Biological and structural properties of MIP-1 alpha expressed in yeast.
The murine macrophage inflammatory proteins-1 alpha (MIP-1 alpha) and MIP-1 beta 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-1 alpha 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-1 alpha 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-1 alpha 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 micrograms/ml MIP-1 alpha. We have also demonstrated that MIP-1 alpha is active in vivo: 5 micrograms of MIP-1 alpha per mouse given as a bolus injection, protects stem cells from subsequent in-vitro killing by tritiated thymidine. MIP-1 alpha 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).
Clements
J M
JM
British Bio-technology, Cowley, Oxford.
Craig
S
S
Gearing
A J
AJ
Hunter
M G
MG
Heyworth
C M
CM
Dexter
T M
TM
Lord
B I
BI
D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D054407:Chemokine CCL4; D002942:Circular Dichroism; D003114:Colony-Forming Units Assay; D016207:Cytokines / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D004357:Drug Synergism; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000187:drug effects; D019402:Macrophage Inflammatory Proteins; D051379:Mice; D008969:Molecular Sequence Data; D015846:Monokines / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics
D054407:Chemokine CCL4; D016207:Cytokines; D019402:Macrophage Inflammatory Proteins; D015846:Monokines; D011993:Recombinant Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor