Structural elements of kallistatin required for inhibition of angiogenesis.
Miao, Robert Q., Vincent Chen, Lee Chao, and Julie Chao.
Department of Biochemistry and Molecular Biology, Medical University of South
Carolina, Charleston, South Carolina 29425-2211
APStracts 10:0065C, 2003.
Kallistatin is a serpin first identified as a specific inhibitor of tissue kallikrein. Our recent
studies showed that kallikrein promoted angiogenesis, whereas kallistatin inhibited
angiogenesis and tumor growth. This study is aimed to identify the structural elements of
kallistatin essential for its antiangiogenic function. Kallistatin mutants at the hinge region
(A377T) and a major heparin-binding domain (K312A/K313A) were created by site-
directed mutagenesis. Recombinant kallistatin mutant A377T did not bind nor inhibit
tissue kallikrein activity. Wild-type kallistatin and kallistatin mutant A377T, but not
kallistatin mutant K312A/K313A lacking heparin-binding activity, inhibited VEGF-
induced proliferation, growth, and migration of human microvascular endothelial cells.
Similarly, wild-type kallistatin and kallistatin mutant A337T, but not kallistatin mutant
K312A/K313A, significantly inhibited VEGF-induced capillary tube formation of
cultured endothelial cells in Matrigel and capillary formation in Matrigel implants in
mice. To elucidate the role of the heparin-binding domain in modulating angiogenesis,
we showed that wild-type kallistatin interrupted the binding of 125I-labeled VEGF to
endothelial cells, whereas kallistatin mutant K312A/K313A did not interfere with VEGF
binding. Consequently, wild-type kallistatin, but not kallistatin mutant K312A/K313A,
suppressed VEGF-induced phosphorylation of Akt. Taken together, these results indicate
that the heparin-binding domain, but not the reactive site loop of kallistatin, is essential
for inhibiting VEGF-induced angiogenesis.
Received 12 November 2002; accepted in final form 12 February 2003
APS Manuscript Number C524-2.
Article publication pending Am J Physiol Cell Physiol
ISSN 1080-4757 Copyright 2003 The American Physiological Society.
Published in APStracts on 25 March 2003