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1.
Enzyme Microb Technol ; 150: 109862, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489021

RESUMO

Glycosylation and methylation of flavonoids are the main types of structural modifications and can endow flavonoids with greater stability, bioactivity, and bioavailability. In this study, five types of O-methyltransferases were screened for producing O-methylated luteolin, and the biosynthesis strategy of 3'-O-methylisoorientin from luteolin was determined. To improve the production of 3'-O-methylluteolin, the S-adenosyl-l-methionine synthesis pathway was reconstructed in the recombinant strain by introducing S-adenosyl-l-methionine synthetase genes. After optimizing the conversion conditions, maximal 3'-O-methylluteolin production reached 641 ± 25 mg/L with a corresponding molar conversion of 76.5 %, which was the highest titer of methylated flavonoids reported to date in Escherichia coli. 3'-O-Methylluteolin (127 mg) was prepared from 250 mL of the broth by silica gel column chromatography and preparative HPLC with a yield of 79.4 %. Subsequently, we used the biocatalytic cascade of Gentiana triflora C-glycosyltransferase (Gt6CGT) and Glycine max sucrose synthase (GmSUS) to biosynthesize 3'-O-methylisoorientin from 3'-O-methylluteolin in vitro. By optimizing the coupled reaction conditions and using the fed-batch operation, maximal 3'-O-methylisoorientin production reached 226 ± 8 mg/L with a corresponding molar conversion of 98 %. Therefore, this study provides an efficient method for the production of novel 3'-O-methylisoorientin and the biosynthesis strategy for methylated C-glycosylation flavonoids by selective O-methylation/C-glycosylation motif on flavonoids.


Assuntos
Flavonoides , Luteolina , Glicosilação , Metilação , Metiltransferases/metabolismo
2.
Se Pu ; 39(10): 1086-1093, 2021 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-34505430

RESUMO

Many secreted proteins, including cytokines, growth factors and hormones, are crucial in processes like intercellular signaling. Dynamic changes in secreted proteins usually reflect the growth and pathological state of the cells. Many drug targets are secretory proteins. The proteins are also important biomarkers. Conditioned cell culture media are important samples for secretory proteomic studies. Biomass spectrometry-based proteomic analysis enables the systematic study of secretory proteins. The main problem in analyzing secretory proteins in conditioned culture media is the low concentration of these proteins and the presence of serum, amino acids, and additives in culture media that may interfere with the protein analysis. Conventional secretory proteome analysis uses serum-free cell culture to reduce sample complexity, and typically involves protein concentration, purification, and desalting using ultrafiltration, dialysis, lyophilization, and trichloroacetic acid (TCA) or acetone precipitation, followed by enzymatic digestion and mass spectrometry analysis. This analytical process does not allow specific enrichment of secreted proteins. Thus, only a few secreted proteins can be identified. In addition, prolonged serum-free incubation of cells also tends to lead to unexpected changes in their activity status. A bioorthogonal-based enrichment approach can effectively avoid this problem. In recent years, unnatural sugars containing bio-orthologous groups, such as azide groups, have been used to metabolically label glycosylated proteins, enabling cellular imaging or selective enrichment of glycoproteins and their use for proteomic analysis. The strategy is a two-step process. First, azide-based sugar analogues are added to the cell culture medium and introduced to glycoproteins via the intracellular glycan biosynthesis pathway. Second, they are specifically covalently labeled with imaging probes or affinity probes via click chemistry. Since secreted proteins are usually glycoproteins, this glycolytic labeling has been used to label and enrich secreted proteins. N-Azidoacetylgalactosamine (GalNAz), N-azidoacetylglucosamine (GlcNAz), and N-azidoacetylmannosamine (ManNAz) are classical azide-based sugar analogues. Their effects on cytoplasmic membrane proteins have been compared. However, only ManNAz has been used for metabolic labeling of secreted proteins. No other glyco-analogues that label secreted proteins have been reported. Here, the bio-orthogonal chemical biology technology achieved highly selective labeling and enriched secreted proteins. In combination with click chemistry, different sugar analogues were evaluated for metabolic labeling of secreted proteins. HeLa cells were metabolically labeled by ManNAz, GalNAz, and GlcNAz (the three most commonly used commercial sugar analogues). These glycolytic markers can selectively label specific types of glycosylation. For example, ManNAz, an analogue of the biosynthetic precursor of sialic acid, N-acetylmannosamine (ManNAc), can label sialylated N- or O-glycoproteins. GalNAz, an analogue of N-acetylgalactosamine (GalNAc), can replace GalNAc as a core residue of mucin-type O-glycans and thus label O-glycoproteins. In addition, the intracellular metabolic intermediate of GalNAz (pyrophosphate) UDP-GalNAz can be interconverted with UDP-GlcNAz catalyzed by UDP-galactose-4-differential isomerase (GALE) and thus can also label N-glycoproteins and O-GlcNAc glycoproteins instead of GlcNAc. The GlcNAz analogue is commonly used to label nuclear and cytoplasmic glycoproteins with β-O-GlcNAc residues, but can also label N-glycoproteins with mucin-type O-glycoproteins by converting GALE to GalNAz, followed by enrichment using a biotin-alkynyl probe. Label-free quantitative proteomic analysis was performed to evaluate their labeling efficiency. ManNAz-based secretory protein labeling identified 282 secretory proteins, 224 plasma membrane proteins, and 846 N-glycosites. Compared with GalNAz and GlcNAz, the enrichment of secreted proteins was increased 130% and 67.2%, respectively, and the enrichment of plasma membrane proteins was increased 273.3% and 148.7%, respectively. This study provides a useful comparative analysis and new strategies for highly selective enrichment and systematic secretome analysis.


Assuntos
Proteômica , Açúcares , Glicoproteínas , Glicosilação , Células HeLa , Humanos
3.
Adv Exp Med Biol ; 1325: 3-24, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495528

RESUMO

N-glycosylation is a highly conserved glycan modification, and more than 7000 proteins are N-glycosylated in humans. N-glycosylation has many biological functions such as protein folding, trafficking, and signal transduction. Thus, glycan modification to proteins is profoundly involved in numerous physiological and pathological processes. The N-glycan precursor is biosynthesized in the endoplasmic reticulum (ER) from dolichol phosphate by sequential enzymatic reactions to generate the dolichol-linked oligosaccharide composed of 14 sugar residues, Glc3Man9GlcNAc2. The oligosaccharide is then en bloc transferred to the consensus sequence N-X-S/T (X represents any amino acid except proline) of nascent proteins. Subsequently, the N-glycosylated nascent proteins enter the folding step, in which N-glycans contribute largely to attaining the correct protein fold by recruiting the lectin-like chaperones, calnexin, and calreticulin. Despite the N-glycan-dependent folding process, some glycoproteins do not fold correctly, and these misfolded glycoproteins are destined to degradation by proteasomes in the cytosol. Properly folded proteins are transported to the Golgi, and N-glycans undergo maturation by the sequential reactions of glycosidases and glycosyltransferases, generating complex-type N-glycans. N-Acetylglucosaminyltransferases (GnT-III, GnT-IV, and GnT-V) produce branched N-glycan structures, affording a higher complexity to N-glycans. In this chapter, we provide an overview of the biosynthetic pathway of N-glycans in the ER and Golgi.


Assuntos
Glicoproteínas , Dobramento de Proteína , Retículo Endoplasmático/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Humanos , Lectinas , Polissacarídeos/metabolismo
4.
Adv Exp Med Biol ; 1325: 151-171, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495534

RESUMO

Although changes in protein glycosylation are observed in a wide range of diseases and pathological states, the examples of use of glycans as biomarkers and therapeutic targets are limited. This is not in small part because the understanding of human glycome regulation in vivo is incomplete and fragmented. Combination of human glycomics and genomics offers a powerful "data-driven hypotheses" approach to dissect the complex human glycobiology in vivo in an agnostic manner.In this chapter we review a decade of quantitative genetic studies of human N-glycome, including studies of its heritability and gene-mapping via genome-wide association studies (GWASs). We show that GWASs of human N-glycome start revealing regulators of the biochemical network of N-glycosylation. Some of these regulators demonstrate pleiotropic effects on human disease, especially autoimmune and inflammatory. We emphasize the use of in silico functional methods and multi-omics approaches to prioritize functional mechanisms to be further validated in laboratory experiments. This combined approach will lead to better understanding of mechanisms of regulation of human protein glycosylation and will provide a rich source of etiologic insight, therapeutic interventions, and biomarkers.


Assuntos
Estudo de Associação Genômica Ampla , Glicômica , Genômica , Glicosilação , Humanos , Polissacarídeos
5.
Adv Exp Med Biol ; 1325: 117-135, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495532

RESUMO

O-Linked glycosylation such as O-fucose, O-glucose, and O-N-acetylglucosamine are considered to be unusual. As suggested by the high levels of evolutional conservation, these O-glycans are fundamentally important for life. In the last two decades, our understanding of the importance of these glycans has greatly advanced. In particular, identification of the glycosyltransferases responsible for the biosynthesis of these glycans has accelerated basic research on the functional significance and molecular mechanisms by which these O-glycans regulate protein functions as well as clinical research on human diseases due to changes in these types of O-glycosylation. Notably, Notch receptor signaling is modified with and regulated by these types of O-glycans. Here, we summarize the current view of the structures and the significance of these O-glycans mainly in the context of Notch signaling regulation and human diseases.


Assuntos
Fucose , Receptores Notch , Glucose , Glicosilação , Humanos , Polissacarídeos , Receptores Notch/metabolismo , Transdução de Sinais
6.
Adv Exp Med Biol ; 1325: 137-149, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495533

RESUMO

Extracellular vesicles (EVs), a generic term for any vesicles or particles that are released from cells, play an important role in modulating numerous biological and pathological events, including development, differentiation, aging, thrombus formation, immune responses, neurodegenerative diseases, and tumor progression. During the biogenesis of EVs, they encapsulate biologically active macromolecules (i.e., nucleotides and proteins) and transmit signals for delivering them to neighboring or cells that are located some distance away. In contrast, there are receptor molecules on the surface of EVs that function to mediate EV-to-cell and EV-to-matrix interactions. A growing body of evidence indicates that the EV surface is heavily modified with glycans, the function of which is to regulate the biogenesis and extracellular behaviors of EVs. In this chapter, we introduce the current status of our knowledge concerning EV glycosylation and discuss how it influences EV biology, highlighting the potential roles of EV glycans in clinical applications.


Assuntos
Exossomos , Vesículas Extracelulares , Doenças Neurodegenerativas , Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Glicosilação , Humanos , Doenças Neurodegenerativas/metabolismo
7.
Adv Exp Med Biol ; 1325: 25-60, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495529

RESUMO

Mucin-type GalNAc O-glycosylation is one of the most abundant and unique post-translational modifications. The combination of proteome-wide mapping of GalNAc O-glycosylation sites and genetic studies with knockout animals and genome-wide analyses in humans have been instrumental in our understanding of GalNAc O-glycosylation. Combined, such studies have revealed well-defined functions of O-glycans at single sites in proteins, including the regulation of pro-protein processing and proteolytic cleavage, as well as modulation of receptor functions and ligand binding. In addition to isolated O-glycans, multiple clustered O-glycans have an important function in mammalian biology by providing structural support and stability of mucins essential for protecting our inner epithelial surfaces, especially in the airways and gastrointestinal tract. Here the many O-glycans also provide binding sites for both endogenous and pathogen-derived carbohydrate-binding proteins regulating critical developmental programs and helping maintain epithelial homeostasis with commensal organisms. Finally, O-glycan changes have been identified in several diseases, most notably in cancer and inflammation, where the disease-specific changes can be used for glycan-targeted therapies. This chapter will review the biosynthesis, the biology, and the translational perspectives of GalNAc O-glycans.


Assuntos
Estudo de Associação Genômica Ampla , Mucinas , Animais , Glicosilação , Humanos , Mucinas/genética , Mucinas/metabolismo , Polissacarídeos , Processamento de Proteína Pós-Traducional
8.
Adv Exp Med Biol ; 1325: 173-186, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495535

RESUMO

Expression of glycosylation-related genes (or glycogenes) is strictly regulated by transcription factors and epigenetic processes, both in normal and in pathological conditions. In fact, glycosylation is an essential mechanism through which proteins and lipids are modified to perform a variety of biological events, to adapt to environment, and to interact with microorganisms.Many glycogenes with a role in normal development are epigenetically regulated. Essential studies were performed in the brain, where expression of glycogenes like MGAT5B, B4GALNT1, and ST8Sia1 are under the control of histone modifications, and in the immune system, where expression of FUT7 is regulated by both DNA methylation and histone modifications. At present, epigenetic regulation of glycosylation is still poorly described under physiological conditions, since the majority of the studies were focused on cancer. In fact, virtually all types of cancers display aberrant glycosylation, because of both genetic and epigenetic modifications on glycogenes. This is also true for many other diseases, such as inflammatory bowel disease, diabetes, systemic lupus erythematosus, IgA nephropathy, multiple sclerosis, and cardiovascular diseases.A deeper knowledge in epigenetic regulation of glycogenes is essential, since research in this field could be helpful in finding novel and personalized therapeutics.


Assuntos
Metilação de DNA , Epigênese Genética , Glicosilação , Código das Histonas , Processamento de Proteína Pós-Traducional
9.
Adv Exp Med Biol ; 1325: 205-218, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495537

RESUMO

Autoimmune diseases are accompanied by changes in protein glycosylation, in both the immune system and target tissues. The best-studied alteration in autoimmunity is agalactosylation of immunoglobulin G (IgG), characterized primarily in rheumatoid arthritis (RA), and then detected also in systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), and multiple sclerosis (MS). The rebuilding of IgG N-glycans in RA correlates with the relapses and remissions of the disease, is associated with physiological states such as pregnancy but also depends on applied anti-inflammatory therapy. In turn, a decreased core fucosylation of the whole pool of IgG N-glycans is a serum glycomarker in autoimmune thyroid diseases (AITD) encompassing Hashimoto's thyroiditis (HT) and Grave's disease (GD). However, fucosylation of anti-thyroglobulin IgG (an immunological marker of HT) was elevated in HT serum. Core fucosylation of IgG oligosaccharides was also lowered in MS and SLE. In AITD and IBD, chronic inflammation T lymphocytes showed the reduced expression of MGAT5 gene encoding ß1,6-N-acetylglucosaminyltransferase V (GnT-V) responsible for ß1,6-branching of N-glycans, which is important for T cell receptor activation. Structural changes of glycans have a profound effect on the pro-inflammatory activity of immune cells and serum immune proteins, including IgG in autoimmunity.


Assuntos
Doenças Autoimunes , Doença de Hashimoto , Lúpus Eritematoso Sistêmico , Glicosilação , Humanos , Imunoglobulina G
10.
Adv Exp Med Biol ; 1325: 219-237, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495538

RESUMO

Glycosylation plays an important role in infectious diseases. Many important interactions between pathogens and hosts involve their carbohydrate structures (glycans). Glycan interactions can mediate adhesion, recognition, invasion, and immune evasion of pathogens. To date, changes in many protein N/O-linked glycosylation have been identified as biomarkers for the development of infectious diseases and cancers. In this review, we will discuss the principal findings and the roles of glycosylation of both pathogens and host cells in the context of human important infectious diseases. Understanding the role and mechanism of glycan-lectin interaction between pathogens and hosts may create a new paradigm for discovering novel glycan-based therapies that can lead to eradication or functional cure of pathogens infection.


Assuntos
Doenças Transmissíveis , Lectinas , Glicosilação , Humanos , Evasão da Resposta Imune , Polissacarídeos
11.
Adv Exp Med Biol ; 1325: 265-283, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495540

RESUMO

The diversity of glycan presentation in a cell, tissue and organism is enormous, which reflects the huge amount of important biological information encoded by the glycome which has not been fully understood. A compelling body of evidence has been highlighting the fundamental role of glycans in immunity, such as in development, and in major inflammatory processes such as inflammatory bowel disease, systemic lupus erythematosus and other autoimmune disorders. Glycans play an instrumental role in the immune response, integrating the canonical circuits that regulate innate and adaptive immune responses. The relevance of glycosylation in immunity is demonstrated by the role of glycans as important danger-associated molecular patterns and pathogen-associated molecular patterns associated with the discrimination between self and non-self; also as important regulators of the threshold of T cell activation, modulating receptors signalling and the activity of both T and other immune cells. In addition, glycans are important determinants that regulate the dynamic crosstalk between the microbiome and immune response. In this chapter, the essential role of glycans in the immunopathogenesis of inflammatory disorders will be presented and its potential clinical applications (diagnosis, prognosis and therapeutics) will be highlighted.


Assuntos
Doenças Autoimunes , Lúpus Eritematoso Sistêmico , Glicosilação , Humanos , Ativação Linfocitária , Polissacarídeos
12.
Adv Exp Med Biol ; 1325: 285-305, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495541

RESUMO

Diabetes mellitus is a group of metabolic disorders characterized by the presence of hyperglycaemia. Due to its high prevalence and substantial heterogeneity, many studies have been investigating markers that could identify predisposition for the disease development, differentiate between the various subtypes, establish early diagnosis, predict complications or represent novel therapeutic targets. N-glycans, complex oligosaccharide molecules covalently linked to proteins, emerged as potential markers and functional effectors of various diabetes subtypes, appearing to have the capacity to meet these requirements. For instance, it has been shown that N-glycome changes in patients with type 2 diabetes and that N-glycans can even identify individuals with an increased risk for its development. Moreover, genome-wide association studies identified glycosyltransferase genes as candidate causal genes for both type 1 and type 2 diabetes. N-glycans have also been suggested to have a major role in preventing the impairment of glucose-stimulated insulin secretion by modulating cell surface expression of glucose transporters. In this chapter we aimed to describe four major diabetes subtypes: type 1, type 2, gestational and monogenic diabetes, giving an overview of suggested role for N-glycosylation in their development, diagnosis and management.


Assuntos
Diabetes Mellitus Tipo 2 , Hiperglicemia , Biomarcadores/metabolismo , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/genética , Estudo de Associação Genômica Ampla , Glicosilação , Humanos , Polissacarídeos
13.
Adv Exp Med Biol ; 1325: 239-264, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495539

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently one of the major health problems worldwide. SARS-CoV-2 survival and virulence are shown to be impacted by glycans, covalently attached to proteins in a process of glycosylation, making glycans an area of interest in SARS-CoV-2 biology and COVID-19 infection. The SARS-CoV-2 uses its highly glycosylated spike (S) glycoproteins to bind to the cell surface receptor angiotensin-converting enzyme 2 (ACE2) glycoprotein and facilitate host cell entry. Viral glycosylation has wide-ranging roles in viral pathobiology, including mediating protein folding and stability, immune evasion, host receptor attachment, and cell entry. Modification of SARS-CoV-2 envelope membrane with glycans is important in host immune recognition and interaction between S and ACE2 glycoproteins. On the other hand, immunoglobulin G, a key molecule in immune response, shows a distinct glycosylation profile in COVID-19 infection and with increased disease severity. Hence, further studies on the role of glycosylation in SARS-CoV-2 infectivity and COVID-19 infection are needed for its successful prevention and treatment. This chapter focuses on recent findings on the importance of glycosylation in COVID-19 infection.


Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Glicosilação , Humanos , Ligação Proteica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
14.
Adv Exp Med Biol ; 1325: 307-319, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495542

RESUMO

Cardiovascular disease (CVD) is the leading cause of death worldwide, accounting for approximately 18 million deaths in 2017. Coronary artery disease is the predominant cause of death from CVD, followed by stroke. Owing to recent technological advancements, glycans and glycosylation patterns of proteins have been investigated in association with CVD risk factors and clinical events. These studies have found significant associations of glycans as biomarkers of systemic inflammation and major CVD risk factors and events. While more limited, studies have also shown that glycans may be useful for monitoring response to anti-inflammatory therapies and may be responsive to changes in lifestyle, particularly in patients with chronic inflammatory diseases. Glycans capture summative risk information related to inflammatory, immune, and signaling pathways and are promising biomarkers for CVD risk prediction and therapeutic monitoring.


Assuntos
Doenças Cardiovasculares , Doença da Artéria Coronariana , Biomarcadores , Glicosilação , Humanos , Fatores de Risco
15.
Adv Exp Med Biol ; 1325: 321-339, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495543

RESUMO

WHO defines health as "a state of complete physical, mental, and social well-being and not merely the absence of disease or infirmity." We coined and defined suboptimal health status (SHS) as a subclinical, reversible stage of the pre-chronic disease. SHS is a physical state between health and disease, characterized by health complaints, general weakness, chronic fatigue, and low energy levels. We have developed an instrument to measure SHS, Suboptimal Health Status Questionnaire-25 (SHSQ-25), a self-reported survey assessing five health components that has been validated in various ethnical populations. Our studies suggest that SHS is associated with the major components of cardiovascular health and the early onset of metabolic diseases. Besides subjective measure of health (SHS), glycans are conceived as objective biomarkers of SHS. Glycans are complex and branching carbohydrate moieties attached to proteins, participating in inflammatory regulation and chronic disease pathogenesis. We have been investigating the role of glycans and SHS in multiple cardiometabolic diseases in different ethnical populations (African, Chinese, and Caucasian). Here we present case studies to prove that a combination of subjective health measure (SHS) with objective health measure (glycans) represents a window of opportunity to halt or reverse the progression of chronic diseases.


Assuntos
Nível de Saúde , Biomarcadores , Doença Crônica , Glicosilação , Humanos , Inquéritos e Questionários
16.
Adv Exp Med Biol ; 1325: 341-373, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495544

RESUMO

Human lifespan has increased significantly in the last 200 years, emphasizing our need to age healthily. Insights into molecular mechanisms of aging might allow us to slow down its rate or even revert it. Similar to aging, glycosylation is regulated by an intricate interplay of genetic and environmental factors. The dynamics of glycopattern variation during aging has been mostly explored for plasma/serum and immunoglobulin G (IgG) N-glycome, as we describe thoroughly in this chapter. In addition, we discuss the potential functional role of agalactosylated IgG glycans in aging, through modulation of inflammation level, as proposed by the concept of inflammaging. We also comment on the potential to use the plasma/serum and IgG N-glycome as a biomarker of healthy aging and on the interventions that modulate the IgG glycopattern. Finally, we discuss the current knowledge about animal models for human plasma/serum and IgG glycosylation and mention other, less explored, instances of glycopattern changes during organismal aging and cellular senescence.


Assuntos
Envelhecimento , Polissacarídeos , Animais , Glicosilação , Humanos , Imunoglobulina G , Longevidade
17.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445522

RESUMO

Crocetin is an apocarotenoid formed from the oxidative cleavage of zeaxanthin, by the carotenoid cleavage enzymes CCD2 (in Crocus species) and specific CCD4 enzymes in Buddleja davidii and Gardenia jasminoides. Crocetin accumulates in the stigma of saffron in the form of glucosides and crocins, which contain one to five glucose molecules. Crocetin glycosylation was hypothesized to involve at least two enzymes from superfamily 1 UDP-sugar dependent glycosyltransferases. One of them, UGT74AD1, produces crocins with one and two glucose molecules, which are substrates for a second UGT, which could belong to the UGT79, 91, or 94 families. An in silico search of Crocus transcriptomes revealed six candidate UGT genes from family 91. The transcript profiles of one of them, UGT91P3, matched the metabolite profile of crocin accumulation, and were co-expressed with UGT74AD1. In addition, both UGTs interact in a two-hybrid assay. Recombinant UGT91P3 produced mostly crocins with four and five glucose molecules in vitro, and in a combined transient expression assay with CCD2 and UGT74AD1 enzymes in Nicotiana benthamiana. These results suggest a role of UGT91P3 in the biosynthesis of highly glucosylated crocins in saffron, and that it represents the last missing gene in crocins biosynthesis.


Assuntos
Carotenoides/metabolismo , Crocus/enzimologia , Perfilação da Expressão Gênica/métodos , Glicosiltransferases/genética , Vias Biossintéticas , Simulação por Computador , Crocus/química , Crocus/genética , Regulação da Expressão Gênica de Plantas , Glicosilação , Proteínas de Plantas/genética , Técnicas do Sistema de Duplo-Híbrido
18.
Molecules ; 26(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34443345

RESUMO

Protein glycosylation that mediates interactions among viral proteins, host receptors, and immune molecules is an important consideration for predicting viral antigenicity. Viral spike proteins, the proteins responsible for host cell invasion, are especially important to be examined. However, there is a lack of consensus within the field of glycoproteomics regarding identification strategy and false discovery rate (FDR) calculation that impedes our examinations. As a case study in the overlap between software, here as a case study, we examine recently published SARS-CoV-2 glycoprotein datasets with four glycoproteomics identification software with their recommended protocols: GlycReSoft, Byonic, pGlyco2, and MSFragger-Glyco. These software use different Target-Decoy Analysis (TDA) forms to estimate FDR and have different database-oriented search methods with varying degrees of quantification capabilities. Instead of an ideal overlap between software, we observed different sets of identifications with the intersection. When clustering by glycopeptide identifications, we see higher degrees of relatedness within software than within glycosites. Taking the consensus between results yields a conservative and non-informative conclusion as we lose identifications in the desire for caution; these non-consensus identifications are often lower abundance and, therefore, more susceptible to nuanced changes. We conclude that present glycoproteomics softwares are not directly comparable, and that methods are needed to assess their overall results and FDR estimation performance. Once such tools are developed, it will be possible to improve FDR methods and quantify complex glycoproteomes with acceptable confidence, rather than potentially misleading broad strokes.


Assuntos
Algoritmos , Glicopeptídeos/análise , Glicoproteínas/análise , COVID-19/metabolismo , Bases de Dados de Proteínas , Glicopeptídeos/química , Glicoproteínas/química , Glicosilação , Humanos , Proteômica/métodos , Proteômica/normas , SARS-CoV-2/metabolismo , Software , Glicoproteína da Espícula de Coronavírus/análise , Glicoproteína da Espícula de Coronavírus/química , Espectrometria de Massas em Tandem/métodos , Proteínas Virais de Fusão/análise , Proteínas Virais de Fusão/química
19.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360826

RESUMO

Glycosylation is a complex post-translational modification that conveys functional diversity to glycoconjugates. Cell surface glycosylation mediates several biological activities such as induction of the intracellular signaling pathway and pathogen recognition. Red blood cell (RBC) membrane N-glycans determine blood type and influence cell lifespan. Although several proteomic studies have been carried out, the glycosylation of RBC membrane proteins has not been systematically investigated. This work aims at exploring the human RBC N-glycome by high-sensitivity MALDI-MS techniques to outline a fingerprint of RBC N-glycans. To this purpose, the MALDI-TOF spectra of healthy subjects harboring different blood groups were acquired. Results showed the predominant occurrence of neutral and sialylated complex N-glycans with bisected N-acetylglucosamine and core- and/or antennary fucosylation. In the higher mass region, these species presented with multiple N-acetyllactosamine repeating units. Amongst the detected glycoforms, the presence of glycans bearing ABO(H) antigens allowed us to define a distinctive spectrum for each blood group. For the first time, advanced glycomic techniques have been applied to a comprehensive exploration of human RBC N-glycosylation, providing a new tool for the early detection of distinct glycome changes associated with disease conditions as well as for understanding the molecular recognition of pathogens.


Assuntos
Antígenos de Grupos Sanguíneos/metabolismo , Eritrócitos/metabolismo , Glicômica , Polissacarídeos/análise , Processamento de Proteína Pós-Traducional , Glicosilação , Humanos , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
20.
Postepy Biochem ; 67(2): 104-116, 2021 06 30.
Artigo em Polonês | MEDLINE | ID: mdl-34378887

RESUMO

Changes in glycosylation pattern of cell surface, body fluids and extracellular matrix glycoconjugates is a characteristic feature of tumor cell malignancy. These changes are the result of mutations of tumor-associated genes as well as epigenetic changes in the tumor environment, including nutrient influx, hypoxia, cytokine expression and stimulation of chronic inflammation. The unique set of cell surface glycoantigens on neoplastic cells is recognized by endogenous lectins located in the extracellular matrix, vascular endothelium, on leukocytes or platelets, and has an impact on disrupting basic cellular processes, such as intercellular recognition, cell-cell adhesion or cell-ECM interaction. These changes have a critical impact on the migration, invasive and metastatic potential of neoplastic cells and modulate the immune response. This unique pattern of sugar antigens on the cancer cells can be a vaulable marker to identify them, determine the stage of the disease as well as be a target of anti-cancer therapy.


Assuntos
Neoplasias , Adesão Celular , Matriz Extracelular/metabolismo , Glicosilação , Humanos , Neoplasias/metabolismo , Polissacarídeos/metabolismo
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