We have continued to study the cellular and molecular processes that control chemokine structure and function with an emphasis on chemokines that effect HIV-1 replication. Natural forms of chemokines secreted from normal lymphocytes and macrophages under different cell activation conditions are being purified from cells and culture supernatants for further characterization. The fine structure of these proteins are then analyzed using several techniques including gel electrophoresis, peptide mapping and mass spectrometry. From these studies we have characterized several common modifications to chemokines and have identified key enzyme dependent pathways that are responsible fo the N-terminal processing. Processed forms of chemokines show changes in receptor interactions compared with full-length forms. Chemokines can either lose activity or in some cases gain activity after processing. One key enzyme controlling chemokine structure is the dipeptidase CD26, which removes two amino acids from the protein. We have studied the cellular expression pattern of this enzyme and have correlated enzyme expression with changes in secreted chemokine structure. Other peptidases are being studied that function together with CD26 in regulating chemokine activity. These pathways regulate differential cell migration during inflammatory and immune responses as well as control the structure and function of chemokines that inhibit HIV-1 infection. We have also continued studies on the quaternary structures of naturally secreted chemokines and its relevance to chemokine function. Based on the observation that macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta are secreted as heterodimers, we have compared function of heterodimeric complexes with individual proteins. Heterodimers have more activity on CCR5 receptor activation than the individual MIP-1alpha or MIP-1beta chemokines. This may be due to an ability to crosslink receptors on the cell surface more efficiently and thereby generate unique intracellular signals. The results suggest that heterodimeric forms of chemokines may be useful as novel therapeutic agents for controlling immune functions.
