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.
Isokawa
K
K
Department of Cellular Biology and Anatomy, Medical College of Wisconsin, Milwaukee 53226.
Rezaee
M
M
Wunsch
A
A
Markwald
R R
RR
Krug
E L
EL
D000818:Animals; D000941:Antigens / Q000097:blood; D002642:Chick Embryo; D004492:Edetic Acid; D016326:Extracellular Matrix Proteins / Q000032:analysis*; D006321:Heart / Q000196:embryology*; D009024:Morphogenesis / Q000502:physiology; D009206:Myocardium / Q000737:chemistry*; D014168:Transferrin / Q000032:analysis*
D000941:Antigens; D016326:Extracellular Matrix Proteins; D014168:Transferrin; D004492:Edetic Acid