Enzyme replacement therapy
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.
Please also check our Fabry disease webpage.
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.
Quinate metabolism and lignin formation in the loblolly pine
Quinic acid is one of the most abundant natural products in the biosphere. It is accumulated in some tissues of woody plants, e.g., newly forming needles of conifers, in amounts equaling up to 10% of the total dry weight. Quinic acid is exceedingly active as an intermediate which possesses alternative metabolic fates. In xylem-forming cells it is mainly transformed to phenylpropanoid precursors of lignin. In autotrophic cells, on the other hand, quinic acid is an effective precursor of other phenolic compounds - the most noteworthy ones being chlorogenic acid, gallic acid and protocatechuic acid. Chlorogenic acid and gallic acid confer resistance to herbivore predation and phytopathogenic microorganisms. Protocatechuic acid is catabolized to central intermediates and can serve as a carbon-source reserve during dark metabolism. Finally, quinic acid is a highly soluble readily transported metabolite that can serve as a versatile precursor of aromatic amino acids in different tissue compartments. Loblolly pine (Pinus taeda), our experimental system, is under intensive study by others in order to understand and manipulate lignin content. Therefore many biological and molecular-genetic resources are available to support our project, including a tissue-culture system that is lignin-inducible. In contrast to the intensity of ongoing research on the downstream metabolism to lignin, work on the nature of the interfacing upstream metabolism that delivers phenylalanine to the phenylpropanoid section has been minimal.
We propose to focus upon three quinate dehydrogenase proteins: NQDH-NADP and NQDH- NAD in needle tissue and XQDH-NADP in xylem-forming cells. NQDH-NADP appears to be trifunctional, having catalytic domains that carry out the overall conversion of quinate to protocatechuate. The enzymes will be purified, used to raise specific antibodies, and characterized for physical and catalytic properties. cDNA clones corresponding to each QDH will be obtained by immunoscreening. Specific antibody will be used for immunogold EM cytolocalization. Regulation will be examined by monitoring levels of enzyme activity, amounts of protein, and transcript abundance in response to developmental and environmental cues. The longterm objective is to understand how the entire metabolic network is differentially regulated to accomplish the dynamic alternative molecular fates of quinate. This is expected to provide ultimately a rational basis for biotechnological manipulations designed to alter flux toward and away from quinate in different cellular compartments and in different specialized tissues.
Hong, Y.L., P.A. Hossleer, D.H. Calhoun, and S.R. Meshnick (1995) Inhibition of recombinant Pneumocystis carinii dihydropteroate synthetase by sulfa drugs. Antimicrob. Agents Chemother. 39:1756-1763.
Lee, S.L, and Calhoun, D.H. (1997) Urease from a potentially pathogenic coccoid isolate: Purification, characterization, and comparison to other microbial ureases. Infect. Immun. 65: 3991-3996. (PDF)
Y. Chen, Ming Jin, M., Leo Goodrich, Gale Smith, George Coppola, and D. H. Calhoun (2000) Purification and characterization of human a-galactosidase A expressed in insect cells using a baculovirus vector. Protein Expression and Purification, 20: 228-236. (PDF)
Chen, Y, M. Jin, T. Egborge, G. Coppola, J. Andre, and D. H. Calhoun (2000) Expression and Characterization of Glycosylated and Catalytically Active Recombinant Human a-galactosidase A Produced in Pichia pastoris. Protein Expression and Purification, 20: 472-484. (PDF)
Lee, S.G. and D. H. Calhoun. (2000) Adhesin from potentially pathogenic coccoid isolate: Purification, characterization, and comparison to other microbial adhesins (in review)
Calhoun, D. H., C. A. Bonner, W. Gu, G. Xie and R.A. Jensen. (2001) The emerging periplasm-localized subclass of AroQ chorismate mutases, exemplified by those from Salmonella typhimurium and Pseudomonas aeruginosa, Genome Biology, 2: research0030:1-16. (PDF)
Jensen, R. A., G. Xie, D. H. Calhoun, and C. A. Bonner (2002) The correct phylogenetic relationship of KdsA (3-Deoxy-D-manno-octulosonate 8-phosphate synthase) with one of two independently evolved classes of AroA (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase), J. Mol. Evol. 54: 416-423. (PDF)
Hubbard, K., DeJesus, V., C. Davis, Y. Chen, D.H. Calhoun, and Z. Zakeri (2002) Cellular aging and lysosomal expression during cell death, Experimental Cell Research 274: 92-99.
Jin M, W. Rosario, E. Watler, and D. H. Calhoun. Development of a large-scale HPLC-based purification for the urease from Staphylococcus leei and determination of subunit structure. Protein Expr Purif. (2004) 34:111-117 (PDF)
Kalyankar, N. D., Sharma, M. K., Vaidya, S., Calhoun, D. H., Maldarelli, C, Couzis, A., and Gilchrist, -L. (2006) Arraying of Intact Liposomes Into Chemically Functionalized Microwell Surfaces, Langmuir, 22, 5403-5411. (PDF)