Biological and structural properties of MIP-1α expressed in yeast

• Published in: Cytokine (Philadelphia, Pa.) Vol. 4; no. 1; pp. 76 - 82
• Main Authors: Clements, John M., Craig, Stewart, Gearing, Andrew J.H., Hunter, Michael G., Heyworth, Clare M., Michael Dexter, T., Lord, Brian I.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 1992
• Subjects: Animals; Base Sequence; Cell Division - drug effects; Cells, Cultured; CFU-S inhibition; Chemokine CCL4; Circular Dichroism; Colony-Forming Units Assay; Cytokines - genetics; Cytokines - metabolism; Cytokines - pharmacology; Drug Synergism; Genetic Vectors; Granulocyte-Macrophage Colony-Stimulating Factor - pharmacology; Hematopoietic Stem Cells - drug effects; Macrophage Inflammatory Proteins; Mice; MIP-1α; Molecular Sequence Data; Monokines - genetics; Monokines - metabolism; Monokines - pharmacology; Protein Conformation; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - pharmacology; Saccharomyces cerevisiae - genetics; structure; yeast; CFU-S inhibition; structure; yeast; MIP-1α
• ISSN: 1043-4666; 1096-0023
• DOI: 10.1016/1043-4666(92)90040-X

The murine macrophage inflammatory proteins-1 alpha (MIP-1α) and MIP-1β are distinct but closely related cytokines. Partially purified mixtures of the two proteins affect neutrophil function and cause local inflammation and fever. The particular properties of MIP-1α have not been well studied, although it has been identified as being identical to an inhibitor of haemopoietic stem cell growth. We have expressed MIP-1α in yeast cells and purified it to sequence homogeneity. Structural analysis of this biologically active material by circular dichroism and fluorescence spectroscopy confirms that MIP-1α has a very similar secondary and tertiary structure to platelet factor 4 and interleukin 8 with which it shares limited sequence homology. The in-vitro stem cell inhibitory properties have been confirmed using a range of murine progenitor cells including purified bone marrow progenitor cells (FACS-1), the FDCP-mix A4 cell line, and spleen colony forming unit (CFU-S) populations. Plateau levels of inhibition of stem cell growth were achieved using concentrations of 0.15 μg/ml MIP-1α. We have also demonstrated that MIP-1α is active in vivo: 5 μg of MIP-1α per mouse given as a bolus injection, protects stem cells from subsequent in-vitro killing by tritiated thymidine. MIP-1α was also shown to enhance the proliferation of more committed progenitor granulocyte macrophage-colony forming cells (GM-CFC) in response to granulocyte macrophage-colony stimulating factor (GM-CSF).