Abstract
Activated lymphocytes synthesize and secrete substantial amounts of the beta-chemokines macrophage inflammatory protein (MIP)-1 alpha/CCL3 and MIP-1 beta/CCL4, both of which inhibit infection of cells with human immunodeficiency virus type 1 (HIV-1). The native form of MIP-1 beta secreted by activated human peripheral blood lymphocytes (MIP-1 beta(3-69)) lacks the two NH(2)-terminal amino acids of the full-length protein. This truncated form of MIP-1 beta has now been affinity-purified from the culture supernatant of such cells, and its structure has been confirmed by mass spectrometry. Functional studies of the purified protein revealed that MIP-1 beta(3-69) retains the abilities to induce down-modulation of surface expression of the chemokine receptor CCR5 and to inhibit the CCR5-mediated entry of HIV-1 in T cells. Characterization of the chemokine receptor specificity of MIP-1 beta(3-69) showed that the truncated protein not only shares the ability of intact MIP-1 beta to induce Ca(2+) signaling through CCR5, but unlike the full-length protein, it also triggers a Ca(2+) response via CCR1 and CCR2b. These results demonstrate that NH(2)-terminally truncated MIP-1 beta functions as a chemokine agonist with expanded receptor reactivity, which may represent an important mechanism for regulation of immune cell recruitment during inflammatory and antiviral responses.
Highlights
Activated lymphocytes synthesize and secrete substantial amounts of the -chemokines macrophage inflammatory protein (MIP)-1␣/CCL3 and MIP-1/CCL4, both of which inhibit infection of cells with human immunodeficiency virus type 1 (HIV-1)
Purification and Characterization of Native Secreted MIP1—We have recently shown that human peripheral blood lymphocytes (PBLs) stimulated by the combination either of IL-2 and IL-12 or of IL-2 and PHA produce a truncated form (MIP-1(3– 69)) of MIP-1 (7658 Da) that lacks the two NH2-terminal amino acids of the full-length protein and is complexed with a truncated form (MIP-1␣(5–70)) of MIP-1␣ (7459 Da) that lacks the four NH2-terminal residues [14]
The removal of the Ala-Pro dipeptide from the NH2 terminus of MIP-1 did not impair its biological activities mediated by CCR5; through its interaction with this receptor, the truncated protein induced down-modulation of the surface expression of CCR5, increased the cytosolic-free Ca2ϩ concentration, and inhibited CCR5-mediated entry of HIV-1
Summary
Vol 277, No 35, Issue of August 30, pp. 32348 –32352, 2002 Printed in U.S.A. Natural Truncation of the Chemokine MIP-1/CCL4 Affects Receptor Specificity but Not Anti-HIV-1 Activity*. The native form of MIP-1 secreted by activated human peripheral blood lymphocytes (MIP-1(3– 69)) lacks the two NH2-terminal amino acids of the full-length protein. This truncated form of MIP-1 has been affinity-purified from the culture supernatant of such cells, and its structure has been confirmed by mass spectrometry. Characterization of the chemokine receptor specificity of MIP-1(3– 69) showed that the truncated protein shares the ability of intact MIP-1 to induce Ca2؉ signaling through CCR5, but unlike the full-length protein, it triggers a Ca2؉ response via CCR1 and CCR2b These results demonstrate that NH2-terminally truncated MIP-1 functions as a chemokine agonist with expanded receptor reactivity, which may represent an important mechanism for regulation of immune cell recruitment during inflammatory and antiviral responses. Our results demonstrate that the endogenous form of MIP-1, which lacks the first two amino acid residues of the encoded protein, is active at CCR1 and CCR2 in addition to CCR5 and retains anti-HIV-1 activity
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