title: Alternative splicing of beta-galactosidase mRNA generates the classic lysosomal enzyme and a beta-galactosidase-related protein.
abstract: 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.
authors.affiliation: Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands.
authors.forename: H
authors.initials: H
authors.lastname: Morreau
authors.forename: N J
authors.initials: NJ
authors.lastname: Galjart
authors.forename: N
authors.initials: N
authors.lastname: Gillemans
authors.forename: R
authors.initials: R
authors.lastname: Willemsen
authors.forename: G T
authors.initials: GT
authors.lastname: van der Horst
authors.forename: A
authors.initials: A
authors.lastname: d'Azzo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005091:Exons; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D005696:Galactosidases / Q000235:genetics*; D005733:Gangliosidoses / Q000201:enzymology / Q000235:genetics; D015723:Gene Library; D005796:Genes; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011506:Proteins / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics*; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D012867:Skin / Q000201:enzymology; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics*
chemical_list: D011506:Proteins; D012333:RNA, Messenger; D004247:DNA; D005696:Galactosidases; C061901:GLB1 protein, human; D001616:beta-Galactosidase