Analysis of Site-specific Glycosylation of Renal and Hepatic γ-Glutamyl Transpeptidase from Normal Human Tissue

The cell surface glycoprotein γ-glutamyl transpeptidase (GGT) was isolated from healthy human kidney and liver to characterize its glycosylation in normal human tissue in vivo. GGT is expressed by a single cell type in the kidney. The spectrum of N-glycans released from kidney GGT constituted a subset of the N-glycans identified from renal membrane glycoproteins. Recent advances in mass spectrometry enabled us to identify the microheterogeneity and relative abundance of glycans on specific glycopeptides and revealed a broader spectrum of glycans than was observed among glycans enzymatically released from isolated GGT. A total of 36 glycan compositions, with 40 unique structures, were identified by site-specific glycan analysis. Up to 15 different glycans were observed at a single site, with site-specific variation in glycan composition. N-Glycans released from liver membrane glycoproteins included many glycans also identified in the kidney. However, analysis of hepatic GGT glycopeptides revealed 11 glycan compositions, with 12 unique structures, none of which were observed on kidney GGT. No variation in glycosylation was observed among multiple kidney and liver donors. Two glycosylation sites on renal GGT were modified exclusively by neutral glycans. In silico modeling of GGT predicts that these two glycans are located in clefts on the surface of the protein facing the cell membrane, and their synthesis may be subject to steric constraints. This is the first analysis at the level of individual glycopeptides of a human glycoprotein produced by two different tissues in vivo and provides novel insights into tissue-specific and site-specific glycosylation in normal human tissues.     

Definitive evidence that a single N-glycan among three glycans on inducible costimulator is required for proper protein trafficking and ligand binding

Glycosylation is a widespread post-translational modification found in glycoproteins. Glycans play key roles in protein folding, quality control in the endoplasmic reticulum (ER) and protein trafficking within cells. However, it remains unclear whether all positions of protein glycosylation are involved in glycan functions, or if specific positions have individual roles. Here we demonstrate the integral involvement of a specific N-glycan from amongst the three glycans present on inducible costimulator (ICOS), a T-cell costimulatory molecule, in proper protein folding and intracellular trafficking to the cell surface membrane. We found that glycosylation-defective mutant proteins lacking N-glycan at amino-acid position 89 (N89), but not proteins lacking either N23 or N110, were retained within the cell and were not detected on the cell surface membrane. Additional evidence suggested that N89 glycosylation was indirectly involved in ICOS ligand binding. These data suggest that amongst the three putative ICOS glycosylation sites, N89 is required for proper ICOS protein folding in the ER, intracellular trafficking and ligand binding activity. This study represents a substantial contribution to the current mechanistic understanding of the necessity and potential functions of a specific N-glycan among the multiple glycans of glycoproteins.     

Serum protein N-glycosylation changes in multiple myeloma

BackgroundMultiple myeloma is characterized by clonal proliferation of malignant plasma cells in the bone marrow that produce monoclonal immunoglobulins. N-glycosylation changes of these monoclonal immunoglobulins have been reported in multiple myeloma, but previous studies only detected limited serum N-glycan features.MethodsHere, a more detailed study of the human serum N-glycome of 91 multiple myeloma patients and 51 controls was performed. We additionally analyzed sequential samples from patients (n = 7) which were obtained at different time points during disease development as well as 16 paired blood serum and bone marrow plasma samples. N-glycans were enzymatically released and measured by mass spectrometry after linkage specific derivatization of sialic acids.ResultsA decrease in both α2,3- and α2,6-sialylation, galactosylation and an increase in fucosylation within complex-type N-glycans were found in multiple myeloma patients compared to controls, as well as a decrease in difucosylation of diantennary glycans. The observed glycosylation changes were present in all ISS stages, including the “low-risk” ISS I. In individual patients, difucosylation of diantennary glycans decreased with development of the disease. Protein N-glycosylation features from blood and bone marrow showed strong correlation. Moreover, associations of monoclonal immunoglobulin (M-protein) and albumin levels with glycan traits were discovered in multiple myeloma patients.Conclusions & general significanceIn conclusion, serum protein N-glycosylation analysis could successfully distinguish multiple myeloma from healthy controls. Further studies are needed to assess the potential roles of glycan trait changes and the associations of glycans with clinical parameters in multiple myeloma early detection and prognosis.     

Structural analysis of <Emphasis Type="Italic">N</Emphasis>-/<Emphasis Type="Italic">O</Emphasis>-glycans assembled on proteins in yeasts

Protein glycosylation, the most universal and diverse post-translational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells. Thus, a comprehensive understanding of protein glycosylation in various yeast species and defining glycan structure characteristics can provide useful information for their biotechnological and clinical implications. Yeast-specific glycans are a target for glyco-engineering; implementing human-type glycosylation pathways in yeast can aid the production of recombinant glycoproteins with therapeutic potential. The virulenceassociated glycans of pathogenic yeasts could be exploited as novel targets for antifungal agents. Nowadays, several glycomics techniques facilitate the generation of species-and strain-specific glycome profiles and the delineation of modified glycan structures in mutant and engineered yeast cells. Here, we present the protocols employed in our laboratory to investigate the N-and O-glycan chains released from purified glycoproteins or cell wall mannoproteins in several yeast species.     

Ligand Binding and Signaling of Dendritic Cell Immunoreceptor (DCIR) Is Modulated by the Glycosylation of the Carbohydrate Recognition Domain

C-type lectins are innate receptors expressed on antigen-presenting cells that are involved in the recognition of glycosylated pathogens and self-glycoproteins. Upon ligand binding, internalization and/or signaling often occur. Little is known on the glycan specificity and ligands of the Dendritic Cell Immunoreceptor (DCIR), the only classical C-type lectin that contains an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM). Here we show that purified DCIR binds the glycan structures Lewis^b and Man₃. Interestingly, binding could not be detected when DCIR was expressed on cells. Since DCIR has an N-glycosylation site inside its carbohydrate recognition domain (CRD), we investigated the effect of this glycan in ligand recognition. Removing or truncating the glycans present on purified DCIR increased the affinity for DCIR-binding glycans. Nevertheless, altering the glycosylation status of the DCIR expressing cell or mutating the N-glycosylation site of DCIR itself did not increase glycan binding. In contrast, cis and trans interactions with glycans induced DCIR mediated signaling, resulting in a decreased phosphorylation of the ITIM sequence. These results show that glycan binding to DCIR is influenced by the glycosylation of the CRD region in DCIR and that interaction with its ligands result in signaling via its ITIM motif.     

Diversity and functions of protein glycosylation in insects

The majority of proteins is modified with carbohydrate structures. This modification, called glycosylation, was shown to be crucial for protein folding, stability and subcellular location, as well as protein-protein interactions, recognition and signaling. Protein glycosylation is involved in multiple physiological processes, including embryonic development, growth, circadian rhythms, cell attachment as well as maintenance of organ structure, immunity and fertility.Although the general principles of glycosylation are similar among eukaryotic organisms, insects synthesize a distinct repertoire of glycan structures compared to plants and vertebrates. Consequently, a number of unique insect glycans mediate functions specific to this class of invertebrates. For instance, the core α1,3-fucosylation of N-glycans is absent in vertebrates, while in insects this modification is crucial for the development of wings and the nervous system.At present, most of the data on insect glycobiology comes from research in Drosophila. Yet, progressively more information on the glycan structures and the importance of glycosylation in other insects like beetles, caterpillars, aphids and bees is becoming available. This review gives a summary of the current knowledge and recent progress related to glycan diversity and function(s) of protein glycosylation in insects. We focus on N- and O-glycosylation, their synthesis, physiological role(s), as well as the molecular and biochemical basis of these processes.     

Neurons and Glia Modify Receptor Protein-tyrosine Phosphatase ζ (RPTPζ)/Phosphacan with Cell-specific O-Mannosyl Glycans in the Developing Brain

Protein O-mannosylation is a glycan modification that is required for normal nervous system development and function. Mutations in genes involved in protein O-mannosyl glycosylation give rise to a group of neurodevelopmental disorders known as congenital muscular dystrophies (CMDs) with associated CNS abnormalities. Our previous work demonstrated that receptor protein-tyrosine phosphatase ζ (RPTPζ)/phosphacan is hypoglycosylated in a mouse model of one of these CMDs, known as muscle-eye-brain disease, a disorder that is caused by loss of an enzyme (protein O-mannose β-1,2-N-acetylglucosaminyltransferase 1) that modifies O-mannosyl glycans. In addition, monoclonal antibodies Cat-315 and 3F8 were demonstrated to detect O-mannosyl glycan modifications on RPTPζ/phosphacan. Here, we show that O-mannosyl glycan epitopes recognized by these antibodies define biochemically distinct glycoforms of RPTPζ/phosphacan and that these glycoforms differentially decorate the surface of distinct populations of neural cells. To provide a further structural basis for immunochemically based glycoform differences, we characterized the O-linked glycan heterogeneity of RPTPζ/phosphacan in the early postnatal mouse brain by multidimensional mass spectrometry. Structural characterization of the O-linked glycans released from purified RPTPζ/phosphacan demonstrated that this protein is a significant substrate for protein O-mannosylation and led to the identification of several novel O-mannose-linked glycan structures, including sulfo-N-acetyllactosamine containing modifications. Taken together, our results suggest that specific glycan modifications may tailor the function of this protein to the unique needs of specific cells. Furthermore, their absence in CMDs suggests that hypoglycosylation of RPTPζ/phosphacan may have different functional consequences in neurons and glia.     

Hitting the sweet spot with capillary electrophoresis: advances in N-glycomics and glycoproteomics

CE–MS glycoproteomics and glycomics. In intact methods, the glycans are identified while attached to the entire protein backbone, exposing the glycosylation profile and providing information on the macro-heterogeneity and site occupancy. In middle-up and bottom-up analysis, the protein is digested into sububits or smaller peptide fragments, revealing specific glycoforms and elucidating micro-heterogeneity in a site-specific approach. In released glycans techniques, the glycans are completely released from the protein molecule through chemical or enzymatic means. Unlike the middle-up and bottom-up techniques, released glycans do not offer site-specific information, but can achieve excellent levels of sensitivity in glycan microheterogeneity identification.

1. Download : Download high-res image (82KB)
2. Download : Download full-size image

     

Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment

Exosomes, also known as microvesicles (EMVs), are nano-sized membranous particles secreted from nearly all mammalian cell types. These nanoparticles play critical roles in many physiological processes including cell-cell signaling, immune activation, and suppression and are associated with disease states such as tumor progression. The biological functions of EMVs are highly dependent on their protein composition, which can dictate pathogenicity. Although some mechanisms have been proposed for the regulation of EMV protein trafficking, little attention has been paid to N-linked glycosylation as a potential sorting signal. Previous work from our laboratory found a conserved glycan signature for EMVs, which differed from that of the parent cell membranes, suggesting a potential role for glycosylation in EMV biogenesis. In this study, we further explore the role of glycosylation in EMV protein trafficking. We identify EMV glycoproteins and demonstrate alteration of their recruitment as a function of their glycosylation status upon pharmacological manipulation. Furthermore, we show that genetic manipulation of the glycosylation levels of a specific EMV glycoprotein, EWI-2, directly impacts its recruitment as a function of N-linked glycan sites. Taken together, our data provide strong evidence that N-linked glycosylation directs glycoprotein sorting into EMVs.     

Quantitative site-specific analysis of protein glycosylation by LC-MS using different glycopeptide-enrichment strategies

A common technique for analysis of protein glycosylation is HPLC coupled to mass spectrometry (LC-MS). However, analysis is challenging due to a low abundance of glycopeptides in complex protein digests, microheterogeneity at the glycosylation site, ion suppression effects, and competition for ionization by coeluting peptides. Specific sample preparation is necessary for a comprehensive and site-specific glycosylation analysis by MS. In this study we qualitatively compared hydrophilic interaction chromatography (HILIC) and hydrazine chemistry for the enrichment of all N-linked glycopeptides and titanium dioxide for capturing sialylated glycopeptides from a complex peptide mixture. Bare silica, microcrystalline cellulose, amino-, amide- (TSKgel Amide-80), and sulfobetaine-(ZIC-HILIC) bonded phases were evaluated for HILIC enrichment. The experiments revealed that ZIC-HILIC and TSKgel Amide-80 are very specific for capturing glycopeptides under optimized conditions. Quantitative analysis of N-glycosidase F-released and 2-aminobenzamide-labeled glycans of a ZIC-HILIC-enriched monoclonal antibody demonstrated that glycopeptides could be enriched without bias for particular glycan structures and without significant losses. Sialylated glycopeptides could be efficiently enriched by titanium dioxide and in addition to HILIC both methods enable a comprehensive analysis of protein glycosylation by MS. Enrichment of N-linked glycopeptides by hydrazine chemistry resulted in lower peptide recovery using a more complex enrichment scheme.     

The cell surface protein MUL_3720 confers binding of the skin pathogen Mycobacterium ulcerans to sulfated glycans and keratin

Mycobacterium ulcerans is the causative agent of the chronic, necrotizing skin disease Buruli ulcer. Modes of transmission and molecular mechanisms involved in the establishment of M. ulcerans infections are poorly understood. Interactions with host glycans are often crucial in bacterial pathogenesis and the 22 kDa M. ulcerans protein MUL_3720 has a putative role in host cell attachment. It has a predicted N-terminal lectin domain and a C-terminal peptidoglycan-binding domain and is highly expressed on the surface of the bacilli. Here we report the glycan-binding repertoire of whole, fixed M. ulcerans bacteria and of purified, recombinant MUL_3720. On an array comprising 368 diverse biologically relevant glycan structures, M. ulcerans cells showed binding to 64 glycan structures, representing several distinct classes of glycans, including sulfated structures. MUL_3720 bound only to glycans containing sulfated galactose and GalNAc, such as glycans known to be associated with keratins isolated from human skin. Surface plasmon resonance studies demonstrated that both whole, fixed M. ulcerans cells and MUL_3720 show high affinity interactions with both glycans and human skin keratin extracts. This MUL_3720-mediated interaction with glycans associated with human skin keratin may contribute to the pathobiology of Buruli ulcer.     

Micro‐ and macroheterogeneity of N‐glycosylation yields size and charge isoforms of human sex hormone binding globulin circulating in serum

Human sex hormone binding globulin (hSHBG) is a serum glycoprotein central to the transport and targeted delivery of sex hormones to steroid‐sensitive tissues. Several molecular mechanisms of action of hSHBG, including the function of its attached glycans remain unknown. Here, we perform a detailed site‐specific characterization of the N‐ and O‐linked glycosylation of serum‐derived hSHBG. MS‐driven glycoproteomics and glycomics combined with exoglycosidase treatment were used in a bottom‐up and top‐down manner to determine glycosylation sites, site‐specific occupancies and monosaccharide compositions, detailed glycan structures, and the higher level arrangement of glycans on intact hSHBG. It was found that serum‐derived hSHBG is N‐glycosylated at Asn³⁵¹ and Asn³⁶⁷ with average molar occupancies of 85.1 and 95.3%, respectively. Both sites are occupied by the same six sialylated and partly core fucosylated bi‐ and triantennary N‐Glycoforms with lactosamine‐type antennas of the form (±NeuAcα6)Galβ4GlcNAc. N‐Glycoforms of Asn³⁶⁷ were slightly more branched and core fucosylated than Asn³⁵¹ N‐glycoforms due probably to a more surface‐exposed glycosylation site. The N‐terminal Thr⁷ was fully occupied by the two O‐linked glycans NeuAcα3Galβ3(NeuAcα6)GalNAc (where NeuAc is N‐acetylneuraminic acid and GalNAc is N‐acetylgalactosamine) and NeuAcα3Galβ3GalNAc in a 1:6 molar ratio. Electrophoretic analysis of intact hSHBG revealed size and charge heterogeneity of the isoforms circulating in blood serum. Interestingly, the size and charge heterogeneity were shown to originate predominantly from differential Asn³⁵¹ glycan occupancies and N‐glycan sialylation that may modulate the hSHBG activity. To date, this work represents the most detailed structural map of the heterogeneous hSHBG glycosylation, which is a prerequisite for investigating the functional aspects of the hSHBG glycans.     

A filamentous phage display system for N-linked glycoproteins

We have developed a filamentous phage display system for the detection of asparagine-linked glycoproteins in Escherichia coli that carry a plasmid encoding the protein glycosylation locus (pgl) from Campylobacter jejuni. In our assay, fusion of target glycoproteins to the minor phage coat protein g3p results in the display of glycans on phage. The glyco-epitope displayed on phage is the product of biosynthetic enzymes encoded by the C. jejuni pgl pathway and minimally requires three essential factors: a pathway for oligosaccharide biosynthesis, a functional oligosaccharyltransferase, and an acceptor protein with a D/E-X₁-N-X₂-S/T motif. Glycosylated phages could be recovered by lectin chromatography with enrichment factors as high as 2 × 10⁵ per round of panning and these enriched phages retained their infectivity after panning. Using this assay, we show that desired glyco-phenotypes can be reliably selected by panning phage-displayed glycoprotein libraries on lectins that are specific for the glycan. For instance, we used our phage selection to identify permissible residues in the −2 position of the bacterial consensus acceptor site sequence. Taken together, our results demonstrate that a genotype–phenotype link can be established between the phage-associated glyco-epitope and the phagemid-encoded genes for any of the three essential components of the glycosylation process. Thus, we anticipate that our phage display system can be used to isolate interesting variants in any step of the glycosylation process, thereby making it an invaluable tool for genetic analysis of protein glycosylation and for glycoengineering in E. coli cells.     

Human disease glycomics: technology advances enabling protein glycosylation analysis – part 1

Introduction: Protein glycosylation is recognized as an important post-translational modification, with specific substructures having significant effects on protein folding, conformation, distribution, stability and activity. However, due to the structural complexity of glycans, elucidating glycan structure-function relationships is demanding. The fine detail of glycan structures attached to proteins (including sequence, branching, linkage and anomericity) is still best analysed after the glycans are released from the purified or mixture of glycoproteins (glycomics). The technologies currently available for glycomics are becoming streamlined and standardized and many features of protein glycosylation can now be determined using instruments available in most protein analytical laboratories.

Areas covered: This review focuses on the current glycomics technologies being commonly used for the analysis of the microheterogeneity of monosaccharide composition, sequence, branching and linkage of released N- and O-linked glycans that enable the determination of precise glycan structural determinants presented on secreted proteins and on the surface of all cells.

Expert commentary: Several emerging advances in these technologies enabling glycomics analysis are discussed. The technological and bioinformatics requirements to be able to accurately assign these precise glycan features at biological levels in a disease context are assessed.     

Unusual <Emphasis Type="Italic">N</Emphasis>-type glycosylation of salivary prolactin-inducible protein (PIP): multiple Lewis<Superscript>Y</Superscript> epitopes generate highly-fucosylated glycan structures

Prolactin-inducible protein (PIP) is a glycoprotein found in body secretions from exocrine glands like saliva and seminal plasma. Important biological functions of PIP concentrations have been demonstrated, e.g. in tumor diagnosis and progression. PIP quantity has been also found useful to determine the success of chemotherapy of mammary carcinoma. Here, we present the analysis of the N-glycosylation of PIP isolated from different sources by LC-MS(/MS) and ¹H-NMR. We found a very uncommon N-type glycosylation of PIP in healthy individuals from both, seminal fluid and saliva. PIP carries unusual highly fucosylated N-linked glycans with multiple Lewis^y (Le^y) epitopes on bi-, tri- and tetraantennary structures resulting in up to nine fucosyl residues on a tetraantennary glycan. In most organs, Le^y epitopes are not present on N-glycans except in case of a tumor when it is highly up-regulated and important for prognosis. Here, for the first time on a specific glycoprotein Le^y antigens are unambiguously characterized on an N-type glycan by NMR spectroscopy. So far, for specific glycoproteins Le^y epitopes had only been reported on O-glycans. Furthermore, a correlation between a nonsynonymous single nucleotide polymorphism (SNP) and glycosylation pattern was detected: individuals heterozygous for the SNP causing the amino acid exchange ⁵¹Gln to ⁵¹His have glycan structures with a higher degree of sialylation compared to individuals lacking the SNP.     

Extended glycan diversity in a bacterial protein glycosylation system linked to allelic polymorphisms and minimal genetic alterations in a glycosyltransferase gene

Glycans manifest in conjunction with the broad spectrum O‐linked protein glycosylation in species within the genus Neisseria display intra‐ and interstrain diversity. Variability in glycan structure and antigenicity are attributable to differences in the content and expression status of glycan synthesis genes. Given the high degree of standing allelic polymorphisms in these genes, the level of glycan diversity may exceed that currently defined. Here, we identify unique protein‐associated disaccharide glycoforms that carry N‐acetylglucosamine (GlcNAc) at their non‐reducing end. This altered structure was correlated with allelic variants of pglH whose product was previously demonstrated to be responsible for the expression of glucose (Glc)‐containing disaccharides. Allele comparisons and site‐specific mutagenesis showed that the presence of a single residue, alanine at position 303 in place of a glutamine, was sufficient for GlcNAc versus Glc incorporation. Phylogenetic analyses revealed that GlcNAc‐containing disaccharides may be widely distributed within the pgl systems of Neisseria particularly in strains of N. meningitidis. Although analogous minimal structural alterations in glycosyltransferases have been documented in association with lipopolysaccharide and capsular polysaccharide variability, this appears to be the first example in which such changes have been implicated in glycan diversification within a bacterial protein glycosylation system.     

Impact of cultivation conditions on N‐glycosylation of influenza virus a hemagglutinin produced in MDCK cell culture

Manufacturers worldwide produce influenza vaccines in different host systems. So far, either fertilized chicken eggs or mammalian cell lines are used. In all these vaccines, hemagglutinin (HA) and neuraminidase are the major components. Both are highly abundant glycoproteins in the viral envelope, and particularly HA is able to induce a strong and protective immune response. The quality characteristics of glycoproteins, such as specific activity, antigenicity, immunogenicity, binding avidity, and receptor‐binding specificity can strongly depend on changes or differences in their glycosylation pattern (potential N‐glycosylation occupancy as well as glycan composition). In this study, capillary gel electrophoresis with laser‐induced fluorescence detection (CGE‐LIF) based glycoanalysis (N‐glycan fingerprinting) was used to determine the impact of cultivation conditions on the HA N‐glycosylation pattern of Madin–Darby canine kidney (MDCK) cell‐derived influenza virus A PR/8/34 (H1N1). We found that adaptation of adherent cells to serum‐free growth has only a minor impact on the HA N‐glycosylation pattern. Only relative abundances of N‐glycan structures are affected. In contrast, host cell adaptation to serum‐free suspension growth resulted in significant changes in the HA N‐glycosylation pattern regarding the presence of specific N‐glycans as well as their abundance. Further controls such as different suppliers for influenza virus A PR/8/34 (H1N1) seed strains, different cultivation scales and vessels in standard or high cell density mode, different virus production media varying in either composition or trypsin activity, different temperatures during virus replication and finally, the impact of β‐propiolactone inactivation resulted—at best—only in minor changes in the relative N‐glycan structure abundances of the HA N‐glycosylation pattern. Surprisingly, these results demonstrate a rather stable HA N‐glycosylation pattern despite various (significant) changes in upstream processing. Only the adaptation of the production host cell line to serum‐free suspension growth significantly influenced HA N‐glycosylation regarding both, the type of attached glycan structures as well as their abundances. Biotechnol. Bioeng. 2013; 110: 1691–1703. © 2013 Wiley Periodicals, Inc.