Aggregation of the chemokine MIP-1 alpha is a dynamic and reversible phenomenon. Biochemical and biological analyses.
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.
Graham
G J
GJ
Beatson Institute for Cancer Research, Cancer Research Campaign, Beatson Laboratories, Bearsden, Glasgow, United Kingdom.
MacKenzie
J
J
Lowe
S
S
Tsang
M L
ML
Weatherbee
J A
JA
Issacson
A
A
Medicherla
J
J
Fang
F
F
Wilkinson
P C
PC
Pragnell
I B
IB
D000595:Amino Acid Sequence; D001483:Base Sequence; D002454:Cell Differentiation / Q000187:drug effects; D054407:Chemokine CCL4; D003114:Colony-Forming Units Assay; D016207:Cytokines / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D017931:DNA Primers; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D046911:Macromolecular Substances; D019402:Macrophage Inflammatory Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009000:Monocytes / Q000166:cytology / Q000187:drug effects; D015846:Monokines / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D013329:Structure-Activity Relationship
D054407:Chemokine CCL4; D016207:Cytokines; D017931:DNA Primers; D046911:Macromolecular Substances; D019402:Macrophage Inflammatory Proteins; D015846:Monokines; D011994:Recombinant Proteins