IDENTIFICATION OF TRANSFERRIN AS ONE OF MULTIPLE EDTA-EXTRACTABLE EXTRACELLULAR PROTEINS INVOLVED IN EARLY CHICK HEART MORPHOGENESIS

It was demonstrated previously that a polyclonal antibody (ES1) raised against EDTA extractable proteins from embryonic chicken heart blocks cardiac endothelial-mesenchymal transformation in a culture bioassay and stains extracellular matrix at sites of embryonic inductive intera ctions, e.g., developing heart, limb buds, and neural crest forming re gion [Krug et al., 1987, Dev Biol 120:348-355; Mjaatvedt et al., 1991, Dev Biol 145:219-230). In the present study, by using an antiserum (E S3) to a similar immunogen, we affinity purified four major EDTA-solub le proteins. These proteins migrated as 27, 44, 63, and 70kD molecules under reduced conditions and 27, 41, 52, and 59kD under nonreduced co nditions, respectively, on SDS-PAGE. Based on several criteria, the pr otein migrating at 70/59 kD (reduced/nonreduced) was indistinguishable from chicken transferrin (conalbumin): 1) amino acid sequencing showe d that eight N-terminal residues were identical to those of chicken tr ansferrin, 2) acid hydrolysates of both proteins had nearly identical compositions, 3) the protein co-migrated exactly with chicken transfer rin under both reduced and nonreduced conditions, and 4) ES3 IgG recog nized both the 70/59 kD protein and chicken transferrin by western blo t analysis of nonreduced samples, but not with reduced samples. Immuno histochemistry of chicken embryonic heart with antibodies against tran sferrin demonstrated that anti-transferrin immunoreactivity is present in myocardium but absent in cardiac endothelium before the initiation of cardiac endothelial-mesenchymal formation. However, both cardiac e ndothelium and migrating mesenchymal cells became immunoreactive with anti-transferrin at the time transformation occurred. These findings s uggest a possible involvement of transferrin in the inductive process of cardiac endothelial-mesenchymal transformation. (C) 1994 Wiley-Liss , Inc.