Separation and characterization of the two Asn-linked glycosylation sites of chicken serum riboflavin-binding protein. Glycosylation differences despite similarity of primary structure.
Ontology highlight
ABSTRACT:
SUBMITTER: Rohrer JS
PROVIDER: S-EPMC1132777 | biostudies-other | 1992 Jul
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Numerous protists and rare fungi have truncated Asn-linked glycan precursors and lack N-glycan-dependent quality control (QC) systems for glycoprotein folding in the endoplasmic reticulum. Here, we show that the abundance of sequons (NXT or NXS), which are sites for N-glycosylation of secreted and membrane proteins, varies by more than a factor of 4 among phylogenetically diverse eukaryotes, based on a few variables. There is positive correlation between the density of sequons and the AT content of coding regions, although no causality can be inferred. In contrast, there appears to be Darwinian selection for sequons containing Thr, but not Ser, in eukaryotes that have N-glycan-dependent QC systems. Selection for sequons with Thr, which nearly doubles the sequon density in human secreted and membrane proteins, occurs by an increased conditional probability that Asn and Thr are present in sequons rather than elsewhere. Increasing sequon densities of the hemagglutinin (HA) of influenza viruses A/H3N2 and A/H1N1 during the past few decades of human infection also result from an increased conditional probability that Asn, Thr, and Ser are present in sequons rather than elsewhere. In contrast, there is no selection on sequons by this mechanism in HA of A/H5N1 or 2009 A/H1N1 (Swine flu). Very strong selection for sequons with both Thr and Ser in glycoprotein of M(r) 120,000 (gp120) of HIV and related retroviruses results from this same mechanism, as well as amino acid composition bias and increases in AT content. We conclude that there is Darwinian selection for sequons in phylogenetically disparate eukaryotes and viruses.
| S-EPMC2726397 | biostudies-literature
Project description:To study the sequence requirements for addition of O-linked N-acetylgalactosamine to proteins, amino acid distributions around 174 O-glycosylation sites were compared with distributions around non-glycosylated sites. In comparison with non-glycosylated serine and threonine residues, the most prominent feature in the vicinity of O-glycosylated sites is a significantly increased frequency of proline residues, especially at positions -1 and +3 relative to the glycosylated residues. Alanine, serine and threonine are also significantly increased. The high serine and threonine content of O-glycosylated regions is due to the presence of clusters of several closely spaced glycosylated hydroxy amino acids in many O-glycosylated proteins. Such clusters can be predicted from the primary sequence in some cases, but there is no apparent possibility of predicting isolated O-glycosylation sites from primary sequence data.
| S-EPMC1150083 | biostudies-other
Project description:Mycobacterium tuberculosis (Mtb) causes tuberculosis, one of the leading causes of fatal infectious diseases worldwide. Cell-cell recognition between the pathogen Mtb and its host is mediated in part by glycosylated proteins. So far, glycoproteins in Mtb are understudied and for only very few glycoproteins glycosylation sites have been described, e.g., alanine and proline rich secreted protein apa, superoxide dismutase SODC, lipoprotein lpqH and MPB83/MPT83. In this study, glycosylated proteins in Mtb culture filtrate were investigated using liquid chromatography-mass spectrometry approaches and bioinformatic analyses. To validate the presence of glycoproteins, several strategies were pursued including collision induced dissociation, high energy collision dissociation and electron transfer dissociation techniques, and bioinformatics analyses involving a neutral loss search for glycosylated moieties. After extensive data curation, we report glycosylation sites for thirteen Mtb glycoproteins using a combination of mass spectrometry techniques on a dataset collected from culture filtrate proteins. This is the first glycoproteomics study identifying glycosylation sites on mycobacterial culture filtrate proteins (CFP) on a global scale.Biological significanceIn this study, glycosylation sites in Mtb were characterized by collision-induced dissociation, electron-transfer dissociation and high energy collision dissociation techniques. The identification of glycosylation sites is important for our understanding of the physiology and pathophysiology of Mtb. Glycoproteins are often responsible for protein-protein interactions between host and pathogen and thus represent interesting targets for vaccine development. In addition, our strategy is not limited to Mtb, but could be extended to other organisms. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.
| S-EPMC3806883 | biostudies-literature
Project description:The addition of asparagine (N)-linked polysaccharide chains (i.e., glycans) to the gp120 and gp41 glycoproteins of human immunodeficiency virus type 1 (HIV-1) envelope is not only required for correct protein folding, but also may provide protection against neutralizing antibodies as a "glycan shield." As a result, strong host-specific selection is frequently associated with codon positions where nonsynonymous substitutions can create or disrupt potential N-linked glycosylation sites (PNGSs). Moreover, empirical data suggest that the individual contribution of PNGSs to the neutralization sensitivity or infectivity of HIV-1 may be critically dependent on the presence or absence of other PNGSs in the envelope sequence. Here we evaluate how glycan-glycan interactions have shaped the evolution of HIV-1 envelope sequences by analyzing the distribution of PNGSs in a large-sequence alignment. Using a "covarion"-type phylogenetic model, we find that the rates at which individual PNGSs are gained or lost vary significantly over time, suggesting that the selective advantage of having a PNGS may depend on the presence or absence of other PNGSs in the sequence. Consequently, we identify specific interactions between PNGSs in the alignment using a new paired-character phylogenetic model of evolution, and a Bayesian graphical model. Despite the fundamental differences between these two methods, several interactions are jointly identified by both. Mapping these interactions onto a structural model of HIV-1 gp120 reveals that negative (exclusive) interactions occur significantly more often between colocalized glycans, while positive (inclusive) interactions are restricted to more distant glycans. Our results imply that the adaptive repertoire of alternative configurations in the HIV-1 glycan shield is limited by functional interactions between the N-linked glycans. This represents a potential vulnerability of rapidly evolving HIV-1 populations that may provide useful glycan-based targets for neutralizing antibodies.
| S-EPMC1779302 | biostudies-literature
Project description:Glycosylation is one of the most complex post translation modification in eukaryotic cells. Almost 50% of the human proteome is glycosylated as glycosylation plays a vital role in various biological functions such as antigen's recognition, cell-cell communication, expression of genes and protein folding. It is a significant challenge to identify glycosylation sites in protein sequences as experimental methods are time taking and expensive. A reliable computational method is desirable for the identification of glycosylation sites. In this study, a comprehensive technique for the identification of N-linked glycosylation sites has been proposed using machine learning. The proposed predictor was trained using an up-to-date dataset through back propagation algorithm for multilayer neural network. The results of ten-fold cross-validation and other performance measures such as accuracy, sensitivity, specificity and Mathew's correlation coefficient inferred that the accuracy of proposed tool is far better than the existing systems such as Glyomine, GlycoEP, Ensemble SVM and GPP.
| S-EPMC5552137 | biostudies-literature
Project description:The vitamin K-dependent carboxylase is an integral membrane protein which is required for the post-translational modification of a variety of vitamin K-dependent proteins. Previous studies have suggested carboxylase is a glycoprotein with N-linked glycosylation sites. In this study, we identify the N-glycosylation sites of carboxylase by mass spectrometric peptide mapping analyses combined with site-directed mutagenesis. Our mass spectrometric results show that the N-linked glycosylation in carboxylase occurs at positions N459, N550, N605, and N627. Eliminating these glycosylation sites by changing asparagine to glutamine caused the mutant carboxylase to migrate faster on SDS-PAGE gels, adding further evidence that these sites are glycosylated. In addition, the mutation studies identified N525, a site that cannot be recovered by mass spectroscopy analysis, as a glycosylation site. Furthermore, the potential glycosylation site at N570 is glycosylated only if all five natural glycosylation sites are simultaneously mutated. Removal of the oligosaccharides by glycosidase from wild-type carboxylase or by elimination of the functional glycosylation sites by site-directed mutagenesis did not affect either the carboxylation or epoxidation activity when the small FLEEL pentapeptide was used as a substrate, suggesting that N-linked glycosylation is not required for the enzymatic function of carboxylase. In contrast, when site N570 and the five natural glycosylation sites were mutated simultaneously, the resulting carboxylase protein was degraded. Our results suggest that N-linked glycosylation is not essential for carboxylase enzymatic activity but is important for protein folding and stability.
| S-EPMC3956053 | biostudies-literature
Project description:Follicle-stimulating hormone (FSH) is a gonadotrope-derived heterodimeric glycoprotein. Both the common α- and hormone-specific β subunits contain Asn-linked N-glycan chains. Recently, macroheterogeneous FSH glycoforms consisting of β-subunits that differ in N-glycan number were identified in pituitaries of several species and subsequently the recombinant human FSH glycoforms biochemically characterized. Although chemical modification and in vitro site-directed mutagenesis studies defined the roles of N-glycans on gonadotropin subunits, in vivo functional analyses in a whole-animal setting are lacking. Here, we have generated transgenic mice with gonadotrope-specific expression of either an HFSHB(WT) transgene that encodes human FSHβ WT subunit or an HFSHB(dgc) transgene that encodes a human FSHβ(Asn7Δ 24Δ) double N-glycosylation site mutant subunit, and separately introduced these transgenes onto Fshb null background using a genetic rescue strategy. We demonstrate that the human FSHβ(Asn7Δ 24Δ) double N-glycosylation site mutant subunit, unlike human FSHβ WT subunit, inefficiently combines with the mouse α-subunit in pituitaries of Fshb null mice. FSH dimer containing this mutant FSHβ subunit is inefficiently secreted with very low levels detectable in serum. Fshb null male mice expressing HFSHB(dgc) transgene are fertile and exhibit testis tubule size and sperm number similar to those of Fshb null mice. Fshb null female mice expressing the mutant, but not WT human FSHβ subunit-containing FSH dimer are infertile, demonstrate no evidence of estrus cycles, and many of the FSH-responsive genes remain suppressed in their ovaries. Thus, HFSHB(dgc) unlike HFSHB(WT) transgene does not rescue Fshb null mice. Our genetic approach provides direct in vivo evidence that N-linked glycans on FSHβ subunit are essential for its efficient assembly with the α-subunit to form FSH heterodimer in pituitary. Our studies also reveal that N-glycans on FSHβ subunit are essential for FSH secretion and FSH in vivo bioactivity to regulate gonadal growth and physiology.
| S-EPMC5130991 | biostudies-literature
Project description:N-Glycans of Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, are of great interest for multiple reasons. E. histolytica makes an unusual truncated N-glycan precursor (Man(5)GlcNAc(2)), has few nucleotide sugar transporters, and has a surface that is capped by the lectin concanavalin A. Here, biochemical and mass spectrometric methods were used to examine N-glycan biosynthesis and the final N-glycans of E. histolytica with the following conclusions. Unprocessed Man(5)GlcNAc(2), which is the most abundant E. histolytica N-glycan, is aggregated into caps on the surface of E. histolytica by the N-glycan-specific, anti-retroviral lectin cyanovirin-N. Glc(1)Man(5)GlcNAc(2), which is made by a UDP-Glc: glycoprotein glucosyltransferase that is part of a conserved N-glycan-dependent endoplasmic reticulum quality control system for protein folding, is also present in mature N-glycans. A swainsonine-sensitive alpha-mannosidase trims some N-glycans to biantennary Man(3)GlcNAc(2). Complex N-glycans of E. histolytica are made by the addition of alpha1,2-linked Gal to both arms of small oligomannose glycans, and Gal residues are capped by one or more Glc. In summary, E. histolytica N-glycans include unprocessed Man(5)GlcNAc(2), which is a target for cyanovirin-N, as well as unique, complex N-glycans containing Gal and Glc.
| S-EPMC2440624 | biostudies-literature
Project description:The asparagine-X-serine/threonine (NXS/T) motif, where X is any amino acid except proline, is the consensus motif for N-linked glycosylation. Significant numbers of high-resolution crystal structures of glycosylated proteins allow us to carry out structural analysis of the N-linked glycosylation sites (NGS). Our analysis shows that there is enough structural information from diverse glycoproteins to allow the development of rules which can be used to predict NGS. A Python-based tool was developed to investigate asparagines implicated in N-glycosylation in five species: Homo sapiens, Mus musculus, Drosophila melanogaster, Arabidopsis thaliana and Saccharomyces cerevisiae. Our analysis shows that 78% of all asparagines of NXS/T motif involved in N-glycosylation are localized in the loop/turn conformation in the human proteome. Similar distribution was revealed for all the other species examined. Comparative analysis of the occurrence of NXS/T motifs not known to be glycosylated and their reverse sequence (S/TXN) shows a similar distribution across the secondary structural elements, indicating that the NXS/T motif in itself is not biologically relevant. Based on our analysis, we have defined rules to determine NGS. Using machine learning methods based on these rules we can predict with 93% accuracy if a particular site will be glycosylated. If structural information is not available the tool uses structural prediction results resulting in 74% accuracy. The tool was used to identify glycosylation sites in 108 human proteins with structures and 2247 proteins without structures that have acquired NXS/T site/s due to non-synonymous variation. The tool, Structure Feature Analysis Tool (SFAT), is freely available to the public at http://hive.biochemistry.gwu.edu/tools/sfat.
| S-EPMC3914773 | biostudies-literature
Project description:Site-directed mutagenesis has been used to remove 15 of the 18 potential N-linked glycosylation sites, in 16 combinations, from the human exon 11-minus receptor isoform. The three glycosylation sites not mutated were asparagine residues 25, 397 and 894, which are known to be important in receptor biosynthesis or function. The effects of these mutations on proreceptor processing into alpha and beta subunits, cell-surface expression, insulin binding and receptor autophosphorylation were assessed in Chinese hamster ovary cells. The double mutants 16+78, 16+111, 16+215, 16+255, 337+418, the triple mutants 295+337+418, 295+418+514, 337+418+514 and 730+743+881 and the quadruple mutants 606+730+743+881 and 671+730+743+881 seemed normal by all criteria examined. The triple mutant 16+215+255 showed only low levels of correctly processed receptor on the cell surface, this processed receptor being autophosphorylated in response to insulin. The quadruple mutant 624+730+743+881 showed normal processing and ligand binding but exhibited a constitutively active tyrosine kinase as judged by autophosphorylation. Three higher-order mutants were constructed, two of which, 16+337+418+730+743+881 (Delta6) and 16+295+337+418+730+743+881 (Delta7a), seemed normal. The third construct, 16+337+418+514+730+743+881 (Delta7b), was expressed at high levels on the cell surface, essentially as uncleaved proreceptor with only the small proportion of Delta7b that was correctly processed showing insulin-stimulated autophosphorylation. The mutations of Delta6 and Delta7a were incorporated into soluble ectodomains, which had affinities for insulin that were 4-fold that of wild-type ectodomain. The Delta6 ectodomain expressed in Lec8 cells was produced in quantity in a bioreactor for subsequent structural analysis.
| S-EPMC1221015 | biostudies-other