Localization of the heparin-binding site of glia-derived nexin/protease nexin-1 by site-directed mutagenesis.
Recombinant rat glia-derived nexin was expressed in insect cells using the baculovirus system. The kinetics for the inhibition of thrombin by this recombinant material were indistinguishable from those observed with natural glia-derived nexin and recombinant nexin expressed in yeast. In addition, the dependence of the rate of inactivation on the concentration of heparin was similar for the three preparations. At the optimal heparin concentration, the association rate constant was 330-fold higher than that observed in the absence of heparin. A putative heparin-binding site is found in glia-derived nexin between residues 71 and 86; heparin-binding sites are found in homologous regions of antithrombin III and heparin cofactor II. Lysines in this region were mutated to glutamates, and the kinetics for the inhibition of thrombin by mutant proteins were determined. Concurrent mutation of all seven lysines in this region (residues 71, 74, 75, 78, 83, 84, and 86) did not affect the rate constant for the association of glia-derived nexin with thrombin in the absence of heparin, but it resulted in complete loss of the heparin acceleration of the rate of association. Mutations of residues 83, 84, and 86 together also caused a marked decrease in the acceleration by heparin of the reaction between glia-derived nexin and thrombin. These results support the hypothesis that the heparin-binding sites of glia-derived nexin, antithrombin III, and heparin cofactor II are found in homologous regions of the molecules. Heparin was also found to potentiate the ability of wild-type glia-derived nexin to inhibit the thrombin-induced retraction of neurites from neuroblastoma NB2a cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Stone
S R
SR
Department of Haematology, University of Cambridge, MRC Centre, U.K.
Brown-Luedi
M L
ML
Rovelli
G
G
Guidolin
A
A
McGlynn
E
E
Monard
D
D
D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor; D000818:Animals; D000990:Antithrombin III / Q000737:chemistry; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D002460:Cell Line; D005822:Genetic Vectors; D006493:Heparin / Q000378:metabolism*; D015844:Heparin Cofactor II / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009036:Moths; D016297:Mutagenesis, Site-Directed; D009457:Neuroglia / Q000737:chemistry*; D058255:Protease Nexins; D017433:Protein Structure, Secondary; D051381:Rats; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D017385:Sequence Homology; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors
D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D015844:Heparin Cofactor II; D000990:Antithrombin III; D006493:Heparin; D013917:Thrombin; D008239:Lysine