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

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 K_m decreasing from 3.0 to 0.6 mm upon titration from 0 to 0.3 m Na⁺ In the presence of 5 mm Ca²⁺, K_m 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 K_m 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.