RESUMO
N-linked glycoproteins are rich in seminal plasma, playing essential roles in supporting sperm function and fertilization process. The alteration of seminal plasma glycans and its correspond glycoproteins may lead to sperm dysfunction and even infertility. In present study, an integrative analysis of glycoproteomic and proteomic was performed to investigate the changes of site-specific glycans and glycoptoteins in seminal plasma of asthenozoospermia. By large scale profiling and quantifying 5,018 intact N-glycopeptides in seminal plasma, we identified 92 intact N-glycopeptides from 34 glycoproteins changed in asthenozoospermia. Especially, fucosylated glycans containing lewis x, lewis y and core fucosylation were significantly up-regulated in asthenozoospermia compared to healthy donors. The up-regulation of fucosylated glycans in seminal plasma may interfere sperm surface compositions and regulation of immune response, which subsequently disrupts sperm function. Three differentiated expression of seminal vesicle-specific glycoproteins (fibronectin, seminogelin-2, and glycodelin) were also detected with fucosylation alteration in seminal plasma of asthenozoospermia. The interpretation of the altered site-specific glycan structures provides data for the diagnosis and etiology analysis of male infertility, as well as providing new insights into the potential therapeutic targets for male infertility.
Assuntos
Astenozoospermia , Fucose , Sêmen , Humanos , Masculino , Astenozoospermia/metabolismo , Sêmen/metabolismo , Sêmen/química , Fucose/metabolismo , Glicoproteínas/metabolismo , Proteômica , Adulto , Regulação para Cima , Polissacarídeos/metabolismo , Polissacarídeos/química , Glicosilação , Glicopeptídeos/metabolismo , Glicopeptídeos/análiseRESUMO
Precision mapping of glycans at structural and site-specific level is still one of the most challenging tasks in the glycobiology field. Here, we describe a modularization strategy for de novo interpretation of N-glycan structures on intact glycopeptides using tandem mass spectrometry. An algorithm named StrucGP is also developed to automate the interpretation process for large-scale analysis. By dividing an N-glycan into three modules and identifying each module using distinct patterns of Y ions or a combination of distinguishable B/Y ions, the method enables determination of detailed glycan structures on thousands of glycosites in mouse brain, which comprise four types of core structure and 17 branch structures with three glycan subtypes. Owing to the database-independent glycan mapping strategy, StrucGP also facilitates the identification of rare/new glycan structures. The approach will be greatly beneficial for in-depth structural and functional study of glycoproteins in the biomedical research.
Assuntos
Algoritmos , Glicopeptídeos/análise , Glicoproteínas/análise , Polissacarídeos/análise , Animais , Glicopeptídeos/química , Glicoproteínas/química , Glicosilação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Polissacarídeos/químicaRESUMO
Spermatozoon represents a very special cell type in human body, and glycosylation plays essential roles in its whole life including spermatogenesis, maturation, capacitation, sperm-egg recognition, and fertilization. In this study, by mapping the most comprehensive N-glycoproteome of human spermatozoa using our recently developed site-specific glycoproteomic approaches, we show that spermatozoa contain a number of distinctive glycoproteins, which are mainly involved in spermatogenesis, acrosome reaction and sperm:oocyte membrane binding, and fertilization. Heavy fucosylation is observed on 14 glycoproteins mostly located at extracellular and cell surface regions in spermatozoa but not in other tissues. Sialylation and Lewis epitopes are enriched in the biological process of immune response in spermatozoa, while bisected core structures and LacdiNAc structures are highly expressed in acrosome. These data deepen our knowledge about glycosylation in spermatozoa and lay the foundation for functional study of glycosylation and glycan structures in male infertility.
Assuntos
Reação Acrossômica , Espermatozoides , Acrossomo/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Humanos , Masculino , Proteômica , Capacitação Espermática , Espermatozoides/metabolismoRESUMO
Aging-related salivary gland degeneration usually causes poor oral health. Periductal fibrosis frequently occurs in the submandibular gland of the elderly. Transforming growth factor ß1 (TGF-ß1) is the primary driving factor for fibrosis, which exhibits an increase in the fibrotic submandibular gland tissue. This study aimed to investigate the effects of TGF-ß1 on the human submandibular gland (HSG) cell secretory function and its influences on aquaporin 5 (AQP5) expressions and distribution. We found that TGF-ß1 reduces the protein secretion amount of HSG and leads to the abundance alteration of 151 secretory proteins. Data are available via ProteomeXchange with the identifier PXD043185. The majority of HSG secretory proteins (84.11%) could be matched to the human saliva proteome. Meanwhile, TGF-ß1 enhances the expression of COL4A2, COL5A1, COL7A1, COL1A1, COL2A1, and α-SMA, hinting that TGF-ß1 possesses the potential to drive HSG fibrosis-related events. Besides, TGF-ß1 also attenuates the AQP5 expression and its membrane distribution in HSGs. The percentage for TGF-ß1-induced AQP5 reduction (52.28%) is much greater than that of the TGF-ß1-induced secretory protein concentration reduction (16.53%). Taken together, we concluded that TGF-ß1 triggers salivary hypofunction via attenuating protein secretion and AQP5 expression in HSGs, which may be associated with TGF-ß1-driven fibrosis events in HSGs.
Assuntos
Aquaporina 5 , Glândula Submandibular , Fator de Crescimento Transformador beta1 , Humanos , Aquaporina 5/genética , Aquaporina 5/metabolismo , Colágeno Tipo VII/metabolismo , Saliva/metabolismo , Glândula Submandibular/citologia , Glândula Submandibular/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
O-Acetylation is a common modification of sialic acid, playing a significant role in glycoprotein stability, immune response, and cell development. Due to the lack of efficient methods for direct analysis of O-acetylated sialoglycopeptides (O-AcSGPs), the majority of identified O-acetylated sialic acids (O-AcSia) until now had no glycosite/glycoprotein information. Herein, we introduced a new workflow for precise interpretation of O-AcSGPs with probability estimation by recognizing the characteristic B and Y ions of O-AcSias. With further optimization of mass spectrometry parameters, the method allowed us to identify a total of 171 unique O-AcSGPs in mouse serum. Although the majority of these O-AcSGPs were at a relatively low abundance compared with their non-O-acetylated states, they were mainly involved in peptidase/endopeptidase inhibitor activities. The method paves the way for large-scale structural and functional analyses of site-specific O-AcSias in various complex samples as well as further identification of many other similar chemical modifications on glycoproteins.
RESUMO
Normal liquefaction of semen is one of the key steps to ensure the smooth progress of fertilization, and glycosylation has been reported to be involved in the whole process of fertilization. Till now, it is still unclear whether and how glycosylation changes during the liquefaction process of semen. In this study, by performing a glycoproteomic analysis of human semen with the liquefaction process (liquefaction time of semen: 0 min vs 30 min) using our recently developed StrucGP software combined with the Tandem Mass Tags (TMT) based quantification, we identified 25 intact glycopeptides (IGPs) from 10 glycoproteins in semen that were significantly changed during liquefaction, including 23 up-regulated and two down-regulated. Among the 23 up-regulated glycopeptides, half were modified with sialylated glycans, suggesting that sialylated glycans may play a key role in the semen liquefaction process. The data provide an invaluable resource for further studies on the role of glycosylation during semen liquefaction.
Assuntos
Líquidos Corporais , Sêmen , Humanos , Glicopeptídeos , Glicosilação , PolissacarídeosRESUMO
N-Linked glycoproteins are rich in seminal plasma, playing various essential roles in supporting sperm function and the fertilization process. However, the detailed information on these glycoproteins, particularly site-specific glycan structures, is still limited. In this study, a precision site-specific N-glycoproteome map of human seminal plasma was established by employing the site-specific glycoproteomic approach and a recently developed glycan structure interpretation software, StrucGP. A total of 9567 unique glycopeptides identified in human seminal plasma were composed of 773 N-linked glycan structures and 1019 N-glycosites from 620 glycoproteins. These glycans were comprised of four types of core structures and 13 branch structures. The majority of identified glycoproteins functioned in response to stimulus and immunity. As we reported in human spermatozoa, heavy fucosylation (fucose residues ≥6 per glycan) was also detected on seminal plasma glycoproteins such as clusterin and galectin-3-binding protein, which were involved in the immune response of biological processes and reactome pathways. Comparison of site-specific glycans between seminal plasma and spermatozoa revealed more complicated glycan structures in seminal plasma than in spermatozoa, even on their shared glycoproteins. These present data will be greatly beneficial for the in-depth structural and functional study of glycosylation in the male reproduction system.
Assuntos
Polissacarídeos , Sêmen , Glicopeptídeos/química , Glicoproteínas/metabolismo , Glicosilação , Humanos , Masculino , Polissacarídeos/química , Sêmen/metabolismoRESUMO
The spike (S) protein plays a key role in COVID-19 (SARS-CoV-2) infection and host-cell entry. Previous studies have systematically analyzed site-specific glycan compositions as well as many important structural motifs of the S protein. Here, we further provide structural-clear N-glycosylation of the S protein at a site-specific level by using our recently developed structural- and site-specific N-glycoproteomics sequencing algorithm, StrucGP. In addition to the common N-glycans as detected in previous studies, many uncommon glycosylation structures such as LacdiNAc structures, Lewis structures, Mannose 6-phosphate (M6P) residues, and bisected core structures were unambiguously mapped at a total of 20 glycosites in the S protein trimer and protomer. These data further support the glycosylation structural-functional investigations of the COVID-19 virus spike.
Assuntos
COVID-19 , SARS-CoV-2 , Glicosilação , Humanos , Polissacarídeos/químicaRESUMO
Accurate identification of core fucosylation on N-glycopeptides remains challenging due to fucose migration during mass spectrometry analysis. Here, we introduce a simple and straightforward method for core-fucosylated glycopeptide recognition based on the relative intensities of Y1+Fuc ions compared with their corresponding Y1 ions (labeled as Y1+Fuc/Y1 or simply Y1F/Y1 ratio > 0.1) in low-energy HCD-based spectra. The method was first developed by systematically evaluating the influence of fucose migration on the Y1F ion from antenna fucoses based on the distribution of the Y1F/Y1 ratios in the MS/MS spectra of antenna-fucosylated glycopeptides from Fut8-/- mouse brain. The feasibility of the method was then confirmed by using two standard glycoproteins, comparison with glycopeptides in Fut8+/+ mouse brain with/without in silico core-fucosylation removal, and Y1F/Y1 ratio alterations under a lower HCD energy. This method will be applicable to the manual interpretation and software-based high-throughput analysis of core-fucosylated glycopeptides.
Assuntos
Glicopeptídeos , Espectrometria de Massas em Tandem , Animais , Camundongos , Glicopeptídeos/análise , Espectrometria de Massas em Tandem/métodos , Fucose/química , Glicosilação , Glicoproteínas/químicaRESUMO
In glycomic and glycoproteomic studies, solutions containing diluted organic acids such as formic acid (FA) have been widely used for dissolving intact glycopeptide and glycan samples prior to mass spectrometry analysis. Here, we show that an undesirable + 28 Da modification occurred in a time-dependent manner when the glycan and glycopeptide samples were stored in FA solution at - 20 °C. We confirmed that this unexpected modification was caused by formylation between the hydroxyl groups of glycans and FA with a relatively low reaction rate. As this incomplete modification affected the glycan and glycopeptide identification and quantification in glycomic and glycoproteomic studies, the storage at - 20 °C should be avoided once the glycan and glycopeptide samples have been dissolved in FA solution.
Assuntos
Glicômica , Glicopeptídeos , Formiatos , Glicômica/métodos , Glicopeptídeos/química , Espectrometria de Massas , Polissacarídeos/químicaRESUMO
Selecting proper and efficient glycopeptide enrichment approaches are essential for mass spectrometry-based glycoproteomics since glycopeptides are usually with microheterogeneity and low abundance in most biological samples. Herein, we introduced a cotton hydrophilic interaction liquid chromatography (HILIC) approach for large-scale glycopeptide enrichment with 80% acetonitrile/1% trifluoroacetic acid as the optimal sample loading buffer. The comparison of cotton HILIC with Venusil HILIC and mixed anion-exchange (MAX) approaches indicated that cotton HILIC was superior in overall glycopeptide enrichment, whereas Venusil HILIC preferred in complex glycan structures and MAX performed better with high mannose glycans. Exploration of capacity and recovery rate of cotton HILIC illustrated that 5mg cotton packed in a 200µL tip achieved a reasonable glycopeptide enrichment performance (~6% recovery) from ~0.5mg peptides. In conclusion, cotton HILIC can be used as an optional glycopeptide enrichment approach in glycosylation analysis with its specific merit.
Assuntos
Glicopeptídeos , Polissacarídeos , Glicopeptídeos/química , Cromatografia Líquida/métodos , Glicosilação , Interações Hidrofóbicas e HidrofílicasRESUMO
Myocardial infarction (MI) is one of the leading causes of deaths worldwide. Because of the incapability of regeneration, the cardiomyocyte loss with MI is replaced by fibrotic scar tissue, which eventually leads to heart failure. Reconstructing regeneration of an adult human heart has been recognized as a promising strategy for cardiac therapeutics. A neonatal mouse heart, which possesses transient regenerative capacity at the first week after birth, represents an ideal model to investigate processes associated with cardiac regeneration. In this work, an integrated glycoproteomic and proteomic analysis was performed to investigate the differences in glycoprotein abundances and site-specific glycosylation between postneonatal day 1 (P1) and day 7 (P7) of mouse hearts. By large-scale profiling and quantifying more than 2900 intact N-glycopeptides in neonatal mouse hearts, we identified 227 altered N-glycopeptides between P1 and P7 hearts. By extracting protein changes from the global proteome data, the normalized glycosylation changes for site-specific glycans were obtained, which showed heterogeneity on glycosites and glycoproteins. Systematic analysis of the glycosylation changes demonstrated an overall upregulation of sialylation and core fucosylation in P7 mice. Notably, the upregulated sialylation was a comprehensive result of increased sialylated glycans with Neu5Gc, with both Neu5Gc and core fucose, and decreased sialylated glycans with Neu5Ac. The upregulated core fucosylation resulted from the increase of glycans containing both core fucose and Neu5Gc but not glycans containing sole core fucose. These data provide a valuable resource for future functional and mechanism studies on heart regeneration and discovery of novel therapeutic targets. All mass spectrometry proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD017139.
Assuntos
Glicopeptídeos , Proteômica , Animais , Animais Recém-Nascidos , Glicosilação , Camundongos , RegeneraçãoRESUMO
Influenza H1N1 virus has posed a serious threat to human health. The glycosylation of neuraminidase (NA) could affect the infectivity and virulence of the influenza virus, but detailed site-specific glycosylation information of NA is still missing. In this study, intact glycopeptide analysis is performed on an influenza NA (A/H1N1/California/2009) that is expressed in human 293T and insect Hi-5 cells. The data indicate that three of four potential N-linked glycosylation sites are glycosylated, including one partial glycosylation site from both cell lines. The NA expressed in human cells has more complex glycans than that of insect cells, suggesting the importance of selecting an appropriate expression system for the production of functional glycoproteins. Different types of glycans are identified from different glycosites of NA expressed in human cells, which implies the site-dependence of glycosylation on NA. This study provides valuable information for the research of influenza virus as well as the functions of viral protein glycosylation.
Assuntos
Glicopeptídeos/análise , Vírus da Influenza A Subtipo H1N1/enzimologia , Influenza Humana/virologia , Neuraminidase/química , Polissacarídeos/análise , Proteínas Virais/química , Animais , Linhagem Celular , Glicosilação , Humanos , Vírus da Influenza A Subtipo H1N1/química , Insetos , Infecções por Orthomyxoviridae/virologiaRESUMO
Bisecting N-glycan represents one of the most important modifications to the N-glycan core, and it is involved in various biological processes. Despite many studies on the biological roles of bisecting N-glycans, current approaches for bisecting N-glycan analysis mainly rely on the use of the lectin PHA-E, which are of low specificity and sensitivity. Here, we describe a straightforward method for the recognition of bisecting N-glycans on intact glycopeptides using two characteristic Y ions [peptide+HexNAc3Hex1] and [peptide+HexNAc3Hex1Fuc1] in low energy fragmented MS/MS spectra under higher energy collisional dissociation (HCD) mode. The critical aspect of the method is the combination use of low energy HCD fragmentation and intact glycopeptide analysis. With samples from rat renal tissues, we determined the optimal fragmentation energies and analyzed the influence of core fucosylation on the intensity of the [peptide+HexNAc3Hex1] ion. Using the method, we identified 183 intact glycopeptides with bisecting N-glycans and investigated the primary bisecting N-glycan structures and the possible biological roles of these identified proteins.
Assuntos
Glicopeptídeos/química , Polissacarídeos/química , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Animais , RatosRESUMO
N-linked glycoprotein is a highly interesting class of proteins for clinical and biological research. Over the last decade, large-scale profiling of N-linked glycoproteins and glycosylation sites from biological and clinical samples has been achieved through mass spectrometry-based glycoproteomic approaches. In this paper, we reviewed the human glycoproteomic profiles that have been reported in more than 80 individual studies, and mainly focused on the N-glycoproteins and glycosylation sites identified through their deglycosylated forms of glycosite-containing peptides. According to our analyses, more than 30,000 glycosite-containing peptides and 7,000 human glycoproteins have been identified from five different body fluids, twelve human tissues (or related cell lines), and four special cell types. As the glycoproteomic data is still missing for many organs and tissues, a systematical glycoproteomic analysis of various human tissues and body fluids using a uniform platform is still needed for an integrated map of human N-glycoproteomes.
RESUMO
A terrestrial biotic ligand model (t-BLM) was developed to predict nickel toxicity to wheat (Triticum aestivum) root elongation in hydroponic solutions. The competitive effects of five major cations (Ca2+, Mg2+, Na+, K+ and H+) on Ni toxicity were investigated and Mg2+was found to be a strong competitor, while H+ showed less competing effect. Besides free Ni2+, the toxicity induced by the species NiHCO3+ was non-neglect able at pH>7 because NiHCO3+ occupied a significant fraction of total Ni under such condition. Thus, a t-BLM including Ni2+, NiHCO3+, Mg2+, and H+ could successfully predict the nickel toxicity to wheat root elongation and it performed better prediction than the conventional free ion activity model. In addition, the model was examined with two sets of independent experiments, which contained multiple cations and low-molecular-weight organic acids to mimic the rhizosphere condition. The developed t-BLM well predicted nickel toxicity in both experiments since it can account in both complexation and competition effects, suggesting its potential to be used in a complicated matrix like soil solution. This study provides direct evidence that the t-BLM is a reliable method for the risk assessment of nickel in terrestrial system.
Assuntos
Níquel/toxicidade , Poluentes do Solo/toxicidade , Testes de Toxicidade/métodos , Ligantes , Modelos Biológicos , Solo , TriticumRESUMO
Bisecting N-glycan is known to be a metastasis suppressor and plays a regulatory role in the biosynthesis of N-glycans. Previous studies have shown that bisecting N-glycans are capable of modulating both the branching and terminal modifications of glycans. However, these effects have been investigated mainly by glycomic approaches and it remains unclear how they alter when glycans are attached to different glycosites of proteins. Here, we systematically investigated the regulatory roles of bisecting N-glycans in human HK-2 cells using StrucGP, a strategy we developed for structural interpretation of site-specific N-glycans on glycoproteins. The glycoproteomics analysis showed that most of bisecting N-glycans are complex type and often occur in company with core fucosylation. With the overexpression and knockdown of MGAT3, the only enzyme responsible for bisecting N-glycan synthesis, we found that bisecting N-glycans can impact the biosynthesis of N-glycans from multiple aspects, including glycan types, branching, sialylation, fucosylation (different effects for core and terminal fucosylation) as well as the presence of terminal N-acetylglucosamine. Furthermore, gene ontology analysis suggested that most proteins with bisecting N-glycans located in the extracellular region or membrane, where they function mostly in cell adhesion, extracellular matrix regulation and cell signaling. Finally, we showed that overexpression of bisecting N-glycans had a broad impact on the protein expressions of HK-2 cells, involving multiple biological processes. Taken together, our work systematically demonstrated the expression profiles of bisecting N-glycans, and their regulatory effects on the biosynthesis of N-glycans and protein expressions, which provide valuable information for the functional elucidation of bisecting N-glycans.
Assuntos
Glicoproteínas , Polissacarídeos , Humanos , Glicosilação , Glicoproteínas/química , Polissacarídeos/químicaRESUMO
The structure of N-glycans on specific proteins can regulate innate and adaptive immunity via sensing environmental signals. Meanwhile, the structural diversity of N-glycans poses analytical challenges that limit the exploration of specific glycosylation functions. In this work, we used THP-1-derived macrophages as examples to show the vast potential of a N-glycan structural interpretation tool StrucGP in N-glycoproteomic analysis. The intact glycopeptides of macrophages were enriched and analyzed using mass spectrometry (MS)-based glycoproteomic approaches, followed by the large-scale mapping of site-specific glycan structures via StrucGP. Results revealed that bisected GlcNAc, core fucosylated, and sialylated glycans (e.g., HexNAc4Hex5Fuc1Neu5Ac1, N4H5F1S1) were increased in M1 and M2 macrophages, especially in the latter. The findings indicated that these structures may be closely related to macrophage polarization. In addition, a high level of glycosylated PD-L1 was observed in M1 macrophages, and the LacNAc moiety was detected at Asn-192 and Asn-200 of PD-L1, and Asn-200 contained Lewis epitopes. The precision structural interpretation of site-specific glycans and subsequent intervention of target glycoproteins and related glycosyltransferases are of great value for the development of new diagnostic and therapeutic approaches for different diseases.
Assuntos
Antígeno B7-H1 , Polissacarídeos , Humanos , Glicosilação , Polissacarídeos/química , Polissacarídeos/metabolismoRESUMO
BACKGROUND: Osteoarthritis (OA) is the most common form of arthritis, affecting millions of aging people. Investigation of abnormal glycosylation is essential for the understanding of pathological mechanisms of OA. METHODS: The total protein was isolated from OA (n = 13) and control (n = 11) cartilages. Subsequently, glycosylation alterations of glycoproteins in OA cartilage were investigated by lectin microarrays and intact glycopeptides analysis. Finally, the expression of glycosyltransferases involved in the synthesis of altered glycosylation was assessed by qPCR and GEO database. RESULTS: Our findings revealed that several glycopatterns, such as α-1,3/6 fucosylation and high-mannose type of N-glycans were altered in OA cartilages. Notably, over 27% of identified glycopeptides (109 glycopeptides derived from 47 glycoproteins mainly located in the extracellular region) disappeared or decreased in OA cartilages, which is related to the cartilage matrix degradation. Interestingly, the microheterogeneity of N-glycans on fibronectin and aggrecan core protein was observed in OA cartilage. Our results combined with GEO data indicated that the pro-inflammatory cytokines altered the expression of glycosyltransferases (ALG3, ALG5, MGAT4C, and MGAT5) which may contribute to the alterations in glycosylation. CONCLUSION: Our study revealed the abnormal glycopatterns and heterogeneities of site-specific glycosylation associated with OA. To our knowledge, it is the first time that the heterogeneity of site-specific N-glycans was reported in OA cartilage. The results of gene expression analysis suggested that the expression of glycosyltransferases was impacted by pro-inflammatory cytokines, which may facilitate the degradation of protein and accelerate the process of OA. Our findings provide valuable information for the understanding of molecular mechanisms in the pathogenesis of OA.
Assuntos
Cartilagem , Glicômica , Glicosilação , Osteoartrite , Humanos , Glicômica/métodos , Glicoproteínas , Cartilagem/metabolismo , CitocinasRESUMO
Sialic acid, a common terminal monosaccharide on many glycoconjugates, plays essential roles in many biological processes such as immune responses, pathogen recognition, and cancer development. For various purposes, sialic acids may need to be removed from glycopeptides or glycans, mainly using enzymatical or chemical approaches. In this study, we found that most commonly used chemical methods couldn't completely remove sialic acids from glycopeptides. Although the de-sialylation efficiency could be further enhanced by increasing the treatment time or acid concentration, the undesirable side reactions on the peptide portion would decrease glycopeptide identification. By adding the deamidation on carbamidomethyl-cysteine (C), asparagine (N), and glutamine (Q) residues as a variable modification during database search, most of the unidentified spectra could be recovered. This optional acid-treatment and database search method for the complete removal of sialic acids without losing much spectral identification should be quite useful for many glycomic and glycoproteomic studies.