title: Molecular characterization of EAP-300: a high molecular weight, embryonic polypeptide containing an amino acid repeat comprised of multiple leucine-zipper motifs.
abstract: In this study we report the biochemical and initial molecular characterization of EAP-300, a developmentally regulated embryonal protein that has been shown previously to be expressed by radial glia in various regions of the CNS, including putative glial barriers. In the present study we have shown that the 300 kDa EAP-300 polypeptide is developmentally regulated in all tissues expressing the protein, which include various PNS and CNS tissues and muscle. In neural tissue the protein is readily detected during early embryogenesis, subsequently down-regulated at later stages, and is not detected in the adult. In contrast to neural tissue, small amounts of the protein are expressed in heart, consistent with earlier studies which showed that EAP-300 expression was maintained in the Purkinje cells of the heart conduction system. Metabolic labeling demonstrates that EAP-300 is a phosphoprotein, and is fatty acylated based on incorporation of [3H]palmitate. We also show that the normal developmental down-regulation of EAP-300 by glia does not occur in vitro, and these data therefore suggest that the signal(s) that regulates EAP-300 gene expression during development in vivo is absent in dissociated cell cultures. We have also initiated molecular studies of EAP-300 by screening embryonic brain cDNA expression libraries with a mixture of EAP-300 monoclonal antibodies. Sequence analysis of partial EAP-300 cDNAs indicate that the protein is related, if not identical, to IFAPa-400, a developmentally regulated intermediate filament-associated protein in chick that is proposed to participate in cell differentiation. These studies also indicate that EAP-300 mRNA is developmentally regulated and is expressed by glial cells in putative CNS barrier structures. Our studies also suggest that two pools of EAP-300 may exist in cells, implying that unlike IFAPa-400 the EAP-300 protein may not always be associated with intermediate filaments. Interestingly, our studies demonstrate that EAP-300 contains a novel repeat amino acid domain comprised of multiple leucine-zipper motifs, which may contribute to its function during glial differentiation.
authors.affiliation: Department of Pharmacology, Medical University of South Carolina, Charleston 29425, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Kelly
authors.forename: C
authors.initials: C
authors.lastname: Phanhthourath
authors.forename: D K
authors.initials: DK
authors.lastname: Brees
authors.forename: C F
authors.initials: CF
authors.lastname: McCabe
authors.forename: G J
authors.initials: GJ
authors.lastname: Cole
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001253:Astrocytes / Q000737:chemistry; D001483:Base Sequence; D002478:Cells, Cultured / Q000737:chemistry; D002642:Chick Embryo; D003001:Cloning, Molecular; D005326:Fetal Proteins / Q000235:genetics* / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D005796:Genes / Q000235:genetics*; D017403:In Situ Hybridization; D016350:Leucine Zippers / Q000235:genetics*; D008969:Molecular Sequence Data; D009132:Muscles / Q000737:chemistry; D009420:Nervous System / Q000737:chemistry; D009474:Neurons / Q000737:chemistry; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*
chemical_list: D005326:Fetal Proteins; D012333:RNA, Messenger