title: 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.affiliation: Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn, AL 36849, USA. samoiti@vetmed.auburn.edu
authors.forename: Tatiana I
authors.initials: TI
authors.lastname: Samoylova
authors.forename: Douglas R
authors.initials: DR
authors.lastname: Martin
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Morrison
authors.forename: Misako
authors.initials: M
authors.lastname: Hwang
authors.forename: Anna M
authors.initials: AM
authors.lastname: Cochran
authors.forename: Alexandre M
authors.initials: AM
authors.lastname: Samoylov
authors.forename: Henry J
authors.initials: HJ
authors.lastname: Baker
authors.forename: Nancy R
authors.initials: NR
authors.lastname: Cox
mesh_terms: D000818:Animals; D000918:Antibody Specificity / Q000276:immunology; D016466:CHO Cells; D002415:Cats; D002847:Chromatography, Agarose; D003001:Cloning, Molecular / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D017077:Culture Media, Conditioned / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004195:Disease Models, Animal; D005677:G(M1) Ganglioside / Q000096:biosynthesis* / Q000235:genetics; D016537:Gangliosidosis, GM1 / Q000201:enzymology* / Q000235:genetics / Q000628:therapy; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D008970:Molecular Weight; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014162:Transfection / Q000379:methods; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D011994:Recombinant Proteins; D005677:G(M1) Ganglioside; D001616:beta-Galactosidase