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Identification of human macrophage inflammatory proteins 1alpha and 1beta as a native secreted heterodimer
1Laboratory of Gene Regulation, Division of Therapeutic Proteins, Center for Biologics Evaluation and Research, Food and Drug Administration, National Institutes of Health, Bethesda, Maryland 20892, USA.
Insights
Macrophage inflammatory protein 1alpha and 1beta (MIP-1alpha and MIP-1beta) form heterodimers from activated human monocytes and lymphocytes. This discovery provides biochemical evidence for naturally occurring chemokine heterodimers and their potential role in receptor signaling.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Chemokines are crucial chemoattractants for leukocytes, mediating immune responses.
- Macrophage inflammatory protein 1alpha (MIP-1alpha) and MIP-1beta (MIP-1beta) are key chemokines involved in monocyte and lymphocyte activation.
- The specific molecular interactions and assembly of these chemokines were not fully understood.
Purpose of the Study:
- To investigate the formation and biochemical characteristics of macrophage inflammatory protein 1alpha and 1beta (MIP-1alpha/beta) complexes.
- To provide the first biochemical evidence for the existence of naturally occurring chemokine heterodimers.
- To explore the potential functional implications of MIP-1alpha/beta heterodimerization on chemokine receptor signaling.
Main Methods:
- Immunoprecipitation and immunoblot analysis were used to detect MIP-1alpha/beta complexes in cell lysates and culture supernatants.
- Mass spectrometry was employed to identify the specific components and modifications within the precipitated heterodimers.
- In vitro mixing of recombinant chemokines and cell treatments (e.g., monensin) were used to study heterodimerization dynamics and localization.
Main Results:
- Activated human monocytes and peripheral blood lymphocytes (PBLs) secrete MIP-1alpha and MIP-1beta as a heterodimer.
- Mass spectrometry identified NH(2)-terminal truncated MIP-1alpha complexed with either intact or truncated MIP-1beta.
- Heterodimers were stable at physiological conditions (pH 7.4, nanomolar concentrations) but dissociated under acidic conditions.
Conclusions:
- The study provides the first biochemical evidence for the natural formation of MIP-1alpha/beta chemokine heterodimers.
- Heterodimerization is specific and occurs under physiological conditions, suggesting a significant biological role.
- The formation of MIP-1alpha/beta heterodimers may influence intracellular signaling pathways, impacting chemokine receptor (e.g., CCR5) function.
Abstract:
Chemokines are secreted proteins that function as chemoattractants for leukocytes. The chemokines macrophage inflammatory protein 1alpha and 1beta (MIP-1alpha and MIP-1beta) now have been shown to be secreted from activated human monocytes and peripheral blood lymphocytes (PBLs) as a heterodimer. Immunoprecipitation and immunoblot analysis revealed that antibodies to either MIP-1alpha or MIP-1beta precipitated a protein complex containing both MIP-1alpha and MIP-1beta under normal conditions from culture supernatants and lysates of these cells. Mass spectrometry of the complexes, precipitated from the culture supernatants of monocytes and PBLs, revealed the presence of NH(2)-terminal truncated MIP-1alpha (residues 5-70) together with either intact MIP-1beta or NH(2)-terminal truncated MIP-1beta (residues 3-69), respectively. The secreted MIP-1alpha/beta heterodimers were dissociated into their component monomers under acidic conditions. Exposure of monocytes or PBLs to monensin induced the accumulation of heterodimers composed of NH(2)-terminal truncated MIP-1alpha and full-length MIP-1beta in the Golgi complex. The mixing of recombinant chemokines in vitro demonstrated that heterodimerization of MIP-1alpha and MIP-1beta is specific and that it occurs at physiological conditions, pH 7.4, and in the range of nanomolar concentrations. The data presented here provide the first biochemical evidence for the existence of chemokine heterodimers under natural conditions. Formation of heterodimers of MIP-1alpha/beta may have an impact on intracellular signaling events that contribute to CCR5 and possibly to other chemokine receptor functions.
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