Dr. David Calhoun

Enzyme replacement therapy for Fabry disease patients

Mutations in the a-galactosidase A gene result in the sphingolipidosis called Fabry disease. The enzymatic defect is inherited as an X-linked recessive disorder and is associated with a progressive deposition of the glycosphingolipids, including globotriaosylceramide, galabioasylceramide, and blood group B substance. In affected males this leads to early death due to occlusive disease of the heart, kidney, and brain. Attempts have been made in vivo and in vitro to replace the defective enzyme with normal enzyme obtained from various human sources. The lack of sufficient quantities of purified human a-galactosidase A has prevented a complete evaluation of the potential efficacy of enzyme replacement in Fabry disease. De Duve first suggested that enzyme replacement therapy might be a successful approach to the treatment of lysosomal storage defects such as Gaucher’s and Fabry disease. For Gaucher’s disease enzyme therapy produced unequivocal clinical responses that were subsequently confirmed by others. These first trials used enzyme derived from human placenta, and recombinant enzyme has since been made available. The successful use of enzyme therapy for Gaucher patients provides a strong precedent for this approach for Fabry disease. In order to obtain large quantities of this human enzyme for physical characterization and for the development of new approaches for enzyme therapy, we previously constructed baculovirus derivatives that produce the human enzyme. The recombinant a-galactosidase A is stable, is produced at high levels, and is active with both the artificial substrate, 4-methylumbelliferyl-a-D-galactopyranoside, and the natural in vivo substrate, trihexosylceramide. The purified recombinant a-galactosidase A purified from the culture supernatant is glycosylated and is taken up by normal and Fabry fibroblasts in cell culture. We have recently expressed the human a-galactosidase A in the methylotrophic yeast, Pichia pastoris, which is expected to produce substantially higher enzyme levels.


Potentially Pathogenic Gastric Coccus
A Gram positive coccoid strain was recently isolated from biopsy material obtained from patients in Korea suffering from gastritis. This organism is a novel stomach adapted Staphylococcus species most closely related to S. cohnii or S. xylosus. It possesses an adhesin specific for gastric mucin. We recently purified and characterized the urease from the coccus and found that it is very similar to the urease of the stomach adapted pathogen, Helicobacter pylori, a Gram negative curved bacillary organism. The urease of H. pylori is a virulence factor essential for colonization of the stomach, and is the basis for a widely used diagnostic ELISA In preliminary experiments we recently found that 20% of a randomly selected group of ulcer patients in the New York City area (who are not infected with H. pylori) have high titers of antibodies that react specifically in an ELISA with the urease purified from the coccus. All control sera from normal individuals screened to date were negative in this ELISA with the urease from the coccus. One major goal of our current work is to increase the number of sera from patients and controls, and to correlate this data with other criteria, including results from biopsy.




Dr. David Calhoun
Department of Chemistry
Tel: (212) 650-6934 (off), -6936, -6001 (lab)
Fax: (212) 650-7974