Immortalization and characterization of a cell line exhibiting a severe multiple sulphatase deficiency phenotype.

Abstract

Multiple sulphatase deficiency (MSD) is a rare genetic defect that causes a simultaneous deficiency of all known sulphatases. All available evidence suggests that the deficient gene product is normally responsible for the post-translational modification of a conserved cysteine residue to 2-amino-3-oxopropionic acid and that this modification is necessary for sulphatase activity. MSD often has an enzymically mild phenotype, with significant levels of residual sulphatase activity being detectable. Here we identify an MSD cell line in which the residual activity of the sulphatases assayed was generally very low. To characterize the phenotype of this cell line further, immortalized lines were established after transformation with simian virus 40 (SV40) T antigen. Immortalized cell lines representing normal and MSD phenotypes were then transduced with a retroviral vector carrying the gene encoding human N-acetylgalactosamine-4-sulphatase. Analysis of N-acetylgalactosamine-4-sulphatase protein synthesis and enzyme activity showed that transduced cell lines expressed large amounts of enzyme and that the specific activity of this enzyme was approx. 0.5-1.5% of normal, confirming that this cell line defines a severe phenotype for MSD. N-Acetylgalactosamine-4-sulphatase purified from a transduced MSD cell line seemed normal on denaturing PAGE. Kinetic analysis of the purified enzyme suggests that the residual activity is due to small amounts of normal enzyme rather than unmodified enzyme with low levels of residual activity. These cell lines and the availability of large amounts of inactive N-acetylgalactosamine-4-sulphatase from MSD cells should facilitate the further study of this disorder.