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

J Biol Chem 2017 04 27;292(15):6381-6388. Epub 2017 Feb 27.

From the Department of Plasma Proteins, Sanquin Research, 1066 CX Amsterdam, The Netherlands and

HABP2 (hyaluronan-binding protein 2) is a Ca-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 Ca, S-2288 cleavage by wild-type HABP2 was Na-dependent, with decreasing from 3.0 to 0.6 mm upon titration from 0 to 0.3 m Na In the presence of 5 mm Ca, was further reduced to 0.05 mm, but without an appreciable contribution of Na At physiological concentrations of Na and Ca, the three HABP2 variants, and particularly G221E, displayed a major 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.

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Source
http://dx.doi.org/10.1074/jbc.M116.757849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391765PMC
April 2017
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