We have previuosly demonstrated that CD26 dipeptidyl-peptidase IV is a key protease regulating the structure and function of chemokines, including chemokines with activity on HIV-1 replication. We have further characterized chemokine processing by studying chemokines secreted from normal T cells and macrophages. Natural forms of chemokines produced in response to stimulation have been affinity purified from cell lysates and culture supernatants, and then analyzed by mass spectrometry, peptide mapping and biological testing. Initial studies focused on chemokines specific for the HIV coreceptor, CCR5, including MIP-1alpha, MIP-1beta, and RANTES. Chemokines with N-terminal motifs consistent with CD26 dipeptidase susceptibility (MIP-1beta and RANTES) were found to have truncated N-termini missing the first two amino-acids. In contrast, MIP-1alpha, which does not contain the CD26 motif, was secreted from T cells as a truncated form missing the first 4 amino-acids. The truncated MIP-1alpha showed intact CCR5 stimulation including Ca++ signalling and anti-HIV activity. The trucated MIP-1alpha had enhanced activity on the CCR1 receptor compared with the full length form. These findings demonstrate that at least two chemokine processing pathways are active in T cells; one which depends on the expression of the CD26 dipeptidase and another that modifies chemokines not susceptible to CD26 activity. Candidate enzymes involved in the non-CD26 processing pathways are being identified. The fine structures of chemokines isolated from monocytes and macrophages were found to differ from T cell produced chemokines. Overall, the results show that chemokine structure and function are controlled by a complex system of proteases that specifically modify the N-termini of these proteins. These studies should provide a biochemical foundation for understanding the impact of chemokine protein structure on chemokine-dependent cell activation and migration. .

National Institute of Health (NIH)
Food and Drug Administration (FDA)
Intramural Research (Z01)
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