Abstract
C-reactive protein (CRP) is normally synthesized by hepatocytes at relatively low rates and is retained within the endoplasmic reticulum (ER) via interaction with two carboxylesterases (termed gp60a and gp60b), which themselves are restricted to the ER by their COOH-terminal retention signals (HIEL and HTEL). During the acute phase response, an increase in CRP synthesis is accompanied by a decrease in its ER retention as a result of a decrease in the CRP binding affinity of gp60b. Our previous data indicated that the esterase active site, the CRP binding site, and the ER retention signal are functionally distinct. In the present studies, we have identified CRP-binding peptides produced by proteolytic cleavage of gp60a. The sequence shared by two CRP-binding peptides indicated that the CRP binding site of gp60a is contained within residues 477-499. These results were confirmed by expression of cDNAs coding for gp60a and b as bacterial fusion proteins. Fusion proteins containing the complete esterase COOH terminus bound CRP, whereas those truncated at residue 477 (or the homologous site in gp60b) did not. Based on the known crystal structures of three homologous hydrolases, the putative CRP-binding site of the gp60s is located on the surface and is physically distant from the esterase active site and the COOH-terminal ER retention signal.
Highlights
We have previously shown that retention of C-reactive protein (CRP) within the endoplasmic reticulum (ER) is the result of its association with two 60-kDa glycosylated microsomal carboxylesterases [5, 6], which we have termed gp60a and gp60b [7]. gp60a is an abundant ER protein with a relatively low affinity for CRP (Kd ϭ 120 nM), whereas gp60b is a less abundant protein, but with a higher binding affinity for CRP (Kd ϭ 1 nM) [3, 7]
Nitrocellulose blots probed with radiolabeled CRP revealed several CRP-binding peptides ranging in size from 6 to about 20 kDa (Fig. 1, B and C), whereas no CRPbinding material was detected in control lanes containing enzyme alone (Fig. 1B)
To identify the 6 kDa CRP-binding peptide (Fig. 1, arrowheads), chymotryptic digests of gp60a were fractionated by gel filtration and peptides of less than 10 kDa were resolved by HPLC on a C4 reverse phase column (Fig. 3A)
Summary
Our previous findings indicate that the three activities of gp60a and b, namely the esterase active site, the carboxyl-terminal ER retention signal, and the CRP binding site, are functionally independent. We have determined that the CRP binding site of these esterases is contained within a sequence of 23 amino acids near the carboxyl terminus. On the basis of the known structure of acetylcholinesterase [15] and its relevance to related hydrolases [16], the CRP binding site of gp60a and b is predicted to be located on the surface of the molecule, on the opposite side of the molecule from the access gorge to the esterase active site [15], and at a distance of more than 20 Å from the carboxyl-terminal ER retention signal
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