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 interactions, e.g., developing heart, limb buds, and neural crest forming region (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 (ES3) to a similar immunogen, we affinity purified four major EDTA‐soluble proteins. These proteins migrated as 27, 44, 63, and 70 kD molecules under reduced conditions and 27, 41, 52, and 59 kD under nonreduced conditions, respectively, on SDS‐PAGE. Based on several criteria, the protein migrating at 70/59 kD (reduced/nonreduced) was indistinguishable from chicken transferrin (conalbumin): (1) amino acid sequencing showed that eight N‐terminal residues were identical to those of chicken transferrin, (2) acid hydrolysates of both proteins had nearly identical compositions, (3) the protein co‐migrated exactly with chicken transferrin under both reduced and nonreduced conditions, and (4) ES3 IgG recognized both the 70/59 kD protein and chicken transferrin by western blot analysis of nonreduced samples, but not with reduced samples. Immunohistochemistry of chicken embryonic heart with antibodies against transferrin 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 endothelium and migrating mesenchymal cells became immunoreactive with anti‐transferrin at the time transformation occurred. These findings suggest a possible involvement of transferrin in the inductive process of cardiac endothelial‐mesenchymal transformation.