RESUMO
Human chorionic gonadotropin (hCG) is a member of the family of glycoprotein hormones containing a common alpha-subunit and distinct beta-subunits that confer hormonal specificity. hCG binds to the relatively large ectodomain of the human luteinizing hormone receptor (hLHR), a member of the G protein-coupled receptor superfamily, leading to increased intracellular production of cAMP. Using protein engineering, two miniaturized versions of hCGbeta have been separately fused to the N-terminus of the alpha-subunit to give N-des[1-91]hCGbeta-alpha-C and N-des[1-91,110-114]hCGbeta-alpha-C, i.e. fusion proteins of the hCGbeta determinant loop (extended to include the complete seat belt and carboxy-terminal peptide) coupled to the alpha-subunit. Bioactivity of these single-chain gonadotropin analogs was assessed in two systems following transient transfections into HEK 293 cells and subsequent cAMP measurements. In one, each mini-beta-alpha cDNA was fused to that of hLHR and transfected into cells to create yoked miniaturized hCG-hLHR complexes; in the other, the cDNA of each single chain mini-beta-alpha was co-transfected with that of hLHR in an effort to produce non-covalent miniaturized hCG-hLHR complexes. Using yoked hCG-hLHR and hLHR as positive and negative controls respectively, expression of each mini-hCG-hLHR complex was confirmed using antibody and ligand binding assays. The two mini-hCGs led to minimal activation of hLHR, suggesting weak intrinsic activity of the mini-beta-alpha fusion proteins. These results suggest that potent agonists and antagonists will require the presence of other portions of hCGbeta in addition to the determinant loop/seat belt.
Assuntos
Gonadotropina Coriônica/análogos & derivados , Gonadotropina Coriônica/química , Sítios de Ligação , Linhagem Celular , Gonadotropina Coriônica/genética , Gonadotropina Coriônica/metabolismo , Cristalografia por Raios X , AMP Cíclico/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores do LH/metabolismo , TransfecçãoAssuntos
Sulfatos de Condroitina/metabolismo , Sulfatos de Condroitina/farmacologia , Dermatan Sulfato/metabolismo , Dermatan Sulfato/farmacologia , Sulfonamidas/síntese química , Sequência de Carboidratos , Proteínas Inativadoras do Complemento/síntese química , Proteínas Inativadoras do Complemento/farmacologia , Inibidores do Fator Xa , Glicosaminoglicanos/química , Glicosaminoglicanos/isolamento & purificação , Glicosaminoglicanos/farmacologia , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , Protrombina/antagonistas & inibidores , Sulfonamidas/farmacologiaRESUMO
A method for the preparation of heparan sulfate from peptidoglycan heparin is described. The objective of this research was to provide a basis for the development and validation of an industrial process to support the preclinical development of heparan sulfate and/or heparan sulfate derivatives. In the preparation of heparan sulfate, heparin was recovered by alcohol fractionation and dermatan sulfate was isolated by selective precipitation. The remaining crude heparan sulfate was fractionated by anion-exchange chromatography into five subfractions. The biological activities of these subfractions were examined by anticoagulant and amidolytic assays. Molecular weight and molecular size were determined using capillary viscometry and polyacrylamide gel electrophoresis. Charge density and degree of sulfation were determined by cellulose acetate electrophoresis and elemental analysis. Oligosaccharide and disaccharide analysis relied on enzymatic depolymerization using heparin lyases followed by polyacrylamide gel and capillary electrophoresis. 1H NMR analysis provided detailed structural information on each subfraction. Crude heparin sulfate and its subfractions showed significant differences in physical, structural and biological properties.
