GM1-gangliosidosis Fibroblasts Research Articles

Kinetic mechanism and characterization of human β-galactosidase precursor secreted by permanently transfected Chinese hamster ovary cells

Chinese hamster ovary cell clones permanently transfected with the cDNA for human lysosomal beta-galactosidase secrete the enzyme precursor into the cell medium, from which it is purified to apparent homogeneity in a single step by affinity chromatography. The purified precursor is fully active, displays the same pH optimum and Km values as the mature placental enzyme, and has an intact C-terminus. The intact enzyme when chromatographed on a Sephacryl S-200 molecular-sieve column elutes as a 105,500 Da monomer, whereas on SDS/PAGE gels the polypeptide migrates as an 88 kDa polypeptide. A time course of digestion with glycopeptide-N-glycanase shows the gradual conversion of the precursor from an 88 to a 72 kDa protein, suggesting the presence of five N-linked oligosaccharides in the protein. The precursor is readily taken up in a mannose-6-phosphate-dependent manner into beta-galactosidase-deficient, GM1-gangliosidosis fibroblasts, and the enzyme activity is returned to normal levels. We show that the stereochemical course of enzymic hydrolysis involves the retention of the beta-configuration at the anomeric centre, suggesting a double-displacement mechanism. Furthermore, the enzyme is rapidly and irreversibly inactivated in the presence of the mechanism-based inactivator 2,4-dinitrophenyl-2-deoxy-2-fluoro-beta-D-galactopyranoside, which implicates a covalent intermediate. The enzyme is also inactivated by 1-ethyl-3(3-dimethylamino-propyl)carbodi-imide and by phenylglyoxal, which implicates carboxylate and arginine residues respectively in the active site. We conclude that the beta-galactosidase precursor is functionally identical to the mature lysosomal form of the enzyme and serves as an excellent enzyme source for investigation of structure-function relationships in the protein.

Alternative splicing of β-galactosidase mRNA generates the classic lysosomal enzyme and a β-galactosidase-related protein

We have isolated two cDNAs encoding human lysosomal beta-galactosidase, the enzyme deficient in GM1-gangliosidosis and Morquio B syndrome, and a beta-galactosidase-related protein. In total RNA from normal fibroblasts a major mRNA of about 2.5 kilobases (kb) is recognized by cDNA probes. A minor transcript of about 2.0 kb is visible only in immunoselected polysomal RNA. A heterogeneous pattern of expression of the 2.5-kb beta-galactosidase transcript is observed in fibroblasts from different GM1-gangliosidosis patients. The nucleotide sequences of the two cDNAs are extensively colinear. However, the short cDNA misses two noncontiguous protein-encoding regions (1 and 2) present in the long cDNA. The exclusion of region 1 in the short molecule introduces a frameshift in its 3'-flanking sequence, which is restored by the exclusion of region 2. These findings imply the existence of two mRNA templates, which are read in a different frame only in the nucleotide stretch between regions 1 and 2. Sequence analysis of genomic exons of the beta-galactosidase gene shows that the short mRNA is generated by alternative splicing. The long and short cDNAs direct the synthesis in COS-1 cells of beta-galactosidase polypeptides of 85 and 68 kDa, respectively. Only the long protein is catalytically active under the assay conditions used, and it is capable of correcting beta-galactosidase activity after endocytosis by GM1-gangliosidosis fibroblasts. The subcellular localization of cDNA-encoded beta-galactosidase and beta-galactosidase-related proteins is different.

Acid beta-galactosidase from human fibroblasts. A microscale purification method monitored by a highly sensitive enzyme assay.

A highly sensitive microassay method and a microscale purification system were developed to isolate the residual acid beta-galactosidase in GM1-gangliosidosis fibroblasts. The sensitivity of the microassay system, composed of a 96-well microplate and a microplate fluorometer, was 100-fold higher than that of the conventional system and the response was linear in the pmole range. Acid beta-galactosidase was characterized as a thiol enzyme which was inactivated by a mercuric compound. This enzyme was completely adsorbed on an Hg-agarose column and was easily eluted from the column by 10 mM 2-mercaptoethanol. The microscale purification system using Con A-Sepharose, PAT-Sepharose, and Hg-agarose column chromatography achieved 565- and 7,970-fold purifications of acid beta-galactosidase with an overall yields of 44% and 45% from normal and GM1-gangliosidosis fibroblasts, respectively. The purified enzyme fractions did not contain any other lysosomal enzyme activities except for a small amount of beta-N-acetylhexosaminidase activity.

GM1-Gangliosidosis: a molecular abnormality of acid beta-galactosidase in fibroblasts.

G~l-Gangliosidosis is an inherited lysosomal disease with a deficiency of acid ~-gatactosidase activity causing generalized accumulation of ganglioside GM~ in somatic cells (Okada and O'Brien, 1968). The molecular structure of [3-galactosidase A was reported to be altered in this disease since liver samples from patients contained more than normal quantities of cross-reacting materials to antinormal liver [3-galactosidase A antibodies (Meisler and Rattazzi, 1974; O'Brien, 1975; BenYoseph et al., 1977). Recently, acid [3-galactosidase from human placenta was found to have highly reactive sulphydryl groups in the molecule closely associated with the enzyme activity (Furuya et al., unpublished data). Subsequently, a purification procedure was developed with Hg-agarose affinity chromatography utilizing a specific affinity to sulphydryl groups of this enzyme molecule. In this report acid J3-galactosidase was studied in GM1gangliosidosis fibroblasts with regard to -SH specificity for the investigation of molecular abnormality in this disease.

Enzyme replacement using liposome carriers in feline Gm1 gangliosidosis fibroblasts.

CURRENT emphasis in research on lysosomal storage diseases is directed towards enzyme replacement therapy (ERT) for these inherited enzyme deficiencies1–3. The rationale for ERT is based on the assumption that endocytosed enzyme would enter lysosomes and function in the degradation of accumulated substrates. However, inactivation of enzyme, immunological reactions and low cellular uptake have prevented successful application of this promising approach to therapy. Circumvention of these limitations requires the use of carriers which protect and stabilise replacement enzyme while enhancing uptake and subcellular distribution2–5. We report here the use of lipid vesicles (liposomes)6–9 as carriers for replacement enzyme in feline GM1 gangliosidosis fibroblasts which have deficient β-galactosidase (β-gal) activity and lysosomal storage of glycopeptides9–11. Our results demonstrate increased β-gal activity and corresponding catabolism of stored glycopeptides in feline GM1 gangliosidosis fibroblasts treated with liposomes containing β-gal.