Identification of a C-reactive protein binding site in two hepatic carboxylesterases capable of retaining C-reactive protein within the endoplasmic reticulum.
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.
Yue
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CC
Department of Medicine, Case Western Reserve University at MetroHeatlh Medical Center, Cleveland, Ohio 44109-1998, USA.
Muller-Greven
J
J
Dailey
P
P
Lozanski
G
G
Anderson
V
V
Macintyre
S
S
D000595:Amino Acid Sequence; D001665:Binding Sites; D002097:C-Reactive Protein / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002918:Chymotrypsin / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004950:Esterases / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D002097:C-Reactive Protein; C060290:60kDa glycoprotein esterase; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; D002918:Chymotrypsin; D014357:Trypsin