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Frontiers in Cellular and Infection... 2017is able to invade, survive and replicate inside a variety of cell types. However, preferentially enters host macrophages where it rapidly escapes to the cytosol to... (Review)
Review
is able to invade, survive and replicate inside a variety of cell types. However, preferentially enters host macrophages where it rapidly escapes to the cytosol to avoid phagosomal stresses and to multiply to high numbers. We previously showed that human monocyte infection by LVS triggered deglycosylation of the glutamine transporter SLC1A5. However, this deglycosylation, specifically induced by infection, was not restricted to SLC1A5, suggesting that host protein deglycosylation processes in general might contribute to intracellular bacterial adaptation. Indeed, we later found that infection modulated the transcription of numerous glycosidase and glycosyltransferase genes in human macrophages and analysis of cell extracts revealed an important increase of N and O-protein glycosylation. In eukaryotic cells, glycosylation has significant effects on protein folding, conformation, distribution, stability, and activity and dysfunction of protein glycosylation may lead to development of diseases like cancer and pathogenesis of infectious diseases. Pathogenic bacteria have also evolved dedicated glycosylation machineries and have notably been shown to use these glycoconjugates as ligands to specifically interact with the host. In this review, we will focus on and summarize our current understanding of the importance of these post-translational modifications on its intracellular niche adaptation.
Topics: Animals; Francisella tularensis; Gene Expression Regulation; Glycoside Hydrolases; Glycosylation; Glycosyltransferases; Host-Pathogen Interactions; Humans; Macrophages
PubMed: 28377902
DOI: 10.3389/fcimb.2017.00071 -
Journal of Translational Medicine Jan 2020Serum protein glycosylation is an area of investigation in inflammatory arthritic disorders such as rheumatoid arthritis (RA). Indeed, some studies highlighted...
Glycosylation deficiency of lipopolysaccharide-binding protein and corticosteroid-binding globulin associated with activity and response to treatment for rheumatoid arthritis.
BACKGROUND
Serum protein glycosylation is an area of investigation in inflammatory arthritic disorders such as rheumatoid arthritis (RA). Indeed, some studies highlighted abnormalities of protein glycosylation in RA. Considering the numerous types of enzymes, monosaccharides and glycosidic linkages, glycosylation is one of the most complex post translational modifications. By this work, we started with a preliminary screening of glycoproteins in serum from RA patients and controls.
METHODS
In order to isolate glycoproteins from serum, lectin wheat germ agglutinin was used and quantitative differences between patients and controls were investigated by LC-MS/MS. Consequently, we focused our attention on two glycoproteins found in this explorative phase: corticosteroid-binding globulin (CBG) and lipopolysaccharide-binding protein (LBP). The subsequent validation with immunoassays was widened to a larger number of early RA (ERA) patients (n = 90) and well-matched healthy controls (n = 90).
RESULTS
We observed a significant reduction of CBG and LBP glycosylation in ERA patients compared with healthy controls. Further, after 12 months of treatment, glycosylated CBG and LBP levels increased both to values comparable to those of controls. In addition, these changes were correlated with clinical parameters.
CONCLUSIONS
This study enables to observe that glycosylation changes of CBG and LBP are related to RA disease activity and its response to treatment.
Topics: Acute-Phase Proteins; Arthritis, Rheumatoid; Carrier Proteins; Chromatography, Liquid; Glycosylation; Humans; Membrane Glycoproteins; Tandem Mass Spectrometry; Transcortin
PubMed: 31907043
DOI: 10.1186/s12967-019-02188-9 -
Journal of Visualized Experiments : JoVE Dec 2011Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell...
Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms. The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCGβ), which carries two N-glycans and four O-glycans. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples. Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), β1-4 Galactosidase, and β-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans. Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (α-N-Acetylgalactosaminidase, α1-2 Fucosidase, α1-3,6 Galactosidase, and β1-3 Galactosidase), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans. SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 μg), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.
Topics: Chorionic Gonadotropin, beta Subunit, Human; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Glycoside Hydrolases; Glycosylation; Humans; Models, Molecular; Proteins; Recombinant Proteins; Substrate Specificity
PubMed: 22230788
DOI: 10.3791/3749 -
Journal of Bacteriology Mar 2023Neisseria meningitidis exhibits a general -linked protein glycosylation system in which pili and other extracytoplasmic proteins are glycosylated. To investigate glycan...
Neisseria meningitidis exhibits a general -linked protein glycosylation system in which pili and other extracytoplasmic proteins are glycosylated. To investigate glycan antigenicity in humans and the significance of high glycan diversity on immune escape mechanisms, we exploited serogroup A meningococcal strains and serum samples obtained from laboratory-confirmed Ethiopian patients with meningococcal disease. The 37 meningococcal isolates were sequenced, and their rotein ycosylation () genotypes and protein glycosylation phenotypes were investigated in detail. An insertion sequence (IS) element in reduced glycan variability in the majority of isolates, while phase variation strengthened glycan variability and microheterogeneity. Homologous recombination events within the genes were identified in eight of the 37 isolates, and the phenotypic consequences ranged from none detected to altered glycoforms in two of the isolates in which the whole locus was exchanged. Immunoblotting of sera against a complete panel of glycan-expressing mutant strains demonstrated that most of these patient sera had IgG antibodies against various neisserial protein glycan antigens. Furthermore, using a bactericidal assay comparing a wild-type meningococcal A strain and a glycosylation-null variant strain, we showed that these protein glycan antigens interfere with bactericidal killing by antibodies in patient sera. Altogether, we were largely able to link genotype with glycosylation phenotype. Our study reveals that protein glycans seem to contribute to the ability of N. meningitidis to resist the bactericidal activity of human serum, possibly by masking protein epitopes important for bactericidal killing and thus protection against meningococcal disease. Bacterial meningitis is a serious global health problem, and one of the major causative organisms is Neisseria meningitidis. Extensive variability in protein glycan structure and antigenicity is due to phase variation of protein glycosylation genes and polymorphic gene content and function. The exact role(s) of glycosylation in remains to be determined, but increasing evidence, supported by this study, suggests that glycan variability can be a strategy to escape the human immune system. The complexity of the -linked protein glycosylation system requires further studies to fully comprehend how these bacteria utilize variation in genes to produce such high glycoform diversity and to evade the human immune response.
Topics: Humans; Glycosylation; Neisseria meningitidis; Bacterial Proteins; Serogroup; Polysaccharides; Meningococcal Infections; Meningococcal Vaccines
PubMed: 36852982
DOI: 10.1128/jb.00458-22 -
ACS Chemical Biology Jan 2012Protein glycosylation is a common and complex posttranslational modification of proteins, which expands functional diversity while boosting structural heterogeneity.... (Review)
Review
Protein glycosylation is a common and complex posttranslational modification of proteins, which expands functional diversity while boosting structural heterogeneity. Glycoproteins, the end products of such a modification, are typically produced as mixtures of glycoforms possessing the same polypeptide backbone but differing in the site of glycosylation and/or in the structures of pendant glycans, from which single glycoforms are difficult to isolate. The urgent need for glycan-defined glycoproteins in both detailed structure-function relationship studies and therapeutic applications has stimulated an extensive interest in developing various methods for manipulating protein glycosylation. This review highlights emerging technologies that hold great promise in making a variety of glycan-defined glycoproteins, with a particular emphasis in the following three areas: specific glycoengineering of host biosynthetic pathways, in vitro chemoenzymatic glycosylation remodeling, and chemoselective and site-specific glycosylation of proteins.
Topics: Animals; CHO Cells; Carbohydrate Conformation; Carbohydrate Sequence; Cricetinae; Escherichia coli; Gene Expression; Glycoconjugates; Glycoproteins; Glycosylation; HEK293 Cells; Humans; Isomerism; Models, Molecular; Molecular Sequence Data; Plant Cells; Polysaccharides; Protein Engineering; Protein Processing, Post-Translational; Protein Structure, Secondary; Structure-Activity Relationship; Yeasts
PubMed: 22141574
DOI: 10.1021/cb200429n -
PloS One 2023Monitoring human circulating N-glycome could provide valuable insight into an individual's metabolic status. Therefore, we examined if aberrant carbohydrate metabolism...
BACKGROUND
Monitoring human circulating N-glycome could provide valuable insight into an individual's metabolic status. Therefore, we examined if aberrant carbohydrate metabolism in gestational diabetes mellitus (GDM) associates with alterations in plasma protein, immunoglobulin G (IgG) and immunoglobulin A (IgA) N-glycosylation.
METHODS
Plasma protein, IgG and IgA N-glycans were enzymatically released, purified and chromatographically profiled in 48 pregnant women with normal glucose tolerance and 41 pregnant women with GDM, all sampled at 24-28 weeks of gestation. Linear mixed models adjusting for age and multiple testing (FDR<0.05) were used to investigate the associations between glycosylation features, metabolic markers and GDM status.
RESULTS
Fasting insulin exhibited significant associations to numerous glycan traits, including plasma protein galactosylation, sialylation, branching, core fucosylation and bisection, to IgG core fucosylated, bisected (FA2B) and afucosylated disialylated (A2G2S2) glycan and to IgA trisialylated triantennary (A3G3S3) glycan (padj range: 4.37x10-05-4.94x10-02). Insulin resistance markers HOMA2-IR and HOMA2-%B were mostly associated to the same glycan structures as fasting insulin. Both markers showed positive association with high-branched plasma glycans (padj = 1.12x10-02 and 2.03x10-03) and negative association with low-branched plasma glycans (padj = 1.21x10-02 and 2.05x10-03). Additionally, HOMA2-%B index was significantly correlated with glycosylation features describing IgG sialylation. Multiple plasma protein IgG and IgA glycans showed significant associations with total cholesterol and triglyceride levels. None of the tested glycan traits showed a significant difference between GDM and normoglycemic pregnancies.
CONCLUSION
Markers of glucose homeostasis and lipid metabolism in pregnancy show extensive associations to various N-glycosylation features. However, plasma protein, IgG and IgA N-glycans were not able to differentiate pregnant women with and without GDM, possibly due to numerous physiological changes accompanying pregnancy, which confound the impact of GDM on protein glycosylation.
Topics: Humans; Female; Pregnancy; Glycosylation; Diabetes, Gestational; Immunoglobulin A; Polysaccharides; Immunoglobulin G; Insulin; Blood Proteins; Glucose
PubMed: 37079606
DOI: 10.1371/journal.pone.0284838 -
Ultrasonics Sonochemistry Sep 2022This study investigated the effects of different ultrasonic power and ultrasonic time on the structure and emulsifying properties of pea protein isolate (PPI)-arabinose...
This study investigated the effects of different ultrasonic power and ultrasonic time on the structure and emulsifying properties of pea protein isolate (PPI)-arabinose conjugates. An examination of the absorbance and color development of PPI-d-arabinose (Ara) conjugates found that compared with traditional heating, the degree of glycosylation of protein reached the maximum when the ultrasonic treatment power was 150 and the treatment time was 30 min. Structural analysis revealed that the content of disordered structures (β-turn and random coil) of the protein conjugates increased, the maximum emission wavelength of the fluorescence spectrum was red-shifted, and the UV second-order derivative values decreased. The protein structure unfolded, exposing more hydrophobic groups on the molecular surface. Ultrasonic treatment improved the emulsification of protein conjugates. The emulsifying activity index (EAI) increased to 19.7 and 19.3 m/g, and the emulsifying stability index (ESI) also increased. The contact angle and zeta potential also demonstrate that ultrasonic power has a positive effect on emulsion stability. Based on examining the thermal stability of the emulsion, the ultrasonic treatment increased the thermal denaturation resistance of the protein. This result confirms that mild sonication can increase the degree of glycosylation reaction and improve the emulsification properties of protein-Ara conjugates, providing a theoretical basis for developing foods with excellent emulsification properties.
Topics: Arabinose; Emulsifying Agents; Emulsions; Glycosylation; Hydrophobic and Hydrophilic Interactions; Maillard Reaction; Pea Proteins; Solubility
PubMed: 36088895
DOI: 10.1016/j.ultsonch.2022.106157 -
International Journal of Molecular... Mar 2023Protein glycosylation, including sialylation, involves complex and frequent post-translational modifications, which play a critical role in different biological... (Review)
Review
Protein glycosylation, including sialylation, involves complex and frequent post-translational modifications, which play a critical role in different biological processes. The conjugation of carbohydrate residues to specific molecules and receptors is critical for normal hematopoiesis, as it favors the proliferation and clearance of hematopoietic precursors. Through this mechanism, the circulating platelet count is controlled by the appropriate platelet production by megakaryocytes, and the kinetics of platelet clearance. Platelets have a half-life in blood ranging from 8 to 11 days, after which they lose the final sialic acid and are recognized by receptors in the liver and eliminated from the bloodstream. This favors the transduction of thrombopoietin, which induces megakaryopoiesis to produce new platelets. More than two hundred enzymes are responsible for proper glycosylation and sialylation. In recent years, novel disorders of glycosylation caused by molecular variants in multiple genes have been described. The phenotype of the patients with genetic alterations in and is consistent with syndromic manifestations, severe inherited thrombocytopenia, and hemorrhagic complications.
Topics: Humans; Glycosylation; Thrombocytopenia; Blood Platelets; Megakaryocytes; Thrombopoiesis; Thrombopoietin; Nucleotide Transport Proteins
PubMed: 36982178
DOI: 10.3390/ijms24065109 -
Science China. Life Sciences Jan 2016The study of human microbiota is an emerging research topic. The past efforts have mainly centered on studying the composition and genomic landscape of bacterial species... (Review)
Review
The study of human microbiota is an emerging research topic. The past efforts have mainly centered on studying the composition and genomic landscape of bacterial species within the targeted communities. The interaction between bacteria and hosts is the pivotal event in the initiation and progression of infectious diseases. There is a great need to identify and characterize the molecules that mediate the bacteria-host interaction. Bacterial surface exposed proteins play an important role in the bacteria- host interaction. Numerous surface proteins are glycosylated, and the glycosylation is crucial for their function in mediating the bacterial interaction with hosts. Here we present an overview of surface glycoproteins from bacteria that inhabit three major mucosal environments across human body: oral, gut and skin. We describe the important enzymes involved in the process of protein glycosylation, and discuss how the process impacts the bacteria-host interaction. Emerging molecular details underlying glycosylation of bacterial surface proteins may lead to new opportunities for designing anti-infective small molecules, and developing novel vaccines in order to treat or prevent bacterial infection.
Topics: Bacterial Proteins; Gastrointestinal Microbiome; Glycosylation; Host-Pathogen Interactions; Humans; Microbiota; Mouth Mucosa; Protein Processing, Post-Translational; Skin
PubMed: 26712033
DOI: 10.1007/s11427-015-4980-7 -
The FEBS Journal Jan 2014Glycosylation is one of the most common, and the most complex, forms of post-translational modification of proteins. This review serves to highlight the role of protein... (Review)
Review
Glycosylation is one of the most common, and the most complex, forms of post-translational modification of proteins. This review serves to highlight the role of protein glycosylation in Alzheimer disease (AD), a topic that has not been thoroughly investigated, although glycosylation defects have been observed in AD patients. The major pathological hallmarks in AD are neurofibrillary tangles and amyloid plaques. Neurofibrillary tangles are composed of phosphorylated tau, and the plaques are composed of amyloid β-peptide (Aβ), which is generated from amyloid precursor protein (APP). Defects in glycosylation of APP, tau and other proteins have been reported in AD. Another interesting observation is that the two proteases required for the generation of amyloid β-peptide (Aβ), i.e. γ-secretase and β-secretase, also have roles in protein glycosylation. For instance, γ-secretase and β-secretase affect the extent of complex N-glycosylation and sialylation of APP, respectively. These processes may be important in AD pathogenesis, as proper intracellular sorting, processing and export of APP are affected by how it is glycosylated. Furthermore, lack of one of the key components of γ-secretase, presenilin, leads to defective glycosylation of many additional proteins that are related to AD pathogenesis and/or neuronal function, including nicastrin, reelin, butyrylcholinesterase, cholinesterase, neural cell adhesion molecule, v-ATPase, and tyrosine-related kinase B. Improved understanding of the effects of AD on protein glycosylation, and vice versa, may therefore be important for improving the diagnosis and treatment of AD patients.
Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Glycosylation; Humans; Protein Processing, Post-Translational; Reelin Protein
PubMed: 24279329
DOI: 10.1111/febs.12590