While adaptive immunity recognizes a nearly infinite range of antigenic determinants, immune tolerance renders adaptive immunity vulnerable to microbes decorated in self-like antigens. Recent studies suggest that sugar-binding proteins galectin-4 and galectin-8 bind microbes expressing blood group antigens. However, the binding profile and potential antimicrobial activity of other galectins, particularly galectin-9 (Gal-9), has remained incompletely defined. Here we demonstrate that while Gal-9 possesses strong binding preference for ABO(H) blood group antigens, each domain exhibits distinct binding patterns, with the C-terminal domain (Gal-9C) exhibiting higher binding to blood group B than the N-terminal domain (Gal-9N). Despite this binding preference, while Gal-9 readily killed blood group B-positive Escherichia coli, Gal-9N displayed higher killing activity against this microbe than Gal-9C. Utilization of microarrays populated with blood group O antigens from a diverse array of microbes revealed that while Gal-9 can bind various microbial glycans, Gal-9N and Gal-9C displayed distinct and overlapping binding preferences. Flow cytometric examination of intact microbes corroborated the microbial glycan microarray findings, demonstrating that Gal-9, Gal-9N, and Gal-9C also possess the capacity to recognize distinct strains of Providencia alcalifaciens and Klebsiella pneumoniae that express mammalian blood group-like antigens while failing to bind related strains that do not express mammalian-like glycans. In each case of microbial binding, Gal-9, Gal-9N and Gal-9C induced microbial death. In contrast, while Gal-9, Gal-9N, and Gal-9C engaged red blood cells, each failed to induce hemolysis. These data suggest that Gal-9 recognition of distinct microbial strains may provide antimicrobial activity against molecular mimicry.
The jawless vertebrates (lamprey and hagfish) evolved a novel adaptive immune system with many similarities to that found in the jawed vertebrates, including the production of antigen-specific circulating antibodies in response to immunization. However, the jawless vertebrates use leucine-rich repeat (LRR)-based antigen receptors termed variable lymphocyte receptors (VLRs) for immune recognition, instead of immunoglobulin (Ig)-based receptors. VLR genes are assembled in developing lymphocytes through a gene conversion-like process, in which hundreds of LRR gene segments are randomly selected as template donors to generate a large repertoire of distinct antigen receptors, similar to that found within the mammalian adaptive immune system. Here we describe the development of a robust platform using immunized lampreys (Petromyzon marinus) for generating libraries of anti-carbohydrate (anti-glycan) variable lymphocyte receptor B, or VLRBs. The anti-carbohydrate VLRBs are isolated using a yeast surface display (YSD) expression platform and enriched by binding to glycan microarrays through the anti-glycan VLRB. This enables both the initial identification and enrichment of individual yeast clones against hundreds of glycans simultaneously. Through this enrichment strategy a broad array of glycan-specific VLRs can be isolated from the YSD library. Subsequently, the bound yeast cells are directly removed from the microarray, the VLR antibody clone is sequenced, and the end product is expressed as a VLR-IgG-Fc fusion protein that can be used for ELISA, Western blotting, flow cytometry, and immunomicroscopy. Thus, by combining yeast surface display with glycan microarray technology, we have developed a rapid, efficient, and novel method for generating chimeric VLR-IgG-Fc proteins that recognize a broad array of unique glycan structures with exquisite specificity.
Glycosylation is essential to brain development and function, but prior studies have often been limited to a single analytical technique and excluded region- and sex-specific analyses. Here, using several methodologies, we analyze Asn-linked and Ser/Thr/Tyr-linked protein glycosylation between brain regions and sexes in mice. Brain N-glycans are less complex in sequence and variety compared to other tissues, consisting predominantly of high-mannose and fucosylated/bisected structures. Most brain O-glycans are unbranched, sialylated O-GalNAc and O-mannose structures. A consistent pattern is observed between regions, and sex differences are minimal compared to those in plasma. Brain glycans correlate with RNA expression of their synthetic enzymes, and analysis of glycosylation genes in humans show a global downregulation in the brain compared to other tissues. We hypothesize that this restricted repertoire of protein glycans arises from their tight regulation in the brain. These results provide a roadmap for future studies of glycosylation in neurodevelopment and disease.
Glycans are critical to every facet of biology and medicine, from viral infections to embryogenesis. Tools to study glycans are rapidly evolving; however, the majority of our knowledge is deeply dependent on binding by glycan binding proteins (e.g., lectins). The specificities of lectins, which are often naturally isolated proteins, have not been well-defined, making it difficult to leverage their full potential for glycan analysis. Herein, we use a combination of machine learning algorithms and expert annotation to define lectin specificity for this important probe set. Our analysis uses comprehensive glycan microarray analysis of commercially available lectins we obtained using version 5.0 of the Consortium for Functional Glycomics glycan microarray (CFGv5). This data set was made public in 2011. We report the creation of this data set and its use in large-scale evaluation of lectin-glycan binding behaviors. Our motif analysis was performed by integrating 68 manually defined glycan features with systematic probing of computational rules for significant binding motifs using mono- and disaccharides and linkages. Combining machine learning with manual annotation, we create a detailed interpretation of glycan-binding specificity for 57 unique lectins, categorized by their major binding motifs: mannose, complex-type N-glycan, O-glycan, fucose, sialic acid and sulfate, GlcNAc and chitin, Gal and LacNAc, and GalNAc. Our work provides fresh insights into the complex binding features of commercially available lectins in current use, providing a critical guide to these important reagents.
Alpha-1-acid glycoprotein (AGP-1) is a positive acute phase glycoprotein with uncertain functions. Serum AGP-1 (sAGP-1) is primarily derived from hepatocytes and circulates as 12-20 different glycoforms. We isolated a glycoform secreted from platelet-activating factor (PAF)-stimulated human neutrophils (nAGP-1). Its peptide sequence was identical to hepatocyte-derived sAGP-1, but nAGP-1 differed from sAGP-1 in its chromatographic behavior, electrophoretic mobility, and pattern of glycosylation. The function of these 2 glycoforms also differed. sAGP-1 activated neutrophil adhesion, migration, and neutrophil extracellular traps (NETosis) involving myeloperoxidase, peptidylarginine deiminase 4, and phosphorylation of ERK in a dose-dependent fashion, whereas nAGP-1 was ineffective as an agonist for these events. Furthermore, sAGP-1, but not nAGP-1, inhibited LPS-stimulated NETosis. Interestingly, nAGP-1 inhibited sAGP-1-stimulated neutrophil NETosis. The discordant effect of the differentially glycosylated AGP-1 glycoforms was also observed in platelets where neither of the AGP-1 glycoforms alone stimulated aggregation of washed human platelets, but sAGP-1, and not nAGP-1, inhibited aggregation induced by PAF or ADP, but not by thrombin. These functional effects of sAGP-1 correlated with intracellular cAMP accumulation and phosphorylation of the protein kinase A substrate vasodilator-stimulated phosphoprotein and reduction of Akt, ERK, and p38 phosphorylation. Thus, the sAGP-1 glycoform limits platelet reactivity, whereas nAGP-1 glycoform also limits proinflammatory actions of sAGP-1. These studies identify new functions for this acute phase glycoprotein and demonstrate that the glycosylation of AGP-1 controls its effects on 2 critical cells of acute inflammation.
Skeletal muscle has the intrinsic ability to self-repair through a multifactorial process, but many aspects of its cellular and molecular mechanisms are not fully understood. There is increasing evidence that some members of the mammalian β-galactoside-binding protein family (galectins) are involved in the muscular repair process (MRP), including galectin-3 (Gal-3). However, there are many questions about the role of this protein on muscle self-repair. Here, we demonstrate that endogenous Gal-3 is required for: (i) muscle repair in vivo by using a chloride-barium myolesion mouse model and (ii) mouse primary myoblasts myogenic programming. Injured muscle from Gal-3 knockout mice (GAL3KO) showed persistent inflammation associated with compromised muscle repair and the formation of fibrotic tissue on the lesion site. In GAL3KO mice, osteopontin expression remained high even after 7 and 14 d of the myolesion, while Myoblast differentiation transcription factor (MyoD) and myogenin had decreased their expression. In GAL3KO mouse primary myoblast cell culture, Paired Box 7 (Pax7) detection seems to sustain even when cells are stimulated to differentiation and MyoD expression is drastically reduced. The detection and temporal expression levels of these transcriptional factors appear to be altered in Gal-3-deficient myoblast. Gal-3 expression in wild-type mice for GAL3KO states, both in vivo and in vitro, in sarcoplasm/cytoplasm and myonuclei; as differentiation proceeds, Gal-3 expression is drastically reduced, and its location is confined to the sarcolemma/plasma cell membrane. We also observed a change in the temporal-spatial profile of Gal-3 expression and muscle transcription factors levels during the myolesion. Overall, these results demonstrate that endogenous Gal-3 is required for the skeletal muscle repair process.
While adaptive immunity enables the recognition of a wide range of microbial antigens, immunological tolerance limits reactively toward self to reduce autoimmunity. Some bacteria decorate themselves with self-like antigens as a form of molecular mimicry to limit recognition by adaptive immunity. Recent studies suggest that galectin-4 (Gal-4) and galectin-8 (Gal-8) may provide a unique form of innate immunity against molecular mimicry by specifically targeting microbes that decorate themselves in self-like antigens. However, the binding specificity and antimicrobial activity of many human galectins remain incompletely explored. In this study, we defined the binding specificity of galectin-3 (Gal-3), the first galectin shown to engage microbial glycans. Gal-3 exhibited high binding toward mammalian blood group A, B, and αGal antigens in a glycan microarray format. In the absence of the N-terminal domain, the C-terminal domain of Gal-3 (Gal-3C) alone exhibited a similar overall binding pattern, but failed to display the same level of binding for glycans over a range of concentrations. Similar to the recognition of mammalian glycans, Gal-3 and Gal-3C also specifically engaged distinct microbial glycans isolated and printed in a microarray format, with Gal-3 exhibiting higher binding at lower concentrations toward microbial glycans than Gal-3C. Importantly, Gal-3 and Gal-3C interactions on the microbial microarray accurately predicted actual interactions toward intact microbes, with Gal-3 and Gal-3C displaying carbohydrate-dependent binding toward distinct strains of Providentia alcalifaciens and Klebsiella pneumoniae that express mammalian-like antigens, while failing to recognize similar strains that express unrelated antigens. While both Gal-3 and Gal-3C recognized specific strains of P. alcalifaciens and K. pneumoniae, only Gal-3 was able to exhibit antimicrobial activity even when evaluated at higher concentrations. These results demonstrate that while Gal-3 and Gal-3C specifically engage distinct mammalian and microbial glycans, Gal-3C alone does not possess antimicrobial activity.
Bladder cancer is the ninth most frequently diagnosed cancer worldwide, and there is a need to develop new biomarkers for staging and prognosis of this disease. Here we report that cell lines derived from low-grade and high-grade bladder cancers exhibit major differences in expression of glycans in surface glycoproteins. We analyzed protein glycosylation in three low-grade bladder cancer cell lines RT4 (grade-1-2), 5637 (grade-2), and SW780 (grade-1), and three high-grade bladder cancer cell lines J82COT (grade-3), T24 (grade-3) and TCCSUP (grade-4), with primary bladder epithelial cells, A/T/N, serving as a normal bladder cell control. Using a variety of approaches including flow cytometry, immunofluorescence, glycomics and gene expression analysis, we observed that the low-grade bladder cancer cell lines RT4, 5637 and SW780 express high levels of the fucosylated Lewis-X antigen (Lex, CD15) (Galβ1-4(Fucα1-3)GlcNAcβ1-R), while normal bladder epithelial A/T/N cells lack Lex expression. T24 and TCCSUP cells also lack Lex, whereas J82COT cells express low levels of Lex. Glycomics analyses revealed other major differences in fucosylation and sialylation of N-glycans between these cell types. O-glycans are highly differentiated, as RT4 cells synthesize core 2-based O-glycans that are lacking in the T24 cells. These differences in glycan expression correlated with differences in RNA expression levels of their cognate glycosyltransferases, including α1-3/4-fucosyltransferase genes. These major differences in glycan structures and gene expression profiles between low- and high-grade bladder cancer cells suggest that glycans and glycosyltransferases are candidate biomarkers for grading bladder cancers.
Mucus is a densely populated ecological niche that coats all non-keratinized epithelia, and plays a critical role in protecting the human body from infections. Although traditionally viewed as a physical barrier, emerging evidence suggests that mucus can directly suppress virulence-associated traits in opportunistic pathogens including Pseudomonas aeruginosa. However, the molecular mechanisms by which mucus affords this protection are unclear. Here, we show that mucins, and particularly their associated glycans, signal through the Dismed2 domain of the sensor kinase RetS in P. aeruginosa. We find that this RetS-dependent signaling leads to the direct inhibition of the GacS-GacA two-component system, the activity of which is associated with a chronic infection state. This signaling includes downregulation of the type VI secretion system (T6SS), and prevents T6SS-dependent bacterial killing by P. aeruginosa. Overall, these results shed light on how mucus impacts P. aeruginosa behavior, and may inspire novel approaches for controlling P. aeruginosa infections.
The pleiotropic functions of macrophages in immune defense, tissue repair, and maintenance of tissue homeostasis are supported by the heterogeneity in macrophage sub-populations that differ both in ontogeny and polarization. Although glycans and glycan-binding proteins (GBPs) are integral to macrophage function and may contribute to macrophage diversity, little is known about the factors governing their expression. Here, we provide a resource for characterizing the N-/O-glycomes of various murine peritoneal macrophage sub-populations, demonstrating that glycosylation primarily reflects developmental origin and, to a lesser degree, cellular polarization. Furthermore, comparative analysis of GBP-coding genes in resident and elicited macrophages indicated that GBP expression is consistent with specialized macrophage functions and correlates with specific types of displayed glycans. An integrated, semi-quantitative approach was used to confirm distinct expression patterns of glycans and their binding proteins across different macrophages. The data suggest that regulation of glycan-protein complexes may be central to macrophage residence and recruitment.
SARS-CoV, MERS-CoV, and potentially SARS-CoV-2 emerged as novel human coronaviruses following cross-species transmission from animal hosts. Although the receptor binding characteristics of human coronaviruses are well documented, the role of carbohydrate binding in addition to recognition of proteinaceous receptors has not been fully explored. Using natural glycan microarray technology, we identified N-glycans in the human lung that are recognized by various human and animal coronaviruses. All viruses tested, including SARS-CoV-2, bound strongly to a range of phosphorylated, high mannose N-glycans and to a very specific set of sialylated structures. Examination of two linked strains, human CoV OC43 and bovine CoV Mebus, reveals shared binding to the sialic acid form Neu5Gc (not found in humans), supporting the evidence for cross-species transmission of the bovine strain. Our findings, revealing robust recognition of lung glycans, suggest that these receptors could play a role in the initial stages of coronavirus attachment and entry.
Cellular interactions between endothelial cells and macrophages regulate macrophage localization and phenotype, but the mechanisms underlying these interactions are poorly understood. Here we explored the role of sialoglycans on lymphatic endothelial cells (LEC) in interactions with macrophage-expressed Siglec-1 (CD169). Lectin-binding assays and mass spectrometric analyses revealed that LEC from human skin express more sialylated glycans than the corresponding blood endothelial cells. Higher amounts of sialylated and/or sulfated glycans on LEC than BEC were consistently observed in murine skin, lung and lymph nodes. The floor LEC of the subcapsular sinus (SCS) in murine lymph nodes (LN) displayed sialylated glycans at particularly high densities. The sialoglycans of LN LEC were strongly bound by Siglec-1. Such binding plays an important role in the localization of Siglec-1+ LN-SCS macrophages, as their numbers are strongly reduced in mice expressing a Siglec-1 mutant that is defective in sialoglycan binding. The residual Siglec-1+ macrophages are less proliferative and have a more anti-inflammatory phenotype. We propose that the densely clustered, sialylated glycans on the SCS floor LEC are a key component of the macrophage niche, providing anchorage for the Siglec-1+ LN-SCS macrophages.
Lupus nephritis (LN) is a serious complication occurring in 50% of patients with systemic lupus erythematosus (SLE) for which there is a lack of biomarkers, a lack of specific medications, and a lack of a clear understanding of its pathogenesis. The expression of calcium/calmodulin kinase IV (CaMK4) is increased in podocytes of patients with LN and lupus-prone mice, and its podocyte-targeted inhibition averts the development of nephritis in mice. Nephrin is a key podocyte molecule essential for the maintenance of the glomerular slit diaphragm. Here, we show that the presence of fucose on N-glycans of IgG induces, whereas the presence of galactose ameliorates, podocyte injury through CaMK4 expression. Mechanistically, CaMK4 phosphorylates NF-kappaB, upregulates the transcriptional repressor SNAIL, and limits the expression of nephrin. In addition, we demonstrate that increased expression of CaMK4 in biopsy specimens and in urine podocytes from people with LN is linked to active kidney disease. Our data shed light on the role of IgG glycosylation in the development of podocyte injury and propose the development of "liquid kidney biopsy" approaches to diagnose LN.
Mucin-type O-glycosylation (O-glycans, O-glycome) is among the most biologically important post-translational modification in glycoproteins but O-glycan structural diversity and expression are poorly understood due to the inadequacy of current analytical methods. We recently developed a new tool termed cellular O-glycome reporter/amplification (CORA), which uses O-glycan precursors, benzyl-alpha-GalNAc (Bn-alpha-GalNAc) or azido-Bn-alpha-GalNAc (N3 -Bn-alpha-GalNAc), as surrogates of protein O-glycosylation. Living cells metabolically convert these precursors to all types of O-GalNAc glycans representative of the cells' capabilities. The amplification and secretion of the O-glycome products greatly facilitates their analysis and functional studies. Here we describe protocols for analytical and preparative applications. (c) 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Cellular O-glycome reporter/amplification for the analysis of mucin-type O-glycans from living cells Basic Protocol 2: Preparation of cellular O-glycans from living cells for functional glycomics and glycan microarrays Basic Protocol 3: Conjugation of cellular O-glycans with a bifunctional fluorescent tag Basic Protocol 4: 2D-HPLC purification and MALDI-TOF/MS identification of individual PYAB-Bn-O-glycan.
The recognition of oligomannose-type glycans in innate and adaptive immunity is elusive due to multiple closely related isomeric glycan structures. To explore the functions of oligomannoses, we developed a multifaceted approach combining mass spectrometry assignments of oligomannose substructures and the development of a comprehensive oligomannose microarray. This defined microarray encompasses both linear and branched glycans, varying in linkages, branching patterns, and phosphorylation status. With this resource, we identified unique recognition of oligomannose motifs by innate immune receptors, including DC-SIGN, L-SIGN, Dectin-2, and Langerin, broadly neutralizing antibodies against HIV gp120, N-acetylglucosamine-1-phosphotransferase, and the bacterial adhesin FimH. The results demonstrate that each protein exhibits a unique specificity to oligomannose motifs and suggest the potential to rationally design inhibitors to selectively block these protein-glycan interactions.
The terminal galactose residues of N- and O-glycans in animal glycoproteins are often sialylated and/or fucosylated, but sulfation, such as 3-O-sulfated galactose (3-O-SGal), represents an additional, but poorly understood modification. To this end, we have developed a novel sea lamprey variable lymphocyte receptor (VLR) termed O6 to explore 3-O-SGal expression. O6 was engineered as a recombinant murine IgG chimera and its specificity and affinity to the 3-O-SGal epitope was defined using a variety of approaches, including glycan and glycoprotein microarray analyses, isothermal calorimetry, ligand-bound crystal structure, FACS, and immunohistochemistry of human tissue macroarrays. 3-O-SGal is expressed on N-glycans of many plasma and tissue glycoproteins, but recognition by O6 is often masked by sialic acid and thus exposed by treatment with neuraminidase. O6 recognizes many human tissues, consistent with expression of the cognate sulfotransferases (GAL3ST-2 and GAL3ST-3). The availability of O6 for exploring 3-O-SGal expression could lead to new biomarkers for disease and aid in understanding the functional roles of terminal modifications of glycans and relationships between terminal sulfation, sialylation and fucosylation.