Abstract
We have previously reported that C-reactive protein (CRP) is normally synthesized by rabbit hepatocytes at relatively low rates and is retained in the endoplasmic reticulum (ER), apparently by specific interaction with a 60-kDa lumenal ER protein. During the acute phase response to tissue injury, a marked increase in CRP synthesis is associated with a decrease in the CRP binding capacity of the 60-kDa protein, with accompanying rapid secretion of CRP. In the present studies, we purified two 60-kDa ER lumenal glycoproteins (referred to as gp60a and gp60b) capable of binding CRP. gp60b, though present at only 5% the level of gp60a, was found to account for 80% of the total CRP binding capacity. Amino-terminal amino acid sequence analysis and biochemical characterization identified gp60a and gp60b as two microsomal carboxylesterases previously reported by others to contain COOH-terminal ER retention signals (HIEL and HTEL). The CRP binding activities of gp60a and gp60b were found to be independent of their esterase activities. In animals undergoing the acute phase response, the levels of gp60a and gp60b were diminished by about 50%, but the CRP binding capacities were reduced by 4-6-fold for gp60a and 25-30-fold for gp60b. These findings indicate that CRP is normally retained within the ER via interaction with gp60a and gp60b, while during the acute phase response a decrease in the CRP binding affinity of these proteins, particularly gp60b, results in efficient secretion of CRP.
Highlights
We have previously reported that C-reactive protein folding andlor assembly(Ref. 1and reviewed in Refs. 2-5)
During the acute phase The proteins that mediate these processes are themselves response to tissue injury, a marked increase in C R P syn- synthesized within andconfined t o the endoplasmic reticulum (ER) and musbt e selecthesis is associated with a decrease in theCRP binding tively retained in the face of the continuous and rapid exit of capacity of the 60-kDa protein, with accompanying membrane andvesicular content [7].Recent studies have idenrapid secretion of C-reactive protein (CRP)
We puri- tified sorting signals that retain ER-resident proteins by a fied two 60-kDa ER lumenal glycoproteins capable of binding CRP.gp60b, ments of the secretory pathway [8,9,10,11,12,13,14,15,16,17].While much progress though present at only 5% the level of gp6Oa, was found has been made understanding the retention of ER-resident to account for 80% of the total CRP binding capacity. proteins, comparatively little is known of the mechanisms re
Summary
Reducing sample buffer, and aliquots of the supernatants were run on Fractions containing gp60a were applied to a1.6 x 85-cm column of duplicate 12.5% SDSgels, one for staining and one for transferto. Were removedby chromatography on concanavalin A-Sepharose (Phar- Enzymatic Digestions-For treatment with endoglycosidase H, mimacia), which had been cross-linkedby exposure to 0.06% glutaralde- crosomal samples were suspendedin 0.5%SDS, 5%mercaptoethanol a t hyde for 30 min a t room temperature, followed by exhaustive washing 5 mg of proteidml, boiled for 10 min,cooled, and centrifugedat 10,000 in 20 mM Hepes-NaOH, 30 mM NaCl, 0.1% CHAPS, 10% glycerol, pH x g for 10 min. The sample was applied to a 1 x 25-cm Hydropore SEC (Rainin Instruments) column equilibrated in 200mhl ammonium acetate (pH 7.01, and the peak binding activity detected on subsequent ligand hlotting (Fig. 2 A , lane 3 ), consistent with our previous ohservatinn that the 60-kDa protein is confined to the ER [24]. Treatmentof intact microsomes withtrypsinhadnoeffect on 60-kDaCRPbindingactivity, whereas trypsin treatment of deterRent-permeahilized microsomes greatly reduced CRP hinding activity (Fig. 21.1).These findings, together with our previous observations that the 60-
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