Aggregation of the chemokine MIP-1 alpha is a dynamic and reversible phenomenon. Biochemical and biological analyses

• Published in: The Journal of biological chemistry Vol. 269; no. 7; pp. 4974 - 4978
• Main Authors: GRAHAM, G. J, MACKENZIE, J, LOWE, S, LIK-SHING TSANG, M, WEATHERBEE, J. A, ISSACSON, A, MEDICHERLA, J, FANG, F, WILKINSON, P. C, PRAGNELL, I. B
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 18-02-1994
• Subjects: Amino Acid Sequence; Analysis of the immune response. Humoral and cellular immunity; Base Sequence; Biological and medical sciences; Cell Differentiation - drug effects; Chemokine CCL4; Colony-Forming Units Assay; Cytokines - biosynthesis; Cytokines - isolation & purification; Cytokines - pharmacology; DNA Primers; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Hematopoietic Stem Cells - cytology; Hematopoietic Stem Cells - drug effects; Humans; Immunobiology; Lymphokines, interleukins ( function, expression); Macromolecular Substances; Macrophage Inflammatory Proteins; Molecular Sequence Data; Molecular Weight; Monocytes - cytology; Monocytes - drug effects; Monokines - biosynthesis; Monokines - isolation & purification; Monokines - pharmacology; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Protein Processing, Post-Translational; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Recombinant Proteins - pharmacology; Regulatory factors and their cellular receptors; Structure-Activity Relationship; Aggregation; Cell proliferation; Structure activity relation; Cytokine; Site directed mutagenesis; Inhibition; Macrophage
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)37641-X

Macrophage inhibitory protein (MIP)-1 alpha is a potent inhibitor of hemopoietic stem cell proliferation and is a member of
a family of pro-inflammatory mediators, the chemokine family. This molecule along with other members of the chemokine family
exists as a peptide of 8 kDa but has a strong tendency for noncovalent extensive self-aggregation. As this aggregation may
interfere with biological activity, we have produced nonaggregating variants of MIP-1 alpha which display a range of molecular
sizes. The mutants, produced by sequential neutralization of carboxyl-terminal acidic residues, display native molecular masses
representative of tetramers, dimers, and monomers. Intriguingly when these mutants are assessed in comparison with native
MIP-1 alpha for bioactivity in vitro, they are seen to be equipotent in both stem cell assays and in monocyte shape-change
assays, suggesting that there is no requirement for aggregation in either of these biological contexts. This indicates that
the aggregated MIP-1 alpha and the aggregated mutants spontaneously disaggregate under assay conditions and ultimately function
as monomers. We have further demonstrated the ability of MIP-1 alpha to disaggregate spontaneously in dilute solution by enzyme-linked
immunosorbent assay analysis of fractions obtained from gel filtration of varying concentrations of MIP-1 alpha. The aggregation
of MIP-1 alpha is therefore a dynamic and reversible phenomenon which has little, if any, impact on bioactivity in vitro.