Cloning and expression of the gene involved in Sanfilippo B syndrome (mucopolysaccharidosis III B)

Sanfilippo B syndrome is caused by a deficiency of alpha-N- acetylglucosaminidase, a lysosomal enzyme involved in the degradation of heparan sulphate. Accumulation of the substrate in lysosomes results in degeneration of the central nervous system with progressive dementia often combined with hyperactivity and aggressive behaviour. In order to clone the deficient gene, we purified the enzyme from human placenta and obtained amino acid sequence information. Alignment of one of the CNBr generated internal peptides to sequence from the database revealed the chromosomal location of the gene in the 5' upstream flanking region of the gene for 17-beta-hydroxysteroid-dehydrogenase at 17q21.1. The available DNA sequence was used to clone the cDNA coding for alpha-N- acetylglucosaminidase and analyse its gene structure. The gene is fully contained in the 5' upstream flanking region of the gene for 17-beta- hydroxysteroid-dehydrogenase and interrupted by five introns. The cDNA clone has a length of 2575 bp and encodes a protein of 743 amino acids. Chinese hamster ovary cells transfected with the cDNA construct show alpha-N-acetylglucosaminidase activity about 17-fold over background. This will allow correction studies with NAG deficient Sanfilippo B cell lines and facilitate the development of enzyme replacement therapy for these patients.      

A reversible monoamine oxidase inhibitor, toloxatone: Structural and electronic properties

Toloxatone is a reversible MAO_A-inhibitor, marketed as antidepressant (Humoryl^®), with an original chemical structure. It differs from first generation irreversible MAOIs, known to induce covalent bonds with the enzyme active site. In order to understand the mechanism of the reversible inactivation of the MAO, as a first step, a detailed structural and electronic analysis was undertaken. An X-ray diffraction-crystallographic study showed that toloxatone is a planar molecule and brought to light hydrogen bonds and π-π interactions. MO calculations confirmed the planar structure as energetically favoured. Electronic analysis demonstrated a delocalization of both ring systems. The combined results give evidence for the potential of toloxatone to participate in reversible, long distance interactions with an appropriate partner.