Apoptotic cells of an epithelial cell line, AsPC-1, release monocyte chemotactic S19 ribosomal protein dimer

A pancreatic carcinoma cell line, AsPC-1, underwent apoptosis in vitro when heat-treated for 60 min at 43 degreesC. Apoptotic AsPC-1 cells liberated a monocyte chemotactic factor into the culture supernatant 24 to 30 h after the heat-treatment. This factor was immunologically identified as the cross -linked homodimer of S19 ribosomal protein (RP Sig), since the majority of the chemotactic activity was absorbed by both anti-RP S19 rabbit antibodies and an anti-isopeptide bond monoclonal antibody immobilized on agarose bea ds, Intracellular transglutaminase activity increased during the apoptotic process, reaching the peak strength between 18 and 24 h after the heat-trea tment. A recombinant RP S19 acquired the monocyte chemotactic capacity when incubated with the apoptotic cell extract obtained at the 18th hour. The c hemotactic activity acquirement as well as the transglutaminase activity we re blocked by treatment of the extract with anti-type II transglutaminase r abbit antibodies. When the recombinant RP S19 was treated with an authentic type II transglutaminase, the dimerization of RP S19 concomitant with the generation of the monocyte chemotactic activity was observed. Peptide-map a nalyses involving amino acid sequencing demonstrated that the inter-molecul ar isopeptide bond was heterogenous: Gln12 or Gln137 and Lys29 or Lys122 we re cross-linked, Site-directed mutagenic analysis indicated that the cross- linking of Gln137, but not other residues such as Gln12, Lys29, and Lys122, was essential for expression of the chemotactic activity.