Identification of cytokine-induced neutrophil chemoattractants (CINC), rat GRO/CINC-2 alpha and CINC-2 beta, produced by granulation tissue in culture: purification, complete amino acid sequences and characterization.
Four basic neutrophil chemotactic factors (chemokines) have been purified from conditioned medium of granulation tissue obtained from carrageenin-induced inflammation in the rat. On the basis of their N-terminal amino acid sequences, one of the chemokines was identical with rat GRO/cytokine-induced neutrophil chemoattractant (CINC) which we reported previously, and another was identical with rat macrophage inflammatory protein-2 (MIP-2). Two other chemokines were novel chemoattractants related to MIP-2. The novel chemokines are referred to as rat GRO/CINC-2 alpha and CINC-2 beta, and consequently CINC and rat MIP-2 are renamed rat GRO/CINC-1 and CINC-3 respectively. The complete amino acid sequences of purified CINC-2 alpha and CINC-3 were determined by analysis of the fragments isolated from proteinase V8-treated CINCs. The cDNA for CINC-2 beta was cloned by reverse transcription/PCR amplification using specific primers starting with total RNA extracted from lipopolysaccharide-stimulated rat macrophages. A comparison of the amino acid sequence encoded by the cDNA with the N-terminal amino acid sequence of purified CINC-2 beta revealed that mature CINC-2 beta is a 68-residue chemoattractant produced by cleavage of a 32-residue signal peptide. The difference in amino acid sequences between CINC-2 alpha and CINC-2 beta consisted of only three C-terminal residues. Rat GRO/CINC-2 alpha is a major chemokine, and the four purified chemokines have similar chemotactic activity, suggesting that they contribute to neutrophil infiltration into inflammatory sites in rats.
Nakagawa
H
H
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Japan.
Komorita
N
N
Shibata
F
F
Ikesue
A
A
Konishi
K
K
Fujioka
M
M
Kato
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D054360:Chemokine CXCL1; D054426:Chemokine CXCL2; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017077:Culture Media, Conditioned; D046508:Culture Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D006097:Granulation Tissue / Q000378:metabolism*; D006133:Growth Substances / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D036341:Intercellular Signaling Peptides and Proteins; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012697:Serine Endopeptidases / Q000378:metabolism
D054360:Chemokine CXCL1; D054426:Chemokine CXCL2; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D017077:Culture Media, Conditioned; C513297:Cxcl1 protein, rat; C513296:Cxcl2 protein, rat; C513294:Cxcl3 protein, rat; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase