Antibody Raised to AKAAAKAAAKA Sequence on Tropoelastin Recognizes Tropoelastin but not Mature Crosslinked Elastin: A New Tool in Metabolic and Structural Studies of Elastogenesis

Abstract

Tropoelastin, which is secreted from the cell in a soluble form, contains specific alanine rich repeat domains that are destined to form covalent desmosine and isodesmosine crosslinks in mature insoluble elastin. We raised a monospecific polyclonal antibody to a AKAAAKAAAKA synthetic peptide (AKA) which represents this alanine rich region of tropoelastin. The antibody was reactive with the original peptide antigen and purified tropoelastin, but not with mature crosslinked elastin isolated from several animal species. Conditioned media from chick aorta smooth muscle cells in culture reacted in an ELISA with the AKA antibody, but only in the presence of BAPN to block the conversion of the σ-amino groups to aldehydes. Immunofluorescence demonstrated that the AKA antibody decorated newly deposited tropoelastin assembled in fine fibrils in matrix produced by cultured human skin fibroblasts. EM-immunogold specifically localized this antibody to the immature elastic fibers present in fetal sheep ductus arteriosus. Moreover, immunohistochemistry demonstrated that the antibody recognized nonpolymerized tropoelastin assembled on the periphery of elastic fibers in the aorta of chicks raised on copper deficient and BAPN containing diets. These studies demonstrate that this new anti-tropoelastin antibody can be used as a useful tool to investigate elastin metabolism where it is important to distinguish between tropoelastin and mature crosslinked elastin.