Generation and characterization of recombinant feline beta-galactosidase for preclinical enzyme replacement therapy studies in GM1 gangliosidosis.

Abstract

Lysosomal beta-galactosidase is required for the degradation of GM1 ganglioside and other glycolipids and glycoproteins with a terminal galactose moiety. Deficiency of this enzyme leads to the lysosomal storage disorder, GM1 gangliosidosis, marked by severe neurodegeneration resulting in premature death. As a step towards preclinical studies for enzyme replacement therapy in an animal model of GM1 gangliosidosis, a feline beta-galactosidase cDNA was cloned into a mammalian expression vector and subsequently expressed in Chinese hamster ovary (CHO-K1) cells. The enzyme secreted into culture medium exhibited specific activity on two synthetic substrates as well as on the native beta-galactosidase substrate, GM1 ganglioside. The enzyme was purified from transfected CHO-K1 cell culture medium by chromatography on PATG-agarose. The affinity-purified enzyme preparation consisted mainly of the protein with approximate molecular weight of 94 kDa and displayed immunoreactivity with antibodies raised against a 16-mer synthetic peptide corresponding to C-terminal amino acid sequence deduced from the feline beta-galactosidase cDNA.

• Authors: Tatiana I Samoylova, Douglas R Martin, Nancy E Morrison, Misako Hwang, Anna M Cochran, Alexandre M Samoylov, Henry J Baker, Nancy R Cox
• Journal: Metabolic brain disease (Metab Brain Dis) Vol. 23 Issue 2 Pg. 161-73 (Jun 2008) ISSN: 0885-7490 [Print] United States
• PMID: 18421424 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Culture Media, Conditioned
• DNA, Complementary
• Recombinant Proteins
• G(M1) Ganglioside
• beta-Galactosidase

Topics

• Animals
• Antibody Specificity (immunology)
• CHO Cells
• Cats
• Chromatography, Agarose
• Cloning, Molecular (methods)
• Cricetinae
• Cricetulus
• Culture Media, Conditioned (chemistry)
• DNA, Complementary (genetics)
• Disease Models, Animal
• G(M1) Ganglioside (biosynthesis, genetics)
• Gangliosidosis, GM1 (enzymology, genetics, therapy)
• Genetic Therapy (methods)
• Genetic Vectors (genetics)
• Molecular Weight
• Protein Structure, Tertiary (physiology)
• Recombinant Proteins (genetics, isolation & purification, metabolism)
• Transfection (methods)
• beta-Galactosidase (genetics, isolation & purification, metabolism)