Dataset Information

Role of glycine 221 in catalytic activity of hyaluronan-binding protein 2.

ABSTRACT: HABP2 (hyaluronan-binding protein 2) is a Ca2+-dependent serine protease with putative roles in blood coagulation and fibrinolysis. A G221E substitution, known as the Marburg I polymorphism, reportedly affects HABP2 function and has been associated with increased risk for cardiovascular disease. However, the importance of Gly-221 for HABP2 activity is unclear. Here, we used G221E, G221A, and G221S mutants to assess the role of Gly-221 in HABP2 catalysis. The G221E variant failed to activate the single-chain urokinase-type plasminogen activator, and the G221A and G221S variants displayed moderately reduced single-chain urokinase-type plasminogen activator activation. Activity toward the peptide substrate S-2288 was markedly decreased in all HABP2 variants, with G221E being the most defective and G221A being the least defective. In the absence of Ca2+, S-2288 cleavage by wild-type HABP2 was Na+-dependent, with Km decreasing from 3.0 to 0.6 mm upon titration from 0 to 0.3 m Na+ In the presence of 5 mm Ca2+, Km was further reduced to 0.05 mm, but without an appreciable contribution of Na+ At physiological concentrations of Na+ and Ca2+, the three HABP2 variants, and particularly G221E, displayed a major Km increase for S-2288. Chemical footprinting revealed that Ile-16 is significantly less protected from chemical modification in G221E than in wild-type HABP2, suggesting impaired insertion of the N terminus into the G221E protease domain, with a concomitant impact on catalytic activity. Homology modeling suggested that the Glu-221 side chain could sterically hinder insertion of the N terminus into the HABP2 protease domain, helping to explain the detrimental effects of Glu-221 substitution on HABP2 activity.

SUBMITTER: Stavenuiter F

PROVIDER: S-EPMC5391765 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Role of glycine 221 in catalytic activity of hyaluronan-binding protein 2.

Stavenuiter Fabian F Ebberink Eduard H T M EHTM Mertens Koen K Meijer Alexander B AB

The Journal of biological chemistry 20170227 15

HABP2 (hyaluronan-binding protein 2) is a Ca<sup>2+</sup>-dependent serine protease with putative roles in blood coagulation and fibrinolysis. A G221E substitution, known as the Marburg I polymorphism, reportedly affects HABP2 function and has been associated with increased risk for cardiovascular disease. However, the importance of Gly-221 for HABP2 activity is unclear. Here, we used G221E, G221A, and G221S mutants to assess the role of Gly-221 in HABP2 catalysis. The G221E variant failed to ac ...[more]

PMID: 28246168

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Project description:BackgroundThe characteristics of human asthma are chronic inflammation and airway remodeling. Hyaluronan, a major extracellular matrix component, accumulates during inflammatory lung diseases, including asthma. Hyaluronan fragments stimulate macrophages to produce inflammatory cytokines. We hypothesized that hyaluronan and its receptors would play a role in human asthma.ObjectiveTo investigate the role of hyaluronan and hyaluronan-binding proteins in human asthma.MethodsTwenty-one subjects with asthma and 25 healthy control subjects underwent bronchoscopy with endobronchial biopsy and bronchoalveolar lavage. Fibroblasts were cultured, and hyaluronan and hyaluronan synthase expression was determined at baseline and after exposure to several mediators relevant to asthma pathobiology. The expression of hyaluronan-binding proteins CD44, TLR (Toll-like receptor)-2, and TLR4 on bronchoalveolar lavage macrophages was determined by flow cytometry. IL-8 production by macrophages in response to hyaluronan fragment stimulation was compared.ResultsAirway fibroblasts from patients with asthma produced significantly increased concentrations of lower-molecular-weight hyaluronan compared with those of normal fibroblasts. Hyaluronan synthase 2 mRNA was markedly increased in asthmatic fibroblasts. Asthmatic macrophages showed a decrease in cell surface CD44 expression and an increase in TLR2 and TLR4 expression. Macrophages from subjects with asthma showed an increase in responsiveness to low-molecular-weight hyaluronan stimulation, as demonstrated by increased IL-8 production.ConclusionHyaluronan homeostasis is deranged in asthma, with increased production by fibroblasts and decreased CD44 expression on alveolar macrophages. Upregulation of TLR2 and TLR4 on macrophages with increased sensitivity to hyaluronan fragments suggests a novel proinflammatory mechanism by which persistence of hyaluronan fragments could contribute to chronic inflammation and airway remodeling in asthma.

Project description:GC-C (guanylate cyclase C) is the receptor for heat-stable enterotoxins, guanylin and uroguanylin peptides. Ligand binding to the extracellular domain of GC-C activates the guanylate cyclase domain leading to accumulation of cGMP. GC-C is expressed as differentially glycosylated forms in HEK-293 cells (human embryonic kidney-293 cells). In the present study, we show that the 145 kDa form of GC-C contains sialic acid and galactose residues and is present on the PM (plasma membrane) of cells, whereas the 130 kDa form is a high mannose form that is resident in the endoplasmic reticulum and serves as the precursor for the PM-associated form. Ligand-binding affinities of the differentially glycosylated forms are similar, indicating that glycosylation of GC-C does not play a role in direct ligand interaction. However, ligand-stimulated guanylate cyclase activity was observed only for the fully mature form of the receptor present on the PM, suggesting that glycosylation had a role to play in imparting a conformation to the receptor that allows ligand stimulation. Treatment of cells at 20 degrees C led to intracellular accumulation of a mature glycosylated form of GC-C that now showed ligand-stimulated guanylate cyclase activity, indicating that localization of GC-C was not critical for its catalytic activity. To determine if complex glycosylation was required for ligand-stimulated activation of GC-C, the receptor was expressed in HEK-293 cells that were deficient in N -acetylglucosaminyltransferase 1. This minimally glycosylated form of the receptor was expressed on the cell surface and could bind a ligand with an affinity comparable with the 145 kDa form of the receptor. However, this form of the receptor was poorly activated by the ligand. Therefore our studies indicate a novel role for glycosidic modification of GC-C during its biosynthesis, in imparting subtle conformational changes in the receptor that allow for ligand-mediated activation and perhaps regulation of basal activity.

Dataset Information

Role of glycine 221 in catalytic activity of hyaluronan-binding protein 2.

Publications

Role of glycine 221 in catalytic activity of hyaluronan-binding protein 2.

Similar Datasets

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