[Show abstract][Hide abstract] ABSTRACT: The serpin heparin cofactor II (HCII) is a glycosaminoglycan-activated inhibitor of thrombin that circulates at a high concentration
in the blood. The antithrombotic effect of heparin, however, is due primarily to the specific interaction of a fraction of
heparin chains with the related serpin antithrombin (AT). What currently prevents selective therapeutic activation of HCII
is the lack of knowledge of the determinants of glycosaminoglycan binding specificity. In this report we investigate the heparin
binding properties of HCII and conclude that binding is nonspecific with a minimal heparin length of 13 monosaccharide units
required and affinity critically dependent on ionic strength. Rapid kinetics of heparin binding indicate an induced fit mechanism
that involves a conformational change in HCII. Thus, HCII binds to heparin in a manner analogous to the interaction of AT
with low affinity heparin. A fully allosteric 2000-fold heparin activation of thrombin inhibition by HCII is demonstrated
for heparin chains up to 26 monosaccharide units in length. We conclude that the heparin-binding mechanism of HCII is closely
analogous to that of AT and that the induced fit mechanism suggests the potential design or discovery of specific HCII agonists.
[Show abstract][Hide abstract] ABSTRACT: We have reported previously that Noggin is a heparin-binding protein and associates with the cell surface through heparan
sulfate proteoglycans, where it remains functional for the binding of bone morphogenetic proteins (BMPs). Here we report that
the binding of Noggin to the cell surface is highly selective for heparan sulfate and that specific structural features are
required for the interaction. Noggin binds most efficiently to heparin sequences composed of 10 or more monosaccharides; N-, 6-O-, and 2-O-sulfates contribute to this interaction. In addition, we have shown that the developmentally regulated endosulfatase Qsulf1
selectively removes sulfate groups from the 6-O position of sugars within the most highly sulfated S domains of heparan sulfate, whereas 6-O-sulfates in the NA/NS domains are not substrates for the enzyme. The activity of Qsulf1 in cells in culture results in the
release of Noggin from the cell surface and a restoration of BMP responsiveness to the cells. This shows that Noggin binds
to the S domains of heparan sulfate and provides evidence that, in addition to modulating Wnt signaling in vivo by the release of heparan sulfate bound Wnt, Qsulf1 also modulates BMP signaling by the release of surface-bound Noggin.
[Show abstract][Hide abstract] ABSTRACT: Neuroplin-1 (NRP1), a receptor for vascular endothelial growth factor (VEGF) family members, has three distinct extracellular domains, a1a2, b1b2, and c. To determine the VEGF(165) and placenta growth factor 2 (PlGF-2)-binding sites of NRP1, recombinant NRP1 domains were expressed in mammalian cells as Myc-tagged, soluble proteins, and used in co-precipitation experiments with 125I-VEGF165 and 125I-PlGF-2. Anti-Myc antibodies immunoprecipitated 125I-VEGF165 and 125I-PlGF-2 in the presence of the b1b2 but not of the a1a2 and c domains. Neither b1 nor b2 alone was capable of binding 125I-VEGF165. In competition experiments, VEGF165 competed PlGF-2 binding to the NRP1 b1b2 domain, suggesting that the binding sites of VEGF165 and PlGF-2 overlap. The presence of the a1a2 domain greatly enhanced VEGF165, but not PlGF-2 binding to b1b2. Heparin enhanced the binding of both 125I-VEGF165 and 125I-PlGF-2 to the b1b2 domain by 20- and 4-fold, respectively. A heparin chain of at least 20-24 monosaccharides was necessary for binding. In addition, the b1b2 domain of NRP1 could bind heparin directly, requiring heparin oligomers of at least 8 monosaccharide units. It was concluded that an intact b1b2 domain serves as the VEGF165-, PlGF-2-, and heparin-binding sites in NRP1, and that heparin is a critical component for regulating VEGF165 and PlGF-2 interactions with NRP1 by physically interacting with both receptor and ligands.