Human cystathionine β-synthase cDNA: sequence, alternative splicing and expression in cultured cells

Abstract

Cystathionine beta-synthase (CBS) deficiency is the major cause of homocystinuria in humans. The most frequent symptoms of homocystinuria include: dislocated optic lenses, vascular disorders, skeletal abnormalities and mental retardation. Patients with this deficiency have elevated levels of homocyst(e)ine, methionine and low cysteine in their body fluids. These abnormal levels often partially or fully normalize upon treatment with pharmacological doses of vitamin B6. To investigate the molecular and biochemical basis for these conditions, it was necessary to determine the nucleotide and polypeptide sequence of CBS. We report here the human CBS cDNA sequence of 2,554 nucleotides encoding the CBS subunit of 551 amino acids. An intron of 214 bp appears to be retained in the 3'-untranslated region of most of the fibroblast and liver mRNA. We also report a frequent Mspl polymorphism in the 3'-untranslated sequence and two synonymous mutations in the coding region: 699C/T (Y233Y) and 1080C/T (A360A). The amino acid sequence similarity of human and rat CBS is greater than 90%; the enzyme also exhibits 52% similarity to O-acetylserine(thiol)-lyase from bacteria and plants. Lastly, we demonstrate that expression of the human enzyme in CHO cells yields enzymatically active protein of the expected size with a half-life of approximately 14 hrs.

Yeast Cystathionine β-Synthase Is a Pyridoxal Phosphate Enzyme but, Unlike the Human Enzyme, Is Not a Heme Protein

“…Removal of the PLP resulted in an apoenzyme having no absorbance in the visible range. 4 In contrast, the absorption spectra of rat and human CBS (10 -12, 23-26) exhibit a visible absorption band at 428 nm, which is approximately equal in intensity to the 280 nm absorption band. The 428 nm band in rat and human CBS is attributed to the presence of both heme and PLP, which have overlapping visible absorption spectra in the 410 -430 nm region.…”

“…Work is in progress to investigate the reaction kinetics, domain composition, oligomeric structure, and substrate and nucleophile specificity of yeast CBS. 4 The absence of heme in the functional yeast enzyme shows that heme is not essential for catalysis and suggests that heme does not play an essential catalytic role in the rat and human enzymes. The results are consistent with the absence of heme in the closely related enzymes O-acetylserine sulfhydrylase, threonine deaminase, and tryptophan synthase.…”

“…4 CBS activity was determined by a modification of a standard method (20). The reaction mixture, which contained 200 mM Tris-HCl, pH 8.6, 20 M PLP, 0.25 mg/ml bovine serum albumin, 5 mM L-[U-…”

“…The deduced sequences of human (4,5), rat (6), and Saccharomyces cerevisiae (yeast) (7,8) CBS are similar. The finding that human CBS complements the cysteine auxotrophy of a yeast strain lacking CBS (5) demonstrates the functional conservation of the human and yeast genes.…”

Defective cystathionine beta-synthase regulation by S-adenosylmethionine in a partially pyridoxine responsive homocystinuria patient.

“…The mutations described so far, are located in the catalytic domain of the protein (amino acids 1 to 418; J.P. Kraus, unpublished results), with some clusters of mutations in exon 3 and 8 (10). No mutations have been reported in the regulatory domain of this protein.…”