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

• Published in: The Journal of biological chemistry Vol. 264; no. 34; pp. 20655 - 20663
• Main Authors: Morreau, H, Galjart, N.J., Gillemans, N, Willemsen, R, van der Horst, G.T., d'Azzo, A
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 05-12-1989; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Base Sequence; beta -galactosidase; beta-Galactosidase - genetics; Cell Line; Cells, Cultured; Cloning, Molecular; DNA - genetics; Exact sciences and technology; Exons; Female; fibroblasts; Fibroblasts - enzymology; Galactosidases - genetics; Gangliosidoses - enzymology; Gangliosidoses - genetics; Gene Library; Genes; Humans; lysosomes; Lysosomes - enzymology; Mathematical analysis; Mathematics; Molecular Sequence Data; mRNA; Placenta - enzymology; Potential theory; Pregnancy; Proteins - genetics; Restriction Mapping; RNA Splicing; RNA, Messenger - genetics; Sciences and techniques of general use; Sequence Homology, Nucleic Acid; Skin - enzymology; Transfection; Complementary DNA; Nucleotide sequence; Enzyme; Morquio disease; Lysosome; Gene library; β-D-Galactosidase; GM1 gangliosidosis; Aminoacid sequence
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)47114-7

We have isolated two cDNAs encoding human lysosomal β-galactosidase, the enzyme deficient in GM1-gangliosidosis and Morquio B syndrome, and a β-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 β-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 β-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 β-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 β-galactosidase activity after endocytosis by GM1-gangliosidosis fibroblasts. The subcellular localization of cDNA-encoded β-galactosidase and β-galactosidase-related proteins is different.