Abstract: Human corticosteroid-binding globulin (CBG), a heavily glycosylated protein containing six N-linked glycosylation sites, transports cortisol and other corticosteroids in blood circulation. Here, we investigate the biological importance of the N-glycans of CBG derived from human serum by performing a structural and functional characterization of CBG N-glycosylation. Liquid chromatography-tandem MS-based glycoproteomics and glycomics combined with exoglycosidase treatment revealed 26 complex type N-glycoforms, all of which were terminated with α2,3-linked neuraminic acid (NeuAc) residues. The CBG N-glycans showed predominantly bi- and tri-antennary branching, but higher branching was also observed. N-glycans from all six N-glycosylation sites were identified with high site occupancies (70.5-99.5%) and glycoforms from all sites contained a relatively low degree of core-fucosylation (0-34.9%). CBG showed site-specific glycosylation and the site-to-site differences in core-fucosylation and branching could be in silico correlated with the accessibility to the individual glycosylation sites on the maturely folded protein. Deglycosylated and desialylated CBG analogs were generated to investigate the biological importance of CBG N-glycans. As a functional assay, MCF-7 cells were challenged with native and glycan-modified CBG and the amount of cAMP, which is produced as a quantitative response upon CBG binding to its cell surface receptor, was used to evaluate the CBG:receptor interaction. The removal of both CBG N-glycans and NeuAc residues increased the production of cAMP significantly. This confirms that N-glycans are involved in the CBG:receptor interaction and indicates that the modulation is performed by steric and/or electrostatic means through the terminal NeuAc residues.
Abstract: Protein phosphorylation and glycosylation are the most common post-translational modifications observed in biology, frequently on the same protein. Assembly protein AP180 is a synapse-specific phosphoprotein and O-linked beta-N-acetylglucosamine (O-GlcNAc) modified glycoprotein. AP180 is involved in the assembly of clathrin coated vesicles in synaptic vesicle endocytosis. Unlike other types of O-glycosylation, O-GlcNAc is nucleocytoplasmic and reversible. It was thought to be a terminal modification, that is, the O-GlcNAc was not found to be additionally modified in any way. We now show that AP180 purified from rat brain contains a phosphorylated O-GlcNAc (O-GlcNAc-P) within a highly conserved sequence. O-GlcNAc or O-GlcNAc-P, but not phosphorylation alone, was found at Thr-310. Analysis of synthetic GlcNAc-6-P produced identical fragmentation products to GlcNAc-P from AP180. Direct O-linkage of GlcNAc-P to a Thr residue was confirmed by electron transfer dissociation MS. A second AP180 tryptic peptide was also glycosyl phosphorylated, but the site of modification was not assigned. Sequence similarities suggest there may be a common motif within AP180 involving glycosyl phosphorylation and dual flanking phosphorylation sites within 4 amino acid residues. This novel type of protein glycosyl phosphorylation adds a new signaling mechanism to the regulation of neurotransmission and more complexity to the study of O-GlcNAc modification.
Abstract: Calreticulin is a chaperone of the endoplasmic reticulum (ER) assisting proteins in achieving the correctly folded structure. Details of the binding specificity of calreticulin are still a matter of debate. Calreticulin has been described as an oligosaccharide-binding chaperone but data are also accumulating in support of calreticulin as a polypeptide binding chaperone. In contrast to mammalian immunoglobulin G (IgG), which has complex type N-glycans, chicken immunoglobulin Y (IgY) possesses a monoglucosylated high mannose N-linked glycan, which is a ligand for calreticulin. Here, we have used solid and solution-phase assays to analyze the in vitro binding of calreticulin, purified from human placenta, to human IgG and chicken IgY in order to compare the interactions. In addition, peptides from the respective immunoglobulins were included to further probe the binding specificity of calreticulin. The experiments demonstrate the ability of calreticulin to bind to denatured forms of both IgG and IgY regardless of the glycosylation state of the proteins. Furthermore, calreticulin exhibits binding to peptides (glycosylated and non-glycosylated) derived from trypsin digestion of both immunoglobulins. Additionally, calreticulin peptide binding was examined with synthetic peptides covering the IgG Cγ2 domain demonstrating interaction with approximately half the peptides. Our results show that the dominant binding activity of calreticulin in vitro is toward the polypeptide moieties of IgG and IgY even in the presence of the monoglucosylated high mannose N-linked oligosaccharide on IgY.
Abstract: Glycopeptide enrichment is a prerequisite to enable structural characterization of protein glycosylation in glycoproteomics. Here we present an improved method for glycopeptide enrichment based on zwitter-ionic hydrophilic interaction chromatography solid phase extraction (ZIC-HILIC SPE) in a microcolumn format. The method involves TFA ion pairing (IP) to increase the hydrophilicity difference between glycopeptides and nonglycosylated peptides. Three mobile phases were investigated, i.e., 2% formic acid (defined as IP(2% FA) ZIC-HILIC SPE), 0.1% TFA and 1% TFA (defined as IP(0.1% TFA) and IP(1% TFA) ZIC-HILIC SPE) all containing 80% acetonitrile. Samples of increasing complexities, i.e., digests of single glycoproteins, a five-glycoprotein mixture, and depleted plasma, were used in the study. The presence of TFA in the mobile phase significantly improved the glycopeptide enrichment for all complexities, as evaluated by enhanced glycopeptide detection using MALDI-TOF MS and RP-LC-ESI-MS/MS, e.g., the glycopeptide ion signals were increased by up to 3.7-fold compared to IP(2% FA) conditions. The enhanced glycopeptide detection was promoted by a substantial depletion of nonglycosylated peptides, offering an almost complete isolation of IgG glycopeptides using a single SPE enrichment step and a reduction from 711 nonglycosylated peptides observed in the IP(2% FA) ZIC-HILIC SPE retained plasma fraction, to only 157 and 97 when 0.1% and 1% TFA was used in the mobile phase. In conclusion, this systematic study has shown that TFA-containing mobile phases increase glycopeptide enrichment efficiency considerably for a broad range of sample complexities when using ZIC-HILIC SPE.
Abstract: Characterization of low microgram levels of glycoprotein remains a challenge due to extensive heterogeneity of the conjugated N-glycans at each individual glycosylation site. We present an optimized, sensitive workflow for glycopeptide isolation and characterization that exploits the complementary features of RP (Poros R2) and hydrophilic (zwitter-ionic hydrophilic interaction chromatography) chromatographic resins. The glycopeptide analysis workflow was applied to human beta2-glycoprotein I (beta2-GPI, apolipoprotein H), which contains multiple N-glycosylation sites. Conditions for rapid proteolytic digestion of beta2-GPI using low-specificity proteases were optimized to detect beta2-GPI glycopeptides by MS. We demonstrate the importance of ensuring sufficient column capacity of both hydrophobic and hydrophilic stationary phases for optimal glycoprofiling by MS. The enriched glycopeptides were characterized using MALDI quadrupole TOF MS/MS. A total of 23 glycan structures, including sialylated bi- and tri-antennary complex type glycans, were characterized at three N-glycosylation sites, namely Asn-143, Asn-174 and Asn-234, of beta2-GPI. Further exploration of the complementary nature of RP and HILIC stationary phases for glycopeptide isolation prior to MS analysis may eventually enable systematic analysis of complex glycoprotein samples in functional proteomic research and advance our understanding of the biological role of protein glycosylation.
Abstract: TAFI is a plasma protein assumed to be an important link between coagulation and fibrinolysis. The three-dimensional crystal structures of authentic mature bovine TAFI (TAFIa) in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI), and recombinant human TAFI have recently been solved. In light of these recent advances, we have characterized authentic bovine TAFI biochemically and compared it to human TAFI.
Abstract: Site-specific glycoprofiling of N-linked glycopeptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technique, but its quantitative accuracy lacks documentation. Thus, a systematic study of widely different glycopeptides was performed to determine the relationship between the relative abundances of the individual glycoforms and the MALDI-TOF MS signal strength. Glycopeptides derived from glycoproteins containing neutral glycans (ribonuclease B, IgG, and ovalbumin) were initially profiled and yielded excellent and reproducible quantitation (correlation coefficient r = 0.9958, n = 5) when evaluated against a normal phase HPLC 2-AB glycan profile. Similarly, precise quantitation was observed for various forms of N-glycans (free, permethylated, and fluorescence-labeled) using MS. In addition, three different sialoglycopeptides from fetuin were site-specifically profiled, and good correlation between peak intensities and relative abundances was found with only a minor loss of sialic acids (r = 0.9664, n = 5). For glycopeptide purification, a range of hydrophilic and graphite materials packed in microcolumn format proved capable of performing desalting without loss of quantitative information, but highlighted the column capacity as a critical parameter. In conclusion, MALDI-TOF MS signal strength of glycopeptides has been found to accurately reflect the relative quantities of glycoforms, providing that certain technical issues are considered, i.e., nonbiased sample handling, matrix choice, and instrumental settings. This enables rapid and sensitive site-specific glycoprofiling of N-glycan populations to promote biomarker discovery and elucidation of glycan structure/function relationships.
Abstract: The interaction of calreticulin with native and denatured forms and polypeptides in proteolytic digests of proteins representing structural classes of all-alpha-helix (hemoglobin, serum albumin), all-beta-sheet (IgG) and alpha-helix + beta-sheets (lysozyme, ovalbumin) was investigated. The binding of calreticulin to denatured proteins was found to depend on conformation and structural class of the protein. No interaction was observed with the native proteins, whereas binding was seen for the denatured proteins, the order of interaction being lysozyme = IgG > ovalbumin >> hemoglobin = serum albumin. Moreover, the interaction between calreticulin and the heat-denatured proteins depended on the temperature and time used for denaturation and the degree of proteolytic fragmentation. Calreticulin bound well to peptides in proteolytic digests from protease K or chymotrypsin treatment of lysozyme, IgG and ovalbumin but weakly or not at all to peptides in proteolytic digests of hemoglobin and serum albumin. Synthetic peptides from lysozyme and ovalbumin confirmed binding to hydrophobic peptides from these proteins. These results show that calreticulin has the ability to interact with denatured and fragmented forms of proteins with a preference for beta-strand structure and hydrophobicity.
Abstract: Recombinant human interferon-beta (rhIFN-beta) is the leading therapeutic intervention shown to change the cause of relapsing-remitting multiple sclerosis, and both a nonglycosylated and a significantly more active glycosylated variant of rhIFN-beta are used in treatment. This study investigates the function of the rhIFN-beta1a glycan moiety and its individual carbohydrate residues, using the myxovirus resistance (Mx) mRNA as a biomarker in Mx-congenic mice. We showed that the Mx mRNA level in blood leukocytes peaked 3 h after s.c. administration of rhIFN-beta1a. In addition, a clear dose-response relationship was confirmed, and the Mx response was shown to be receptor-mediated. Using specific glycosidases, different glycosylation analogs of rhIFN-beta1a were obtained, and their activities were determined. The glycosylated rhIFN-beta1a showed significantly higher activity than its deglycosylated counterpart, due to a protein stabilization/solubilization effect of the glycan. It is interesting to note that the terminating sialic acids were essential for these effects. Conclusively, the structure/bioactivity relationship of rhIFN-beta1a was determined in vivo, and it provided a novel insight into the role of the rhIFN-beta1a glycan and its carbohydrate residues. The possibilities of improving the pharmacological properties of rhIFN-beta1a using glycoengineering are discussed.
Abstract: Tissue inhibitor of metalloproteinases 1 (TIMP-1) has been identified as a potential biomarker in diseases such as cancer, cardiovascular diseases and diabetes. Since TIMP-1 resides in most tissues and bodily fluids, we evaluated the potential of using saliva to obtain reproducible TIMP-1 measurements in a non-invasive manner.
Abstract: Cancer-induced alterations of protein glycosylations are well-known phenomena. Hence, the glycoprofile of certain glycoproteins can potentially be used as biomarkers for early diagnosis. However, there are a substantial number of candidates and the techniques for measuring their biomarker potential are limited, calling for new methods. Here, we have investigated the cancer marker potential of the glycoprofile of tissue inhibitor of metalloproteinase-1 (TIMP-1) using a method for comparative glycoprofiling. Glycoprofiles were obtained from plasma TIMP-1 of five healthy donors and five colorectal cancer (CRC) patients showing increased amounts of TIMP-1. Furthermore, the TIMP-1 glycoprofiles of media from two colon cancer cell lines (CCC) and a prostate cancer cell line were determined as disease references. TIMP-1 was purified from IgG-depleted samples using immuno affinity and gel electrophoresis and the glycoprofiling was performed using glycopeptide enrichment and mass spectrometry. The heterogeneous glycoprofiles of TIMP-1 were found to be highly conserved among the healthy donors, proving an ideal candidate marker and showed high reproducibility of the method. Numerous CCC-specific TIMP-1 glycans were observed illustrating cancer-induced changes. Unexpectedly, quantitation revealed that the glycoprofiles of healthy donors and CRC patients varied minimally. Considering the increased CRC TIMP-1 levels and the observed CCC-specific glycans, the lack of variation indicates that the increased amount of CRC TIMP-1 is not a direct product of the cancer cells. Hence, the TIMP-1 glycoprofile holds no biomarker potential for CRC when using plasma as the sample origin. This study clearly illustrates that the technique is capable of performing individualised site-specific glycan analysis and representing a new tool for biomarker investigation of low-abundant glycoproteins.
Abstract: A gel-based method for a mass spectrometric site-specific glycoanalysis was developed using a recombinant glycoprotein expressed in two different cell lines. Hydrophilic interaction liquid chromatography at nanoscale level was used to enrich for glycopeptides prior to MS. The glycoprofiling was performed using matrix-assisted laser desorption/ionization MS and MS/MS. The method proved to be fast and sensitive and furthermore yielded a comprehensive site-specific glycan analysis, allowing a differentiation of the glycoprofiles of the two sources of recombinant protein, both comprising N-glycans of a highly heterogeneous nature. To test the potential of the method, tissue inhibitor of metalloproteinases-1 (TIMP-1), a secreted low abundance N-glycosylated protein and a cancer marker, was purified in an individual-specific manner from plasma of five healthy individuals using IgG depletion and immunoaffinity chromatography. The corresponding TIMP-1 glycoprofiles were determined to be highly similar, comprising mainly bi- and triantennary complex oligosaccharides. Additionally it was shown that platelet-derived TIMP-1 displayed a similar glycoprofile. This is the first study to investigate the glycosylation of naturally occurring human TIMP-1, and the high similarity of the glycoprofiles showed that individual-specific glycosylation variations of TIMP-1 are minimal. In addition, the results showed that TIMP-1 derived from platelets and plasma is similarly glycosylated. This comprehensive and rapid glycoprofiling of a low abundance glycoprotein performed in an individual-specific manner allows for future studies of glycosylated biomarkers for person-specific detection of altered glycosylation and may thus allow early detection and monitoring of diseases.
Abstract: The tetanus vaccine is based on the extremely potent tetanus neurotoxin (TeNT), which is converted by treatment with formaldehyde and lysine into the non-toxic, but still immunogenic tetanus toxoid (TTd). This formaldehyde-induced detoxification, which to a large extend determines the quality and properties of the vaccine component, occurs through partly unknown chemical modifications of the toxin. The aim of this study was to gain knowledge of the detoxification mechanism in the generation of the tetanus vaccine. Two approaches were chosen: (i) the effect of changes in the concentrations of lysine and formaldehyde in the detoxification process and (ii) characterisation of the chemically detoxified TTd. (i) We examined a number of TTd components that was produced by varying the concentrations of formaldehyde and lysine during the inactivation. Toxicity tests showed that the detoxification failed when the lysine or formaldehyde concentration was < or =1/5 or < or =1/10, respectively, of the standard level. Gel-electrophoretic analyses showed that inter-chain cross-linking was formaldehyde-dependent and, furthermore, revealed that inter-chain cross-linking was not the only requirement for the inactivation. In addition, the measurable amount of tyrosine correlated inversely with the degree of inter-chain cross-linking. (ii) To study the formaldehyde-induced chemical modifications, the TTd was investigated using protein chemical techniques in combination with mass spectrometry (MS). Using off-line liquid chromatography (LC)-MS, the most pronounced chemical modifications were characterised as unstable Schiff-bases (+12 Da) located on lysine residues and the N-termini of peptides throughout the molecule. Several arginine residues were also found with +12 Da modifications due to Schiff-base formation or as a consequence of degenerative fragmentation of lysine/formaldehyde adducts or cross-links during MS. A few tyrosine residues were similarly observed with a mass increase of 12 Da. Even though it cannot be ruled out that this is a residual mass of higher molecule adducts or cross-links to tyrosine, amino acid analysis and MS data indicated that the modification forms a ring structure from a carbon in the aromatic ring to the backbone N(alpha). In addition, several mono-epsilon-methyllysines (+14 Da) were observed as a likely consequence of reductive methylation of the Schiff-bases. A substantial part (87%) of the known TeNT sequence, including the active site, was covered using the off-line LC-MS approach to investigate the tryptic digested TTd. In contrast to the results obtained from the gel-electrophoretic experiments, neither intra/inter-chain cross-links nor cross-links to external lysines were observed in the MS analysis. Instability of the cross-links during separation and/or MS is likely to explain their absence in the analyses. The biological relevance of the observed modifications is discussed in relation to 3D mapping analyses. Proposals for the TeNT detoxification are discussed, although no direct evidence for the exact mechanism could be obtained.
