Structure?Function Studies Using Deletion Mutants Identify Domains of gC1qR/p33 as Potential Therapeutic Targets for Vascular Permeability and Inflammation

Abstract

The endothelial cell receptor complex for kininogen (HK) comprises gC1qR, cytokeratin 1, and urokinase-type plasminogen activator receptor and is essential for activation of the kinin system that leads to bradykinin (BK) generation. Of these, gC1qR/p33 constitutes a high affinity site for HK – the BK precursor – and is therefore critical for the assembly of the kinin-generating cascade. Previous studies have identified a putative HK site within the C-terminal domain (residues 204–218) of gC1qR recognized by mAb 74.5.2. In these studies, we used information from the crystal structure of gC1qR, to engineer several deletion (Δ) mutants and test their ability to bind and/or support BK generation. While deletion of residues 204–218 (gC1qRΔ204–218), showed significantly reduced binding to HK, BK generation was not affected when tested by a sensitive bradykinin immunoassay. In fact, all of the gC1qR deletion mutants supported BK generation with the exception of gC1qRΔ154–162 and a point mutation in which Trp 233 was substituted with Gly. Binding studies also identified the existence of two additional sites at residues 144–162 and 190–202. Moreover, binding of HK to a synthetic peptide 190–202 was inhibited by mAbs 48 and 83, but not by mAb 74.5.2. Since a single residue separates domains 190–202 and 204–218, they may be part of a highly stable HK binding pocket and therefore a potential target for drug design to prevent vascular permeability and inflammation.

Identification of the gC1qR sites for the HIV-1 viral envelope protein gp41 and the HCV core protein: Implications in viral-specific pathogenesis and therapy

“…The strategy for the construction of plasmids containing the glutathione-S-transferase (GST)-gC1qR wild type (WT), as well as several important deletion (Δ) mutants lacking highly charged domains selected on the basis of their prominent position in the crystal structure (Jiang et al, 1999) have been described in detail in our earlier publications (Ghebrehiwet et al, 2011; Ghebrehiwet et al, 2002). In all cases, the final constructs were sequenced to verify sequence integrity.…”

“…After verification by ELISA and visualization by SDS-PAGE (Laemmli, 1970), the single peak containing the gC1qR protein was pooled, concentrated to 1–2 mg/ml, and stored at −80 °C in the presence of 50 nM PPACK (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone)—a specific thrombin inhibitor. To estimate the organization and integrity of each recombinant protein, gel filtration of purified gC1qR (wild Type, WT) as well the deletion mutants was carried out by analytical gel filtration on Superose column (Ghebrehiwet et al, 2011). With the exception of those lacking residues 74–95, 204–218, and 212–223, all of the deletions mutants displayed a properly folded trimer as described in detail earlier (Ghebrehiwet et al, 2011).…”

“…To estimate the organization and integrity of each recombinant protein, gel filtration of purified gC1qR (wild Type, WT) as well the deletion mutants was carried out by analytical gel filtration on Superose column (Ghebrehiwet et al, 2011). With the exception of those lacking residues 74–95, 204–218, and 212–223, all of the deletions mutants displayed a properly folded trimer as described in detail earlier (Ghebrehiwet et al, 2011). …”

“…The cell lines, MOLT-4 and U937 – representing CD4+ T cell and monocytic cell – were grown in suspension in RPMI 1640 containing 10% heat inactivated fetal bovine serum and 100 units/ml penicillin and 100 μg/ml streptomycin (GIBCO-Invitrogen, Grand Island NY) and maintained in a humidified air consisting of 5% CO 2 and 95% air as described (Ghebrehiwet et al, 2011). Prior to each experiment, the viability of cells was verified by Trypan blue exclusion and only cultures with ≥95% viability were used for experiments.…”

“…The receptor for the globular heads of C1q, gC1qR/p33 (also know as p32, HABP-1), is a ubiquitously distributed highly acidic (pI 4.15), multicompartmental and multifunctional cellular protein, which modulates a plethora of immunological functions including infection, inflammation, autoimmunity, and cancer (Ghebrehiwet et al, 2011). In addition to its known plasma ligands [C1q, high molecular weight kininogen (HK), fibrinogen, thrombin and multimeric vitronectin], gC1qR is also able to bind a diverse array of bacterial- (Braun et al, 2000; Nguyen et al, 2000; Ghebrehiwet et al, 2007) and viral-associated molecular ligands (Wang et al, 1997; Yu et al, 1995; Luo et al, 1994; Szabo et al, 2001; Fausther-Bonvendo et al, 2010; Bruni and Roizman, 1996; Mathews and Russell, 1998; Kittlesen et al, 2000; Mohan et al, 2002; Choi et al, 2009; Watthanasurorot et al, 2012) and as such is considered to be a significant pathogen recognition receptor (PRR).…”