Identification of amino acid residues critical for LD78beta, a variant of human macrophage inflammatory protein-1alpha, binding to CCR5 and inhibition of R5 human immunodeficiency virus type 1 replication.
In an attempt to determine which amino acid(s) of LD78beta, a variant of human macrophage inflammatory protein-1alpha, plays a critical role in the interaction with CCR5, we generated six LD78beta variants with an amino acid substituted to Ala at the NH(2) terminus of LD78beta. There was no significant difference in eliciting Ca(2+) flux and chemotaxis among the variants with the exception of LD78beta(T9A) showing a substantially reduced activity. The comparative order for human immunodeficiency virus type 1 (HIV-1) replication inhibition was: LD78beta(P8A) > LD78beta(D6A) > LD78beta(WT), LD78beta(L3A) > LD78beta(T7A), LD78beta(P2A) > LD78beta(T9A). In binding inhibition assays of LD78beta variants using 2D7 monoclonal antibody and (125)I-labeled macrophage inflammatory protein-1alpha, the comparative order was: LD78beta(P8A), LD78beta(D6A) > LD78beta(WT) > LD78beta(L3A) > LD78beta(T7A) > LD78beta(T9A), LD78betaP2A). The order for CCR5 down-regulation induction was comparable to that for binding inhibition. The present data suggest that Pro-2, Asp-6, Pro-8, and Thr-9 are critical for LD78beta binding to CCR5 and HIV-1 replication inhibition, and that LD78beta binding to CCR5, regardless of affinity, is sufficient for the initial signal transduction of LD78beta, whereas the greater anti-HIV-1 activity requires the greater magnitude of binding. The data also suggest that LD78beta variants with appropriate amino acid substitution(s) such as LD78beta(D6A) and LD78beta(P8A) may represent effective chemokine-based anti-HIV-1 therapeutics while preserving LD78beta-CCR5 interactions.
Miyakawa
Toshikazu
T
Department of Internal Medicine II, Kumamoto University School of Medicine, Honjo 1-1-1, Kumamoto 860-0811, Japan.
Obaru
Kenshi
K
Maeda
Kenji
K
Harada
Shigeyoshi
S
Mitsuya
Hiroaki
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002469:Cell Separation; D054407:Chemokine CCL4; D018925:Chemokines / Q000378:metabolism; D002633:Chemotaxis; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D005434:Flow Cytometry; D015723:Gene Library; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D019402:Macrophage Inflammatory Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010920:Placenta / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019713:Receptors, CCR5 / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014779:Virus Replication
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D054407:Chemokine CCL4; D018925:Chemokines; D018076:DNA, Complementary; D019402:Macrophage Inflammatory Proteins; D019713:Receptors, CCR5; D011994:Recombinant Proteins; D002118:Calcium