Abstract
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.
Highlights
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
The Effect of pH on HPRG Binding to Immobilized Heparin—The amount of HPRG bound at equilibrium to immobilized heparin at physiological ionic strength was markedly influenced by pH (Fig. 1A)
To examine HPRG binding to heparin under conditions more like those found in vivo, rabbit serum was added to the immobilized heparin cuvette at different pH values, followed by affinity purified anti-rabbit HPRG IgG (Fig. 2)
Summary
(Received for publication, September 16, 1997, and in revised form, December 18, 1997). 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-prolinerich domain. 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 HPRG binds. 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
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