Recent advances in the understanding of the mechanisms regulating protein secretion have indicated that secretory proteins may require differing periods of time to traverse the secretary pathway. Studies from our laboratory have shown that an individual protein, C-reactive protein (CRP), is secreted markedly faster when its synthesis in the liver is induced during the acute phase response to inflammation in the rabbit. This is the result of a great reduction in the time required for newly- synthesized CRP to exit from the rough endoplasmic reticulum (RER) during phase response (APR). In control animals CRP is actively retained within the RER and is secreted much less efficiently. In preliminary studies we have demonstrated specific binding of CRP to detergent-permeabilized rough microsomes. The overall goal of this proposal is to elucidate the mechanism by which CRP is retained within the RER and how this process is regulated during the APR.
Specific aims i nclude 1) characterization of the binding reaction of CRP to microsomes, 2) localization of the binding site within both microsomes and hepatocytes, 3) determination of changes in microsomal binding during the APR, and 4) identification and isolation of the microsomal binding site for CRP. Specifically, conditions for binding will be further optimized with respect to osmotic and ionic conditions, pH, and detergent-protein ratio. The relevancy of this reaction to biologically synthesized CRP will be assessed by studies of the competition of purified CRP for pulse-labelled CRP contained in microsomes prepared from cultured hepatocytes. The distribution of the binding site within microsomes and cultured hepatocytes will be determined by incubating detergent-permeabilized preparations with biotinylated CRP followed by peroxidase-conjugated Streptavidin. The affinity and number of binding sites will be determined by Scatchard analysis of microsomes prepared from rabbits manifesting varying degrees of the APR. Identification of the binding site will involve affinity chromatography of detergent extracts on CRP-Sepharose, chemical crosslinking of CRP to microsomes followed by immunochemical isolation of CRP-linked complexes, as well as the production of antibodies against the idiotype of affinity purified anti-CRP immunoglobulin. The serum concentration of CRP is markedly elevated in a number of inflammatory diseases. CRP itself appears to play a role in the modulation of the inflammatory response. Investigation of mechanisms which control CRP secretion will contribute to our understanding of the host response to a wide variety of inflammatory conditions, including many of the rheumatic diseases.
