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

J Biol Chem. 2017 Apr 14;292(15):6381-6388. doi: 10.1074/jbc.M116.757849. Epub 2017 Feb 27.


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.

Keywords: Marburg I polymorphism; allosteric regulation; coagulation factor; enzyme mechanism; factor seven activating protease (FSAP); fibrinolysis; hyaluronan-binding protein 2 (HABP2); serine protease; sodium binding.

MeSH terms

  • Amino Acid Substitution
  • Calcium / chemistry
  • Catalysis
  • Glycine / chemistry
  • Glycine / genetics
  • Mutation, Missense
  • Protein Domains
  • Serine Endopeptidases / chemistry*
  • Serine Endopeptidases / genetics
  • Urokinase-Type Plasminogen Activator / chemistry
  • Urokinase-Type Plasminogen Activator / genetics


  • HABP2 protein, human
  • Serine Endopeptidases
  • Urokinase-Type Plasminogen Activator
  • Calcium
  • Glycine

Associated data

  • PDB/1YC0
  • PDB/2BOK