Identification of the Antithrombin III Heparin Binding Site

Human group IIa phospholipase A 2 (hIIa-PLA2) is a highly basic protein that is secreted from a number of cells during inflammation and may play a role in arachidonate liberation and in destruction of invading bacteria. It has been proposed that rodent group IIa PLA 2 is anchored to cell surfaces via attachment to heparan sulfate proteoglycan and that this interaction facilitates lipolysis. hIIa-PLA2 contains 13 lysines, 2 histidines, and 10 arginines that fall into 10 clusters. A panel of 26 hIIa-PLA2 mutants were prepared in which 1-4 basic residues in each cluster were changed to glutamate or aspartate (charge reversal). A detailed analysis of the affinities of these mutants for anionic vesicles and for heparin and heparan sulfate in vitro and of the specific activities of these proteins for hydrolysis of vesicles in vitro and of living cell membranes reveal the following trends: 1) the affinity of hIIa-PLA2 for heparin and heparan sulfate is modulated not by a highly localized site of basic residues but by diffuse sites that partially overlap with the interfacial binding site. In contrast, only those residues on the interfacial binding site of hIIa-PLA2 are involved in binding to membranes; 2) the relative ability of these mutants to hydrolyze cellular phospholipids when enzymes were added exogenously to CHO-K1, NIH-3T3, and RAW 264.7 cells correlates with their relative in vitro affinity for vesicles and not with their affinity for heparin and heparan sulfate. 3) The rates of exogenous hIIa-PLA2-catalyzed fatty acid release from wild type CHO-K1 cells and two mutant lines, one lacking glycosaminoglycan and one lacking heparan sulfate, were similar. Thus basic residues that modulate interfacial binding are important for plasma membrane fatty acid release by exogenously added hIIa-PLA2. Binding of hIIa-PLA2 to cell surface heparan sulfate does not modulate plasma membrane phospholipid hydrolysis by exogenously added hIIa-PLA2.

Section: Preparation and Kinetic Characterization Of Hiia-pla2contrasting

confidence: 54%

Action of Human Group IIa Secreted Phospholipase A2on Cell Membranes

Koduri¹,

Baker²,

Snitko³

et al. 1998

“…A clear and comparable increase in complex band intensity was observed for the serpin-protease reaction with the addition of a 12.5-fold excess of UFH for the full-length vaspin (left gel) and the N-terminally cleaved variant (right gel). (25), PAI (26), HC II (27), and protease nexin 1 (28), the heparin binding region is located at helix D and at the beginning of helix A, whereas PCI binds heparin primarily with basic residues in helix H and also helix D (29,30 (25,31)), mutation of these residues did not affect heparin binding of vaspin. Also, the mutation of basic residues surrounding helix H and D (as for the PCI heparin binding site) did not result in decreased heparin affinity.…”

Section: Discussionmentioning

confidence: 99%

Basic Residues of β-Sheet A Contribute to Heparin Binding and Activation of Vaspin (Serpin A12)

Ulbricht

Oertwig

Arnsburg

et al. 2017

Vaspin (serpin A12) was identified in adipose tissue of the OLETF rat type-2 diabetes model and functions as an antidiabetic and anti-atherogenic adipokine in obesity-related disorders, such as insulin resistance and inflammation (1, 2). Insulin-resistant mouse models exhibit improved glucose tolerance after intraperitoneal vaspin application (3, 4), and transgenic mice overexpressing vaspin are protected from diet-induced obesity and comorbidities, such as glucose intolerance, insulin resistance, and adipose tissue inflammation (5). We found that intraperitoneal and central administration of vaspin leads to reduced food intake and blood glucose in mice (6). A recent study demonstrated that central vaspin regulates hepatic glucose production and insulin signaling via brain-liver communication through the dorsal vagal complex (7). In endothelial and vascular smooth muscle cells, multiple studies demonstrated anti-apoptotic (8), anti-inflammatory (9), and anti-migratory (10) effects of vaspin, suggesting a protective role in the pathogenesis of atherosclerosis. In skin, vaspin expression by keratinocytes suppresses the expression and release of inflammatory cytokines by immune cells (11) and possibly regulates the activation of proinflammatory cytokines, such as chemerin, via KLK7 (12). Whereas vaspin-mediated improvement of glucose tolerance and reduction of food intake were found to be dependent on protease inhibition (4, 13), anti-inflammatory effects in the liver have been linked to interaction with a membrane-associated endoplasmic reticulum chaperone protein (5).The only protease target of vaspin known so far is human KLK7 (4), a member of a family of 15 serine peptidases with chymotrypsin-like specificity (14). A non-inhibitory vaspin mutant failed to improve glucose tolerance in obese mice, proving that protease inhibition is a prerequisite for vaspin effects on glucose disposal (4). Human insulin is a substrate of KLK7, and vaspin and KLK7 are co-expressed in murine pancreatic islets. We hypothesized that KLK7 inhibition by vaspin may prolong insulin action and increase glucose uptake in insulinresponsive tissues, such as adipose tissue, liver, muscle, and also brain.We have recently reported that the inhibition of KLK7 by vaspin is critically dependent on an arginine residue in proximity to the reactive center loop (RCL), 2 because the native P1Ј glutamate residue is essentially repressing inhibition of KLK7 (15). Inhibition of KLK7 by vaspin is furthermore accelerated by heparin (15). Yet, the nature of the interaction between glycosaminoglycans and vaspin has not been explored in much detail.

“…In one of the most completely studied systems, the binding of heparin to a specific domain on human antithrombin III involves specific basic residues within the molecule (40). Additionally, heparin has been found to bind to a number of other proteins in human plasma (41).…”

Section: Discussionmentioning

confidence: 99%

A Binding Site for Heparin in the Apple 3 Domain of Factor XI

Ho¹,

Badellino²,

Baglia³

et al. 1998

Since heparin potentiates activated factor XI (FXIa) inhibition by protease nexin-2 by providing a template to which both proteins bind (Zhang, Y., Scandura, J. M., Van Factor XI (FXI) 1 is a zymogen that circulates in plasma in a non-covalent complex with high molecular weight kininogen (HMWK) (1, 2) and participates in the contact phase of blood coagulation (3). The active enzyme, activated factor XI (FXIa), is a trypsin-like serine protease that is generated when FXI is cleaved by FXIIa (3), thrombin, or FXIa (4, 5) at an internal Arg 369 -Ile 370 bond (2). Upon activation, FXIa is capable of converting FIX into its active form, FIXa (2, 6). FIXa, in the presence of FVIII and platelets, can activate FX (7,8), resulting in the generation of thrombin and, subsequently, a fibrin clot.The structure of FXI is unique among the plasma coagulation enzymes (9, 10), since it exists as a homodimer consisting of two subunits each of which contains 607 amino acids. During proteolytic activation, each of these subunits is cleaved to generate a heavy chain of 369 amino acids and a light chain or catalytic domain of 238 amino acids. The heavy chain of FXI provides binding surfaces for several blood coagulation proteins and is organized into four tandem repeat Apple domains designated A1, A2, A3, and A4 (9, 10). Each of these four domains contains 90 to 91 amino acids, the sequences of which are 23-34% identical. The A1 domain provides a binding site for HMWK (11), a protein that promotes the activation of FXI by FXIIa (12). Both the A2 domain (13) and the A3 domain (14) have been implicated in the binding of FXIa to its substrate, FIX. The A4 domain contains Cys 321 that is responsible for the dimerization of FXI (15,16). In addition, the A4 domain binds to FXIIa (10).Examination of the cellular site of FXI activation by FXIIa has resulted in evidence that platelets promote the proteolytic activation of FXI by FXIIa (17) and that FXI, in the presence of zinc ions, calcium ions, and HMWK, binds to activated platelets in a specific, reversible, and saturable manner with a dissociation constant (K d ) of ϳ10 nM (18). Previous work from this laboratory showed that a synthetic peptide (Asn 235 -Arg 266 ) from the A3 domain inhibited 125 I-FXI binding to platelets (inhibition constant (K i ) ϭ 10 nM) in the presence of HMWK, ZnCl 2 , and CaCl 2 (19,20). Hence, these studies indicate that the A3 domain mediates FXI binding to platelets.A second possible site of FXI activation is the endothelial cell surface. Berrettini et al. (21) demonstrated that FXI binds to endothelial cells in the presence of HMWK, CaCl 2 , and ZnCl 2 with a K d(app) ϳ4.5 nM, whereas FXIa binds with higher affinity (K d(app) ϳ1.5 nM). The binding of FXI to the endothelial cell surface may be of functional significance in localizing coagulation to the site of vascular injury since activation of FXI can occur on the endothelial cell surface (21), where proteoglycans such as heparan sulfate are known to be present. Both Naito and Fujikawa (4) and Gailani an...