Cloning of Equine CD40 Ligand an Important Molecule in the Regulation of Cellular & Humoral Immune Responses
ACVIM 2008
B.A. Sponseller; S.K. Clark; J.M. Hostetter; D.E. Jones
College of Veterinary Medicine, Iowa State University, Ames, IA

CD40 Ligand, or CD154, is a 33 kD, type II transmembrane protein with sequence homology to the tumor necrosis factor (TNF) superfamily of cell surface signaling molecules. It is expressed on activated B and T lymphocytes, natural killer cells, mast cells, and dendritic cells. CD154 binds to CD40, a 50 kD glycoprotein, also a member of the TNF receptor superfamily. The CD40 receptor is expressed on the surface of immune cells including monocytes, thymic epithelial cells, B lymphocytes, and dendritic cells. Binding of CD154 with CD40 contributes to the activation of antigen presenting cells. Without CD154, antigen presentation by both dendritic cells and macrophages is markedly impaired, as is macrophage-mediated killing of both intracellular and extracellular pathogens. Dendritic cell production of IL-12, an important cytokine in driving a type I response, is induced by the CD154/ CD40 interaction. Type II responses are elicited when CD154 binds cell surface CD40 on mature B cells, resulting in B cell activation. Recent studies comparing expression of CD154 between human adults and neonates have revealed that there is a decrease in expression of CD154 by neonatal CD4+ T lymphocytes, suggesting that decreased expression of CD154 may play a role in acquired diseases of the neonate. Given the importance of the CD40/CD154 interaction, we initiated studies to characterize equine CD154 in order to determine its role in maturation of immune responses in the horse. Here, we report the cloning, sequence analysis, and expression of the equine CD154 in Chinese hamster ovary (CHO) cells and characterization of the recombinant protein by Western blot analysis. Sequence alignments of the translated equine sequence revealed distinct regions of homology with those of ovine, bovine, porcine, feline, canine, murine and human species. Similar to other species, equine CD154 also has a hydrophobic signal anchor region typical of type II membrane proteins. In contrast to human CD154, which contains 2 potential N-linked glycosylation sites, equine CD154 contains but one site, with a similar location as described in other domestic species. In the horse it us unknown as to whether or not the putative N-glycan sites are glycosylated. Nonetheless, the observed cross-reactivity of a murine monoclonal antibody to human CD154 in Western blot analysis suggests that the extracellular protein domain shares considerable homology with human, porcine, and murine CD154. The cloning and sequence analyses of equine CD154 provide the basis for future studies of equine neonatal immunology with regard to intracellular and extracellular infections.

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Brett Sponseller

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