RESUMO
The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has impacted public health globally. As the glycosylation of viral envelope glycoproteins is strongly associated with their immunogenicity, intensive studies have been conducted on the glycans of the glycoprotein of SARS-CoV-2, the spike (S) protein. Here, we conducted intensive glycoproteomic analyses of the SARS-CoV-2 S protein of ancestral and γ-variant strains using a combinatorial approach with two different technologies: mass spectrometry (MS) and lectin microarrays (LMA). Our unique MS1-based glycoproteomic technique, Glyco-RIDGE, in addition to MS2-based Byonic search, identified 1448 (ancestral strain) and 1785 (γ-variant strain) site-specific glycan compositions, respectively. Asparagine at amino acid position 20 (N20) is mainly glycosylated within two successive potential glycosylation sites, N17 and N20, of the γ-variant S protein; however, we found low-frequency glycosylation at N17. Our novel approaches, glycostem mapping and glycoleaf scoring, also illustrate the moderately branched/extended, highly fucosylated, and less sialylated natures of the glycoforms of S proteins. Subsequent LMA analysis emphasized the intensive end-capping of glycans by Lewis fucoses, which complemented the glycoproteomic features. These results illustrate the high-resolution glycoproteomic features of the SARS-CoV-2 S protein, contributing to vaccine design and understanding of viral protein synthesis.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/química , Lectinas , Polissacarídeos/química , Espectrometria de MassasRESUMO
The Human Glycome Atlas (HGA) Project was launched in April 2023, spearheaded by three Japanese institutes: the Tokai National Higher Education and Research System, the National Institutes of Natural Sciences, and Soka University. This was the first time that a field in the life sciences was adopted by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) for a Large-scale Academic Frontiers Promotion Project. This project aims to construct a knowledgebase of human glycans and glycoproteins as a standard for the human glycome. A high-throughput pipeline for comprehensively analyzing 20,000 blood samples in its first five years is planned, at which time an access-controlled version of a human glycomics knowledgebase, called TOHSA, will be released. By the end of the final tenth year, TOHSA will provide a central resource linking human glycan data with other omics data including disease-related information.
RESUMO
Protein O-glycosylation, also known as mucin-type O-glycosylation, is one of the most abundant glycosylation in mammalian cells. It is initially catalyzed by a family of polypeptide GalNAc transferases (ppGalNAc-Ts). The trimeric spike protein (S) of SARS-CoV-2 is highly glycosylated and facilitates the virus's entry into host cells and membrane fusion of the virus. However, the functions and relationship between host ppGalNAc-Ts and O-glycosylation on the S protein remain unclear. Herein, we identify 15 O-glycosites and 10 distinct O-glycan structures on the S protein using an HCD-product-dependent triggered ETD mass spectrometric analysis. We observe that the isoenzyme T6 of ppGalNAc-Ts (ppGalNAc-T6) exhibits high O-glycosylation activity for the S protein, as demonstrated by an on-chip catalytic assay. Overexpression of ppGalNAc-T6 in HEK293 cells significantly enhances the O-glycosylation level of the S protein, not only by adding new O-glycosites but also by increasing O-glycan heterogeneity. Molecular dynamics simulations reveal that O-glycosylation on the protomer-interface regions, modified by ppGalNAc-T6, potentially stabilizes the trimeric S protein structure by establishing hydrogen bonds and non-polar interactions between adjacent protomers. Furthermore, mutation frequency analysis indicates that most O-glycosites of the S protein are conserved during the evolution of SARS-CoV-2 variants. Taken together, our finding demonstrate that host O-glycosyltransferases dynamically regulate the O-glycosylation of the S protein, which may influence the trimeric structural stability of the protein. This work provides structural insights into the functional role of specific host O-glycosyltransferases in regulating the O-glycosylation of viral envelope proteins.
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Wisteria floribunda agglutinin (WFA)-reactive ceruloplasmin (CP) is a candidate marker for ovarian clear cell carcinoma (CCC) reported in our previous paper. Herein, a new measurement system was developed to investigate its potential as a serum marker for CCC. Site-specific glycome analysis using liquid chromatography/mass spectrometry showed that WFA-CP from CCC binds to WFA via the GalNAcß1,4GlcNAc (LDN) structure. We used mutant recombinant WFA (rWFA), which has a high specificity to the LDN structure, instead of native WFA, to increase the specificity of the serum sample measurement. To improve the sensitivity, we used a surface plasmon field-enhanced fluorescence spectroscopy immunoassay system, which is approximately 100 times more sensitive than the conventional sandwich enzyme-linked immunosorbent assay system. With these two improvements, the specificity and sensitivity of the serum rWFA-CP measurement were dramatically improved, clearly distinguishing CCC from endometrioma, from which CCC originates. This rWFA-CP assay can be used clinically for the serodiagnosis of early-stage CCC, which is difficult to detect with existing serum markers.
Assuntos
Carcinoma , Endometriose , Antígenos de Neoplasias , Biomarcadores , Ceruloplasmina/metabolismo , Endometriose/diagnóstico , Humanos , Cirrose Hepática/diagnóstico , Lectinas de Plantas/química , Receptores de N-Acetilglucosamina/metabolismoRESUMO
Isohemagglutinin assays employing red blood cells (RBCs) are the most common assays used to measure antibody titer in ABO-incompatible kidney transplantation (ABOi KTx). However, ABO antigens expressed on RBCs are not identical to those of kidney and antibody titers do not always correlate with clinical outcome. We previously reported that CD31 was the main protein linked to ABO antigens on kidney endothelial cells (KECs), which was different from those on RBCs. We developed a new method to measure antibody titer using a microarray of recombinant CD31 (rCD31) linked to ABO antigens (CD31-ABO microarray). Mass spectrometry analysis suggested that rCD31 and native CD31 purified from human kidney had similar ABO glycan. To confirm clinical use of CD31-ABO microarray, a total of 252 plasma samples including volunteers, hemodialysis patients, and transplant recipients were examined. In transplant recipients, any initial IgG or IgM antibody intensity >30,000 against the donor blood type in the CD31-ABO microarray showed higher sensitivity, specificity, positive predictive value, and negative predictive value of AABMR, compared to isohemagglutinin assays. Use of a CD31-ABO microarray to determine antibody titer specifically against ABO antigens expressed on KECs will contribute to precisely predicting AABMR or preventing over immunosuppression following ABOi KTx.
Assuntos
Transplante de Rim , Sistema ABO de Grupos Sanguíneos , Anticorpos , Incompatibilidade de Grupos Sanguíneos , Carboidratos , Células Endoteliais , Rejeição de Enxerto , Humanos , Transplante de Rim/métodos , Molécula-1 de Adesão Celular Endotelial a PlaquetasRESUMO
The extent of liver fibrosis predicts prognosis and is important for determining treatment strategies for chronic hepatitis. During the fibrosis progression, serum levels of Mac2 binding protein (M2BP) increase and the N-glycan structure changes to enable binding to Wisteria floribunda agglutinin (WFA) lectin. As a novel diagnostic marker, glycosylation isomer of M2BP (M2BPGi) has been developed. However, its glycan structures recognized by WFA are unclear. In this study, we analyzed site-specific N-glycan structures of serum M2BP using Glyco-RIDGE (Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile) method. We evaluated five sample types: (1) M2BP immunoprecipitated from normal healthy sera (NHS-IP(+)), (2) M2BP immunoprecipitated from sera of patients with liver cirrhosis (stage 4; F4-IP(+)), (3) M2BP captured with WFA from serum of patients with liver cirrhosis (stage 4; F4-WFA(+)), (4) recombinant M2BP produced by HEK293 cells (rM2BP) and (5) WFA-captured rM2BP (rM2BP-WFA(+)). In NHS-IP(+) M2BP, bi-antennary N-glycan was the main structure, and LacNAc extended to its branches. In F4-IP(+) M2BP, many branched structures, including tri-antennary and tetra-antennary N-glycans, were found. F4-WFA(+) showed a remarkable increase in branched structures relative to the quantity before enrichment. In recombinant M2BP, both no sialylated-LacdiNAc and -branched LacNAc structures were emerged. The LacdiNAc structure was not found in serum M2BP. Glycosidase-assisted HISCL assays suggest that reactivity with WFA of both serum and recombinant M2BP depends on unsialylated and branched LacNAc and in part of recombinant depends on LacdiNAc. On M2BPGi, the highly branched LacNAc, probably dense cluster of LacNAc, would be recognized by WFA.
Assuntos
Antígenos de Neoplasias/química , Biomarcadores Tumorais/química , Cirrose Hepática/sangue , Lectinas de Plantas/química , Polissacarídeos/química , Receptores de N-Acetilglucosamina/química , Antígenos de Neoplasias/sangue , Biomarcadores Tumorais/sangue , Células HEK293 , Voluntários Saudáveis , Humanos , Lectinas de Plantas/sangue , Polissacarídeos/sangue , Análise Serial de Proteínas , Receptores de N-Acetilglucosamina/sangue , Proteínas Recombinantes/sangue , Proteínas Recombinantes/químicaRESUMO
The type-I LacdiNAc (LDN; GalNAcß1-3GlcNAc) has rarely been observed in mammalian cells except in the O-glycan of α-dystroglycan, in contrast to type-II LDN structures (GalNAcß1-4GlcNAc) in N- and O-glycans that are present in many mammalian glycoproteins, such as pituitary and hypothalamic hormones. Although a ß1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2; type-I LDN synthase) has been cloned, the function of type-I LDN in mammalian cells is still unclear, as its carrier protein(s) has not been identified. In this study, using HeLa cells, we demonstrate that inhibition of Golgi-resident glycosyltransferase increases the abundance of B3GALNT2-synthesized type-I LDN structures, recognized by Wisteria floribunda agglutinin (WFA). Using isotope-coded glycosylation site-specific tagging (IGOT)-LC/MS analysis of Lec8 Chinese hamster cells lacking galactosylation and of cells transfected with the B3GALNT2 gene, we identified the glycoproteins that carry B3GALNT2-generated type-I LDN in their N-glycans. Our results further revealed that LDN presence on low-density lipoprotein receptor-related protein 1 and nicastrin depends on B3GALNT2, indicating the occurrence of type-I LDN in vivo in mammalian cells. Our analysis also uncovered that most of the identified glycoproteins localize to intracellular organelles, particularly to the endoplasmic reticulum. Whereas B4GALNT3 and B4GALNT4 synthesized LDN on extracellular glycoproteins, B3GALNT2 primarily transferred LDN to intracellular glycoproteins, thereby clearly delineating proteins that carry type-I or type-II LDNs. Taken together, our results indicate the presence of mammalian glycoproteins carrying type-I LDN on N-glycans and suggest that type-I and type-II LDNs have different roles in vivo.
Assuntos
Glicoproteínas/química , N-Acetilgalactosaminiltransferases/metabolismo , Animais , Cricetinae , Glicoproteínas/biossíntese , Glicosilação , Células HeLa , Humanos , Organelas/metabolismo , Lectinas de Plantas/metabolismo , Receptores de N-Acetilglucosamina/metabolismoRESUMO
BACKGROUND: One of the pathognomonic features of asthma is epithelial hyperproduction of mucus, which is composed of a series of glycoproteins; however, it remains unclear how glycosylation is induced in lung epithelial cells from asthmatic patients and how glycan residues play a role in the pathogenesis of asthma. OBJECTIVE: The objective of this study was to explore comprehensive epithelial glycosylation status induced by allergic inflammation and reveal its possible role in the pathogenesis of asthma. METHODS: We evaluated the glycosylation status of lung epithelium using a lectin microarray. We next searched for molecular mechanisms underlying epithelial glycosylation. We also examined whether epithelial glycosylation is involved in induction of allergic inflammation. RESULTS: On allergen inhalation, lung epithelial cells were heavily α(1,2)fucosylated by fucosyltransferase 2 (Fut2), which was induced by the IL-13-signal transducer and activator of transcription 6 pathway. Importantly, Fut2-deficient (Fut2-/-) mice, which lacked lung epithelial fucosylation, showed significantly attenuated eosinophilic inflammation and airway hyperresponsiveness in house dust mite (HDM)-induced asthma models. Proteome analyses and immunostaining of the HDM-challenged lung identified that complement C3 was accumulated in fucosylated areas. Indeed, Fut2-/- mice showed significantly reduced levels of C3a and impaired accumulation of C3a receptor-expressing monocyte-derived dendritic cells in the lung on HDM challenge. CONCLUSION: Fut2 induces epithelial fucosylation and exacerbates airway inflammation in asthmatic patients in part through C3a production and monocyte-derived dendritic cell accumulation in the lung.
Assuntos
Asma/imunologia , Células Epiteliais/imunologia , Fucosiltransferases/imunologia , Pulmão/imunologia , Mucosa Respiratória/imunologia , Alérgenos/imunologia , Animais , Complemento C3/imunologia , Modelos Animais de Doenças , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Eosinofilia Pulmonar/imunologia , Pyroglyphidae/imunologia , Células Th17/imunologia , Células Th2/imunologia , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
Cardiac fibrosis is a typical phenomenon in failing hearts for most cardiac diseases, including dilated cardiomyopathy (DCM), and its specific detection and quantification are crucial for the analysis of cardiac remodeling. Since cardiac fibrosis is characterized by extensive remodeling of the myocardial extracellular matrix (ECM), in which glycoproteins are the major components, we assumed that fibrosis-related alterations in the cardiac glycome and glycoproteome would be suitable targets for the detection of cardiac fibrosis. Here, we compared protein glycosylation between heart tissues of normal and DCM model mice by laser microdissection-assisted lectin microarray. Among 45 lectins, Wisteria floribunda agglutinin (WFA) was selected as the most suitable lectin for staining cardiac fibrotic tissues. Although the extent of WFA staining was highly correlated (r > 0.98) with that of picrosirius red staining, a common collagen staining method, WFA did not bind to collagen fibers. Further histochemical analysis with N-glycosidase revealed that WFA staining of fibrotic tissues was attributable to the binding of WFA to N-glycoproteins. Using a mass spectrometry-based approach, we identified WFA-binding N-glycoproteins expressed in DCM hearts, many of which were fibrogenesis-related ECM proteins, as expected. In addition, the identified glycoproteins carrying WFA-binding N-glycans were detected only in DCM hearts, suggesting their cooperative glycosylation alterations with disease progression. Among these WFA-binding ECM N-glycoproteins, co-localization of the collagen α6(VI) chain protein and WFA staining in cardiac tissue sections was confirmed with a double-staining analysis. Collectively, these results indicate that WFA staining is more suitable for the quantitative assessment of cardiac fibrogenic activity than current collagen staining methods. Furthermore, given that plasma WFA-binding glycoprotein levels were significantly correlated with the echocardiographic parameters for left ventricular remodeling, cardiac WFA-binding glycoproteins are candidate circulating glyco-biomarkers for the quantification and monitoring of cardiac fibrogenesis.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Lectinas de Plantas , Receptores de N-Acetilglucosamina , Coloração e Rotulagem/métodos , Animais , Colágeno Tipo VI/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Proteínas da Matriz Extracelular/metabolismo , Fibrose , Glicoproteínas/metabolismo , Glicosilação , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Miocárdio/metabolismo , Miocárdio/patologia , Lectinas de Plantas/farmacocinética , Polissacarídeos/metabolismo , Ligação Proteica , Espectrometria de Massas em TandemRESUMO
A quantitative description of glyco-alteration/differences in diseases can lead to the development of a diagnostic agent for use in vitro to monitor the degree of change in target glycoproteins. Analytical systems have been developed along with the progress of omics-oriented technologies. For clinical implementation, their full automation is required with an apparatus that is simple to operate. Here, we report an automatic analysis system for quantitative characterization of glyco-alteration/differences that depends on the unique strategy of "bead arrays in a single tip." The alternative lectin array can obtain a minimum characterization of the glycan profile for nanogram quantities of an endogenous glycoprotein. A simple autopipetting robot produces the precise chemiluminescence detection of glycan-lectin interactions with a wide dynamic range that is superior to fluorescence-based lectin arrays. The tip-based array format enables automatic glycan profiling from sample pretreatment to detection with low variation and linear detection, which may facilitate the use of this lectin array in clinical practice.
Assuntos
Automação , Glicoproteínas/análise , Lectinas/química , Fluorescência , Espectrometria de Massas , Análise em MicrossériesRESUMO
OBJECTIVE: An increased proportion of circulating follicular helper T (Tfh) cells was reported in rheumatoid arthritis (RA), but it remains uncertain how Tfh cells affect antibody hyposialylation. We investigated the regulation of autoantibody hyposialylation by Tfh cells in RA using murine model. METHODS: Behaviours of Tfh cells and their function on B cell promotion were analysed. Change of arthritogenicity and sialylation of autoantibodies during the course of arthritis was examined by mass spectrometry. Tfh-mediated regulation of hyposialylation was investigated, and the responsible cell surface molecule was specified both in vitro and in vivo. The relation between circulating Tfh cells and hyposialylation was analysed in patients with RA. RESULTS: An increase in Tfh, particularly interleukin-17 producing Tfh (Tfh17) cells, at the onset of arthritis and their enhancement of autoantibody production were found. Autoantibodies at the onset phase demonstrated stronger inflammatory properties than those at the resolution phase, and mass spectrometric analysis revealed their difference in sialylation. In vitro coculture showed enhanced hyposialylation by the Tfh cells via OX40, which was highly expressed in the Tfh and Tfh17 cells. Blockade of OX40 prevented the development of arthritis with reduction in Tfh17 cells and recovery of autoantibody sialylation. Analysis of patients with RA showed abundance of OX40-overexpressing Tfh17 cells, and their proportion correlated negatively with the expression of α2,6-sialyltransferase 1, an enzyme responsible for sialylation. CONCLUSIONS: OX40 expressed on Tfh cells can regulate autoantibody sialylation and play a crucial role in the development of autoimmune arthritis.
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Artrite Reumatoide/imunologia , Autoanticorpos/metabolismo , Receptores OX40/metabolismo , Ácidos Siálicos/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Modelos Animais de Doenças , Imunidade Celular/genética , Masculino , Camundongos , Camundongos Endogâmicos DBARESUMO
Glycoproteomics is an important recent advance in the field of glycoscience. In glycomics, glycan structures are comprehensively analyzed after glycans are released from glycoproteins. However, a major limitation of glycomics is the lack of insight into glycoprotein functions. The Biology/Disease-driven Human Proteome Project has a particular focus on biological and medical applications. Glycoproteomics technologies aimed at obtaining a comprehensive understanding of intact glycoproteins, i.e., the kind of glycan structures that are attached to particular amino acids and proteins, have been developed. This Review focuses on the recent progress of the technologies and their applications. First, the methods for large-scale identification of both N- and O-glycosylated proteins are summarized. Next, the progress of analytical methods for intact glycopeptides is outlined. MS/MS-based methods were developed for improving the sensitivity and speed of the mass spectrometer, in parallel with the software for complex spectrum assignment. In addition, a unique approach to identify intact glycopeptides using MS1-based accurate masses is introduced. Finally, as an advance of glycomics, two approaches to provide the spatial distribution of glycans in cells are described, i.e., MS imaging and lectin microarray. These methods allow rapid glycomic profiling of different types of biological samples and thus facilitate glycoproteomics.
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Glicoproteínas/análise , Proteômica/tendências , Linhagem Celular , Glicômica/métodos , Glicosilação , Humanos , Polissacarídeos/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosAssuntos
Carboidratos/química , Carboidratos/fisiologia , Glicômica , Internet , Bases de Dados FactuaisRESUMO
Secretogranin III (SgIII) is a member of the chromogranin/secretogranin family of neuroendocrine secretory proteins. Granins are expressed in endocrine and neuroendocrine cells and subsequently processed into bioactive hormones. Although granin-derived peptide expression is correlated with neuroendocrine carcinomas, little is known about SgIII. We previously identified SgIII by a comparative glycoproteomics approach for elucidation of glycobiomarker candidates in lung carcinoma. Here, we examined the expression, secretion, and glycosylation of SgIII to identify novel biomarkers of small cell lung carcinoma (SCLC). In comparative immunohistochemical analysis and secretion profiling, SgIII was observed in all types of lung cancer. However, low-molecular-weight SgIII (short-form SgIII) was specifically found in SCLC culture medium. Glycoproteomics analysis showed that a fucosylated glycan was attached to the first of three potential N-glycosylation sites and an unfucosylated glycan was detected on the second site; however, the third site was not glycosylated. Next, we performed lectin capture with a fucose-binding lectin and detected short-form SgIII specifically in the sera of patients with SCLC. The results suggested an association between the fucosylated glycoform of short-form SgIII and SCLC. Thus, fucosylated short-form SgIII may be a valuable biomarker for SCLC and could be used to monitor development of the disease. All MS data are available via ProteomeXchange and jPOST with identifiers PXD007626 and JPST000313, respectively.
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Cromograninas/sangue , Carcinoma de Pequenas Células do Pulmão/sangue , Biomarcadores , Células Cultivadas , Cromograninas/química , Cromograninas/metabolismo , Glicoproteínas/análise , Glicosilação , Humanos , Espectrometria de Massas , Carcinoma de Pequenas Células do Pulmão/diagnósticoRESUMO
Wisteria floribunda agglutinin (WFA) is a useful probe for distinguishing glycan structural alterations in diseases such as intrahepatic bile duct carcinoma and hepatic fibrosis; however, the gene encoding WFA has not been identified. Here, we identified the gene encoding WFA, and recombinant WFA (rWFA) was expressed in Escherichia coli and purified. The natural complementary DNA sequence obtained from wisteria seeds contained an open reading frame of 861 nucleotides encoding a WFA precursor, which included a hydrophobic signal peptide at the N-terminus, a propeptide at the C-terminus and a single cysteine (Cys) residue for dimer formation. We characterized the natural and rWFA by the glycoconjugate microarray and frontal affinity chromatography. rWFA exhibited glycan binding specificity similar to that of natural WFA: both bound to Gal- and N-acetylgalactosamine (GalNAc)-terminated glycans. Moreover, the engineered WFA with an amino acid substitution in Cys-272 yielded a recombinant monomeric lectin with limited binding specificity but wild-type affinity for GalNAc-terminated glycans, specifically GalNAcß1,4GlcNAc. Thus, this engineered lectin may be applied to highly sensitive biomarker detection.
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Cypridina noctiluca luciferase has been utilized for biochemical and molecular biological applications, including bioluminescent enzyme immunoassays, far-red luminescence imaging, and high-throughput reporter assays. Some of these applications require a large amount of purified luciferase. However, conventional protein expression systems are not capable of producing sufficient quantities of protein with a high quality and purity without laborious and costly purification processes. To improve the productivity and expand the breadth of possibilities for Cypridina luciferase applications, we employed a variety of secretion expression systems, including yeast, mammalian cells, and silk worms. In this study, we established a simple production procedure using plant cell cultures. The plant cell culture BY-2 efficiently secreted luciferase, which was easily purified using a simple one-step ion-exchange chromatography method. The production yield was 20-30 mg of luciferase per liter of culture medium, and its Km for the luciferin (0.45 µM) was similar to that of the native protein. Additionally, we characterized its glycosylation pattern and confirmed that the two potential N-glycosylation sites were modified with plant-type oligosaccharide chains. Interestingly, the oligosaccharide chains could be trimmed without any detectable decrease in recombinant protein activity. Therefore, the results of our study indicate that this method offers a more cost-effective production method for Cypridina luciferase than conventional methods.
Assuntos
Arabidopsis/citologia , Arabidopsis/metabolismo , Crustáceos/genética , Luciferases , Células Vegetais/metabolismo , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/genética , Crustáceos/enzimologia , Glicosilação , Luciferases/biossíntese , Luciferases/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
Endoplasmic reticulum (ER)-associated degradation (ERAD) is a proteolytic pathway for handling misfolded or improperly assembled proteins that are synthesized in the ER. Cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme that cleaves N-glycans that are attached to ERAD substrates. While the critical roles of N-glycans in monitoring the folding status of carrier proteins in the ER lumen are relatively well understood, the physiological role of PNGase-mediated deglycosylation in the cytosol remained poorly understood. We report herein the identification of endogenous substrates for the cytoplasmic PNGase in Saccharomyces cerevisiae Using an isotope-coded glycosylation site-speciï¬c tagging (IGOT) method-based LC/MS analysis, 11 glycoproteins were specifically detected in the cytosol of PNGase-deletion cells (png1Δ). Among these molecules, at least five glycoproteins were clearly identified as ERAD substrates in vivo Moreover, four out of the five proteins were found to be either deglycosylated by PNGase in vivo or the overall degradation was delayed in a png1Δ mutant. Our results clearly indicate that the IGOT method promises to be a powerful tool for the identification of endogenous substrates for the cytoplasmic PNGase.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromatografia Líquida , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Espectrometria de Massas em TandemRESUMO
Proteins carrying sulfated glycans (i.e., sulfated glycoproteins) are known to be associated with diseases, such as cancer, cystic fibrosis, and osteoarthritis. Sulfated glycoproteins, however, have not been isolated or characterized from complex biological samples due to lack of appropriate tools for their enrichment. Here, we describe a method to identify and characterize sulfated glycoproteins that are involved in chemical modifications to control the molecular charge of the peptides. In this method, acetohydrazidation of carboxyl groups was performed to accentuate the negative charge of the sulfate group, and Girard's T modification of aspartic acid was performed to assist in protein identification by MS tagging. Using this approach, we identified and characterized the sulfated glycoproteins: Golgi membrane protein 1, insulin-like growth factor binding protein-like 1, and amyloid beta precursor-like protein 1 from H2171 cells, a small cell lung carcinoma cell line. These sulfated glycoproteins carry a complex-type N-glycan with a core fucose and 4'-O-sulfated LacdiNAc as the major glycan.
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Glicoproteínas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Carcinoma de Pequenas Células do Pulmão/metabolismo , Linhagem Celular Tumoral , HumanosRESUMO
The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.
Assuntos
Glicômica/métodos , Espectrometria de Massas/métodos , Técnicas de Diagnóstico Molecular/métodos , Polissacarídeos/química , Biomarcadores/química , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida de Alta Pressão/normas , Glicômica/normas , Glicoproteínas/química , Humanos , Espectrometria de Massas/normas , Técnicas de Diagnóstico Molecular/normas , Proteômica/métodos , Proteômica/normas , Reprodutibilidade dos TestesRESUMO
One of the promising approaches to the development of cancer diagnostic systems is quantification of a specific protein carrying cancerous glycans. Potential utility of Wisteria floribunda agglutinin (WFA) for such assays has been suggested for several cancer types. To develop such diagnostic systems, identification of WFA-recognized glycoproteins is essential. Here, we successfully identified 504 WFA-recognized glycoproteins from the secretome of HEK293T cells. Most of the identified proteins were likely soluble or single-pass transmembrane proteins, which may serve as specific proteins for the diagnosis using biological fluids. Our method may help to discover marker glycoproteins for various cancers generating WFA-recognized glycans.