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 degrees C. 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 S19), 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 beads. Intracellular transglutaminase activity increased during the apoptotic process, reaching the peak strength between 18 and 24 h after the heat-treatment. A recombinant RP S19 acquired the monocyte chemotactic capacity when incubated with the apoptotic cell extract obtained at the 18th hour. The chemotactic activity acquirement as well as the transglutaminase activity were blocked by treatment of the extract with anti--type II transglutaminase rabbit 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 analyses involving amino acid sequencing demonstrated that the inter-molecular isopeptide bond was heterogeneous: Gln12 or Gln137 and Lys29 or Lys122 were 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.
Nishimura
T
T
Division of Molecular Pathology, Graduate School of Medical Sciences, Kumamoto University, Honjo, Kumamoto 860-0811, Japan.
Horino
K
K
Nishiura
H
H
Shibuya
Y
Y
Hiraoka
T
T
Tanase
S
S
Yamamoto
T
T
D000595:Amino Acid Sequence; D017209:Apoptosis; D002457:Cell Extracts; D002634:Chemotaxis, Leukocyte; D002846:Chromatography, Affinity; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D004790:Enzyme Induction; D004847:Epithelial Cells / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology*; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D018945:Monocyte Chemoattractant Proteins / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010190:Pancreatic Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D010449:Peptide Mapping; D011485:Protein Binding; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D011503:Transglutaminases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D002457:Cell Extracts; D017077:Culture Media, Conditioned; D018945:Monocyte Chemoattractant Proteins; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C040343:ribosomal protein S19; D006493:Heparin; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins