ABSTRACT
Chemical synthesis in combination with precision polymer modification allows the systematic exploration of the effect of protein properties, such as charge and hydrodynamic radius, on potency using defined, homogeneous conjugates. A series of polymer-modified synthetic erythropoiesis proteins were constructed that had a polypeptide chain similar to the amino acid sequence of human erythropoietin but differed significantly in the number and type of attached polymers. The analogs differed in charge from +5 to -26 at neutral pH and varied in molecular weight from 30 to 54 kDa. All were active in an in vitro cell proliferation assay. However, in vivo potency was found to be strongly dependent on overall charge and size. The trends observed in this study may serve as starting points for the construction of more potent synthetic EPO analogs in the future.
Subject(s)
Erythropoiesis/physiology , Polymers/chemical synthesis , Proteins/chemical synthesis , Amino Acid Sequence , Animals , Binding Sites/drug effects , Binding Sites/physiology , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Erythropoietin/chemical synthesis , Erythropoietin/metabolism , Erythropoietin/physiology , Humans , Macaca fascicularis , Mice , Molecular Sequence Data , Polymers/metabolism , Polymers/pharmacology , Proteins/metabolism , Proteins/physiology , RatsABSTRACT
Although advances have been made in understanding cell differentiation, only rudimentary knowledge exists concerning how differentiated cells form tissues and organs. We studied liver organogenesis because the cell and tissue architecture of this organ is well defined. Approximately 60% of the adult liver consists of hepatocytes that are arranged as single-cell anastomosing plates extending from the portal region of the liver lobule toward the central vein. The basal surface of the hepatocytes is separated from adjacent sinusoidal endothelial cells by the space of Disse, where the exchange of substances between serum and hepatocytes takes place. The hepatocyte's apical surface forms bile canaliculi that transport bile to the hepatic ducts. Proper liver architecture is crucial for hepatic function and is commonly disrupted in disease states, including cirrhosis and hepatitis. Here we report that hepatocyte nuclear factor 4alpha (Hnf4alpha) is essential for morphological and functional differentiation of hepatocytes, accumulation of hepatic glycogen stores and generation of a hepatic epithelium. We show that Hnf4alpha is a dominant regulator of the epithelial phenotype because its ectopic expression in fibroblasts induces a mesenchymal-to-epithelial transition. Most importantly, the morphogenetic parameters controlled by Hnf4alpha in hepatocytes are essential for normal liver architecture, including the organization of the sinusoidal endothelium.
Subject(s)
DNA-Binding Proteins , Hepatocytes/cytology , Liver/embryology , Phosphoproteins/physiology , Transcription Factors/physiology , Animals , Apoptosis , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Cell Differentiation , Cell Division , Cells, Cultured , Epithelium , Female , Flow Cytometry , Gene Expression , Hepatocyte Nuclear Factor 4 , Immunoblotting , Immunoenzyme Techniques , In Situ Nick-End Labeling , Liver Glycogen/metabolism , Male , Mice , Mice, Knockout , Morphogenesis , Pregnancy , Retroviridae/genetics , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
We report the design and total chemical synthesis of "synthetic erythropoiesis protein" (SEP), a 51-kilodalton protein-polymer construct consisting of a 166-amino-acid polypeptide chain and two covalently attached, branched, and monodisperse polymer moieties that are negatively charged. The ability to control the chemistry allowed us to synthesize a macromolecule of precisely defined covalent structure. SEP was homogeneous as shown by high-resolution analytical techniques, with a mass of 50,825 +/-10 daltons by electrospray mass spectrometry, and with a pI of 5.0. In cell and animal assays for erythropoiesis, SEP displayed potent biological activity and had significantly prolonged duration of action in vivo. These chemical methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications.
