This review summarizes the analytical advances made during the last several years in the structural and quantitative determinations of glycoproteins in complex biological mixtures. The main analytical techniques used in the fields of glycomics and glycoproteomics involve different modes of mass spectrometry and their combinations with capillary separation methods such as microcolumn liquid chromatography and capillary electrophoresis. The needs for high-sensitivity measurements have been emphasized in the oligosaccharide profiling used in the field of biomarker discovery through MALDI mass spectrometry. High-sensitivity profiling of both glycans and glycopeptides from biological fluids and tissue extracts has been aided significantly through lectin preconcentration and the uses of affinity chromatography.
biomolecular mass spectrometry; glycoproteomics; glycomics; affinity chromatography; lectin enrichment; permethylation; sialic acid linkage analysis; capillary electrophoresis
The past 25 years have seen significant advances in understanding the diversity and functions of glycoprotein glycans in Drosophila melanogaster. Genetic screens have captured mutations that reveal important biological activities modulated by glycans, including protein folding and trafficking, as well as cell signaling, tissue morphogenesis, fertility, and viability. Many of these glycan functions have parallels in vertebrate development and disease, providing increasing opportunities to dissect pathologic mechanisms using Drosophila genetics. Advances in the sensitivity of structural analytic techniques have allowed the glycan profiles of wild-type and mutant tissues to be assessed, revealing novel glycan structures that may be functionally analogous to vertebrate glycans. This review describes a selected set of recent advances in understanding the functions of N-linked and O-linked (non-glycosaminoglycan) glycoprotein glycans in Drosophila with emphasis on their relatedness to vertebrate organisms.
Drosophila; glycosylation; N-linked; O-linked
N -acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) is the enzyme that removes sulfate groups from the N-acetylgalactosamine-4-sulfate residue at the non-reducing end of chondroitin-4-sulfate (C4S) and dermatan sulfate (DS). Previous studies demonstrated reduction in cell-bound high molecular weight kininogen in normal rat kidney (NRK) epithelial cells when chondroitin-4-sulfate content was reduced following overexpression of ARSB activity, and chondroitinase ABC produced similar decline in cell-bound kininogen. Reduction in the cell-bound kininogen was associated with increase in secreted bradykinin. In this report, we extend the in vitro findings to in vivo models, and present findings in Dahl salt-sensitive (SS) rats exposed to high (SSH) and low salt (SSL) diets. In the renal tissue of the SSH rats, ARSB activity was significantly less than in the SSL rats, and chondroitin-4-sulfate and total sulfated glycosaminoglycan content were significantly greater. Disaccharide analysis confirmed marked increase in C4S disaccharides in the renal tissue of the SSH rats. In contrast, unsulfated, hyaluronan-derived disaccharides were increased in the rats on the low salt diet. In the SSH rats, with lower ARSB activity and higher C4S levels, cell-bound, high-molecular weight kininogen was greater and urinary bradykinin was lower. ARSB activity in renal tissue and NRK cells declined when exogenous chloride concentration was increased in vitro. The impact of high chloride exposure in vivo on ARSB, chondroitin-4-sulfation, and C4S-kininogen binding provides a mechanism that links dietary salt intake with bradykinin secretion and may be a factor in blood pressure regulation.
Bradykinin; Chondroitin; Disaccharide; Kininogen; Sulfatase; Sulfate
The bisecting GlcNAc is transferred to the core mannose residue of complex or hybrid N-glycans on glycoproteins by the β1,4-N-acetylglucosaminyltransferase III (GlcNAcT-III) or MGAT3. The addition of the bisecting GlcNAc confers unique lectin recognition properties to N-glycans. Thus, LEC10 gain-of-function Chinese hamster ovary (CHO) cells selected for the acquisition of ricin resistance, carry N-glycans with a bisecting GlcNAc, which enhances the binding of the erythroagglutinin E-PHA, but reduces the binding of ricin and galectins-1, -3 and -8. The altered interaction with galactose-binding lectins suggests that the bisecting GlcNAc affects N-glycan conformation. LEC10 mutants expressing polyoma middle T antigen (PyMT) exhibit reduced growth factor signaling. Furthermore, PyMT-induced mammary tumors lacking MGAT3, progress more rapidly than tumors with the bisecting GlcNAc on N-glycans of cell surface glycoproteins. In recent years, evidence for a new paradigm of cell growth control has emerged involving regulation of cell surface residency of growth factor and cytokine receptors via interactions and cross-linking of their branched N-glycans with a lattice of galectin(s). Specific cross-linking of glycoprotein receptors in the lattice regulates their endocytosis, leading to effects on growth factor-induced signaling. This review will describe evidence that the bisecting GlcNAc of N-glycans regulates cellular signaling and tumor progression, apparently through modulating N-glycan/galectin interactions.
Glycosylation; Bisecting GlcNAc; Complex N-glycans; Galectins; Mgat3
A group of fluorescent statistical glycopolymers, prepared via reversible addition–fragmentation chain-transfer (RAFT)-based polymerizations, were successfully employed in lectin-mediated bacterial binding studies. The resultant glycopolymers contained three different monomers: N-(2-hydroxyethyl) acrylamide (HEAA), N-(2-aminoethyl) methacrylamide (AEMA) and N-(2-glyconamidoethyl)-methacrylamides possessing different pendant sugars. Low dispersities (≤1.32) and predictable degrees of polymerization were observed among the products. After the polymerization, the glycopolymers were further modified by different succinimidyl ester fluorophores targeting the primary amine groups on AEMA. With their binding specificities being confirmed by testing with lectin coated agarose beads, the glycopolymers were employed in bacterial binding studies, where polymers containing α-galactose or β-galactose as the pendant sugar were specifically bound by two clinically important pathogens Pseudomonas aeruginosa and Staphylococcus aureus, respectively. This is the first report of using RAFT-based glycopolymers in bacterial binding studies, and the ready access to tri-component statistical glycopolymers also warrants further exploration of their utility in other glycobiological applications.
Electronic supplementary material
The online version of this article (doi:10.1007/s10719-013-9508-4) contains supplementary material, which is available to authorized users.
Glycopolymer; RAFT; Lectin; Bacterial binding
Our study compares the status of human seminal plasma immunoglobulin G (IgG) and IgA secretory component (SC) fucosylation between infertile leukocytospermic and normal, fertile normozoospermic patients. The seminal IgG and SC are decorated with AAL-reactive core fucose, and antennary UEA- and LTA-reactive fucose of Lewisy and Lewisx structures, respectively. However, a correlation between IgG core fucosylation and IgG concentration (r = −0.52; p < 0.0003) was observed. The IgG present in leukocytospermic samples is characterized by lower expression of core fucose than in the normal group (0.82 ± 0.3 AU and 1.2 ± 0.3 AU, respectively; p < 0.002). In seminal plasma the SC is present in two forms: 78-kDa and 63-kDa. The present study has also shown a higher AAL and LTA specific reactivity of glycans expressed in 63-kDa SC, in comparison to 78-kDa SC, in the normal group. In leukocytospermia, the values of specific lectin reactivity for core fucose, fucose α(1-2)- and α(1-3)- linked, were similar for both SC bands. Moreover, the present study has shown that in leukocytospermic samples the mean concentrations of IgG and S-IgA are twice as high (131.68 ± 102.6 mg/l and 36 ± 27 mg/l, respectively) as in the normal group (67.68 ± 29.2 mg/l; p < 0.02, and 19 ± 18 mg/l, p < 0.019, respectively). The analysis of IgG and SC fucosylation status and the determination of IgG and S-IgA concentrations in seminal plasma might constitute a valuable diagnosis tools for the evaluation of male infertility associated with leukocytospermia with accompanying inflammation.
Immunoglobulin G; Secretory component; Fucosylation; Leukocytospermia; Male infertility
UDP-GalNAc:polypeptide GalNAc transferase (ppGalNAcT; EC 18.104.22.168) catalyzes the first step in mucin-type O-glycosylation. To date, several members of this large enzyme family have been analyzed in detail. In this study we present cloning, expression and characterization of the first representative of this type of glycosyltransferase from mollusk origin, namely from Biomphalaria glabrata. The full length sequence of the respective gene was obtained by screening of a cDNA library using homology-based PCR. The entire gene codes for a protein consisting of 600 amino acids comprising the features of a typical type II membrane protein containing a cytoplasmic tail at the N-terminus, a transmembrane and a catalytic domain as well as a ricin-like motif at the C-terminus. Sequence comparison with ppGalNAcTs from various species revealed high similarities in terms of structural architecture. The enzyme is O-glycosylated but does not have any putative N-glycosylation sites. All four tested acceptor peptides were functional substrates, with Muc2 being the best one. Further biochemical parameters tested, confirmed a close relationship to the family of yet known ppGalNAcTs.
ppGalNAcT; GalNAc-transferase; O-glycosylation; Snail; O-glycoprotein; Biomphalaria glabrata
Zebrafish (Danio rerio) remains a versatile model organism for the investigation of early development and organogenesis, and has emerged as a valuable platform for drug discovery and toxicity evaluation [1–6]. Harnessing the genetic power and experimental accessibility of this system, three decades of research have identified key genes and pathways that control the development of multiple organ systems and tissues, including the heart, kidney, and craniofacial cartilage, as well as the hematopoietic, vascular, and central and peripheral nervous systems [7–31]. In addition to their application in large mutagenic screens, zebrafish has been used to model a variety of diseases such as diabetes, polycystic kidney disease, muscular dystrophy and cancer [32–36]. As this work continues to intersect with cellular pathways and processes such as lipid metabolism, glycosylation and vesicle trafficking, investigators are often faced with the challenge of determining the degree to which these pathways are functionally conserved in zebrafish. While they share a high degree of genetic homology with mouse and human, the manner in which cellular pathways are regulated in zebrafish during early development, and the differences in the organ physiology, warrant consideration before functional studies can be effectively interpreted and compared with other vertebrate systems. This point is particularly relevant for glycosylation since an understanding of the glycan diversity and the mechanisms that control glycan biosynthesis during zebrafish embryogenesis (as in many organisms) is still developing.
Nonetheless, a growing number of studies in zebrafish have begun to cast light on the functional roles of specific classes of glycans during organ and tissue development. While many of the initial efforts involved characterizing identified mutants in a number of glycosylation pathways, the use of reverse genetic approaches to directly model glycosylation-related disorders is now increasingly popular. In this review, the glycomics of zebrafish and the developmental expression of their glycans will be briefly summarized along with recent chemical biology approaches to visualize certain classes of glycans within developing embryos. Work regarding the role of protein-bound glycans and glycosaminoglycans (GAG) in zebrafish development and organogenesis will also be highlighted. Lastly, future opportunities and challenges in the expanding field of zebrafish glycobiology are discussed.
Zebrafish; Glycosylation; Development; Sialylation; Glycosaminoglycans; N-glycans; Mucins; Cartilage
Trypanosoma cruzi, an intracellular protozoan etiologic agent of Chagas disease is covered by a dense coat of mucin-type glycoproteins, which is important to promote the parasite entry and persistence in the mammalian host cells. The O-glycosylation of T. cruzi mucins (Tc-mucins) is initiated by enzymatic addition of α-O-N-acetylglucosamine (GlcNAc) to threonine (Thr) by the UDP-GlcNAc:polypeptide α-N-acetylglucosaminyltransferase (pp-α-GlcNAcT) in the Golgi. The Tc-mucin is characterized by the presence of a high structural diversity of O-linked oligosaccharides found among different parasite strains, comprising two O-glycan Cores. In the Core 1, from strains principally associated with the domestic transmission cycle of Chagas disease, the GlcNAc O-4 is substituted with a β-galactopyranose (βGalp) unit, and in the most complex oligosaccharides the GlcNAc O-6 is further processed by the addition of β1 → 2-linked Galp residues creating a short linear Galp-containing chain. In the Core 2 structures, expressed by strains isolated from T. cruzi sylvatic hosts, the GlcNAc O-4 carries a β-galactofuranose (βGalf) unit and the GlcNAc O-6 can carry a branched Galpβ1 → 3[Galpβ1 → 2]Galpβ1 → 6 motif. The O-glycans carrying nonreducing terminal βGalp are available for sialylation by a surface T. cruzi trans-sialidase activity. Based on structural results, this review summarizes available data on the highly conserved process, which adds the GlcNAc unit in α-linkage to Thr residues the basis of the post-translational modification system in T. cruzi mucins. In addition, a mechanism unique employed by the parasite to transfer exogenous sialic acid residues to Tc-mucins is presented.
Trypanosoma cruzi; Posttranslational modification; Mucins; pp-α-GlcNAcT; trans-sialidase
Human β-1,4-galactosyltransferase (β-1,4-GalT) V was shown to be involved in the biosynthesis of N-glycans, O-glycans and lactosylceramide (Lac-Cer) by in vitro studies. To determine its substrate specificity, enzymatic activity and its products were analyzed using mouse embryonic fibroblast (MEF) cells from β-1,4-GalT V (B4galt5)-mutant mice. Analysis of expression levels of the β-1,4-GalT I-VI genes revealed that the expression of the β-1,4-GalT V gene in B4galt5+/−- and B4galt5−/−-derived MEF cells are a half and null when compared to that of B4galt5+/+-derived MEF cells without altering the expression levels of other β-1,4-GalT genes. These MEF cells showed no apparent difference in their growth. When β-1,4-GalT activities were determined towards GlcNAcβ-S-pNP, no significant difference in its specific activity was obtained among B4galt5+/+-, B4galt5+/−- and B4galt5−/−-derived MEF cells. No significant differences were obtained in structures and amounts of N-glycans and lectin bindings to membrane glycoproteins among B4galt5+/+-, B4galt5+/−- and B4galt5−/−-derived MEF cells. However, when cell homogenates were incubated with glucosylcer-amide in the presence of UDP-[3H]Gal, Lac-Cer synthase activity in B4galt5+/−- and B4galt5−/−-derived MEF cells decreased to 41% and 11% of that of B4galt5+/+-derived MEF cells. Consistent with this, amounts of Lac-Cer and its derivative GM3 in B4galt5−/−-derived MEF cells decreased remarkably when compared with those of B4galt5+/+ derived MEF cells. These results indicate that murine β-1,4-GalT V is involved in Lac-Cer biosynthesis.
B4galt5−/− mice; MEF cells; β-1; 4-GalT V; Lactosylceramide
Advances in the glycobiology and immunology fields have provided many insights into the role of carbohydrate-protein interactions in the immune system. We aim to present a comprehensive review of the effects that some plant lectins exert as immunomodulatory agents, showing that they are able to positively modify the immune response to certain pathological conditions, such as cancer and infections. The present review comprises four main themes: (1) an overview of plant lectins that exert immunomodulatory effects and the mechanisms accounting for these activities; (2) general characteristics of the immunomodulatory lectin ArtinM from the seeds of Artocarpus heterophyllus; (3) activation of innate immunity cells by ArtinM and consequent induction of Th1 immunity; (4) resistance conferred by ArtinM administration in infections with intracellular pathogens, such as Leishmania (Leishmania) major, Leishmania (Leishmania) amazonensis, and Paracoccidioides brasiliensis. We believe that this review will be a valuable resource for more studies in this relatively neglected area of research, which has the potential to reveal carbohydrate targets for novel prophylactic and therapeutic strategies.
Plant lectins; ArtinM lectin; Immunomodulation; Toll-like receptor; Leishmania; Paracoccidioides brasiliensis
The sugar moiety of IgA is known to provide a link between the innate and adaptive immune systems. Terminally located glycotopes on IgA are potential ligands engaged in the interactions which may modulate the biological activities of IgA. In the present work the expressions of Maackia amurensis (MAA), Sambucus nigra (SNA), Lens culinaris (LCA), Tetragonolobus purpureus (LTA), and Ulex europaeus (UEA) reactive glycotopes on maternal plasma and amniotic IgA were evaluated in relation to the progression of a normal human pregnancy, from the 2nd trimester, throughout the 3rd trimester, perinatal period, post-date pregnancy and delivery, by lectin-IgA-ELISA, using specific biotinylated lectins. The amniotic and maternal plasma IgA concentrations and a degree of SNA and LCA reactivity of maternal plasma IgA were almost unaltered during the normal pregnancy. The amniotic IgA from the 2nd trimester was decorated by MAA-, SNA-reactive and LCA-, LTA-, and UEA-reactive glycotopes. At the turn of the 2nd and 3rd trimesters the expression of MAA-, SNA-, LTA-, and UEA-reactive glycotopes, except for LCA-reactive, increased and remained almost at unaltered levels throughout the perinatal period and delivery. However, in the post-date pregnancy the expression of LCA-, LTA-, and UEA-reactive and SNA-reactive glycotopes were significantly higher. The unique fucosylated and sialylated glycovariants of amniotic IgA associated with the progression of the normal pregnancy may illustrate a general importance of carbohydrate-lectin receptor interactions in the control and modulation of biological events to ensuring homeostasis during pregnancy, protection and well-being of fetus.
Amniotic fluid; Fucosylation; Glycovariant; Glycotope; Immunoglobulin A; Lectin; Pregnancy; Sialylation
Cytotoxic CD8+ T cells are major players of anti-tumor immune responses, as their functional activity can limit tumor growth and progression. Data show that cytotoxic T cells efficiently control the proliferation of tumor cells through major histocompatibility complex class I-mediated mechanisms; nevertheless, the presence of tumor-infiltrating CD8+ T cells in lesional tissue does not always correlate with better prognosis and increased survival of cancer patients. Similarly, adoptive transfer of tumor-specific cytotoxic T cells has only shown marginal improvement in life spans of patients with metastatic disease. In this report, we discuss experimental evidence showing that expression of tumor-derived galectins, galectin (Gal)-1, Gal-3 and Gal-9, and concomitant presence of their ligands on the surface of anti-tumor immunocytes directly compromise anti-tumor CD8+ T cell immune responses and, perhaps, undermine the promise of adoptive CD8+ T cell immunotherapy. Furthermore, we describe novel strategies designed to counteract Gal-1-, Gal-3- and Gal-9-mediated effects and highlight their targeting potential for creating more effective anti-tumor immune responses. We believe that Gal and their ligands represent an efficacious targeted molecular paradigm that warrants clinical evaluation.
Galectins; Cancer immunotherapy; Carbohydrate therapeutics; Anti-tumor immunity; Glycoimmunology; T cell
The whole tissue of the earthworm (Eisenia andrei) was lyophilized and extracted to purify glycosaminoglycans. Fractions, eluting from an anion-exchange column at 1.0 M and 2.0 M NaCl, showed the presence of acidic polysaccharides on agarose gel electrophoresis. Monosaccharide compositional analysis showed that galactose and glucose were most abundant monosaccharides in both fractions. Depolymerization of the polysaccharide mixture with glycosaminoglycandegrading enzymes confirmed the presence of chondroitin sulfate/dermatan sulfate and heparan sulfate in the 2.0 M NaCl fraction. The content of GAGs (uronic acid containing polysaccharide) in the 2.0 M NaCl fraction determined by carbazole assay was 2%. Disaccharide compositional analysis using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) analysis after chondroitinase digestion (ABC and ACII), showed that the chondroitin sulfate/dermatan sulfate contained a 4-O-sulfo (76%), 2,4-di-O-sulfo (15%), 6-O-sulfo (6%), and unsulfated (4%) uronic acid linked N-acetylgalactosamine residues. LC-ESI-MS analysis of heparin lyase I/II/III digests demonstrated the presence of N-sulfo (69%), N-sulfo-6-O-sulfo (25%) and 2-O-sulfo-N-sulfo-6-O-sulfo (5%) uronic acid linked N-acetylglucosamine residues.
Glycosaminoglycans; Earthworm; Eisenia andrei; Disaccharide compositional analysis; Monosaccharide compositional analysis; Heparan sulfate; Chondroitin sulfate/dermatan sulfate
Brine shrimp are primitive crustacean arthropodal model organisms, second to daphnia, which can survive in high-salinity environments. Their oviposited cysts, cuticle-covered diapausing eggs, are highly resistant to dryness. To elucidate specialties of brine shrimp, this study characterized glycosphingolipids, which are signal transduction-associated material. A group of novel and complex fucosyl glycosphingolipids were separated and identified from cysts of the brine shrimp Artemia franciscana by repeated lipid extraction, alkaline methanolysis, acid treatment, successive column chromatography, and post-source decay measurements by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Structures of the glycosphingolipids were elucidated by conventional structural characterization and mass spectrometry, and the compounds were identified as GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4(Fucα1-3)GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer, and GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer. These compounds also contained a branching, non-arthro-series disaccharide with an α-GlcNAc terminus, similar to that found in a previously reported ceramide hexasaccharide (III3(GlcNAcα2Fucα)-At4Cer). The glycans within these complex GSLs are longer than reported glycans of the animal kingdom containing α-GlcNAc terminus. These complex GSLs as well as the longest GSL with ten sugar residues, ceramide decasaccharide (CDeS), contain the fucosylated LacdiNAc sequence reported to associate with parasitism/immunosuppression and the α-GlcNAc terminus reported to show a certain antibacterial effect in other reports. CDeS, the longest GSL of this species, was found in the highest amount, which indicates that CDeS may be functionally important.
Electronic supplementary material
The online version of this article (doi:10.1007/s10719-012-9436-8) contains supplementary material, which is available to authorized users.
Sphingosine; Fucomannolipid; Terminal α-N-acetylglucosamine residue; Structure characterization; Branchiopoda; Dormant cyst
Despite numerous original publications describing the structural complexity of N- and O-linked glycans on glycoproteins, only very few answer the basic question of which particular glycans are linked to which amino acid residues along the polypeptide chain. Such structural information is of fundamental importance for understanding the biological roles of complex glycosylations as well as deciphering their non-template driven biosynthesis. This review focuses on presenting and commenting on recent strategies, specifically aimed at identifying the glycoproteome of cultured cells and biological samples, using targeted and global enrichment procedures and utilizing the high resolution power, high through-put capacity and complementary fragmentation techniques of tandem mass spectrometry. The goal is to give an update of this emerging field of protein and glyco-sciences and suggest routes to bridge the data gap between the two aspects of glycoprotein characteristics, i.e. glycan structures and their attachment sites.
Glycoproteomics; Glycopeptide; Attachment sites; Liquid chromatography; Tandem mass spectrometry; Enrichment; Lectin affinity; Hydrazide chemistry
Heparin (HP) inhibits the growth of several cell types in vitro including bovine pulmonary artery (BPA) smooth muscle cells (SMCs). In initial studies we discovered that an O-hexanoylated low-molecular-weight (LMW) HP derivative having acyl groups with 6-carbon chain length was more potent inhibitor of BPA-SMCs than the starting HP. We prepared several O-acylated LMWHP derivatives having 4-, 6-, 8-, 10-, 12-, and 18- carbon acyl chain lengths to determine the optimal acyl chain length for maximum anti-proliferative properties of BPA-SMCs. The starting LMWHP was prepared from unfractionated HP by sodium periodate treatment followed by sodium borohydride reduction. The tri-n-butylammonium salt of this LMWHP was O-acylated with butanoic, hexanoic, octanoic, decanoic, dodecanoic, and stearyl anhydrides separately to give respective O-acylated LMWHP derivatives. Gradient polyacrylamide gel electrophoresis (PAGE) was used to examine the average molecular weights of those O-acylated LMWHP derivatives. NMR analysis indicated the presence of one O-acyl group per disaccharide residue. Measurement of the inhibition of BPA-SMCS as a function of O-acyl chain length shows two optima, at a carbon chain length of 6 (O-hexanoylated LMWHP) and at a carbon chain length 12–18 (O-dodecanoyl and O-stearyl LMWHPs). A solution competition SPR study was performed to test the ability of different O-acylated LMWHP derivatives to inhibit fibroblast growth factor (FGF) 1 and FGF2 binding to surface-immobilized heparin. All the LMWHP derivatives bound to FGF1 and FGF2 but each exhibited slightly different binding affinity.
heparin; low molecular weight heparin; O-acylated; smooth muscle cells; surface plasmon resonance
Proteoglycans have been studied to a limited extent in lymphoid cells. In this study we have investigated the expression of proteoglycans in B-cells, CD4+ T-cells, CD8+ T-cells, natural killer cells, as well as in nine different cell lines established from patients with lymphoid malignancies. Serglycin was the major proteoglycan expressed at mRNA level by the primary lymphocytes. None of the syndecans or glycpicans was detected at mRNA level in the primary lymphocytes, except for syndecan-4 in CD4+ T-cells and CD8+ T-cells. All lymphoid cell lines expressed serglycin mRNA, as well as one or several members of the syndecan and glypican families. Further, increased synthesis of proteoglycans was found in the cell lines compared to the primary lymphocytes, as well as the presence of heparan sulfate on the cell surface of five of the cells lines. Western blot analysis showed a close correlation between serglycin mRNA level and expression of serglycin core protein. Our results show that serglycin is a major proteoglycan in all the normal lymphoid cells and that these cells carry little, or none, proteoglycans on the cell surface. Serglycin was also a major proteoglycan in the malignant lymphoid cells, but these also expressed one or more types of cell surface proteoglycans. Thus, malignant transformation of lymphoid cells may be followed by increased synthesis of proteoglycans and expression of cell surface proteoglycans.
Proteoglycan; Serglycin; Syndecan; Glypican; Lymphoid cells
Kashin-Beck Disease (KBD) is an endemic, chronic and degenerative osteoarthropathy principally occurring in children. The characteristic pathological change of KBD is chondrocyte necrosis in hyaline articular cartilage. Proteoglycans are one of the major components in the extracellular matrix of articular cartilage, and disrupted proteoglycan metabolism and loss of proteoglycans in articular cartilage from KBD patients has been observed. In this mini-review, we discuss the close relationship between chondrocyte death including necrosis and loss of proteoglycan, and its potential mechanism during KBD onset and development, which may provide new clues for KBD research.
Kashin-Beck Disease; Proteoglycan; Chondrocyte; Necrosis; Apoptosis; Oxidative stress
The glycosylation abilities of snails deserve attention, because snail species serve as intermediate hosts in the developmental cycles of some human and cattle parasites. In analogy to many other host-pathogen relations, the glycosylation of snail proteins may likewise contribute to these host-parasite interactions. Here we present an overview on the O-glycan structures of 8 different snails (land and water snails, with or without shell): Arion lusitanicus, Achatina fulica, Biomphalaria glabrata, Cepaea hortensis, Clea helena, Helix pomatia, Limax maximus and Planorbarius corneus. The O-glycans were released from the purified snail proteins by β-elimination. Further analysis was carried out by liquid chromatography coupled to electrospray ionization mass spectrometry and – for the main structures – by gas chromatography/mass spectrometry. Snail O-glycans are built from the four monosaccharide constituents: N-acetylgalactosamine, galactose, mannose and fucose. An additional modification is a methylation of the hexoses. The common trisaccharide core structure was determined in Arion lusitanicus to be N-acetylgalactosamine linked to the protein elongated by two 4-O-methylated galactose residues. Further elongations by methylated and unmethylated galactose and mannose residues and/or fucose are present. The typical snail O-glycan structures are different to those so far described. Similar to snail N-glycan structures they display methylated hexose residues.
Gastropod; Methylated glycans; O-glycosylation; Snail
Human immunoglobulin G (IgG) molecules are composed of two Fab portions and one Fc portion. The glycans attached to the Fc portions of IgG are known to modulate its biological activity as they influence interaction with both complement and various cellular Fc receptors. IgG glycosylation changes significantly with pregnancy, showing a vast increase in galactosylation and sialylation and a concomitant decrease in the incidence of bisecting GlcNAc. Maternal IgGs are actively transported to the fetus by the neonatal Fc receptor (FcRn) expressed in syncytiotrophoblasts in the placenta, providing the fetus and newborn with immunological protection. Two earlier reports described significant differences in total glycosylation between fetal and maternal IgG, suggesting a possible glycosylation-selective transport via the placenta. These results might suggest an alternative maternal transport pathway, since FcRn binding to IgG does not depend on Fc-glycosylation. These early studies were performed by releasing N-glycans from total IgG. Here, we chose for an alternative approach analyzing IgG Fc glycosylation at the glycopeptide level in an Fc-specific manner, providing glycosylation profiles for IgG1 and IgG4 as well as combined Fc glycosylation profiles of IgG2 and 3. The analysis of ten pairs of fetal and maternal IgG samples revealed largely comparable Fc glycosylation for all the analyzed subclasses. Average levels of galactosylation, sialylation, bisecting GlcNAc and fucosylation were very similar for the fetal and maternal IgGs. Our data suggest that the placental IgG transport is not Fc glycosylation selective.
Fc receptor; Galactosylation; Glycopeptide; Placenta; Sialylation
Neuroblastoma is the most common extracranial solid tumor in children and tumor ganglioside composition has been linked to its biological and clinical behavior. We recently found that high expression of complex gangliosides that are products of the enzyme GM1a/GD1b synthase predicts a more favorable outcome in human neuroblastoma, and others have shown that complex gangliosides such as GD1a inhibit metastasis of murine tumors. To determine how a switch from structurally simple to structurally complex ganglioside expression affects neuroblastoma cell behavior, we engineered IMR32 human neuroblastoma cells, which contain almost exclusively (89%) the simple gangliosides (SG) GM2, GD2, GM3, and GD3, to overexpress the complex gangliosides (CG) GM1, GD1a, GD1b and GT1b, by stable retroviral-mediated transduction of the cDNA encoding GM1a/GD1b synthase. This strikingly altered cellular ganglioside composition without affecting total ganglioside content: There was a 23-fold increase in the ratio of complex to simple gangliosides in GM1a/GD1b synthase-transduced cells (IMR32-CG) vs. wild type (IMR32) or vector-transfected (IMR32-V) cells with essentially no expression of the clinical neuroblastoma marker, GD2, confirming effectiveness of this molecular switch from simple to complex ganglioside synthesis. Probing for consequences of the switch, we found that among functional properties of IMR32-CG cells, cell migration was inhibited and Rho/Rac1 activities were altered, while proliferation kinetics and cell differentiation were unaffected. These findings further implicate cellular ganglioside composition in determining cell migration characteristics of tumor cells. This IMR32 model system should be useful in delineating the impact of ganglioside composition on tumor cell function.
complex gangliosides; GD2; neuroblastoma; cell migration; GM1a/GD1b synthase; molecular switch
Dramatic changes in glycan biosynthesis during oncogenic transformation result in the emergence of marker glycans on the cell surface. We analysed the N-linked glycans of L1CAM from different stages of melanoma progression, using high-performance liquid chromatography combined with exoglycosidase sequencing, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and lectin probes. L1CAM oligosaccharides are heavily sialylated, mainly digalactosylated, biantennary complex-type structures with galactose β1-4/3-linked to GlcNAc and with or without fucose α1-3/6-linked to GlcNAc. Hybrid, bisected hybrid, bisected triantennary and tetraantennary complex oligosaccharides, and β1-6-branched complex-type glycans with or without lactosamine extensions are expresses at lower abundance. We found that metastatic L1CAM possesses only α2-6-linked sialic acid and the loss of α2-3-linked sialic acid in L1CAM is a phenomenon observed during the transition of melanoma cells from VGP to a metastatic stage. Unexpectedly, we found a novel monoantennary complex-type oligosaccharide with a Galβ1-4Galβ1- epitope capped with sialic acid residues A1G(4)2S2-3. To our knowledge this is the first report documenting the presence of this oligosaccharide in human cancer. The novel and unique N-glycan should be recognised as a new class of human melanoma marker. In functional tests we demonstrated that the presence of cell surface α2-3-linked sialic acid facilitates the migratory behaviour and increases the invasiveness of primary melanoma cells, and it enhances the motility of metastatic cells. The presence of cell surface α2-6-linked sialic acid enhances the invasive potential of both primary and metastatic melanoma cells. Complex-type oligosaccharides in L1CAM enhance the invasiveness of metastatic melanoma cells.
Electronic supplementary material
The online version of this article (doi:10.1007/s10719-012-9374-5) contains supplementary material, which is available to authorized users.
L1CAM; Galβ1-4Galβ1-motif; N-glycolylneuraminic acid; Isogenic melanoma cells; NP HPLC; MALDI MS
Mass spectrometry plays an increasingly important role in structural glycomics. This review provides an overview on currently used mass spectrometric approaches such as the characterization of glycans, the analysis of glycopeptides obtained by proteolytic cleavage of proteins and the analysis of glycosphingolipids. The given examples are demonstrating the application of mass spectrometry to study glycosylation changes associated with congenital disorders of glycosylation, lysosomal storage diseases, autoimmune diseases and cancer.
Cancer; Congentical disorders of glycosylation; Lysosomal storage diseases; MALDI-TOF-MS; Permethylation
Galectin-3 is a β-galactoside-binding protein involved in immunomodulation, cell interactions, cancer progression, and pathogenesis of infectious organisms. We report the identification and characterization of galectin-3 in human semen. In the male reproductive tract, the ~30 kDa galectin-3 protein was identified in testis, epididymis, vas deferens, prostate, seminal vesicle, and sperm protein extracts. In seminal plasma, galectin-3 was identified in the soluble fraction and in prostasomes, cholesterol-rich, membranous vesicles that are secreted by the prostate and incorporated into seminal plasma during ejaculation. Two-dimensional immunoblot analysis of purified prostasomes identified five galectin-3 isoelectric variants with a pI range of 7.0 to 9.2. Affinity purification and tandem mass spectrometry of β-galactoside-binding proteins from prostasomes confirmed the presence of galectin-3 in prostasomes and identified a truncated galectin-3 variant. The intact galectin-3 molecule contains a carbohydrate recognition domain and a non-lectin domain that interacts with protein and lipid moieties. The identification of a monovalent galectin-3 fragment with conserved carbohydrate-binding activity indicates the functional relevance of this truncation and suggests a regulatory mechanism for galectin-3 in prostasomes. Surface biotinylation studies suggested that galectin-3 and the truncated galectin-3 variant are localized to the prostasome surface. Prostasomes are proposed to function in immunosuppression and regulation of sperm function in the female reproductive tract, are implicated in facilitating sexually-transmitted infections, and are indicated in prostate cancer progression. Given the overlap in functional significance, the identification of galectin-3 in prostasomes lays the groundwork for future studies of prostasomes in reproduction, disease transmission, and cancer progression.
galectin-3; prostate; prostasomes; seminal plasma; lectin