanii (RhoII) is fused to a zinc finger domain. Chromosomal deletion of rhoII was successful, indicating that this gene is not essential for this organism; however, the mutant strain (MIG1) showed reduced motility and increased sensitivity to novobiocin. Membrane preparations of MIG1 were enriched in two glycoproteins, identified as the S-layer glycoprotein and an ABC transporter component. The H. volcanii S-layer glycoprotein has been extensively used as a model to study haloarchaeal protein N-glycosylation. HPLC analysis of oligosaccharides released from the S-layer glycoprotein after PNGase treatment revealed that MIG1 was enriched in species with lower retention times than those derived from the parent strain. Mass spectrometry analysis showed that the wild type glycoprotein released a novel oligosaccharide species corresponding to GlcNAc-GlcNAc(Hex)2-(SQ-Hex)6 in contrast to the mutant protein, which contained the shorter form GlcNAc2(Hex)2-SQ-Hex-SQ. A glycoproteomics approach of the wild type glycopeptide fraction revealed Asn-732 peptide fragments linked to the sulfoquinovose-containing oligosaccharide. This work describes a novel N-linked oligosaccharide containing a repeating SQ-Hex unit bound to Asn-732 of the H. volcanii S-layer glycoprotein, a position that had not been reported as glycosylated. Furthermore, this study provides the first insight on the biological role of rhomboid proteases in Archaea, suggesting a link between protein glycosylation and this protease family.

Cardiomyocyte sialylation and complex N‐glycosylation protect against dilated cardiomyopathy and heart failure (697.2)

Human congenital disorders of glycosylation are characterized by variably reduced glycoprotein glycosylation resulting in multi‐system effects, including cardiac dysfunction. We seek to understand whether and how reduced glycosylation contributes to cardiac symptoms and reported previously that the reduced glycoprotein sialylation that results from the global gene deletion of a cardiac expressing sialyltransferase, ST3Gal4, contributed to pressure‐induced heart failure (FASEB J, 2013, 27:595.2). Here we questioned whether cardiomyocyte‐specific (αMHC‐Cre) deletion of mannosyl (alpha‐1,3‐)‐glycoprotein beta‐1,2‐N‐acetylglucosaminyltransferase, Mgat1, responsible for initiating synthesis of complex and hybrid N‐glycans, contributes to the onset of dilated cardiomyopathy. At age 16 weeks (earliest time point tested), homozygous floxed Mgat1 and αMHC‐Cre positive mice, Mgat1F/FαMHC‐Cre, showed significantly reduced ejection fraction (EF) and fractional shortening (FS) compared to littermate controls (in %, for controls (n=8), EF = 73.8 ± 2.1, FS = 42.5 ± 1.8; for Mgat1F/FαMHC‐Cre mice (n=7), EF = 61.4 ± 2.3; FS = 32.8 ± 1.6; p<0.002). Systolic function of Mgat1F/FαMHC‐Cre mice, but not of control mice, continued to deteriorate with age (at 30 weeks, Mgat1F/FαMHC‐Cre mice (n=5) in %: EF = 38.2 ± 4.5; FS = 18.8 ± 2.5; p<0.0005). Mgat1F/FαMHC‐Cre mice presented with apparent dilated cardiomyopathy indicated by reduced systolic function, a thinner wall and larger internal diameter of the left ventricle (p<0.0005) with no apparent increase in left ventricular mass index, and marked right and left ventricular dilatation observed during necropsy. Together, our data suggest sialylation and complex N‐glycosylation are necessary for normal cardiac function, and reduced cardiomyocyte N‐glycosylation contributes to the onset of significant ventricular systolic dysfunction consistent with dilated cardiomyopathy leading to heart failure.Grant Funding Source: Supported by NSF grant # IOS‐1146882

Bauhinia forficata lectin (BfL) induces cell death and inhibits integrin-mediated adhesion on MCF7 human breast cancer cells

BackgroundPlant lectins have attracted great interest in cancer studies due to their antitumor activities. These proteins or glycoproteins specifically and reversibly bind to different types of carbohydrates or glycoproteins. Breast cancer, which presents altered glycosylation of cell surface glycoproteins, is one of the most frequent malignant diseases in women. In this work, we describe the effect of the lectin Bauhinia forficata lectin (BfL), which was purified from B. forficata Link subsp. forficata seeds, on the MCF7 human breast cancer cellular line, investigating the mechanisms involved in its antiproliferative activity. MethodsMCF7 cells were treated with BfL. Viability and adhesion alterations were evaluated using flow cytometry and western blotting. ResultsBfL inhibited the viability of the MCF7 cell line but was ineffective on MDA-MB-231 and MCF 10A cells. It inhibits MCF7 adhesion on laminin, collagen I and fibronectin, decreases α1, α6 and β1 integrin subunit expression, and increases α5 subunit expression. BfL triggers necrosis and secondary necrosis, with caspase-9 inhibition. It also causes deoxyribonucleic acid (DNA) fragmentation, which leads to cell cycle arrest in the G2/M phase and a decrease in the expression of the regulatory proteins pRb and p21. ConclusionBfL shows selective cytotoxic effect and adhesion inhibition on MCF7 breast cancer cells. General significanceCell death induction and inhibition of cell adhesion may contribute to understanding the action of lectins in breast cancer.

Analysis of Glycan Variation on Glycoproteins from Serum by the Reverse Lectin-Based ELISA Assay

Altered glycosylation in glycoproteins is associated with carcinogenesis, and certain glycan structures and glycoproteins are well-known markers for tumor progression. To identify potential diagnostic candidate markers, we have developed a novel method for analysis of glycosylation changes of glycoproteins from crude serum samples using lectin-based glycoprotein capture followed by detection with biotin/HRP-conjugated antibodies. The amount of lectin coated on the microplate well was optimized to achieve low background and improved S/N compared with current lectin ELISA methods. In the presence of competing sugars of lectin AAL or with sialic acid removed from the glycoproteins, we confirmed that this method specifically detects glycosylation changes of proteins rather than protein abundance variation. Using our reverse lectin-based ELISA assay, increased fucosylated haptoglobin was observed in sera of patients with ovarian cancer, while the protein level of haptoglobin remained the same between cancers and noncases. The combination of fucosylated haptoglobin and CA125 (AUC = 0.88) showed improved performance for distinguishing stage-III ovarian cancer from noncases compared with CA125 alone (AUC = 0.86). In differentiating early-stage ovarian cancer from noncases, fucosylated haptoglobin showed comparable performance to CA125. The combination of CA125 and fucosylated haptoglobin resulted in an AUC of 0.855, which outperforms CA125 to distinguish early-stage cancer from noncases. Our study provides an alternative method to quantify glycosylation changes of proteins from serum samples, which will be essential for biomarker discovery and validation studies.

β3GnT8 regulates the metastatic potential of colorectal carcinoma cells by altering the glycosylation of CD147

Aberrant glycosylation of cell surface glycoproteins is commonly associated with the invasion and metastasis of colorectal carcinomas, which can be attributed to the upregulated expression of glycosyltransferases. Therefore, elucidation of glycosyltransferases and their substrates may improve our understanding of their roles in tumor metastasis. β-1,3-N-acetylglucosaminyltransferase-8 (β3GnT8) is a key enzyme that catalyzes the formation of poly-N-acetyllactosamine (polylactosamine) chains on β1,6-branched N-glycans invitro, which is also involved in tumor invasion. In the present study, we analyzed the expression of β3GnT8 and its product polylactosamine in four human colorectal carcinoma cell lines (LS-174T, SW620, SW480 and LoVo) with different metastatic potential. We found that the levels of β3GnT8 and polylactosamine chains were gradually increased in the colorectal cancer cell lines in a trend from low to high metastatic potential. Notably, a significantly positive relationship between β3GnT8 expression and HG-CD147 was noted in the colorectal cancer cell lines. To further investigate their relationships, exogenous β3GnT8 was introduced into the LS-174T cells, while expression of β3GnT8 was downregulated in the LoVo cells. The overexpression of β3GnT8 in LS-174T cells increased the level of HG-CD147. Conversely, downregulation of β3GnT8 expression in LoVo cells significantly decreased the expression of HG-CD147. HG-CD147 is a major carrier of β1,6-branched polylactosamine sugars; therefore, the regulation of β3GnT8 significantly altered the β1,6-branched polylactosamine structures on CD147. Hence, we suggest that β3GnT8 plays a key role in the metastasis of colorectal cancer cells by altering the β1,6-branched polylactosamine sugars of CD147.

Comprehensive Functional Analysis of N-Linked Glycans on Ebola Virus GP1

ABSTRACTEbola virus (EBOV) entry requires the virion surface-associated glycoprotein (GP) that is composed of a trimer of heterodimers (GP1/GP2). The GP1 subunit contains two heavily glycosylated domains, the glycan cap and the mucin-like domain (MLD). The glycan cap contains only N-linked glycans, whereas the MLD contains both N- and O-linked glycans. Site-directed mutagenesis was performed on EBOV GP1 to systematically disrupt N-linked glycan sites to gain an understanding of their role in GP structure and function. All 15 N-glycosylation sites of EBOV GP1 could be removed without compromising the expression of GP. The loss of these 15 glycosylation sites significantly enhanced pseudovirion transduction in Vero cells, which correlated with an increase in protease sensitivity. Interestingly, exposing the receptor-binding domain (RBD) by removing the glycan shield did not allow interaction with the endosomal receptor, NPC1, indicating that the glycan cap/MLD domains mask RBD residues required for binding. The effects of the loss of GP1 N-linked glycans on Ca2+-dependent (C-type) lectin (CLEC)-dependent transduction were complex, and the effect was unique for each of the CLECs tested. Surprisingly, EBOV entry into murine peritoneal macrophages was independent of GP1 N-glycans, suggesting that CLEC-GP1 N-glycan interactions are not required for entry into this important primary cell. Finally, the removal of all GP1 N-glycans outside the MLD enhanced antiserum and antibody sensitivity. In total, our results provide evidence that the conserved N-linked glycans on the EBOV GP1 core protect GP from antibody neutralization despite the negative impact the glycans have on viral entry efficiency.

Importance of altered glycoprotein-bound N- and O-glycans for epithelial-to-mesenchymal transition and adhesion of cancer cells

Aberrant glycosylation of cell surface glycoproteins acquired during malignant progression is a common characteristic of human cancer cells. Several biological processes and molecular mechanisms relevant for tumour progression are accompanied by altered mRNA expression levels of certain glycosyltransferases resulting in unusual ratios of common glycoconjugates present in a cancer cell’s glycocalyx or even in the development of unusual, cancer-characterizing carbohydrates. This mini-review aims to give a concise overview on the current knowledge of the functional relevance of altered O- and N-glycans during two critical steps of tumour progression: (I) epithelial-to-mesenchymal transition of primary tumour cells during intravasation and (II) adhesion of circulating tumour cells towards the vascular wall during extravasation at a distant metastatic site. Characteristic lectin binding patterns reflecting these glycosylation changes and the resulting prognostic impact of certain lectin binding sites in different neoplasias are reviewed as well.

The Fusarium oxysporum gnt2, Encoding a Putative N-Acetylglucosamine Transferase, Is Involved in Cell Wall Architecture and Virulence

With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity.

Sialic acid level reflects the disturbances of glycosylation and acute-phase reaction in rheumatic diseases

In the rheumatic diseases, the changes in the carbohydrate part of serum glycoproteins occur and these abnormalities can be monitored by serum level of total and free sialic acid. The aim of this study was to evaluate the total and free sialic acid level as a marker of inflammation activity (TSA) and the changes in glycosylation of blood glycoproteins (FSA) in rheumatoid arthritis (RA), systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). Studies were carried out in 50 patients with RA, 24 with SLE and 32 with SSc. TSA concentration was measured with an enzymatic, colorimetric method and FSA with a thiobarbituric method. The serum levels of TSA in RA and SLE patients were significantly increased compared to controls and in RA patients were higher than that in SSc patients. The mean serum level of FSA in RA patients was significantly higher, but in SSc patients significantly lower than that in the controls, and in RA patients was significantly higher than in SLE and in SSc patients. All acute-phase proteins were changed: Positive acute-phase proteins were elevated, and the negative protein was decreased. The positive acute-phase proteins positively correlated with the levels of TSA and FSA in RA and SSc patients. In SLE patients, TSA positively correlated with haptoglobin and α1-antitrypsin. In RA patients, there was the positive correlation of TSA and FSA with DAS 28. The changes in the serum levels of TSA and FSA in the course of rheumatic diseases could reflect the abnormalities in glycosylation/sialylation patterns of glycoproteins induced by acute-phase response.

Sialoglycoproteins and N-Glycans from Secreted Exosomes of Ovarian Carcinoma Cells

Exosomes consist of vesicles that are secreted by several human cells, including tumor cells and neurons, and they are found in several biological fluids. Exosomes have characteristic protein and lipid composition, however, the results concerning glycoprotein composition and glycosylation are scarce. Here, protein glycosylation of exosomes from ovarian carcinoma SKOV3 cells has been studied by lectin blotting, NP-HPLC analysis of 2-aminobenzamide labeled glycans and mass spectrometry. An abundant sialoglycoprotein was found enriched in exosomes and it was identified by peptide mass fingerprinting and immunoblot as the galectin-3-binding protein (LGALS3BP). Exosomes were found to contain predominantly complex glycans of the di-, tri-, and tetraantennary type with or without proximal fucose and also high mannose glycans. Diantennary glycans containing bisecting N-acetylglucosamine were also detected. This work provides detailed information about glycoprotein and N-glycan composition of exosomes from ovarian cancer cells, furthermore it opens novel perspectives to further explore the functional role of glycans in the biology of exosomes.

Comparative study of sialyl glycoprotein with multiple glycosylation sites using isotope labeling and capillary liquid chromatography/mass spectrometry

A comparative strategy has been demonstrated using RNase B, a single-site N-linked high-mannose glycoprotein. Glycoproteins are more common with multiple glycosylation sites and with complex glycans. A strategy capable of differentiating the changes caused by glycoprotein concentration, glycosylation site occupancy, and a glycoform profile of complex glycoproteins would be beneficial. Tryptic-digested glycoproteins were labeled using 12 C,H-formaldehyde and 13 C, D-formaldehyde, purified, and then analyzed using capillary reversed-phase liquid chromatography/mass spectrometry (RPLC/MS). The relative intensity of non-glycosylated peptides provided information on glycoprotein concentration variation. A site-specific glycoform profile variation was obtained by comparing the glycoform profile of CH2 and 13 CD2 glycopeptides. Determining the protein concentration and glycoform profile variations allows the glycosylation site occupancy variation to be calculated. A strong correlation between the observed and prepared ratios for fetuin glycopeptides from 0.2 to 5 was obtained. Two fetuin samples with different glycoprotein concentrations (4-fold change), glycoform profiles (normal and modified), and glycosylation site occupancies (100% and 50%) were prepared, labeled, mixed, purified, and analyzed using RPLC/MS. The results of the comparative study had a strong correlation with the prepared values. In this report, we demonstrated a comparative analysis of fetuin, a glycoprotein with multiple glycosylation sites and complex sialyl glycans. Compared to our previous approach, we made several modifications including the use of RPLC, a larger mass difference isotope tag, and isotope overlapping correction. The modified approach is expected to be applicable to most glycoproteins. Copyright © 2013 John Wiley & Sons, Ltd.

A mutation in a ganglioside biosynthetic enzyme, ST3GAL5, results in salt & pepper syndrome, a neurocutaneous disorder with altered glycolipid and glycoprotein glycosylation

'Salt & Pepper' syndrome is an autosomal recessive condition characterized by severe intellectual disability, epilepsy, scoliosis, choreoathetosis, dysmorphic facial features and altered dermal pigmentation. High-density SNP array analysis performed on siblings first described with this syndrome detected four shared regions of loss of heterozygosity (LOH). Whole-exome sequencing narrowed the candidate region to chromosome 2p11.2. Sanger sequencing confirmed a homozygous c.994G>A transition (p.E332K) in the ST3GAL5 gene, which encodes for a sialyltransferase also known as GM3 synthase. A different homozygous mutation of this gene has been previously associated with infantile-onset epilepsy syndromes in two other cohorts. The ST3GAL5 enzyme synthesizes ganglioside GM3, a glycosophingolipid enriched in neural tissue, by adding sialic acid to lactosylceramide. Unlike disorders of glycosphingolipid (GSL) degradation, very little is known regarding the molecular and pathophysiologic consequences of altered GSL biosynthesis. Glycolipid analysis confirmed a complete lack of GM3 ganglioside in patient fibroblasts, while microarray analysis of glycosyltransferase mRNAs detected modestly increased expression of ST3GAL5 and greater changes in transcripts encoding enzymes that lie downstream of ST3GAL5 and in other GSL biosynthetic pathways. Comprehensive glycomic analysis of N-linked, O-linked and GSL glycans revealed collateral alterations in response to loss of complex gangliosides in patient fibroblasts and in zebrafish embryos injected with antisense morpholinos that targeted zebrafish st3gal5 expression. Morphant zebrafish embryos also exhibited increased apoptotic cell death in multiple brain regions, emphasizing the importance of GSL expression in normal neural development and function.

Comparison of Haptoglobin and Alpha1-Acid Glycoprotein Glycosylation in the Sera of Small Cell and Non-Small Cell Lung Cancer Patients

Cancer-related carbohydrate epitopes, which are regarded as potential diagnostic and prognostic biomarkers, are carried on the main acute phase proteins. It is not clear, however, if the glycosylation profile is similar in different glycoproteins, or it is protein specific to some extent. The aim of the study was to compare fucosylation, α2,3 sialylation and expression of sialyl-Lewisx epitopes (sLe(x)) in the serum as a whole, AGP and haptoglobin of small cell (SCLC) and non-small cell lung cancer (NSCLC) patients with respect to healthy subjects as well as the cancer stage and its histological type. Thirty-three NSCLC, 13 SCLC patients and 20 healthy volunteers were included in the study. Carbohydrate epitopes were detected by means of their reactivity with specific lectins and monoclonal anti-sLe(x) antibodies in direct or dual-ligand ELISA tests. Significantly increased fucosylation was found in total serum in both cancer groups and in NSCLC haptoglobin. No difference was observed in SCLC haptoglobin or α₁-acid glycoprotein in both cancer groups. Also α2,3 sialylation was elevated in total serum, but not in α₁-acid glycoprotein. This type of sialylation was undetectable in haptoglobin by means of MAA reactivity, in both healthy and cancer subjects. Complete sLe(x) antigens were overexpressed in total NSCLC serum and SCLC AGP, and their level was considerably lowered in cancer haptoglobin. Typical acute phase proteins, haptoglobin and AGP, exhibit different glycosylation profiles in lung cancer. Alterations observed in haptoglobin reflected the disease process better than those in AGP. Comparison of haptoglobin and AGP glycosylation to that observed in total serum suggests that some efficient carriers of disease-altered glycoproteins still remain unidentified.