Browsing by Subject "Glycosylation"

Now showing 1 - 8 of 8
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    Compound heterozygosity involving Antithrombin Cambridge II (p.Ala416Ser) in antithrombin deficiency.
    (Elsevier, 2013-03-10) Águila Martínez, Sonia; Martínez-Martínez, Irene; Collado, Miriam; Llamas, Pilar; Antón, Ana I.; Martínez-Redondo, Consuelo; Padilla, José; Miñano, Antonia; Morena Barrio, María Eugenia de la; García-Avello, Ángel; Vicente García, Vicente; Corral de la Calle, Javier; Medicina Interna
    Background: The characterization of natural mutants identified in patients with antithrombin deficiency has helped to identify functional domains or regions of this key anticoagulant and the mechanisms involved in the deficiency, as well as to define the clinical prognosis. Recently, we described an abnormal glycosylation in a pleiotropic mutant (K241E) that explained the impaired heparin affinity and the mild risk of thrombosis in carriers. Objectives: To evaluate the effects of different natural pleiotropic mutations on the glycosylation of antithrombin and their functional effects. Methods: Five pleiotropic mutations identified in patients with antithrombin deficiency and located at each one of the strands of the C-sheet were selected (K241E, M251I, M315K, F402L, and P429L). Recombinant mutants were generated and purified. Glycoform heterogeneity and conformational sensitivity were studied with electrophoresis, proteomic analysis, and glycomic analysis. Heparin affinity was evaluated from intrinsic fluorescence. Reactivity assays with factor Xa, thrombin and neutrophil elastase in the presence or absence of heparin were also performed. Results and Conclusions: Pleiotropic mutants, except for that with the M315K mutation, which affects a non-exposed residue, showed two glycoforms. Variant 1, with abnormal glycosylation, had reduced heparin affinity and severely affected reactivity with the target proteases. In contrast, variant 2, with similar electrophoretic mobility and heparin affinity to wild-type antithrombin, had impaired inhibitory activity that was partially compensated for by activation with heparin. Our results suggest the C-sheet of antithrombin as a new region that is relevant for proper maturation of the N-glycans. Therefore, pleiotropic mutations lead to glycosylation defects that are responsible for the reduced heparin affinity
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    Emerging role of tissue lectins as microenvironmental effectors in tumors and wounds
    (F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2015) Smetana, Karel Jr.; Szabo, Pavol; Gál, Peter; André, Sabine; Gabius, Hans-Joachim; Kodet, Ondřej; Dvořánková, Barbora
    Detailed comparative analysis of at first sight not related process cascades is a means toward this aim: to trace common effector mechanisms and hereby eventually inspire innovative routes for therapeutic management. Following this concept, promotion of tumor progression by stroma, especially cancerassociated fibroblasts and smooth muscle actin-positive myofibroblasts, and beneficial activity of respective cells in wound healing have helped to delineate the involvement of endogenous lectins of the family of galectins. In addition to initiating conversion of fibroblasts to myofibroblasts, galectin-1 instructs the cells to produce a structurally complex extracellular matrix. This bioscaffold is useful for keratinocyte culture, also apparently operative in ameliorating wound healing. These functional aspects encourage to study in detail how lectin-(glycan) counterreceptor display is orchestrated. Such insights are assumed to have potential to contribute to rationally manipulate stem/precursor cells as resource in regenerative medicine.
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    How galectins have become multifunctional proteins
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) García Caballero, Gabriel; Kaltner, Herbert; Kutzner, Tanja J.; Ludwig, Anna-Kristin; Manning, Joachim C.; Schmidt, Sebastian; Sinowatz, Fred; Gabius, Hans-Joachim
    Having identified glycans of cellular glycoconjugates as versatile molecular messages, their recognition by sugar receptors (lectins) is a fundamental mechanism within the flow of biological information. This type of molecular interplay is increasingly revealed to be involved in a wide range of (patho)physiological processes. To do so, it is a vital prerequisite that a lectin (and its expression) can develop more than a single skill, that is the general ability to bind glycans. By studying the example of vertebrate galectins as a model, a total of five relevant characteristics is disclosed: i) access to intra- and extracellular sites, ii) fine-tuned gene regulation (with evidence for co-regulation of counterreceptors) including the existence of variants due to alternative splicing or single nucleotide polymorphisms, iii) specificity to distinct glycans from the glycome with different molecular meaning, iv) binding capacity also to peptide motifs at different sites on the protein and v) diversity of modular architecture. They combine to endow these lectins with the capacity to serve as multi-purpose tools. Underscoring the arising broad-scale significance of tissue lectins, their numbers in terms of known families and group members have steadily grown by respective research that therefore unveiled a well-stocked toolbox. The generation of a network of (ga)lectins by evolutionary diversification affords the opportunity for additive/synergistic or antagonistic interplay in situ, an emerging aspect of (ga)lectin functionality. It warrants close scrutiny. The realization of the enormous potential of combinatorial permutations using the five listed features gives further efforts to understand the rules of functional glycomics/lectinomics a clear direction.
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    Identification of Antithrombin-Modulating Genes. Role of LARGE, a Gene Encoding a Bifunctional Glycosyltransferase, in the Secretion of Proteins?
    (Public Library of Science, 2013) Morena-Barrio, María Eugenia de la; Buil, Alfonso; Antón, Ana Isabel; Martínez-Martínez, Irene; Miñano, Antonia; Guriérrez-Gallego, Ricardo; Navarro-Fernandez, José; Águila, Sonia; Souto, Juan Carlos; Vicente, Vicente; Soria, José Manuel; Corral, Javier; Medicina
    The haemostatic relevance of antithrombin together with the low genetic variability of SERPINC1, and the high heritability of plasma levels encourage the search for modulating genes. We used a hypothesis-free approach to identify these genes, evaluating associations between plasma antithrombin and 307,984 polymorphisms in the GAIT study (352 individuals from 21 Spanish families). Despite no SNP reaching the genome wide significance threshold, we verified milder positive associations in 307 blood donors from a different cohort. This validation study suggested LARGE, a gene encoding a protein with xylosyltransferase and glucuronyltransferase activities that forms heparin-like linear polysaccharides, as a potential modulator of antithrombin based on the significant association of one SNPs, rs762057, with anti-FXa activity, particularly after adjustment for age, sex and SERPINC1 rs2227589 genotype, all factors influencing antithrombin levels (p = 0.02). Additional results sustained this association. LARGE silencing inHepG2 and HEK-EBNA cells did not affect SERPINC1 mRNA levels but significantly reduced the secretion of antithrombin with moderate intracellular retention. Milder effects were observed on a1-antitrypsin, prothrombin and transferrin. Our study suggests LARGE as the first known modifier of plasma antithrombin, and proposes a new role for LARGE in modulating extracellular secretion of certain glycoproteins.
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    N-Glycosylation as a Tool to Study Antithrombin Secretion, Conformation, and Function.
    (MDPI, 2021-06-06) Águila Martínez, Sonia; Noto, Rosina; Luengo-Gil, Ginés; Espín, Salvador; Bohdan, Nataliya; Morena Barrio, María Eugenia de la; Peñas, Julia; Rodenas, Maria Carmen; Vicente García, Vicente; Corral de la Calle, Javier; Manno, Mauro; Martínez-Martínez, Irene; Medicina Interna
    N-linked glycosylation is a crucial post-translational modification involved in protein folding, function, and clearance. N-linked glycosylation is also used therapeutically to enhance the half-lives of many proteins. Antithrombin, a serpin with four potential N-glycosylation sites, plays a pivotal role in hemostasis, wherein its deficiency significantly increases thrombotic risk. In this study, we used the introduction of N-glycosylation sites as a tool to explore what effect this glycosylation has on the protein folding, secretion, and function of this key anticoagulant. To accomplish this task, we introduced an additional N-glycosylation sequence in each strand. Interestingly, all regions that likely fold rapidly or were surrounded by lysines were not glycosylated even though an Nglycosylation sequon was present. The new sequon in the strands of the A- and B-sheets reduced secretion, and the B-sheet was more sensitive to these changes. However, the mutations in the strands of the C-sheet allowed correct folding and secretion, which resulted in functional variants. Therefore, our study revealed crucial regions for antithrombin secretion and could potentially apply to all serpins. These results could also help us understand the functional effects of natural variants causing type-I deficiencies.
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    N-linked glycan truncation causes enhanced clearance of plasma-derived von Willebrand factor
    (Elsevier, 2016-12-09) Águila Martínez, Sonia; O'Sullivan, J.M.; McRae, E.; Ward, S.E.; Rawley, O.; Fallon, P.G.; Brophy, T.M.; Preston, R.J.S.; Brady, L.; Sheils, O.; Chion, A.; O'Donnell, J.S.; Medicina
    Background: Enhanced von Willebrand factor (VWF) clearance is important in the etiology of both type 1 and type 2 von Willebrand disease (VWD). In addition, previous studies have demonstrated that VWF glycans play a key role in regulating in vivo clearance. However, the molecular mechanisms underlying VWF clearance remain poorly understood. Objective: To define the molecular mechanisms through which VWF N-linked glycan structures influence in vivo clearance. Methods: By use of a series of exoglycosidases, different plasma-derived VWF (pd-VWF) glycoforms were generated. In vivo clearance of these glycoforms was then assessed in VWF−/− mice in the presence or absence of inhibitors of asialoglycoprotein receptor (ASGPR), or following clodronate-induced macrophage depletion. Results Reduced amounts of N-linked and O-linked sialylation resulted in enhanced pd-VWF clearance modulated via ASGPR. In addition to this role of terminal sialylation, we further observed that progressive N-linked glycan trimming also resulted in markedly enhanced VWF clearance. Furthermore, these additional N-linked glycan effects on clearance were ASGPR-independent, and instead involved enhanced macrophage clearance that was mediated, at least in part, through LDL receptor-related protein 1. Conclusion: The carbohydrate determinants expressed on VWF regulate susceptibility to proteolysis by ADAMTS-13. In addition, our findings now further demonstrate that non-sialic acid carbohydrate determinants expressed on VWF also play an unexpectedly important role in modulating in vivo clearance through both hepatic ASGPR-dependent and macrophage-dependent pathways. In addition, these data further support the hypothesis that variation in VWF glycosylation may be important in the pathophysiology underlying type 1C VWD.
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    Role of the C-sheet in the maturation of N-glycans on antithrombin: functional relevance of pleiotropic mutations
    (2014-04-15) Águila Martínez, Sonia; Navarro Fernández, José Luis; Bohdan, N.; Gutiérrez Gallego, R.; Morena Barrio, María Eugenia de la; Vicente García, Vicente; Corral de la Calle, Javier; Martínez-Martínez, I.; Medicina Interna; Facultad de Medicina
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    The presence of the u-opioid receptor in the isthmus of mare oviduct
    (Murcia : F. Hernández, 2008) Desantis, S.; Albrizio, M.; Ventriglia, G.; Deflorio, M.; Guaricci, A.C.; Minoia, R.; De Metrio, G.
    The presence of the μ-opioid receptor and the type of glycosylation in the third extra-cellular loop of this receptor was investigated in the isthmus of mare oviduct during oestrus by means of immunoblotting and immunohistochemistry combined with enzymatic (Nglycosidase F and O-glycosidase) and chemical (ßelimination) treatments. Immunoblotting analysis showed that the μ -opioid receptor consists of two peptides with molecular weights of around 65 and 50 kDa. After N-deglycosylation with N-glycosidase F an additional immunoreactive peptide was observed at around 30 KDa. The cleavage of O-glycans by Oglycosidase failed in immunoblotting as well as in immunohistochemistry investigations, revealing that the third extra-cellular loop of the μ -opioid receptor expressed in mare isthmus oviduct contains some modifications of the Galß(1-3)GalNAc core binding to serine or threonine. Immunohistochemistry revealed the μ-opioid receptor in the mucosal epithelium, some stromal cells, muscle cells and blood vessels. In ciliated cells the μ-opioid receptor showed N-linked glycans, since the immunoreactivity was abolished after Nglycosidase F treatment, whereas it was preserved in the apical region after ß-elimination. Most non-ciliated cells expressed the μ-opioid receptor with both N- and Olinked oligosaccharides, as revealed by the abolition of immunostaining after N-glycosidase F and ßelimination. Stromal cells, endothelial and muscle cells of blood vessels expressed the μ -opioid receptor containing both N- and O-linked oligosaccharides. Myosalpinx myocytes expressed the μ-opioid receptor with O-linked oligosaccharides. The immunopositive myocytes formed a circular coat in the intrinsic musculature, whereas they were arranged in some isolated, oblique bundles in the extrinsic musculature. In conclusion, the μ-opioid receptor could have a role in the production and the movement of isthmus lumen content that contributes to ensuring the effective condition of the sperm in the mare oviduct.