Modifications of proteins by O-glycosylation determine many of the properties and functions of proteins. We wish to understand the mechanisms of O-glycosylation and develop inhibitors that could affect glycoprotein functions and alter cellular behavior.
We expressed recombinant soluble human Gal- and GlcNAc-transferases that synthesize the O-glycan cores 1 to 4 and are critical for the overall structures of O-glycans. We determined the properties and substrate specificities of these enzymes using synthetic acceptor substrate analogs. Compounds that were inactive as substrates were tested as inhibitors.
Enzymes significantly differed in their recognition of the sugar moieties and aglycone groups of substrates. Core 1 synthase was active with glycopeptide substrates but GlcNAc-transferases preferred substrates with hydrophobic aglycone groups. Chemical modifications of the acceptors shed light on enzyme–substrate interactions. Core 1 synthase was weakly inhibited by its substrate analog benzyl 2-butanamido-2-deoxy-α-D-galactoside while two of the three GlcNAc-transferases were selectively and potently inhibited by bis-imidazolium salts which are not substrate analogs.
This work delineates the distinct specificities and properties of the enzymes that synthesize the common O-glycan core structures 1 to 4. New inhibitors were found that could selectively inhibit the synthesis of cores 1, 2 and 3 but not core 4.
These studies help our understanding of the mechanisms of action of enzymes critical for O-glycosylation. The results may be useful for the re-engineering of O-glycosylation to determine the roles of O-glycans and the enzymes critical for O-glycosylation, and for biotechnology with potential therapeutic applications.
O-Glycans; Specificity; Inhibitors; C1GalT; C3GnT; C2GnT
Assessing interactions of a glycan-binding protein (GBP) or lectin with glycans on a microarray generates large datasets, making it difficult to identify a glycan structural motif or determinant associated with the highest apparent binding strength of the GBP. We have developed a computational method, termed GlycanMotifMiner, that uses the relative binding of a GBP with glycans within a glycan microarray to automatically reveal the glycan structural motifs recognized by a GBP. We implemented the software with a web-based graphical interface for users to explore and visualize the discovered motifs. The utility of GlycanMotifMiner was determined using five plant lectins, SNA, HPA, PNA, Con A, and UEA-I. Data from the analyses of the lectins at different protein concentrations were processed to rank the glycans based on their relative binding strengths. The motifs, defined as glycan substructures that exist in a large number of the bound glycans and few non-bound glycans, were then discovered by our algorithm and displayed in a web-based graphical user interface (http://glycanmotifminer.emory.edu). The information is used in defining the glycan-binding specificity of GBPs. The results were compared to the known glycan specificities of these lectins generated by manual methods. A more complex analysis was also carried out using glycan microarray data obtained for a recombinant form of human galectin-8. Results for all of these lectins show that GlycanMotifMiner identified the major motifs known in the literature along with some unexpected novel binding motifs.
The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3′SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3′SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3′SIA-LeX identified in a previous glycan microarray screen.
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
Helminths are multicellular parasitic worms that comprise a major class of human pathogens and cause an immense amount of suffering worldwide. Helminths possess an abundance of complex and unique glycoconjugates that interact with both the innate and adaptive arms of immunity in definitive and intermediate hosts. These glycoconjugates represent a major untapped reservoir of immunomodulatory compounds, which have the potential to treat autoimmune and inflammatory disorders, and antigenic glycans, which could be exploited as vaccines and diagnostics. This review will survey current knowledge of the interactions between helminth glycans and host immunity and highlight the gaps in our understanding which are relevant to advancing therapeutics, vaccine development, and diagnostics.
glycans; glycoconjugates; helminths; C-type lectin; innate immunity; adaptive immunity; anti-glycan antibodies; schistosomiasis
CD52 is a GPI-anchored glycopeptide antigen found on sperm cells and human lymphocytes. Recent structural studies indicate that sperm-associated CD52 antigen carries both a complex type N-glycan and an O-glycan on the polypeptide backbone. To facilitate functional and immunological studies of distinct CD52 glycoforms, we report in this paper the first chemoenzymatic synthesis of homogeneous CD52 glycoforms carrying both N- and O-glycans. The synthetic strategy consists of two key steps: monosaccharide primers GlcNAc and GalNAc were first installed at the pre-determined N- and O-glycosylation sites by a facile solid-phase peptide synthesis, and then the N- and O-glycans were extended by respective enzymatic glycosylations. It was found that the endoglycosidase-catalyzed transglycosylation allowed efficient attachment of an intact N-glycan in a single step at the N-glycosylation site, while the recombinant human T-synthase could independently extend the O-linked GalNAc to form the core 1 O-glycan. This chemoenzymatic approach is highly convergent and permits easy construction of various homogeneous CD52 glycoforms from a common polypeptide precursor. In addition, the introduction of a latent thiol group in the form of protected cysteamine at the C-terminus of the CD52 glycoforms will enable site-specific conjugation to a carrier protein to provide immunogens for generating CD52 glycoform-specific antibodies for functional studies.
The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR) plays an essential role in the biogenesis of lysosomes by delivering newly synthesized lysosomal enzymes from the trans Golgi network to the endosomal system. The CI-MPR is expressed in most eukaryotes, with Saccharomyces cerevisiae and Caenorhabditis elegans being notable exceptions. Although the repertoire of glycans recognized by the bovine receptor has been studied extensively, little is known concerning the ligand-binding properties of the CI-MPR from non-mammalian species. To assess the evolutionary conservation of the CI-MPR, surface plasmon resonance analyses using lysosomal enzymes with defined N-glycans were carried out to probe the glycan-binding specificity of the Danio rerio CI-MPR. The results demonstrate that the D. rerio CI-MPR harbors three glycan-binding sites that, like the bovine CI-MPR, map to domains 3, 5 and 9 of its 15-domain-containing extracytoplasmic region. Analyses on a phosphorylated glycan microarray further demonstrated the unique binding properties of each of the three sites and showed that, similar to the bovine CI-MPR, only domain 5 of the D. rerio CI-MPR is capable of recognizing Man-P-GlcNAc-containing glycans.
glycans; lectin; lysosome; receptor
It is generally accepted that human influenza viruses bind glycans containing sialic acid linked α2–6 to the next sugar, that avian influenza viruses bind glycans containing the α2–3 linkage, and that mutations that change the binding specificity might change the host tropism. We noted that human H3N2 viruses showed dramatic differences in their binding specificity, and so we embarked on a study of representative human H3N2 influenza viruses, isolated from 1968 to 2012, that had been isolated and minimally passaged only in mammalian cells, never in eggs. The 45 viruses were grown in MDCK cells, purified, fluorescently labeled and screened on the Consortium for Functional Glycomics Glycan Array. Viruses isolated in the same season have similar binding specificity profiles but the profiles show marked year-to-year variation. None of the 610 glycans on the array (166 sialylated glycans) bound to all viruses; the closest was Neu5Acα2–6(Galβ1–4GlcNAc)3 in either a linear or biantennary form, that bound 42 of the 45 viruses. The earliest human H3N2 viruses preferentially bound short, branched sialylated glycans while recent viruses bind better to long polylactosamine chains terminating in sialic acid. Viruses isolated in 1996, 2006, 2010 and 2012 bind glycans with α2–3 linked sialic acid; for 2006, 2010 and 2012 viruses this binding was inhibited by oseltamivir, indicating binding of α2–3 sialylated glycans by neuraminidase. More significantly, oseltamivir inhibited virus entry of 2010 and 2012 viruses into MDCK cells. All of these viruses were representative of epidemic strains that spread around the world, so all could infect and transmit between humans with high efficiency. We conclude that the year-to-year variation in receptor binding specificity is a consequence of amino acid sequence changes driven by antigenic drift, and that viruses with quite different binding specificity and avidity are equally fit to infect and transmit in the human population.
Mucin glycoproteins present a complex structural landscape arising from the multiplicity of glycosylation patterns afforded by their numerous serine and threonine glycosylation sites, often in clusters, and with variations in respective glycans. To explore the structural complexities in such glycoconjugates we used NMR to systematically analyze the conformational effects of glycosylation density within a cluster of sites. This allows correlation with molecular recognition through analysis of interactions between these and other glycopeptides, with antibodies, lectins, and sera, using a glycopeptide microarray. Selective antibody interactions with discrete conformational elements, reflecting aspects of the peptide and disposition of GalNAc residues are observed. Our results help bridge the gap between conformational properties and molecular recognition of these molecules, with implications for their physiological roles. Features of the native mucin motifs impact their relative immunogenicity and are accurately encoded in the antibody binding site, with the conformational integrity being preserved in isolated glycopeptides, as reflected in the antibody binding profile to array components.
glycan microarrays; carbohydrate; glycomics
Major challenges of glycomics are to characterize a glycome and identify functional glycans as ligands for glycan-binding proteins (GBPs). To address these issues we have developed a general strategy termed shotgun glycomics. We focus on glycosphingolipids (GSLs), a challenging class of glycoconjugates recognized by toxins, antibodies, and GBPs. We derivatized GSLs extracted from cells with a heterobifunctional fluorescent tag suitable for covalent immobilization. Fluorescent GSLs were separated by multidimensional chromatography, quantified, and coupled to glass slides to create GSL shotgun microarrays. The microarrays were interrogated with cholera toxin, antibodies, and sera from patients with Lyme disease to identify biologically relevant GSLs that were subsequently characterized by mass spectrometry. Shotgun glycomics incorporating GSLs and potentially glycoprotein-derived glycans provides an approach to accessing the complex glycomes of animal cells and offers a strategy for focusing structural analyses on functionally significant glycans.
glycosphingolipids; glycan array; fluorescent labeling; immobilization; functional glycomics
Diagnostic methods for parasite infections still highly depend on the identification of the parasites by direct methods such as microscopic examination of blood, stool and tissue biopsies. Serodiagnosis is often carried out to complement the direct methods, however few synthetic antigens with sufficient sensitivity and specificity are available. Here we evaluated a glycan microarray approach to select for synthetic glycan antigens that could be used for serodiagnosis of parasitic infections. Using a glycan array containing over 250 different glycan antigens, we identified GalNAcβ1-4(Fucα1-3)GlcNAc-R (LDNF) as a glycan antigen that is recognized by antibodies from Trichinella-infected individuals. We synthesized a neoglycoconjugate, consisting of 5 LDNF molecules covalently coupled to bovine serum albumin (BSA), and used this neoglycoconjugate as an antigen to develop a highly sensitive total-Ig ELISA for serological screening of trichinellosis. The results indicate that glycan microarrays constitute a promising technology for fast and specific identification of parasite glycan antigens to improve serodiagnosis of different parasitic infections, either using an ELISA format, or parasite-specific glycan-arrays.
Glycan microarray technology has become a successful tool for studying protein-carbohydrate interactions, but a limitation has been the laborious synthesis of glycan structures by enzymatic and chemical methods. Here we describe a new method to generate quantifiable glycan libraries from natural sources by combining widely used protease digestion of glycoproteins and Fmoc chemistry. Glycoproteins including chicken ovalbumin, bovine fetuin, and horseradish peroxidase (HRP) were digested by pronase, protected by FmocCl, and efficiently separated by 2D-HPLC. We show that glycans from HRP glycopeptides separated by HPLC and fluorescence monitoring retained their natural reducing end structures, mostly core α1,3-fucose and core α1,2-xylose. After simple Fmoc-deprotection, the glycans were printed on NHS-activated glass slides. The glycans were interrogated using plant lectins and antibodies in sera from mice infected with Schistosoma mansoni, which revealed the presence of both IgM and IgG antibody responses to HRP-glycopeptides. This simple approach to glycopeptide purification and conjugation allows for the development of natural glycopeptide microarrays without the need to remove and derivatize glycans and potentially compromise their reducing end determinants.
Glycan array; fluorescent labeling; immobilization; functional glycomics
Hemolytic transfusion reactions represent one of the most common causes of transfusion-related mortality. Although many factors influence hemolytic transfusion reactions, complement activation represents one of the most common features associated with fatality. In this paper we will focus on the role of complement in initiating and regulating hemolytic transfusion reactions and will discuss potential strategies aimed at mitigating or favorably modulating complement during incompatible red blood cell transfusions.
A novel strategy for creating naturally-derived glycan microarrays has been developed. Glycosylamines are prepared from free reducing glycans and stabilized by reaction with acryloyl chloride to generate a glycosylamide in which the reducing monosaccharide has a closed ring structure. Ozonolysis of the protected glycan yields an active aldehyde, to which a bifunctional fluorescent linker is coupled by reductive amination. The fluorescent derivatives are easily coupled through a residual primary alkylamine to generate glycan microarrays. This strategy preserves structural features of glycans required for antibody recognition, and allows development of natural arrays of fluorescent glycans in which the cyclic pyranose structure of the reducing-end sugar residue is retained.
Fluorescent labeling; Functional glycomics; Glycan array; Glycosylamine; Immobilization
The expression of ABO(H) blood group antigens causes deletion of cells that generate self anti-blood group antibodies, but this deletion limits adaptive immunity toward pathogens bearing cognate blood group antigens. To explore potential defense mechanisms against these pathogens, given such limitations in adaptive immunity, we screened for innate proteins that could recognize human blood group antigens. Here we report that two innate immune lectins, galectins-4 and -8, which are expressed in the intestinal tract, recognize and kill human blood group antigen-expressing E. coli, while failing to alter viability of other E. coli strains or other gram-negative or gram-positive organisms both in vitro and in vivo. Killing by both galectins-4 and -8 resides within their C-terminal domains, occurs rapidly and independently of complement, and is accompanied by disruption of membrane integrity. These results demonstrate that innate defense lectins can provide immunity against pathogens that display blood group self-antigens on their surface.
Many diseases and disorders are characterized by quantitative and/or qualitative changes in complex carbohydrates. Mass spectrometry methods show promise in monitoring and detecting these important biological changes. Here we report a new glycomics method, termed Glycan Reductive Isotope Labeling (GRIL), where free glycans are derivatized by reductive amination with the differentially coded stable isotope tags [12C6]-aniline and [13C6]-aniline. These dual-labeled aniline-tagged glycans can be recovered by reversed-phase chromatography and quantified based on UV-absorbance and relative ion abundances. Unlike previously reported isotopically coded reagents for glycans, GRIL does not contain deuterium, which can be chromatographically resolved. Our method shows no chromatographic resolution of differentially labeled glycans. Mixtures of differentially tagged glycans can be directly compared and quantified using mass spectrometric techniques. We demonstrate the use of GRIL to determine relative differences in glycan amount and composition. We analyze free glycans and glycans enzymatically or chemically released from a variety of standard glycoproteins, as well as human and mouse serum glycoproteins using this method. This technique allows for linear, relative quantitation of glycans over a 10-fold concentration range and can accurately quantify sub-picomole levels of released glycans, providing a needed advancement in the field of Glycomics.
Mass spectrometry; MALDI; glycans; glycoproteins; oligosaccharides; reductive amination
Galectin-1 (Gal-1) and galectin-3 (Gal-3) are widely expressed galectins with immunoregulatory functions in animals. To explore their glycan specificity, we developed microarrays of naturally occurring glycans using a novel bifunctional fluorescent linker, 2-amino-N-(2-aminoethyl)-benzamide (AEAB), directly conjugated through its arylamine group by reductive amination to free glycans to form glycan-AEABs (GAEABs). Glycans from natural sources were used to prepare over 200 GAEABs, which were purified by multidimensional HPLC and covalently immobilized onto NHS-activated glass slides via their free alkylamine. Fluorescence-based screening demonstrated that Gal-1 recognizes a wide variety of complex N-glycans, whereas Gal-3 primarily recognizes poly-N-acetyllactosamine-containing glycans independent of N-glycan presentation. GAEABs provide a general solution to glycan microarray preparation from natural sources for defining the specificity of glycan-binding proteins.
galectin; glycan microarray; fluorescent labeling; immobilization; functional glycomics
To examine the range of selective processes that potentially operate when poorly binding influenza viruses adapt to replicate more efficiently in alternative environments, we passaged a virus containing an attenuating mutation in the hemagglutinin (HA) receptor binding site in mice and characterized the resulting mutants with respect to the structural locations of mutations selected, the replication phenotypes of the viruses, and their binding properties on glycan microarrays. The initial attenuated virus had a tyrosine-to-phenylalanine mutation at HA1 position 98 (Y98F), located in the receptor binding pocket, but viruses that were selected contained second-site pseudoreversion mutations in various structural locations that revealed a range of molecular mechanisms for modulating receptor binding that go beyond the scope that is generally mapped using receptor specificity mutants. A comparison of virus titers in the mouse respiratory tract versus MDCK cells in culture showed that the mutants displayed distinctive replication properties depending on the system, but all were less attenuated in mice than the Y98F virus. An analysis of receptor binding properties confirmed that the initial Y98F virus bound poorly to several different species of erythrocytes, while all mutants reacquired various degrees of hemagglutination activity. Interestingly, both the Y98F virus and pseudoreversion mutants were shown to bind very inefficiently to standard glycan microarrays containing an abundance of binding substrates for most influenza viruses that have been characterized to date, provided by the Consortium for Functional Glycomics. The viruses were also examined on a recently developed microarray containing glycans terminating in sialic acid derivatives, and limited binding to a potentially interesting subset of glycans was revealed. The results are discussed with respect to mechanisms for HA-mediated receptor binding, as well as regarding the species of molecules that may act as receptors for influenza virus on host cell surfaces.
Cells normally undergo physiological turnover through the induction of apoptosis and phagocytic removal, partly through exposure of cell surface phosphatidylserine (PS). In contrast, neutrophils appear to possess apoptosis-independent mechanisms of removal. Here we show that Galectin-1 (Gal-1) induces PS exposure independent of alterations in mitochondrial potential, caspase activation, or cell death. Furthermore, Gal-1–induced PS exposure reverts after Gal-1 removal without altering cell viability. Gal-1–induced PS exposure is uniquely microdomain restricted, yet cells exposing PS do not display evident alterations in membrane morphology nor do they exhibit bleb formation, typically seen in apoptotic cells. Long-term exposure to Gal-1 prolongs PS exposure with no alteration in cell cycle progression or cell growth. These results demonstrate that Gal-1–induced PS exposure and subsequent phagocytic removal of living cells represents a new paradigm in cellular turnover.
Nature possesses an unlimited number and source of biologically-relevant natural glycans, many of which are too complicated to synthesize in the laboratory. To capitalize on the naturally-occurring plethora of glycans, we have developed a method to fluorescently tag the isolated free glycans, which maintains the closed-ring structure. After purification of the labeled glycans, they can be printed on a glass surface to create a natural glycan microarray, available for interrogation with potential glycan-binding proteins. The derivatization of these natural glycans has vastly expanded the number of glycans for functional studies.
Fluorescence; reductive amination; glycan microarray; conjugation
Regulatory pathways for protein glycosylation are poorly understood, but expression of branchpoint enzymes is critical. A key branchpoint enzyme is the T-synthase, which directs synthesis of the common core 1 O-glycan structure (T-antigen), the precursor structure for most mucin-type O-glycans in a wide variety of glycoproteins. Formation of active T-synthase, which resides in the Golgi apparatus, requires a unique molecular chaperone, Cosmc, encoded on Xq24. Cosmc is the only molecular chaperone known to be lost through somatic acquired mutations in cells. We show that Cosmc is an endoplasmic reticulum (ER)–localized adenosine triphosphate binding chaperone that binds directly to human T-synthase. Cosmc prevents the aggregation and ubiquitin-mediated degradation of the T-synthase. These results demonstrate that Cosmc is a molecular chaperone in the ER required for this branchpoint glycosyltransferase function and show that expression of the disease-related Tn antigen can result from deregulation or loss of Cosmc function.
Loss of T-synthase (uridine diphosphate galactose:N-acetylgalactosaminyl-α1-Ser/Thr β3galactosyltransferase), a key enzyme required for the formation of mucin-type core 1 O-glycans, is observed in several human diseases, including cancer, Tn syndrome and IgA nephropathy, but current methods to assay the enzyme use radioactive substrates and complicated isolation of the product. Here we report the development of a novel fluorescent assay to measure its activity in a variety of tumor cell lines. Deficiencies in T-synthase activity correlate with mutations in the gene encoding the molecular chaperone Cosmc that is required for folding the T-synthase. This new high-throughput assay allows for facile screening of tumor specimens and other biological material for T-synthase activity and could be used diagnostically.
Cosmc; fluorescent assay; glycosyltransferase assay; 4-methylumbelliferone; T-synthase
Incomplete or aberrant glycosylation leading to Tn antigen (GalNAcα1-Ser/Thr) expression on human glycoproteins is strongly associated with human pathological conditions, including tumors, certain autoimmune diseases, such as the idiopathic IgA nephropathy, and may modulate immune homeostasis. In addition, the Tn antigen is highly expressed by certain pathogens and plays a role in host–pathogen interactions. To enable experimental approaches to study interactions of the Tn antigen with the immune system and analyse anti-Tn antibody responses in infection or disorders, we generated a Tn-expressing resource that can be used for high-throughput screening. In consideration of IgA nephropathy in which the hinge region is incompletely glycosylated, we used this hinge sequence that encodes five potential glycosylation sites as the ideal template for the synthesis of a Tn antigen expressing glycopeptide. Inclusion of an N-terminal biotin in the peptide enabled binding to streptavidin-coated ELISA plates as monitored using Helix pomatia agglutinin or anti-Tn monoclonal antibody. We also found that the biotinylated IgA-Tn peptide is a functional acceptor for β1-3-galactosylation using recombinant T-synthase (β1-3-galactosyltransferase). Besides its immunochemical functionality as a possible diagnostic tool for IgA nephropathy, the peptide is an excellent substrate for glycan elongation and represents a novel template applicable for glycan–antigen-associated diseases.
glycopeptide synthesis; IgA nephropathy; Tn-antigen
Endoglycan is a mucin-like glycoprotein expressed by endothelial cells and some leukocytes and is recognized by L-selectin, a C-type lectin important in leukocyte trafficking and extravasation during inflammation. Here, we show that recombinant L-selectin and human T lymphocytes expressing L-selectin bind to synthetic glycosulfopeptides (GSPs). These synthetic glycosulfopeptides contain 37 amino acid residues modeled after the N-terminus of human endoglycan and contain one or two tyrosine sulfates (TyrSO3) along with a nearby core-2-based Thr-linked O-glycan with sialyl Lewis x (C2-SLex). TyrSO3 at position Y118 was more critical for binding than at Y97. C2-SLex at T124 was required for L-selectin recognition. Interestingly, under similar conditions, neither L-selectin nor T lymphocytes showed appreciable binding to the sulfated carbohydrate epitope 6-sulfo-SLex. P-selectin also bound to endoglycan-based GSPs but with lower affinity than toward GSPs modeled after PSGL-1, the physiological ligand for P- and L-selectin that is expressed on leukocytes. These results demonstrate that TyrSO3 residues in association with a C2-SLex moiety within endoglycan and PSGL-1 are preferentially recognized by L-selectin.
endoglycan; glycosulfopeptide; L-selectin; O-glycan; tyrosine sulfate