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 chemoki ne, inhibits proliferation of human hematopoietic progenitors responsive to co-stimulation by recombinant steel factor (rSLF), is chemotactic for huma n monocytes and T-lymphocytes, and promotes T-lymphocyte adhesion to endoth elial cells. Because chemokines have four conserved cysteines forming two i ntramolecular disulfide bridges, we decided to investigate their contributi on 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-1 0, they were cloned after an initiating methionine into expression vector p ET-3d. Subsequently rIP-10 was purified by enzymatic cell lysis, solubiliza tion of refractile bodies with guanidine hydrochloride, renaturation by dia lysis against dilute acetic acid, and sequential ion-exchange and reverse-p hase high-performance liquid chromatography. Purified rIP-10 was reduced wi th 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 hu man bone marrow hematopoietic progenitor proliferation and by chemotaxis as says using human T-lymphocytes and monocytes. In both assay systems, the bi ological 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 wa s abolished after modification by NMM. We conclude that disulfide bridges a re not essential for the biological activity of rIP-10.