Structural and functional analysis of lysosomal ss-galactosidase and its relation to the protective protein.

Lysosomal B-galactosidase is the glycosidase, that cleaves B-linked galactosyl mmenes from a variety of natural and synthetic substrates. In normal tissues of various species this enzyme appears to associate with two other hydrolases, N-acetyl-o:-neuraminidase and the protective protein. Mutations at the B-galactosidase locus on chromosome 3 are the basis of two lysosomal storage disorders, GMcgangliosidosis and Morquio B syndrome. The scope of our experimental work is to gain more insights into the structural and functional properties of these proteins, their mutual relation in lysosomes and their involvement in genetic disorders. Knowledge on the primary structure of lysosomal protective protein has recently brought to the identification of its multifunctional activities. This thesis deals with the isolation and characterization of the cDNAs encoding the classic lysosomal B-galactosidase and a £-galactosidase-related protein. The latter is derived from alternatively spliced pre-m.RNA, as deduced from analysis of the genomic organization of the £-galactosidase gene. These studies include the identification of the £-galactosidase promoter region. In order to introduce this part of the experimental work, the thesis starts with an overview of what is known to date about promoter elements of genes encoding lysosomal enzymes and their putative mode of regulation. The occurrence of alternative splicing among pre-mRNAs coding for other lysosomal enzymes is briefly reviewed. The thesis continues with the description of the genetic lesions found in two patients with the severe infantile type of GMcgangliosidosis. Curiously this mutation, affecting both alleles in the patients, impairs correct splicing of premRNA molecules. The final part of the thesis describes on one hand the two separable roles of the protective protein: a protective function towards £-galactosidase and neuraminidase and a catalytic activity resembling that of lysosomal cathepsin A On the other hand it gives new meaning to the association of £-galactosidase and protective protein, that seems to be important for the correct transport of £galactosidase out of the endoplasmic reticulum and to the lysosomes.