Cloning, bacterial expression and biological characterization of recombinant human granulocyte chemotactic protein-2 and differential expression of granulocyte chemotactic protein-2 and epithelial cell-derived neutrophil activating peptide-78 mRNAs.
Human osteosarcoma cells secrete a novel C-X-C chemokine called granulocyte chemotactic protein-2 (GCP-2), which was previously identified by amino acid sequencing of the purified natural protein. In order to understand the role of this new protein in inflammatory reactions, we cloned GCP-2 DNA sequences to generate recombinant protein and specific DNA probes and primers. By means of PCR on cloned cDNA of osteosarcoma cells induced by interleukin-1 beta and fibroblasts induced by lipopolysaccharide plus dsRNA, the complete coding domain of GCP-2 was isolated. This sequence was cloned into the bacterial expression vector pHEN1 and, after induction, GCP-2 was secreted into the periplasm of Escherichia coli. Recombinant GCP-2 (rGCP-2) was purified and characterized by SDS/PAGE as a monomeric 6.5-kDa protein and by amino-terminal sequencing. The chemoattractive potency of GCP-2 for neutrophilic granulocytes was about 10-times less than that of interleukin-8 and the minimal effective dose was 10 ng/ml. However, at optimal dose (100 ng/ml) the maximal chemotactic response was comparable with that of interleukin-8. Both characteristics correspond with those of natural GCP-2. In addition, intracellular calcium release in neutrophils by recombinant GCP-2 was achieved with as little as 10 ng/ml. Quantitation studies using reverse transcriptase and the polymerase chain reaction revealed higher GCP-2 mRNA production in normal fibroblasts than in tumor cells. When compared with epithelial-cell-derived neutrophil-activating peptide-78 (ENA-78) mRNA, the GCP-2 mRNA levels were higher in all cell lines tested. In addition, GCP-2 and ENA-78 expression seem to be differentially regulated in that phorbol ester and lipopolysaccharide have opposing effects on their mRNA induction in diploid fibroblasts and epithelial cells, respectively. Interleukin-1 was demonstrated to be a general inducer for both chemokines, while interferon-gamma down-regulates their mRNA expression. The availability of recombinant GCP-2 together with the quantitation studies on mRNA expression will help to further elucidate the biological role of GCP-2 during the inflammatory response.
Froyen
G
G
Rega Institute for Medical Research, University of Leuven, Belgium.
Proost
P
P
Ronsse
I
I
Mitera
T
T
Haelens
A
A
Wuyts
A
A
Opdenakker
G
G
Van Damme
J
J
Billiau
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D054365:Chemokine CXCL5; D054427:Chemokine CXCL6; D018925:Chemokines / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D019743:Chemokines, CXC; D002634:Chemotaxis, Leukocyte; D003001:Cloning, Molecular; D003062:Codon / Q000302:isolation & purification; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006098:Granulocytes / Q000502:physiology; D006801:Humans; D016209:Interleukin-8 / Q000031:analogs & derivatives / Q000235:genetics*; D008969:Molecular Sequence Data; D018375:Neutrophil Activation; D012333:RNA, Messenger / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry*
C486263:CXCL5 protein, human; C515503:CXCL6 protein, human; D054365:Chemokine CXCL5; D054427:Chemokine CXCL6; D018925:Chemokines; D019743:Chemokines, CXC; D003062:Codon; D016209:Interleukin-8; D012333:RNA, Messenger; D011994:Recombinant Proteins