Biological properties of chemokines are believed to be influenced by their association with glycosaminoglycans. Surface plasmon resonance kinetic analysis shows that the CXC chemokine stromal cell-derived factor-1␣ (SDF-1␣), which binds the CXCR4 receptor, associates with heparin with an affinity constant of 38.4 nM (k on ؍ 2.16 ؋ 10 6 M ؊1 s ؊1 and k off ؍ 0.083 ؋ s ؊1 ). A modified SDF-1␣ (SDF-1 3/6) was generated by combined substitution of the basic cluster of residues Lys 24 , His 25 , and Lys 27 by Ser. SDF-1 3/6 conserves the global native structure and functional properties of SDF-1␣, but it is unable to interact with sensor chip-immobilized heparin. The biological relevance of these in vitro findings was investigated. SDF-1␣ was unable to bind in a CXCR4-independent manner on epithelial cells that were treated with heparan sulfate (HS)-degrading enzymes or constitutively lack HS expression. The inability of SDF-1 3/6 to bind to cells underlines the importance of the identified basic cluster for the physiological interactions of SDF-1␣ with HS. Importantly, the amino-terminal domain of SDF-1␣ which is required for binding to, and activation of, CXCR4 remains exposed after binding to HS and is recognized by a neutralizing monoclonal antibody directed against the first residues of the chemokine. Overall, these findings indicate that the Lys 24 , His 25 , and Lys 27 cluster of residues forms, or is an essential part of, the HS-binding site which is distinct from that required for binding to, and signaling through, CXCR4.
Adenosine deaminase (ADA, EC 3.5.4.4) is an enzyme of the purine metabolism which has been the object of considerable interest mainly because the congenital defect causes severe combined immunodeficiency (SCID). In the last 10 years, ADA, which was considered to be cytosolic, has been found on the cell surface of many cells and, therefore, it can be considered an ecto-enzyme. There is recent evidence about a specific role of ecto-ADA, which is different from that of intracellular ADA. Apart from degrading extracellular adenosine (Ado) or 2'-deoxyadenosine (dAdo), which are toxic for lymphocytes, ecto-ADA has an extraenzymatic function via its interaction with CD26. ADA/CD26 interaction results in co-stimulatory signals in T cells. This co-stimulation is blocked by HIV-1, thus evidencing a role for ecto-ADA in the pathophysiology of AIDS. The fact that, besides CD26, ADA can interact with different cell-surface proteins opens new perspectives in the research for a role of ecto-ADA in the function of the immune system and in the interactions that take place between different cells in the development of the immune system. The most interesting aspect is the possible participation of the ecto-enzyme in cell-to-cell contacts during ontogenesis and maturation of immunocompetent cells.
HIV-1 external envelope glycoprotein gp120 inhibits adenosine deaminase (ADA) binding to its cell surface receptor in lymphocytes, CD26, by a mechanism that does not require the gp120^CD4 interaction. To further characterize this mechanism, we studied ADA binding to murine clones stably expressing human CD26 and/or human CD4, and transiently expressing human CXCR4. In this heterologous model, we show that both recombinant gp120 and viral particles from the X4 HIV-1 isolate IIIB inhibited the binding of ADA to wild-type or catalytically inactive forms of CD26. In cells lacking human CXCR4 expression, this gp120-mediated inhibition of ADA binding to human CD26 was completely dependent on the expression of human CD4. In contrast, when cells were transfected with human CXCR4 the inhibitory effect of gp120 was significantly enhanced and was not blocked by anti-CD4 antibodies. These data suggest that the interaction of gp120 with CD4 or CXCR4 is required for efficient inhibition of ADA binding to CD26, although in the presence of CXCR4 the interaction of gp120 with CD4 may be dispensable. ß 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
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