Convergent inactivation of the skin-specific C-C motif chemokine ligand 27 in mammalian evolution.

The appearance of mammalian-specific skin features was a key evolutionary event contributing for the elaboration of physiological processes such as thermoregulation, adequate hydration, locomotion, and inflammation. Skin inflammatory and autoimmune processes engage a population of skin-infiltrating T cells expressing a specific C-C chemokine receptor (CCR10) which interacts with an epidermal CC chemokine, the skin-specific C-C motif chemokine ligand 27 (CCL27). CCL27 is selectively produced in the skin by keratinocytes, particularly upon inflammation, mediating the adhesion and homing of skin-infiltrating T cells. Here, we examined the evolution and coding condition of Ccl27 in 112 placental mammalian species. Our findings reveal that a number of open reading frame inactivation events such as insertions, deletions, and start and stop codon mutations independently occurred in Cetacea, Pholidota, Sirenia, Chiroptera, and Rodentia, totalizing 18 species. The diverse habitat settings and lifestyles of Ccl27-eroded lineages probably implied distinct evolutionary triggers rendering this gene unessential. For example, in Cetacea, the rapid renewal of skin layers minimizes the need for an elaborate inflammatory mechanism, mirrored by the absence of epidermal scabs. Our findings suggest that the convergent and independent loss of Ccl27 in mammalian evolution concurred with unique adaptive roads for skin physiology.

NMR analysis of the structure, dynamics, and unique oligomerization properties of the chemokine CCL27.

Chemokines have two essential interactions in vivo, with G protein-coupled receptors, which activate intracellular signaling pathways, and with glycosaminoglycans (GAGs), which are involved in cell surface localization and transport. Although it has been shown that chemokines bind and activate their respective G protein-coupled receptors as monomers, many chemokines oligomerize upon GAG binding, and the ability to oligomerize and bind GAGs is required for in vivo function. In this study, we investigated the structure, dynamics, and oligomerization behavior of cutaneous T-cell-attracting chemokine (CTACK, also known as CCL27) by NMR. (15)N relaxation and translational self-diffusion rates indicate that CCL27 oligomerizes, but in contrast to many other chemokines that form relatively discrete oligomers, CCL27 transitions between monomer, dimer, and tetramer species over a relatively narrow concentration range. A three-dimensional structure determination was pursued under conditions where CCL27 is primarily dimeric, revealing the standard motif for a chemokine monomer. Analysis of chemical shift perturbations of (1)H-(15)N HSQC spectra, relaxation-dispersion experiments, and filtered nuclear Overhauser effects suggest that CCL27 does not adopt a discrete CXC or CC dimer motif. Instead, CCL27 has uncommon oligomerization behavior, where several equilibria involving relatively low affinity interactions between different interfaces seem to be simultaneously at work. However, interaction with heparin avidly promotes oligomerization under conditions where CCL27 is monomeric by itself. We hypothesize that the plasticity in the oligomerization state may enable CCL27 to adopt different oligomeric structures, depending on the nature of the GAG binding partner, thereby providing a mechanism for increased diversity and specificity in GAG-binding and GAG-related functions.

Application of a novel thioesterification reaction to the synthesis of chemokine CCL27 by the modified thioester method.

Aryl thioesters of peptide segments were prepared by the conventional 9-fluorenylmethoxycarbonyl (Fmoc) strategy using a novel N-alkyl cysteine (NAC)-assisted thioesterification reaction. The peptide carrying NAC at its C-terminus was prepared by the Fmoc strategy and converted to the aryl thioester by 4-mercaptophenylacetic acid (MPAA) treatment without significant side reactions. The peptide thioester was used for the efficient preparation of 95-amino acid (AA) chemokine CCL27 by an Ag(+)-free thioester method.