On the activation of human leuserpin-2, a thrombin inhibitor, by glycosaminoglycans.

H Ragg,T Ulshöfer,J Gerewitz

doi:10.1016/s0021-9258(19)34108-0

Abstract

Human leuserpin-2 (hLS2) cDNA variants generated by site-directed mutagenesis were expressed in a transient COS cell system. Functional analysis of the mutants revealed two regions in the NH2-terminal half of hLS2 which are essential for glycosaminoglycan-enhanced thrombin inhibition by hLS2. One of these regions, which encompasses a dimeric structure enriched in basic amino acids, is required for both glycosaminoglycan binding and glycosaminoglycan-mediated acceleration of thrombin inhibition. Deletion of another dimeric region, which spans a sequence with a high negative charge density, resulted in a strong reduction in the glycosaminoglycan-enhanced activity of hLS2. This polyanionic region displays structural and functional similarities to the COOH-terminal end of hirudin, another potent thrombin inhibitor, indicating that both inhibitors may have a common binding site on thrombin. Based on our observations we propose a model for the activation of hLS2 by glycosaminoglycans. The key feature of this model is the suggestion that the glycosaminoglycan-enhanced reaction between hLS2 and thrombin is mediated by at least two regions of contact, involving both the reactive center region and the acidic domain of hLS2. Binding of glycosaminoglycans to hLS2 is suggested to result first in the release of the acidic region from intramolecular interactions. Then, amino acid sequences in thrombin are proposed to interact with the acidic dimer of hLS2.

Highlights

This polyanionic region displays structural and functional similarities to the COOH-terminal end of hirudin, another potent thrombin inhibitor, indicating that both inhibitors may have a common binding site on thrombin
Mutations within the Basic Dimer and Their Effects on Heparin Binding-It has been suspected that sequences which are rich in basic amino acids are involved in the binding of negatively charged glycosaminoglycans like heparin or dermatan sulfate (Cardin and Weintraub, 1989). Human leuserpin-2 (hLS2), which is activated by these substances, contains a cluster of arginine and lysine residues between positions 184 and 193 (Fig. 1)
Preliminary data suggest that simultaneous substitution of several acidic amino acids in the acidic dimer by glutamine or asparagine results in a strong reduction of complex formation5 our experiments indicate that the polyanionic dimer including the phenylalanine residue at position 67 in hLS2 is important for the glycosaminoglycan-enhanced activity of hLS2

Summary

Introduction

This polyanionic region displays structural and functional similarities to the COOH-terminal end of hirudin, another potent thrombin inhibitor, indicating that both inhibitors may have a common binding site on thrombin. The key feature of this model is the suggestion that the glycosaminoglycan-enhanced reaction between hLS2 and thrombin is mediated by at least two regions of contact, involving both the reactive center region and the acidic domain of hLS2. Binding of glycosaminoglycans to hLS2 is suggested to result first in the release of the acidic region from intramolecular interactions. HLS2 has been allocated to the serpin superfamily, and it has been postulated that in analogy to other family members hLS2 contains a leucine residue at the Pl position of its reactive center (Ragg, 1986). Al-antitrypsin display equivalent exon-intron structures and constitute a distinct entity within the serpin gene family, the members of which show a highly heterogeneous genomic structure both with respect to number and positions of introns

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Conclusion