Abstract
Abstract: :
We have found that the natural bacterial cell wall component muramyl dipeptide (MDP) can induce apoptotic DNA ladders in rabbit tears. And it can also induce primary cultured rabbit retinal cells and rabbit kidney cell line RK13 cells to go apoptotic in a dose-dependent manner. Moreover, we have already determined that the receptor mediating the MDP-induced apoptosis of RK13 cells is on its cell surface. Purpose: To isolate and identify the MDP receptor from RK13 cells. Methods: Sepharose 4B coupled MDP (MDP-SP4B) was first synthesized. The MDP binding protein (MBP) of RK13 cells was isolated by MDP-SP4B affinity chromatography. Isolated MBP was analyzed by mass spectrometry, and spectra data were analyzed by Auto MS-Fit Software (UCSF) against NCBI and Genpept protein databases (protein identification analysis). Moreover, RK13 cells were surface labeled by Sulfo-NHS-LC-Biotin and calreticulin (CRT) was then purified from the biotinylated cells by mannan-agarose affinity chromatography. Western blotting analysis of purified CRT was performed with both horseradish peroxidase conjugated streptavidin (S-HRP) and anti-CRT antibody/horseradish peroxidase conjugated secondary antibody. Free MDP binding activity of both isolated MBP and purified CRT was determined by apoptosis inhibition method. Results: The molecular weight of the isolated MBP is 56 kilodalton (kD) and it had free MDP binding activity. Protein identification analysis suggested MBP is calreticulin. ELISA first confirmed MBP was calreticulin. Western blotting analysis showed purified CRT had 2 protein bands: 56 kD and 60 kD. And only 56 kD band could be detected by S-HRP. Eluate which just contained 56 kD band could exhibit strong free MDP binding activity while eluate with both bands showed very weak free MDP binding activity. Conclusions: Our isolated MDP binding protein is a special kind of calreticulin with molecular weight of 56 kD, which resides on the RK13 cell surface and can mediate MDP-induced apoptosis.
Keywords: 323 apoptosis/cell death • 541 receptors: pharmacology/physiology • 437 inflammation