epidermal-growth-factor and Homocystinuria

Homocystinuria is an inborn error of methionine metabolism that results in raised serum levels of the highly reactive thiol-containing amino acid homocysteine. Homocystinurics often exhibit phenotypic abnormalities that are similar to those found in Marfan syndrome (MFS), a heritable connective tissue disorder that is caused by reduced levels of, or defects in, the cysteine-rich extracellular matrix (ECM) protein fibrillin-1. The phenotypic similarities between homocystinuria and MFS suggest that elevated homocysteine levels may result in an altered function of fibrillin-1. We have used recombinant calcium binding epidermal growth factor-like (cbEGF) domain fragments from fibrillin-1, and an unrelated protein Notch1, to analyse the effects of homocysteine on the native disulphide (cystine) bonds of these domains. We show using analytical reverse phase, high performance liquid chromatography (HPLC), electrospray ionisation mass spectrometry (ESI-MS) and limited proteolysis that homocysteine attacks intramolecular disulphide bonds causing reduction of cystine and domain misfolding, and that the effects of homocysteine are dependent on its concentration. We also identify the importance of calcium binding to cbEGF domains for their stabilisation and protection against homocysteine attack. Collectively, these data suggest that reduction of intramolecular cbEGF domain disulphide bonds by homocysteine and the resulting disruption of this domain fold may contribute to the change in connective tissue function seen in homocystinuria. Furthermore, since we show that the effects of homocysteine are not unique to fibrillin-1, other cbEGF-containing proteins may be implicated in the pathogenic mechanisms underlying homocystinuria.

Topics: Calcium; Cystathionine beta-Synthase; Epidermal Growth Factor; Fibrillin-1; Fibrillins; Homocysteine; Homocystinuria; Humans; In Vitro Techniques; Marfan Syndrome; Microfilament Proteins; Models, Molecular; Oxidation-Reduction; Peptide Fragments; Protein Folding; Protein Structure, Tertiary; Receptor, Notch1; Receptors, Cell Surface; Recombinant Proteins; Spectrometry, Mass, Electrospray Ionization; Transcription Factors; Trypsin

Homocystinuria, a disorder originating in defects in the methionine metabolism, is characterized by an elevated plasma concentration of homocysteine. Most patients have a defect in the cystathionine-beta-synthase, the key enzyme in the conversion of homocysteine to cysteine. Many abnormalities in the connective tissue of patients with homocystinuria resemble those seen in Marfan syndrome, caused by mutations in fibrillin-1. These observations led to the hypothesis that the structure and function of fibrillin-1 is compromised in patients with homocystinuria. To test this hypothesis we produced recombinant human fibrillin-1 fragments spanning the central portion of the molecule (8-Cys/transforming growth factor-beta binding domain 3 to calcium binding EGF domain 22) and extensively analyzed the potential of homocysteine to modify structural and functional properties of these proteins. Circular dichroism spectroscopy revealed moderate changes of their secondary structures after incubation with homocysteine. Equilibrium dialysis demonstrated a number of high affinity calcium binding sites in the tandemly repeated calcium binding epidermal growth factor-like domains 11-22. Calcium binding of homocysteine-modified fragments was completely abolished. Incubation of the recombinant proteins with homocysteine rendered the analyzed calcium binding EGF domains as well as the 8-Cys/transforming growth factor-beta binding domain 3 significantly more susceptible to proteolytic degradation. Furthermore, data were obtained demonstrating that homocysteine can covalently modify fibrillin-1 via disulfide bonds. These data strongly suggest that structural and functional modifications as well as degradation processes of fibrillin-1 in the connective tissues of patients with homocystinuria play a major role in the pathogenesis of this disorder.

Topics: Amino Acid Sequence; Calcium; Cells, Cultured; Chymotrypsin; Circular Dichroism; Dose-Response Relationship, Drug; Epidermal Growth Factor; Extracellular Matrix; Fibrillin-1; Fibrillins; Fibroblasts; Glycosylation; Homocysteine; Homocystinuria; Humans; Marfan Syndrome; Microfilament Proteins; Microscopy, Fluorescence; Molecular Sequence Data; Peptides; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Sequence Homology, Amino Acid; Transfection; Trypsin

Cystathionine beta-synthase (CBS; EC 4.2.1.22) is a key enzyme in the generation of cysteine from methionine. A deficiency of CBS leads to homocystinuria, an inherited human disease characterized by mental retardation, seizures, psychiatric disturbances, skeletal abnormalities, and vascular disorders; however, the underlying mechanisms remain largely unknown. Here, we show the regional and cellular distribution of CBS in the adult and developing mouse brain. In the adult mouse brain, CBS was expressed ubiquitously, but it is expressed most intensely in the cerebellar molecular layer and hippocampal dentate gyrus. Immunohistochemical analysis revealed that CBS is preferentially expressed in cerebellar Bergmann glia and in astrocytes throughout the brain. At early developmental stages, CBS was expressed in neuroepithelial cells in the ventricular zone, but its expression changed to radial glial cells and then to astrocytes during the late embryonic and neonatal periods. CBS was most highly expressed in juvenile brain, and a striking induction was observed in cultured astrocytes in response to EGF, TGF-alpha, cAMP, and dexamethasone. Moreover, CBS was significantly accumulated in reactive astrocytes in the hippocampus after kainic acid-induced seizures, and cerebellar morphological abnormalities were observed in CBS-deficient mice. Taken together, these results suggest that CBS plays a crucial role in the development and maintenance of the CNS and that radial glia/astrocyte dysfunction might be involved in the complex neuropathological features associated with abnormal homocysteine metabolism.

Topics: Animals; Astrocytes; Brain; Bromodeoxyuridine; Cell Lineage; Central Nervous System; Cerebellum; Cerebral Cortex; Corpus Callosum; Cyclic AMP; Cystathionine beta-Synthase; Dexamethasone; Epidermal Growth Factor; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Glucocorticoids; Heterozygote; Hippocampus; Homocysteine; Homocystinuria; Immunoblotting; Immunohistochemistry; In Situ Hybridization; Kainic Acid; Ligands; Methionine; Mice; Mice, Transgenic; Microscopy, Fluorescence; Models, Biological; Neuroglia; Olfactory Bulb; Oxidative Stress; Transforming Growth Factor alpha; Up-Regulation