a. How is the binding of HPRG to heparin dependent on pH? Give structural reason

Question

a. How is the binding of HPRG to heparin dependent on pH? Give structural reasons for the binding dependence. The structure of heparin is shown in Figure 2.2. b. The same binding studies were carried out in which HPRG was reacted with diethylpyrocarbonate (DEPC), a compound that specifically reacts with histidine residues. The reaction is shown in Figure 2.3. Explain the results.

Explanation / Answer

The middle domain of plasma histidine-proline-rich glycoprotein (HPRG) contains unusual tandem pentapeptide repeats (consensus G(H/P)(H/P)PH) and binds heparin and transition metals. Unlike other proteins that interact with heparin via lysine or arginine residues, HPRG relies exclusively on histidine residues for this interaction. To assess the consequences of this unusual requirement, we have studied the interaction between human plasma HPRG and immobilized glycosaminoglycans (GAGs) using resonant mirror biosensor techniques. HPRG binding to immobilized heparin was strikingly pH-sensitive, producing a titration curve with a midpoint at pH 6.8. There was little binding of HPRG to heparin at physiological pH in the absence of metals, but the interaction was promoted by nanomolar concentrations of free zinc and copper, and its pH dependence was shifted toward alkaline pH by zinc. The affinity of HPRG for various GAGs measured in a competition assay decreased in the following order: heparin > dermatan sulfate > heparan sulfate > chondroitin sulfate A. Binding of HPRG to immobilized dermatan sulfate had a midpoint at pH 6.5, was less influenced by zinc, and exhibited cooperativity. Importantly, plasminogen interacted specifically with GAG-bound HPRG. We propose that HPRG is a physiological pH sensor, interacting with negatively charged GAGs on cell surfaces only when it acquires a net positive charge by protonation and/or metal binding. This provides a mechanism to regulate the function of HPRG (the local pH) and rationalizes the role of its unique, conserved histidine-proline-rich domain. Thus, under conditions of local acidosis (e.g. ischemia or hypoxia), HPRG can co-immobilize plasminogen at the cell surface as well as compete for heparin with other proteins such as antithrombin. HPRG1 is a relatively abundant plasma glycoprotein (125 µg/ml or about 1.5 µM) (1). Its two N-terminal cystatin modules identify it as a member of the cystatin superfamily, along with kininogen and fetuin (2). Based on its interactions with various ligands, HPRG has been proposed to be a regulator of coagulation, fibrinolysis, and the immune response and to play a role in zinc transport. In vitro HPRG binds components of the coagulation and fibrinolytic systems, including heparin (3), plasminogen (4), and fibrinogen (5); cells, viz. T-cells (6), macrophages (7), and platelets (8); and small ligands, such as transition metal ions and heme (9). No hypothesis of the function of HPRG has been able to rationalize these numerous interactions or explain how the activity of HPRG may be regulated in vivo. The most noteworthy feature of HPRG is a very high content of histidine, 13 mol?%, concentrated in the central His-Pro-rich domain which contains a pentapeptide consensus sequence (GHHPH) repeated in tandem 12 times in human HPRG (10) and the sequences GHPPH and GPPPH repeated 18 times in the rabbit protein (11). Thus, the ionic charge of HPRG is exquisitely sensitive to pH in the range of pH 6–7, and an electrophoretic titration curve of rabbit HPRG showed that protonation of histidine side chains spans the physiological pH range, with the maximum change occurring between pH 6.5 and 7 (11). Considering these observations, surprisingly little attention has been given to the effect of pH on the structure and function of HPRG. Histidine residues in the His-Pro-rich domain of HPRG mediate interactions with heparin (12), transition metals, and heme (13). Binding of HPRG to heparin and the subsequent neutralization of heparin’s anticoagulant effect have obvious pharmacological interest. An analysis of proteins from normal human plasma that bound to immobilized heparin indicated that HPRG was the only protein likely to inhibit anticoagulant activity by direct competition with antithrombin (14), although several plasma proteins display this ability in purified systems. Interaction of heparin with heparin-binding proteins is largely electrostatic but requires lysine or arginine side chains in other known cases. It is thus noteworthy that histidine residues are responsible for HPRG binding to heparin, and this leads to the immediate prediction that protonation of histidine side chains at low pH or binding of transition metals such as zinc, which also bestows positive charge on the His-Pro-rich domain, will strengthen heparin binding to HPRG. Indeed, inhibition by HPRG of the heparin-catalyzed thrombin-antithrombin reaction is modulated by pH (15) or zinc (16), and the stoichiometry of the heparin-HPRG interaction is shifted from 1:1 to 1:2 at low pH or in the presence of zinc and copper (12). Here we characterize directly and quantitatively the modulation by pH and transition metals of HPRG-glycosaminoglycan (GAG) interactions, and we propose a new paradigm of the function of HPRG based on these data. We suggest that the unique, conserved His-Pro-rich domain is a pH sensor, providing a plausible mechanism for regulating HPRG’s activity and an approach to understanding its physiological role. In this model, when the local pH drops (e.g. in ischemia or hypoxia) histidine side chains of HPRG are protonated, the His-Pro-rich domain becomes positively charged, and hence HPRG binds strongly to negatively charged GAGs on cell surfaces. As a consequence ligands of HPRG such as plasminogen can be co-immobilized at the cell surface, and other GAG-binding proteins like antithrombin may be displaced. http://www.jbc.org/content/273/10/5493.full

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