RESUMO
Intestinal mucus forms the first line of defense against bacterial invasion while providing nutrition to support microbial symbiosis. How the host controls mucus barrier integrity and commensalism is unclear. We show that terminal sialylation of glycans on intestinal mucus by ST6GALNAC1 (ST6), the dominant sialyltransferase specifically expressed in goblet cells and induced by microbial pathogen-associated molecular patterns, is essential for mucus integrity and protecting against excessive bacterial proteolytic degradation. Glycoproteomic profiling and biochemical analysis of ST6 mutations identified in patients show that decreased sialylation causes defective mucus proteins and congenital inflammatory bowel disease (IBD). Mice harboring a patient ST6 mutation have compromised mucus barriers, dysbiosis, and susceptibility to intestinal inflammation. Based on our understanding of the ST6 regulatory network, we show that treatment with sialylated mucin or a Foxo3 inhibitor can ameliorate IBD.
Assuntos
Microbioma Gastrointestinal , Doenças Inflamatórias Intestinais , Sialiltransferases/genética , Animais , Homeostase , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Camundongos , Muco/metabolismo , Sialiltransferases/metabolismo , SimbioseRESUMO
Glycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammals use RNA as a third scaffold for glycosylation. Using a battery of chemical and biochemical approaches, we found that conserved small noncoding RNAs bear sialylated glycans. These "glycoRNAs" were present in multiple cell types and mammalian species, in cultured cells, and in vivo. GlycoRNA assembly depends on canonical N-glycan biosynthetic machinery and results in structures enriched in sialic acid and fucose. Analysis of living cells revealed that the majority of glycoRNAs were present on the cell surface and can interact with anti-dsRNA antibodies and members of the Siglec receptor family. Collectively, these findings suggest the existence of a direct interface between RNA biology and glycobiology, and an expanded role for RNA in extracellular biology.
Assuntos
Membrana Celular/metabolismo , Polissacarídeos/metabolismo , RNA/metabolismo , Animais , Anticorpos/metabolismo , Sequência de Bases , Vias Biossintéticas , Linhagem Celular , Sobrevivência Celular , Humanos , Espectrometria de Massas , Ácido N-Acetilneuramínico/metabolismo , Poliadenilação , Polissacarídeos/química , RNA/química , RNA/genética , RNA não Traduzido/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Coloração e RotulagemRESUMO
The colony-stimulating factor 3 receptor (CSF3R) controls the growth of neutrophils, the most abundant type of white blood cell. In healthy neutrophils, signaling is dependent on CSF3R binding to its ligand, CSF3. A single amino acid mutation in CSF3R, T618I, instead allows for constitutive, ligand-independent cell growth and leads to a rare type of cancer called chronic neutrophilic leukemia. However, the disease mechanism is not well understood. Here, we investigated why this threonine to isoleucine substitution is the predominant mutation in chronic neutrophilic leukemia and how it leads to uncontrolled neutrophil growth. Using protein domain mapping, we demonstrated that the single CSF3R domain containing residue 618 is sufficient for ligand-independent activity. We then applied an unbiased mutational screening strategy focused on this domain and found that activating mutations are enriched at sites normally occupied by asparagine, threonine, and serine residues-the three amino acids which are commonly glycosylated. We confirmed glycosylation at multiple CSF3R residues by mass spectrometry, including the presence of GalNAc and Gal-GalNAc glycans at WT threonine 618. Using the same approach applied to other cell surface receptors, we identified an activating mutation, S489F, in the interleukin-31 receptor alpha chain. Combined, these results suggest a role for glycosylated hotspot residues in regulating receptor signaling, mutation of which can lead to ligand-independent, uncontrolled activity and human disease.
Assuntos
Leucemia Neutrofílica Crônica , Humanos , Leucemia Neutrofílica Crônica/diagnóstico , Leucemia Neutrofílica Crônica/genética , Leucemia Neutrofílica Crônica/metabolismo , Glicosilação , Ligantes , Mutação , Receptores de Fator Estimulador de Colônias/genética , Receptores de Fator Estimulador de Colônias/metabolismo , Treonina/metabolismo , Fatores Estimuladores de Colônias/genética , Fatores Estimuladores de Colônias/metabolismoRESUMO
Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of healthy and disease-driven biological functions. Previously, we developed a mucin-selective enrichment strategy by employing a catalytically inactive mucinase (StcE) conjugated to a solid support. While this method was effective, it suffered from low throughput and high sample requirements. Further, the elution step required boiling in SDS, thus necessitating an in-gel digest with trypsin. Here, we introduce innovative elution conditions amenable to mucinase digestion and downstream analysis using mass spectrometry. This increased throughput and lowered sample input while maintaining mucin selectivity and enhancing the glycopeptide signal. We then benchmarked this technique against different O-glycan binding moieties for their ability to enrich mucins from various cell lines and human serum. Overall, the new method outperformed our previous procedure and all of the other enrichment techniques tested. This allowed for the effective isolation of more mucin-domain glycoproteins, resulting in a high number of O-glycopeptides, thus enhancing our ability to analyze the mucinome.
Assuntos
Glicoproteínas , Mucinas , Humanos , Mucinas/química , Espectrometria de Massas , Glicosilação , Glicopeptídeos/químicaRESUMO
Depression is a prevalent psychological condition with limited treatment options. While its etiology is multifactorial, both chronic stress and changes in microbiome composition are associated with disease pathology. Stress is known to induce microbiome dysbiosis, defined here as a change in microbial composition associated with a pathological condition. This state of dysbiosis is known to feedback on depressive symptoms. While studies have demonstrated that targeted restoration of the microbiome can alleviate depressive-like symptoms in mice, translating these findings to human patients has proven challenging due to the complexity of the human microbiome. As such, there is an urgent need to identify factors upstream of microbial dysbiosis. Here we investigate the role of mucin 13 as an upstream mediator of microbiome composition changes in the context of stress. Using a model of chronic stress, we show that the glycocalyx protein, mucin 13, is selectively reduced after psychological stress exposure. We further demonstrate that the reduction of Muc13 is mediated by the Hnf4 transcription factor family. Finally, we determine that deleting Muc13 is sufficient to drive microbiome shifts and despair behaviors. These findings shed light on the mechanisms behind stress-induced microbial changes and reveal a novel regulator of mucin 13 expression.
Assuntos
Depressão , Disbiose , Microbioma Gastrointestinal , Estresse Psicológico , Animais , Masculino , Camundongos , Comportamento Animal/fisiologia , Depressão/metabolismo , Depressão/microbiologia , Disbiose/metabolismo , Disbiose/microbiologia , Microbioma Gastrointestinal/fisiologia , Fator 4 Nuclear de Hepatócito/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucinas/metabolismo , Estresse Psicológico/metabolismo , Estresse Psicológico/microbiologiaRESUMO
Glyco-immune checkpoint receptors, molecules that inhibit immune cell activity following binding to glycosylated cell-surface antigens, are emerging as attractive targets for cancer immunotherapy. Defining biologically relevant ligands that bind and activate such receptors, however, has historically been a significant challenge. Here, we present a CRISPRi genomic screening strategy that allowed unbiased identification of the key genes required for cell-surface presentation of glycan ligands on leukemia cells that bind the glyco-immune checkpoint receptors Siglec-7 and Siglec-9. This approach revealed a selective interaction between Siglec-7 and the mucin-type glycoprotein CD43. Further work identified a specific N-terminal glycopeptide region of CD43 containing clusters of disialylated O-glycan tetrasaccharides that form specific Siglec-7 binding motifs. Knockout or blockade of CD43 in leukemia cells relieves Siglec-7-mediated inhibition of immune killing activity. This work identifies a potential target for immune checkpoint blockade therapy and represents a generalizable approach to dissection of glycan-receptor interactions in living cells.
Assuntos
Antígenos de Diferenciação Mielomonocítica/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genoma Humano , Lectinas/metabolismo , Polissacarídeos/metabolismo , Motivos de Aminoácidos , Antígenos de Diferenciação Mielomonocítica/química , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Glicopeptídeos/metabolismo , Humanos , Sinapses Imunológicas/metabolismo , Células Matadoras Naturais/metabolismo , Lectinas/química , Leucossialina/química , Leucossialina/metabolismo , Ligantes , Ligação ProteicaRESUMO
Interleukin (IL)-22 is a cytokine that plays a critical role in intestinal epithelial homeostasis. Its downstream functions are mediated through interaction with the heterodimeric IL-22 receptor and subsequent activation of signal transducer and activator of transcription 3 (STAT3). IL-22 signaling can induce transcription of genes necessary for intestinal epithelial cell proliferation, tissue regeneration, tight junction fortification, and antimicrobial production. Recent studies have also implicated IL-22 signaling in the regulation of intestinal epithelial fucosylation in mice. However, whether IL-22 regulates intestinal fucosylation in human intestinal epithelial cells and the molecular mechanisms that govern this process are unknown. Here, in experiments performed in human cell lines and human-derived enteroids, we show that IL-22 signaling regulates expression of the B3GNT7 transcript, which encodes a ß1-3-N-acetylglucosaminyltransferase that can participate in the synthesis of poly-N-acetyllactosamine (polyLacNAc) chains. Additionally, we find that IL-22 signaling regulates levels of the α1-3-fucosylated Lewis X (Lex) blood group antigen, and that this glycan epitope is primarily displayed on O-glycosylated intestinal epithelial glycoproteins. Moreover, we show that increased expression of B3GNT7 alone is sufficient to promote increased display of Lex-decorated carbohydrate glycan structures primarily on O-glycosylated intestinal epithelial glycoproteins. Together, these data identify B3GNT7 as an intermediary in IL-22-dependent induction of fucosylation of glycoproteins and uncover a novel role for B3GNT7 in intestinal glycosylation.
Assuntos
Células Epiteliais , Glicoproteínas , Interleucinas , Mucosa Intestinal , N-Acetilglucosaminiltransferases , Células Epiteliais/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Mucosa Intestinal/metabolismo , N-Acetilglucosaminiltransferases/biossíntese , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/metabolismo , Interleucina 22RESUMO
Antibodies against posttranslational modifications (PTMs) such as lysine acetylation, ubiquitin remnants, or phosphotyrosine have resulted in significant advances in our understanding of the fundamental roles of these PTMs in biology. However, the roles of a number of PTMs remain largely unexplored due to the lack of robust enrichment reagents. The addition of N-acetylglucosamine to serine and threonine residues (O-GlcNAc) by the O-GlcNAc transferase (OGT) is a PTM implicated in numerous biological processes and disease states but with limited techniques for its study. Here, we evaluate a new mixture of anti-O-GlcNAc monoclonal antibodies for the immunoprecipitation of native O-GlcNAcylated peptides from cells and tissues. The anti-O-GlcNAc antibodies display good sensitivity and high specificity toward O-GlcNAc-modified peptides and do not recognize O-GalNAc or GlcNAc in extended glycans. Applying this antibody-based enrichment strategy to synaptosomes from mouse brain tissue samples, we identified over 1300 unique O-GlcNAc-modified peptides and over 1000 sites using just a fraction of sample preparation and instrument time required in other landmark investigations of O-GlcNAcylation. Our rapid and robust method greatly simplifies the analysis of O-GlcNAc signaling and will help to elucidate the role of this challenging PTM in health and disease.
Assuntos
Anticorpos Monoclonais/imunologia , Glicopeptídeos/imunologia , N-Acetilglucosaminiltransferases/imunologia , Animais , Encéfalo , Camundongos , Células-Tronco Embrionárias MurinasRESUMO
Densely O-glycosylated mucin domains are found in a broad range of cell surface and secreted proteins, where they play key physiological roles. In addition, alterations in mucin expression and glycosylation are common in a variety of human diseases, such as cancer, cystic fibrosis, and inflammatory bowel diseases. These correlations have been challenging to uncover and establish because tools that specifically probe mucin domains are lacking. Here, we present a panel of bacterial proteases that cleave mucin domains via distinct peptide- and glycan-based motifs, generating a diverse enzymatic toolkit for mucin-selective proteolysis. By mutating catalytic residues of two such enzymes, we engineered mucin-selective binding agents with retained glycoform preferences. StcEE447D is a pan-mucin stain derived from enterohemorrhagic Escherichia coli that is tolerant to a wide range of glycoforms. BT4244E575A derived from Bacteroides thetaiotaomicron is selective for truncated, asialylated core 1 structures commonly associated with malignant and premalignant tissues. We demonstrated that these catalytically inactive point mutants enable robust detection and visualization of mucin-domain glycoproteins by flow cytometry, Western blot, and immunohistochemistry. Application of our enzymatic toolkit to ascites fluid and tissue slices from patients with ovarian cancer facilitated characterization of patients based on differences in mucin cleavage and expression patterns.
Assuntos
Mucinas/análise , Polissacarídeo-Liases/metabolismo , Adenocarcinoma/química , Motivos de Aminoácidos , Western Blotting , Feminino , Citometria de Fluxo , Humanos , Neoplasias Ovarianas/química , Mutação Puntual , Polissacarídeo-Liases/química , Polissacarídeo-Liases/genéticaRESUMO
Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.
Assuntos
Acetilgalactosamina/metabolismo , Glicoproteínas/metabolismo , Acetilgalactosamina/química , Regulação Enzimológica da Expressão Gênica , Glicosilação , Humanos , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Especificidade por Substrato , Uridina Difosfato N-Acetilgalactosamina/químicaRESUMO
Currently approved immune checkpoint inhibitor therapies targeting the PD-1 and CTLA-4 receptor pathways are powerful treatment options for certain cancers; however, most patients across cancer types still fail to respond. Consequently, there is interest in discovering and blocking alternative pathways that mediate immune suppression. One such mechanism is an upregulation of sialoglycans in malignancy, which has been recently shown to inhibit immune cell activation through multiple mechanisms and therefore represents a targetable glycoimmune checkpoint. Since these glycans are not canonically druggable, we designed an αHER2 antibody-sialidase conjugate that potently and selectively strips diverse sialoglycans from breast cancer cells. In syngeneic breast cancer models, desialylation enhanced immune cell infiltration and activation and prolonged the survival of mice, an effect that was dependent on expression of the Siglec-E checkpoint receptor found on tumor-infiltrating myeloid cells. Thus, antibody-sialidase conjugates represent a promising modality for glycoimmune checkpoint therapy.
Assuntos
Imunoterapia/métodos , Melanoma Experimental/terapia , Neuraminidase/imunologia , Polissacarídeos/química , Receptor ErbB-2/química , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/imunologia , Aloenxertos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Linhagem Celular Tumoral , Humanos , Hidrólise , Imunoconjugados/química , Imunoconjugados/metabolismo , Imunoconjugados/farmacologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/mortalidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Terapia de Alvo Molecular , Neuraminidase/química , Neuraminidase/genética , Polissacarídeos/imunologia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/química , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Análise de Sobrevida , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
Epithelial surfaces throughout the body are coated by mucins, a class of proteins carrying domains characterized by a high density of O-glycosylated serine and threonine residues. The resulting mucosal layers form crucial host-microbe interfaces that prevent the translocation of microbes while also selecting for distinct bacteria via the presented glycan repertoire. The intricate interplay between mucus production and breakdown thus determines the composition of the microbiota maintained within these mucosal environments, which can have a large influence on the host during both homeostasis and disease. Most research to date on mucus breakdown has focused on glycosidases that trim glycan structures to release monosaccharides as a source of nutrients. More recent work has uncovered the existence of mucin-type O-glycosylation-dependent proteases that are secreted by pathogens, commensals, and mutualists to facilitate mucosal colonization and penetration. Additionally, immunoglobulin A (IgA) proteases promote bacterial colonization in the presence of neutralizing secretory IgA through selective cleavage of the heavily O-glycosylated hinge region. In this review, we summarize families of O-glycoproteases and IgA proteases, discuss known structural features, and review applications of these enzymes to glycobiology.
Assuntos
Proteínas de Bactérias/metabolismo , Metaloendopeptidases/metabolismo , Mucina-1/metabolismo , Mucinas/metabolismo , Sequência de Aminoácidos , Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Sequência de Carboidratos , Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Humanos , Metaloendopeptidases/química , Metaloendopeptidases/classificação , Metaloendopeptidases/genética , Mucina-1/química , Mucina-1/genética , Mucinas/química , Família Multigênica , Domínios Proteicos , Especificidade por SubstratoRESUMO
Mucin domains are densely O-glycosylated modular protein domains that are found in a wide variety of cell surface and secreted proteins. Mucin-domain glycoproteins are known to be key players in a host of human diseases, especially cancer, wherein mucin expression and glycosylation patterns are altered. Mucin biology has been difficult to study at the molecular level, in part, because methods to manipulate and structurally characterize mucin domains are lacking. Here, we demonstrate that secreted protease of C1 esterase inhibitor (StcE), a bacterial protease from Escherichia coli, cleaves mucin domains by recognizing a discrete peptide- and glycan-based motif. We exploited StcE's unique properties to improve sequence coverage, glycosite mapping, and glycoform analysis of recombinant human mucins by mass spectrometry. We also found that StcE digests cancer-associated mucins from cultured cells and from ascites fluid derived from patients with ovarian cancer. Finally, using StcE, we discovered that sialic acid-binding Ig-type lectin-7 (Siglec-7), a glycoimmune checkpoint receptor, selectively binds sialomucins as biological ligands, whereas the related receptor Siglec-9 does not. Mucin-selective proteolysis, as exemplified by StcE, is therefore a powerful tool for the study of mucin domain structure and function.
Assuntos
Antígenos CD/química , Antígenos de Diferenciação Mielomonocítica/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Lectinas/química , Metaloendopeptidases/química , Mucinas/química , Proteínas de Neoplasias/química , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/química , Motivos de Aminoácidos , Humanos , Espectrometria de Massas , Especificidade por SubstratoRESUMO
Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Most progress has occurred in the area of N-glycoproteomics. There is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and determine their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods in terms of identifications, indicating that ETD-based methods are not required for routine N-glycoproteomics even if they can generate higher quality spectra. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.
Assuntos
Glicopeptídeos , Espectrometria de Massas em Tandem , GlicosilaçãoRESUMO
The recently described O-glycoprotease OpeRATOR presents exciting opportunities for O-glycoproteomics. This bacterial enzyme purified from Akkermansia muciniphila cleaves N-terminally to serine and threonine residues that are modified with (preferably asialylated) O-glycans. This provides orthogonal cleavage relative to canonical proteases (e.g., trypsin) for improved O-glycopeptide characterization with tandem mass spectrometry (MS/MS). O-glycopeptides with a modified N-terminal residue, such as those generated by OpeRATOR, present several potential benefits, perhaps the most notable being de facto O-glycosite localization without the need of glycan-retaining fragments in MS/MS spectra. Indeed, O-glycopeptides modified exclusively at the N-terminus would enable O-glycoproteomic methods to rely solely on collision-based fragmentation rather than electron-driven dissociation because glycan-retaining peptide fragments would not be required for localization. The caveat is that modified peptides would need to reliably contain only a single O-glycosite. Here, we use methods that combine collision- and electron-based fragmentation to characterize the number of O-glycosites that are present in O-glycopeptides derived from the OpeRATOR digestion of four known O-glycoproteins. Our data show that over 50% of O-glycopeptides in our sample generated from combined digestion using OpeRATOR and trypsin contain multiple O-glycosites, indicating that collision-based fragmentation alone is not sufficient. Electron-based dissociation methods are necessary to capture the O-glycopeptide diversity present in OpeRATOR digestions.
Assuntos
Elétrons , Glicopeptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , Proteólise , Akkermansia/enzimologia , Sequência de Aminoácidos , Peptídeo Hidrolases/química , Espectrometria de Massas em TandemRESUMO
O-Linked α-N-acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). Despite proven disease relevance, correlating the activity of individual GalNAc-Ts with biological function remains challenging due to a lack of tools to probe their substrate specificity in a complex biological environment. Here, we develop a "bump-hole" chemical reporter system for studying GalNAc-T activity in vitro. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly synthesized collection of 20 (bumped) uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) analogs to identify enzyme-substrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme-substrate pairs. The approach was applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and -T2 that prefer nonglycosylated peptide substrates and GalNAcT-10 that prefers a preglycosylated peptide substrate. A detailed investigation of enzyme kinetics and specificities revealed the robustness of the approach to faithfully report on GalNAc-T activity and paves the way for studying substrate specificities in living systems.
Assuntos
Acetilgalactosamina/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Engenharia de Proteínas , Difosfato de Uridina/metabolismo , Acetilgalactosamina/química , Sequência de Aminoácidos , Domínio Catalítico , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Mutagênese , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/genética , Especificidade por Substrato , Difosfato de Uridina/química , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
The MHC class II (MHCII) processing pathway presents peptides derived from exogenous or membrane-bound proteins to CD4+ T cells. Several studies have shown that glycopeptides are necessary to modulate CD4+ T cell recognition, though glycopeptide structures in these cases are generally unknown. Here, we present a total of 93 glycopeptides from three melanoma cell lines and one matched EBV-transformed line with most found only in the melanoma cell lines. The glycosylation we detected was diverse and comprised 17 different glycoforms. We then used molecular modeling to demonstrate that complex glycopeptides are capable of binding the MHC and may interact with complementarity determining regions. Finally, we present the first evidence of disulfide-bonded peptides presented by MHCII. This is the first large scale study to sequence glyco- and disulfide bonded MHCII peptides from the surface of cancer cells and could represent a novel avenue of tumor activation and/or immunoevasion.
Assuntos
Regiões Determinantes de Complementaridade/química , Glicopeptídeos/química , Antígenos HLA-DR/química , Melanócitos/imunologia , Sequência de Aminoácidos , Sítios de Ligação , Sequência de Carboidratos , Linhagem Celular Tumoral , Regiões Determinantes de Complementaridade/imunologia , Cristalografia por Raios X , Dissulfetos/química , Dissulfetos/imunologia , Glicopeptídeos/genética , Glicopeptídeos/imunologia , Glicosilação , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Humanos , Melanócitos/patologia , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , TermodinâmicaRESUMO
Quantitative peptide-binding motifs of MHC class I alleles provide a valuable tool to efficiently identify putative T cell epitopes. Detailed information on equine MHC class I alleles is still very limited, and to date, only a single equine MHC class I allele, Eqca-1*00101 (ELA-A3 haplotype), has been characterized. The present study extends the number of characterized ELA class I specificities in two additional haplotypes found commonly in the Thoroughbred breed. Accordingly, we here report quantitative binding motifs for the ELA-A2 allele Eqca-16*00101 and the ELA-A9 allele Eqca-1*00201. Utilizing analyses of endogenously bound and eluted ligands and the screening of positional scanning combinatorial libraries, detailed and quantitative peptide-binding motifs were derived for both alleles. Eqca-16*00101 preferentially binds peptides with aliphatic/hydrophobic residues in position 2 and at the C-terminus, and Eqca-1*00201 has a preference for peptides with arginine in position 2 and hydrophobic/aliphatic residues at the C-terminus. Interestingly, the Eqca-16*00101 motif resembles that of the human HLA A02-supertype, while the Eqca-1*00201 motif resembles that of the HLA B27-supertype and two macaque class I alleles. It is expected that the identified motifs will facilitate the selection of candidate epitopes for the study of immune responses in horses.
Assuntos
Epitopos de Linfócito T/imunologia , Genes MHC Classe I , Antígenos de Histocompatibilidade Classe I/metabolismo , Fragmentos de Peptídeos/metabolismo , Motivos de Aminoácidos , Animais , Haplótipos , Antígenos de Histocompatibilidade Classe I/imunologia , Cavalos , Fragmentos de Peptídeos/imunologia , Ligação Proteica , Domínios ProteicosRESUMO
Glycosylation is an incredibly common and diverse post-translational modification that contributes widely to cellular health and disease. Mass spectrometry is the premier technique to study glycoproteins; however, glycoproteomics has lagged behind traditional proteomics due to the challenges associated with studying glycosylation. For instance, glycans dissociate by collision-based fragmentation, thus necessitating electron-based fragmentation for site-localization. The vast glycan heterogeneity leads to lower overall abundance of each glycopeptide, and often, ion suppression is observed. One of the biggest issues facing glycoproteomics is the lack of reliable software for analysis, which necessitates manual validation and serves as a massive bottleneck in data processing. Here, I will discuss each of these challenges and some ways in which the field is attempting to address them, along with perspectives on how I believe we should move forward.