Role of lysine 173 in heparin binding to heparin cofactor II.

摘要: Abstract Heparin cofactor II (HC) is a plasma serine proteinase inhibitor (serpin) that inhibits alpha-thrombin in reaction dramatically enhanced by heparin and other glycosaminoglycans/polyanions. We investigated the glycosaminoglycan binding site HC by: (i) chemical modification with pyridoxal 5'-phosphate (PLP) absence presence of dermatan sulfate; (ii) molecular modeling; (iii) site-directed oligonucleotide mutagenesis. Four lysyl residues (173, 252, 343, 348) were protected from to lesser extent sulfate. Heparin-protected PLPHC retained both sulfate activity while sulfate-protected some little activity. Molecular modeling studies revealed Lys173 Lys252 are within region previously shown contain involved binding. Lys343 Lys348 distant this region, but protection might result conformational change following inhibitor. Site-directed mutagenesis was performed further dissect role these two regions during HC-heparin HC-dermatan interactions. The Lys343----Asn or Thr mutants had normal only slightly reduced eluted heparin-Sepharose at same ionic strength as native recombinant HC. However, Lys173----Gln Leu greatly significantly lower than Our results demonstrate interaction not sulfate, whereas critical for either glycosaminoglycan. These data provide evidence determinants required