A C-terminal carbohydrate-binding domain in the endothelial cell regulatory protein, pigpen: new function for an EWS family member.
The potential for encoding information in carbohydrate (CHO) structures has long been recognized. Selective CHO-binding proteins known as lectins and the biological events they mediate are well known. However, many lectins were originally discovered for biological activities other than saccharide binding, and only subsequently was it realized that one or more of their key functions were mediated by specific CHO recognition. Our previous observations suggested that the nuclear protein pigpen had an affinity for CHO structures. This would represent a new attribute for proteins of the EWS (Ewing's sarcoma) family, of which pigpen is a member. In this study we demonstrate that a CHO-binding domain resides in the C-terminus of the molecule and can be preferentially inhibited by saccharides, most notably N-acetyl-d-galactosamine (GalNAc) and the GalNAc-containing polysaccharide, chondroitin sulfate. Ligand blotting experiments were subsequently performed with fractionated, [(3)H]galactose-labeled cells to demonstrate the presence of chondroitin sulfate-inhibitable endogenous CHO ligands for pigpen in endothelial nuclei. Finally, microinjection of polysaccharide competitor into the nucleus of cultured endothelial cells resulted in a loss of pigpen focal accumulations, suggesting that the CHO-binding activity may be instrumental in subcellular localization of the protein. In summary, our results show ligand preference and domain specificity for pigpen's CHO affinity and provide initial evidence for physiological ligands and function. They may also shed new light on the mechanisms of oncogenic transformation involving EWS proteins.
Alliegro
M C
MC
Department of Cell Biology, Louisiana State University Medical Center, New Orleans, Louisiana, 70112, USA. mallie@Isumc.edu
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002241:Carbohydrates; D002417:Cattle; D004268:DNA-Binding Proteins; D004730:Endothelium, Vascular / Q000378:metabolism*; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D034802:RNA-Binding Protein EWS; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002241:Carbohydrates; D004268:DNA-Binding Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009687:Nuclear Proteins; D034802:RNA-Binding Protein EWS; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; C099524:pigpen protein, bovine