Identification of Amino Acid Residues Critical for Aggregation of Human CC Chemokines Macrophage Inflammatory Protein (MIP)-1α, MIP-1β, and RANTES

Abstract

Human CC chemokines macrophage inflammatory protein (MIP)-1␣, MIP-1␤, and RANTES (regulated on activation normal T cell expressed) self-associate to form high-molecular mass aggregates. To explore the biological significance of chemokine aggregation, nonaggregating variants were sought. The phenotypes of 105 hMIP-1␣ variants generated by systematic mutagenesis and expression in yeast were determined. hMIP-1␣ residues Asp 26 and Glu 66 were critical to the self-association process. Substitution at either residue resulted in the formation of essentially homogenous tetramers at 0.5 mg/ml. Substitution of identical or analogous residues in homologous positions in both hMIP-1␤ and RAN-TES demonstrated that they were also critical to aggregation. Our analysis suggests that a single charged residue at either position 26 or 66 is insufficient to support extensive aggregation and that two charged residues must be present. Solution of the three-dimensional NMR structure of hMIP-1␣ has enabled comparison of these residues in hMIP-1␤ and RANTES. Aggregated and disaggregated forms of hMIP-1␣, hMIP-1␤, and RANTES generally have equivalent G-protein-coupled receptormediated biological potencies. We have therefore generated novel reagents to evaluate the role of hMIP-1␣, hMIP-1␤, and RANTES aggregation in vitro and in vivo. The disaggregated chemokines retained their human immunodeficiency virus (HIV) inhibitory activities. Surprisingly, high concentrations of RANTES, but not disaggregated RANTES variants, enhanced infection of cells by both M-and T-tropic HIV isolates/strains. This observation has important implications for potential therapeutic uses of chemokines implying that disaggregated forms may be necessary for safe clinical investigation.

Antagonist of Secondary Lymphoid-Tissue Chemokine (CCR Ligand 21) Prevents the Development of Chronic Graft-Versus-Host Disease in Mice

“…The naturally cleaved forms of MCP-2 and RANTES are also devoid of bioactivity (31,34). The function of chemokines has also been further clarified from crystal structure determination and nuclear magnetic resonance data (42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56). In several chemokines such as IL-8, MCP-1, MCP-2, MCP-3, eotaxin, RANTES, MIP-1␣, MIP-1␤, fractalkine, and stromal cell-derived factor-1, it has been observed that the N-terminal region is essential for functional activity and that the loop immediately following the first two cysteines in the sequence, as well as the N-terminal region, plays an important role in receptor binding.…”

Unravelling Glycobiology by NMR Spectroscopy

“…Although, the heparin biding site of CCL5 has already been reported [33], comprising the segment 44 RKNR 47 , the potential GAG binding sites of CCL5 are yet undocumented, especially considering chondroitin sulfate which is the most abundant GAG in the human body, and widely spread across surface proteoglycans of blood vessels. The 15 N-HSQC spectrum of the 15 N-labeled E66S CCL5 (Figure 12), which is mostly found as monomers in solution rather than the oligomerization propensity of the wild type [35], showed well-resolved amide signals that allowed easily a near-complete chemical shift assignment (Table 4). These initial assignments are crucial to further inform the chemical shift changes induced by the presence of increasing concentration of the ligants.…”

Control of feeding behavior inC. elegansby human G protein-coupled receptors permits screening for agonist-expressing bacteria

“…The N-terminal pelB leader is removed after causing secretion into the periplasmic space. This construct was further mutagenized to create pSANG10 hMIP-1␣-D26A (aspartate26 substituted with alanine) to reduce aggregation during expression while still maintaining biological activity (4). As with the previously described D26A version, this one lacked the first encoded amino acid and utilized ser2 as the first amino acid.…”

“…S2 for binding curves for other mutants. (4), showing in red side chain residues that have been reported previously to be involved in CCR5 binding to structurally related MIP-1␤ (F12, R17, Q18, P20, K44, R45, and R47). Interactions identified from this study are highlighted in green (T8, A9, N22, and A25).…”

“…The potencies of different N-terminal variants have been reported. In this article, we use a variant with reduced aggregation properties due to a substitution of Asp-26 to alanine (3,4). This version, which also lacks the first alanine residue (with Ser as the first amino acid), has been shown to have compatible potency with full-length MIP-1␣ (5).…”