Human recombinant interferon-inducible protein-10: intact disulfide bridges are not required for inhibition of hematopoietic progenitors and chemotaxis of T lymphocytes and monocytes.
Human recombinant interferon-inducible protein-10 (rIP-10), a C-X-C chemokine, inhibits proliferation of human hematopoietic progenitors responsive to co-stimulation by recombinant steel factor (rSLF), is chemotactic for human monocytes and T-lymphocytes, and promotes T-lymphocyte adhesion to endothelial cells. Because chemokines have four conserved cysteines forming two intramolecular disulfide bridges, we decided to investigate their contribution in the biological activity of rIP-10. Since amino acid residues 22-98 of the sequence predicted by the cDNA constitute the naturally occurring IP-10, they were cloned after an initiating methionine into expression vector pET-3d. Subsequently rIP-10 was purified by enzymatic cell lysis, solubilization of refractile bodies with guanidine hydrochloride, renaturation by dialysis against dilute acetic acid, and sequential ion-exchange and reverse-phase high-performance liquid chromatography. Purified rIP-10 was reduced with 20 mM dithiothreitol, and chemically modified with 100 mM iodoacetamide (IAA), or S-methyl-methanethiosulfonate (MMTS), or N-methylmaleimide (NMM). Radiolabeling experiments demonstrated that 95% of the rIP-10 thiols were modified, and this was confirmed with SDS-PAGE. The biological activity of modified rIP-10 was determined in vitro by inhibition of rSLF-responsive human bone marrow hematopoietic progenitor proliferation and by chemotaxis assays using human T-lymphocytes and monocytes. In both assay systems, the biological activity was evident at rIP-10 concentrations of 20-100 ng/ml. The activity was preserved after modification of rIP-10 by IAA or MMTS, but was abolished after modification by NMM. We conclude that disulfide bridges are not essential for the biological activity of rIP-10.
Crow
M
M
Department of Lymphoma-Myeloma, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
Taub
D D
DD
Cooper
S
S
Broxmeyer
H E
HE
Sarris
A H
AH
D002455:Cell Division / Q000187:drug effects; D054357:Chemokine CXCL10; D019743:Chemokines, CXC / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D002633:Chemotaxis / Q000187:drug effects*; D003001:Cloning, Molecular; D004220:Disulfides / Q000378:metabolism / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D009000:Monocytes / Q000502:physiology; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000494:pharmacology; D013601:T-Lymphocytes / Q000502:physiology
D054357:Chemokine CXCL10; D019743:Chemokines, CXC; D004220:Disulfides; D011994:Recombinant Proteins