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

• Published in: Metabolic brain disease 2008-06, Vol.23 (2), p.161-173
• Main Authors: Samoylova, Tatiana I., Martin, Douglas R., Morrison, Nancy E., Hwang, Misako, Cochran, Anna M., Samoylov, Alexandre M., Baker, Henry J., Cox, Nancy R.
• Format: Article
• Language: English
• Subjects: Animals; Antibody Specificity - immunology; beta-Galactosidase - genetics; beta-Galactosidase - isolation & purification; beta-Galactosidase - secretion; Biochemistry; Biomedical and Life Sciences; Biomedicine; Cats; CHO Cells; Chromatography, Agarose; Cloning, Molecular - methods; Cricetinae; Cricetulus; Culture Media, Conditioned - chemistry; Disease Models, Animal; DNA, Complementary - genetics; G(M1) Ganglioside - biosynthesis; G(M1) Ganglioside - genetics; Gangliosidosis, GM1 - enzymology; Gangliosidosis, GM1 - genetics; Gangliosidosis, GM1 - therapy; Genetic Therapy - methods; Genetic Vectors - genetics; Metabolic Diseases; Molecular Weight; Neurology; Neurosciences; Oncology; Original Paper; Protein Structure, Tertiary - physiology; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - secretion; Transfection - methods
• ISSN: 0885-7490; 1573-7365
• DOI: 10.1007/s11011-008-9086-5

Lysosomal β-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 β-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 β-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 β-galactosidase cDNA.