Structural Analysis of Variable Domain Glycosylation of Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis Reveals the Presence of Highly Sialylated Glycans

Recently, we showed the unexpectedly high abundance of N-linked glycans on the Fab-domain of Anti-Citrullinated Protein Antibodies (ACPA). As N-linked glycans can mediate a variety of biological functions, we now aimed at investigating the structural composition of the Fab-glycans of ACPA-IgG to better understand their mediated biological effects. ACPA-IgG and noncitrulline specific (control) IgG from plasma and/or synovial fluid of nine ACPA positive rheumatoid arthritis patients were affinity purified. The N-linked glycosylation of total, Fc and F(ab′)₂ fragments, as well as heavy and light chains of ACPA-IgG and control IgG were analyzed by UHPLC and MALDI-TOF mass spectrometry. The Fc-glycosylation of ACPA-IgG and IgG was analyzed at the glycopeptide level using LC-MS. The structural analyses revealed that ACPA-IgG molecules contain highly sialylated glycans in their Fab-domain. Importantly, Fab-glycans were estimated to be present on over 90% of ACPA-IgG, which is five times higher than in control IgG isolated from the same patients. This feature was more prominent on ACPA isolated from synovial fluid compared with peripheral blood. These observations provide the first evidence pointing to the ability of ACPA-IgG to mediate novel immunological activities, for example through binding specific lectins via hyper-sialylated Fab-glycans.     

Development and application of mass spectrometric methods for the analysis of progranulin N-glycosylation

PGRN is a modular protein with 7 1/2 repeats of the granulin domain separated by short spacer sequences. Elevated expression of PGRN is associated with cancer growth, while mutations of PGRN cause frontotemporal lobar degeneration (FTLD), an early onset form of dementia. PGRN is a glycoprotein, containing five N-glycosylation consensus sequons, three of which fall within granulin domains. A method tailored to enable detailed analysis of the PGRN oligosaccharides and glycopeptides has been developed. The approach involves in-gel deglycosylation using peptide-N-glycosidase F (PNGase F) followed by permethylation of the released oligosaccharides. Permethylation was applied for rapid sample clean-up and to improve sensitivity of MS detection and mass spectrometric fragmentation. Reversed-phase monolithic LC–ESI–MS/MS was used for analysis of permethylated oligosaccharides, enabling structural characterization of released N-linked glycans in one chromatographic run. In-gel tryptic digestion was further applied to the gel pieces containing deglycosylated protein, for N-glycosylation site determination. In addition, glycopeptides were produced using in-solution pronase digestion to identify species of N-glycan attached at particular sites. The method developed was applied to progranulin (PGRN) to characterize the structures of the released glycans and to identify the sites of glycosylation. Glycosylation of four out of five potential PGRN N-glycosylation consensus sites was demonstrated (the final one remains undetermined), with one of the four observed to be partially occupied. Two of the observed glycosylation sites occur within granulin domains, which may have important implications for understanding the structural basis of PGRN action.     

Distinct glycosylation in membrane proteins within neonatal versus adult myocardial tissue

Mammalian hearts have regenerative potential restricted to early neonatal stage and lost within seven days after birth. Carbohydrates exclusive to cardiac neonatal tissue may be key regulators of regenerative potential. Although cell surface and extracellular matrix glycosylation are known modulators of tissue and cellular function and development, variation in cardiac glycosylation from neonatal tissue to maturation has not been fully examined.In this study, glycosylation of the adult rat cardiac ventricle showed no variability between the two strains analysed, nor were there any differences between the glycosylation of the right or left ventricle using lectin histochemistry and microarray profiling. However, in the Sprague-Dawley strain, neonatal cardiac glycosylation in the left ventricle differed from adult tissues using mass spectrometric analysis, showing a higher expression of high mannose structures and lower expression of complex N-linked glycans in the three-day-old neonatal tissue. Man₆GlcNAc₂ was identified as the main high mannose N-linked structure that was decreased in adult while higher expression of sialylated N-linked glycans and lower core fucosylation for complex structures were associated with ageing. The occurrence of mucin core type 2 O-linked glycans was reduced in adult and one sulfated core type 2 O-linked structure was identified in neonatal tissue. Interestingly, O-linked glycans from mature tissue contained both N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), while all sialylated N-linked glycans detected contained only Neu5Ac.As glycans are associated with intracellular communication, the specific neonatal structures found may indicate a role for glycosylation in the neonatal associated regenerative capacity of the mammalian heart. New strategies targeting tissue glycosylation could be a key contributor to achieve an effective regeneration of the mammalian heart in pathological scenarios such as myocardial infarction.     

Four unreported types of glycans containing mannose-6-phosphate are heterogeneously attached at three sites (including newly found Asn 233) to recombinant human acid alpha-glucosidase that is the only approved treatment for Pompe disease

Myozyme is a recombinant human acid alpha-glucosidase (rhGAA) that is currently the only drug approved for treating Pompe disease, and its low efficacy means that a high dose is required. Mannose-6-phosphate (M6P) glycosylation on rhGAA is a key factor influencing lysosomal enzyme targeting and the efficacy of enzyme replacement therapy (ERT); however, its complex structure and relatively small quantity still remain to be characterized. This study investigated M6P glycosylation on rhGAA using liquid chromatography (LC)–electrospray ionization (ESI)–high-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS). The glycans released from rhGAA were labeled with procainamide to improve mass ionization efficiency and the sensitivity of MS/MS. The relative quantities (%) of 78 glycans were obtained, and 1.0% of them were glycans containing M6P (M6P glycans). These were categorized according to their structure into 4 types: 3 newly found ones, comprising high-mannose-type M6P glycans capped with N-acetylglucosamine (GlcNAc) (2 variants, 17.5%), hybrid-type M6P glycans (2 variants, 11.2%), and hybrid-type M6P glycans capped with GlcNAc (3 variants, 6.9%), as well as high-mannose-type M6P glycans (3 variants, 64.4%). HCD-MS/MS spectra identified six distinctive M6P-derived oxonium ions. The glycopeptides obtained from protease-digested rhGAA were analyzed using nano-LC-ESI-HCD-MS/MS, and the extracted-ion chromatograms of M6P-derived oxonium ions confirmed three M6P glycosylation sites comprising Asn 140, Asn 233 (newly found), and Asn 470 attached heterogeneously to nine M6P glycans (two types), eight M6P glycans (four types), and seven M6P glycans (two types), respectively. This is the first study of rhGAA to differentiate M6P glycans and identify their attachment sites, despite rhGAA already being an approved drug for Pompe disease.     

Nonreductive chemical release of intact N-glycans for subsequent labeling and analysis by mass spectrometry

A novel strategy is proposed, using cost-saving chemical reactions to generate intact free reducing N-glycans and their fluorescent derivatives from glycoproteins for subsequent analysis. N-Glycans without core α-1,3-linked fucose are released in reducing form by selective hydrolysis of the N-type carbohydrate–peptide bond of glycoproteins under a set of optimized mild alkaline conditions and are comparable to those released by commonly used peptide-N-glycosidase (PNGase) F in terms of yield without any detectable side reaction (peeling or deacetylation). The obtained reducing glycans can be routinely derivatized with 2-aminobenzoic acid (2-AA), 1-phenyl-3-methyl-5-pyrazolone (PMP), and potentially some other fluorescent reagents for comprehensive analysis. Alternatively, the core α-1,3-fucosylated N-glycans are released in mild alkaline medium and derivatized with PMP in situ, and their yields are comparable to those obtained using commonly used PNGase A without conspicuous peeling reaction or any detectable deacetylation. Using this new technique, the N-glycans of a series of purified glycoproteins and complex biological samples were successfully released and analyzed by electrospray ionization mass spectrometry (ESI–MS) and tandem mass spectrometry (MS/MS), demonstrating its general applicability to glycomic studies.     

Functional role of N-glycosylation from ADAM10 in processing,localization and activity of the enzyme

A disintegrin and metalloprotease 10 (ADAM10) is a type I transmembrane glycoprotein with four potential N-glycosylation sites (N267, N278, N439 and N551), that cleaves several plasma membrane proteins. In this work, ADAM10 was found to contain high-mannose and complex-type glycans. Individual N-glycosylation site mutants S269A, T280A, S441A, T553A were constructed, and results indicated that all sites were occupied. T280A was found to accumulate in the endoplasmic reticulum as the non-processed precursor of the enzyme. Furthermore, it exhibited only residual levels of metalloprotease activity in vivo towards the L1 cell adhesion molecule, as well as in vitro, using a ProTNF-alpha peptide as substrate. S441A showed increased ADAM10 susceptibility to proteolysis. Mutation of N267, N439 and N551 did not completely abolish enzyme activity, however, reduced levels were found. ADAM10 is sorted into secretory vesicles, the exosomes. Here, a fraction of ADAM10 from exosomes was found to contain more processed N-linked glycans than the cellular enzyme. In conclusion, N-glycosylation is crucial for ADAM10 processing and resistance to proteolysis, and results suggest that it is required for full-enzyme activity.     

Advances in LC–MS/MS-based glycoproteomics: Getting closer to system-wide site-specific mapping of the N- and O-glycoproteome

Site-specific structural characterization of glycoproteins is important for understanding the exact functional relevance of protein glycosylation. Resulting partly from the multiple layers of structural complexity of the attached glycans, the system-wide site-specific characterization of protein glycosylation, defined as glycoproteomics, is still far from trivial leaving the N- and O-linked glycoproteomes significantly under-defined. However, recent years have seen significant advances in glycoproteomics driven, in part, by the developments of dedicated workflows and efficient sample preparation, including glycopeptide enrichment and prefractionation. In addition, glycoproteomics has benefitted from the continuous performance enhancement and more intelligent use of liquid chromatography and tandem mass spectrometry (LC–MS/MS) instrumentation and a wider selection of specialized software tackling the unique challenges of glycoproteomics data. Together these advances promise more streamlined N- and O-linked glycoproteome analysis. Tangible examples include system-wide glycoproteomics studies detecting thousands of intact glycopeptides from hundreds of glycoproteins from diverse biological samples. With a strict focus on the system-wide site-specific analysis of protein N- and O-linked glycosylation, we review the recent advances in LC–MS/MS based glycoproteomics. The review opens with a more general discussion of experimental designs in glycoproteomics and sample preparation prior to LC–MS/MS based data acquisition. Although many challenges still remain, it becomes clear that glycoproteomics, one of the last frontiers in proteomics, is gradually maturing enabling a wider spectrum of researchers to access this new emerging research discipline. The next milestone in analytical glycobiology is being reached allowing the glycoscientist to address the functional importance of protein glycosylation in a system-wide yet protein-specific manner.     

The identification of trimethylsilylated dipeptides with chemical ionization mass spectrometry.

The chemical ionization (CI) and electron impact (EI) mass spectra were compared for over 40 trimethylsilylated (Me₃Si) dipeptides. The dipeptides chosen had all 20 common amino acids represented at amino and carboxyl positions. The CI mass spectra of Me₃Si dipeptides typically contain three ions of high abundance used for dipeptide identification: a sequence-determining ion and two molecular weight-determining ions. The intensity of the molecular weight-determining ions relative to that of the ion that characterizes the N-terminal residue (β-cleavage ion) is greater in the CI mode than in the EI mode. Because the available intensity of the β-cleavage ion is similar in both modes, use of the CI mode will extend the lower limit for Me₃Si dipeptide identification.     

Glycosyl part identified within Balanites aegyptiaca fruit protease

The many milk-clotting proteases from plant are glycosylated; attachment of monosaccharides to enzyme is an advantage for its activity and stability. In this study, gas chromatography coupled to mass spectrometry-electrospray ionization was used to identify glycans bond to proteases purified from Balanites aegyptiaca fruits pulp through cation exchange chromatography. Carbohydrates were identified according to the retention time and the ion at m/z after derivation by heptafluorobutyric acid. The chromatograms obtained from monosaccharides analysis revealed the presence of galactose, mannose, arabinose, xylose, rhamnose and glucuronic acid. The mass spectrometry-electrospray ionization spectra corroborated these findings.     

Galectin-3 Interactions with Glycosphingolipids

Galectins have essential roles in pathological states including cancer, inflammation, angiogenesis and microbial infections. Endogenous receptors include members of the lacto- and neolacto-series glycosphingolipids present on mammalian cells and contain the tetrasaccharides lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) that form their core structural components and also ganglio-series glycosphingolipids. We present crystallographic structures of the carbohydrate recognition domain of human galectin-3, both wild type and a mutant (K176L) that influenced ligand affinity, in complex with LNT, LNnT and acetamido ganglioside a-G_M3 (α2,3-sialyllactose). Key structural features revealed include galectin-3's demonstration of a binding mode towards gangliosides distinct from that to the lacto/neolacto-glycosphingolipids, with its capacity for recognising the core β-galactoside region being challenged when the core oligosaccharide epitope of ganglio-series glycosphingolipids (G_M3) is embedded within particular higher-molecular-weight glycans. The lacto- and neolacto- glycosphingolipids revealed different orientations of their terminal galactose in the galectin-3-bound LNT and LNnT structures that has significant ramifications for the capacity of galectin-3 to interact with higher-order lacto/neolacto-series glycosphingolipids such as ABH blood group antigens and the HNK-1 antigen that is common on leukocytes. LNnT also presents an important model for poly-N-acetyllactosamine-containing glycans and provides insight into galectin-3's accommodation of extended oligosaccharides such as the poly-N-acetyllactosamine-modified N- and O-glycans that, via galectin-3 interaction, facilitate progression of lung and bladder cancers, respectively. These findings provide the first atomic detail of galectin-3's interactions with the core structures of mammalian glycosphingolipids, providing information important in understanding the capacity of galectin-3 to engage with receptors identified as facilitators of major disease.     

Engineering of Sialylated Mucin-type O-Glycosylation in Plants

Proper N- and O-glycosylation of recombinant proteins is important for their biological function. Although the N-glycan processing pathway of different expression hosts has been successfully modified in the past, comparatively little attention has been paid to the generation of customized O-linked glycans. Plants are attractive hosts for engineering of O-glycosylation steps, as they contain no endogenous glycosyltransferases that perform mammalian-type Ser/Thr glycosylation and could interfere with the production of defined O-glycans. Here, we produced mucin-type O-GalNAc and core 1 O-linked glycan structures on recombinant human erythropoietin fused to an IgG heavy chain fragment (EPO-Fc) by transient expression in Nicotiana benthamiana plants. Furthermore, for the generation of sialylated core 1 structures constructs encoding human polypeptide:N-acetylgalactosaminyltransferase 2, Drosophila melanogaster core 1 β1,3-galactosyltransferase, human α2,3-sialyltransferase, and Mus musculus α2,6-sialyltransferase were transiently co-expressed in N. benthamiana together with EPO-Fc and the machinery for sialylation of N-glycans. The formation of significant amounts of mono- and disialylated O-linked glycans was confirmed by liquid chromatography-electrospray ionization-mass spectrometry. Analysis of the three EPO glycopeptides carrying N-glycans revealed the presence of biantennary structures with terminal sialic acid residues. Our data demonstrate that N. benthamiana plants are amenable to engineering of the O-glycosylation pathway and can produce well defined human-type O- and N-linked glycans on recombinant therapeutics.     

Structural determination of N-linked glycans by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

This paper reviews methods for the analysis of N-linked glycans by mass spectrometry with emphasis on studies conducted at the Oxford Glycobiology Institute. Topics covered are the release of glycans from sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels, their purification for analysis by mass spectrometry, methods based on matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization for producing fragment ions, and details of their fragmentation. MALDI mass spectrometry provided a rapid method for profiling neutral N-linked glycans as their [M + Na](+) ions which could be fragmented by collision-induced decomposition to give spectra containing both glycosidic and cross-ring fragments. Electrospray ionization mass spectrometry was more versatile in that it was relatively easy to change the type of ion that was formed and, furthermore, unlike MALDI, electrospray did not cause extensive loss of sialic acids from sialylated glycans. Negative ions formed by addition of anions such as chloride and, particularly, nitrate, to the electrospray solvent were stable and enabled singly charged ions to be obtained from larger glycans than was possible in positive ion mode. Fragmentation of negative ions followed specific pathways that defined structural details of the glycans that were difficult to obtain by classical methods such as exoglycosidase digestion.     

Amino acid sequencing by gas chromatography-mass spectrometry using perfluoro-dideuteroalkylated peptide derivatives: B. Interpretation of the mass spectra

The mass spectra of the O-trimethylsilylated trifluoro-dideuteroethyl polyamino alcohols, produced by LiAlD₄-reduction and O-trimethylsilylation of N-trifluororacetyl oligopeptide methyl esters, are evaluated. Characteristic mass spectra of derivatives are shown which are derived from peptides containing all protein amino acids including Arg, His, Trp, Gln, Asn and carboxyl terminal amides as well as modified Cys-residues. The mass spectra of these derivatives can be easily interpreted in terms of the amino acid sequence of the original peptides since they contain abundant and intensity-balanced sequence-determining ions.     

Hemocytes and Plasma of the Eastern Oyster (Crassostrea virginica) Display a Diverse Repertoire of Sulfated and Blood Group A-modified N-Glycans

The eastern oyster (Crassostrea virginica) has become a useful model system for glycan-dependent host-parasite interactions due to the hijacking of the oyster galectin CvGal1 for host entry by the protozoan parasite Perkinsus marinus, the causative agent of Dermo disease. In this study, we examined the N-glycans of both the hemocytes, which via CvGal1 are the target of the parasite, and the plasma of the oyster. In combination with HPLC fractionation, exoglycosidase digestion, and fragmentation of the glycans, mass spectrometry revealed that the major N-glycans of plasma are simple hybrid structures, sometimes methylated and core α1,6-fucosylated, with terminal β1,3-linked galactose; a remarkable high degree of sulfation of such glycans was observed. Hemocytes express a larger range of glycans, including core-difucosylated paucimannosidic forms, whereas bi- and triantennary glycans were found in both sources, including structures carrying sulfated and methylated variants of the histo-blood group A epitope. The primary features of the oyster whole hemocyte N-glycome were also found in dominin, the major plasma glycoprotein, which had also been identified as a CvGal1 glycoprotein ligand associated with hemocytes. The occurrence of terminal blood group moieties on oyster dominin and on hemocyte surfaces can account in part for their affinity for the endogenous CvGal1.     

Quantification of urinary S- and N-homocysteinylated protein and homocysteine-thiolactone in mice

Homocysteine (Hcy) and its metabolites Hcy-thiolactone, N-Hcy-protein, and S-Hcy-protein are implicated in vascular and neurological diseases. However, quantification of these metabolites remains challenging. Here I describe streamlined assays for these metabolites based on their conversion to Hcy-thiolactone. Free Hcy-thiolactone is extracted from the urine with chloroform/methanol. Total Hcy is converted to Hcy-thiolactone in the presence of 1 N HCl. Major urinary protein (MUP)-bound S-linked Hcy is liberated from the protein by reduction with dithiothreitol and converted to Hcy-thiolactone. Acid hydrolysis of MUP with 6 N HCl liberates N-linked Hcy as Hcy-thiolactone, which is then extracted with chloroform/methanol. Ferritin is used as an N-Hcy-protein standard and an authentic Hcy-thiolactone is used to monitor the efficiency of extraction. Hcy-thiolactone (free, derived from total Hcy, or from MUP-bound N-linked or S-linked Hcy) is separated by a cation exchange high-performance liquid chromatography, post-column derivatized with o-phthaldialdehyde, and quantified by fluorescence. Using these assays with as little as 2–20 μL of urine I show that MUP carry N-linked and S-linked Hcy and that N-Hcy-MUP and S-Hcy-MUP and Hcy-thiolactone are severely elevated in cystathionine β-synthase-deficient mice. These assays will facilitate examination of the role of protein-related Hcy metabolites in health and disease.     

Non-lysosomal degradation pathway for N-linked glycans and dolichol-linked oligosaccharides

There is growing evidence that asparagine (N)-linked glycans play pivotal roles in protein folding and intra- or intercellular trafficking of N-glycosylated proteins. During the N-glycosylation of proteins, significant amounts of free oligosaccharides (fOSs) and phosphorylated oligosaccharides (POSs) are generated at the endoplasmic reticulum (ER) membrane by unclarified mechanisms. fOSs are also formed in the cytosol by the enzymatic deglycosylation of misfolded glycoproteins destined for proteasomal degradation. This article summarizes the current knowledge of the molecular and regulatory mechanisms underlying the formation of fOSs and POSs in mammalian cells and Saccharomyces cerevisiae.     

Dual-gradient high-performance liquid chromatography for identification of cytosolic high-mannose-type free glycans

It has been shown that free oligosaccharides derived from N-linked glycans accumulate in the cytosol of animal cells. Most of the glycans have only a single GlcNAc at their reducing termini (Gn1 glycans), whereas the original N-glycans retain N,N′-diacetylchitobiose at their reducing termini (Gn2 glycans). Under the conditions of high-performance liquid chromatography (HPLC) mapping established for pyridylamine (PA)-labeled Gn2 N-glycans, Gn1 glycans are not well retained on reversed-phase HPLC, making simultaneous analysis of Gn1 and Gn2 glycans problematic. We introduced a dual gradient (i.e., pH and butanol gradient) for the separation of Gn1 and Gn2 glycans in a single reversed-phase HPLC. Determination of elution time for various standard Gn2 high-mannose-type glycans, as well as Gn1 glycans found in the cytosol of animal cells, showed that elution of Gn1 and Gn2 glycans could be separated. Sufficient separation for most of the structural isomers could be achieved for Gn1 and Gn2 glycans. This HPLC, therefore, is a powerful method for identification of the structures of PA-labeled glycans, especially Gn1-type glycans, isolated from the cytosol of animal cells.     

Immunosilencing a Highly Immunogenic Protein Trimerization Domain

Many therapeutic proteins and protein subunit vaccines contain heterologous trimerization domains, such as the widely used GCN4-based isoleucine zipper (IZ) and the T4 bacteriophage fibritin foldon (Fd) trimerization domains. We found that these domains induced potent anti-IZ or anti-Fd antibody responses in animals when fused to an HIV-1 envelope glycoprotein (Env) immunogen. To dampen IZ-induced responses, we constructed an IZ domain containing four N-linked glycans (IZN4) to shield the underlying protein surface. When fused to two different vaccine antigens, HIV-1 Env and influenza hemagglutinin (HA), IZN4 strongly reduced the antibody responses against the IZ, but did not affect the antibody titers against Env or HA. Silencing of immunogenic multimerization domains with glycans might be relevant for therapeutic proteins and protein vaccines.     

N-Glycosylation pattern of recombinant human CD82 (KAI1), a tumor-associated membrane protein

The membrane glycoprotein CD82 (KAI1) has attracted increasing attention as a suppressor of cell migration, related tumor invasion, as well as metastasis. The glycosylation of CD82 has been shown to be involved in a correlative cell adhesion and motility. However, the N-glycosylation pattern of CD82 has not been described yet. In the current study, a detailed characterization of the recombinant human CD82 N-linked glycosylation pattern was conducted by employing an integrative proteomic and glycomic approach, including glycosidase and protease digestions, glycan permethylation, MS analyses, site-directed mutagenesis, and lectin blots. The results reveal three N-glycosylation sites, and further demonstrate a putative glycosylation site at Asn₁₅₇ for the first time. A highly heterogeneous pattern of N-linked glycans is described, which express distinct carbohydrate epitopes, such as bisecting N-acetylglucosamine, (α-2,6) N-acetylneuraminic acid, and core fucose. These epitopes are highly associated with various biological functions, including cell adhesion and cancer metastasis, and can possibly influence the anti-cancer inhibition ability of CD82.