TY - JOUR
T1 - Characterization of synthetic human granulocyte chemotactic protein 2
T2 - Usage of chemokine receptors CXCR1 and CXCR2 and in vivo inflammatory properties
AU - Wuyts, Anja
AU - Van Osselaer, Nancy
AU - Haelens, Annemie
AU - Samson, Isabelle
AU - Herdewijn, Piet
AU - Ben-Baruch, Adit
AU - Oppenheim, Joost J.
AU - Proost, Paul
AU - Van Damme, Jo
PY - 1997/3/4
Y1 - 1997/3/4
N2 - Human granulocyte chemotactic protein 2 (GCP-2) has originally been isolated from cytokine-stimulated osteosarcoma cells as a chemokine coproduced in minute amounts together with interleukin 8. Human GCP-2 (75 residues) was synthesized on a 0.25-mmol scale using Fmoc chemistry. After disulfide bridge formation and purification, monomeric GCP-2 was recovered as a 6-kDa protein; the pure synthetic protein showed a molecular mass of 8076 Da as determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The exact amino acid sequence of synthetic GCP-2 was confirmed by Edman degradation. Synthetic GCP-2 was an equally active (minimal effective concentration of 1-3 nM) chemoattractant for neutrophilic granulocytes as was natural 75-residue GCP-2. At concentrations up to 30 nM, synthetic GCP-2 did not stimulate eosinophil, monocyte, or lymphocyte chemotaxis. GCP-2 induced a dose-dependent increase in [Ca2+](i) in neutrophils, 1 nM being the minimal effective concentration. The GCP-2- induced [Ca2+](i) increase was completely prevented by pertussis toxin. Prestimulation of neutrophils with equimolar concentrations of purified natural IL-8, GROα, GROγ and ENA-78 abolished the [Ca2+](i) increase in response to 1 nM GCP-2. Alternatively, the [Ca2+](i) rise induced by these CXC chemokines was inhibited by pretreatment of neutrophils with GCP-2. GCP- 2 stimulated [Ca2+](i) increases in CXCR1- and CXCR2-transfected cells, demonstrating that GCP-2 binds to both IL-8 receptors. Intradermal injection of synthetic GCP-2 resulted in a dose-dependent neutrophil accumulation and plasma extravasation in rabbit skin. To provoke this skin reaction, GCP-2 (10 pmol/site) was nearly as effective as IL-8, indicating that it is an important complementary mediator of the inflammatory response.
AB - Human granulocyte chemotactic protein 2 (GCP-2) has originally been isolated from cytokine-stimulated osteosarcoma cells as a chemokine coproduced in minute amounts together with interleukin 8. Human GCP-2 (75 residues) was synthesized on a 0.25-mmol scale using Fmoc chemistry. After disulfide bridge formation and purification, monomeric GCP-2 was recovered as a 6-kDa protein; the pure synthetic protein showed a molecular mass of 8076 Da as determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The exact amino acid sequence of synthetic GCP-2 was confirmed by Edman degradation. Synthetic GCP-2 was an equally active (minimal effective concentration of 1-3 nM) chemoattractant for neutrophilic granulocytes as was natural 75-residue GCP-2. At concentrations up to 30 nM, synthetic GCP-2 did not stimulate eosinophil, monocyte, or lymphocyte chemotaxis. GCP-2 induced a dose-dependent increase in [Ca2+](i) in neutrophils, 1 nM being the minimal effective concentration. The GCP-2- induced [Ca2+](i) increase was completely prevented by pertussis toxin. Prestimulation of neutrophils with equimolar concentrations of purified natural IL-8, GROα, GROγ and ENA-78 abolished the [Ca2+](i) increase in response to 1 nM GCP-2. Alternatively, the [Ca2+](i) rise induced by these CXC chemokines was inhibited by pretreatment of neutrophils with GCP-2. GCP- 2 stimulated [Ca2+](i) increases in CXCR1- and CXCR2-transfected cells, demonstrating that GCP-2 binds to both IL-8 receptors. Intradermal injection of synthetic GCP-2 resulted in a dose-dependent neutrophil accumulation and plasma extravasation in rabbit skin. To provoke this skin reaction, GCP-2 (10 pmol/site) was nearly as effective as IL-8, indicating that it is an important complementary mediator of the inflammatory response.
UR - http://www.scopus.com/inward/record.url?scp=0031017657&partnerID=8YFLogxK
U2 - 10.1021/bi961999z
DO - 10.1021/bi961999z
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AN - SCOPUS:0031017657
SN - 0006-2960
VL - 36
SP - 2716
EP - 2723
JO - Biochemistry
JF - Biochemistry
IS - 9
ER -