Role of lysine 173 in heparin binding to heparin cofactor II

H. C. Whinna, M. A. Blinder, M. Szewczyk, D. M. Tollefsen, F. C. Church

Research output: Contribution to journalArticle

57 Scopus citations

Abstract

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

Original languageEnglish
Pages (from-to)8129-8135
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number13
StatePublished - Aug 13 1991

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