Mannose-6-Phosphate (Man6P) on the oligosaccharides of newly synthesized mammalian lysosomal enzymes is important for their targeting to the lysosome. Severe genetic lesions in its addition are fatal in humans. Two enzymes are needed to synthesize Man6P in mammalian cells. The first transfers GlcNAc-alpha-l-P to the 6-position of selected Man residues forming a phosphodiester (Man6P-l-GlcNAc), and the second is a phosphoglycosidase that cleaves GlcNAc to generate the phosphomonoester, Man6P. Although the enzymes were identified a dozen years ago, and in spite of considerable effort, neither of them has been purified nor have their genes been cloned. A better understanding of these enzymes and their genes could, in time, provide reagents for enzyme replacement therapy. The soil amoeba, Dictyostelium discoideum, also synthesizes lysosomal enzymes with Man6P residues by using the same pathway as mammalian cells. In addition, the stringent structural requirements for the acceptor oligosaccharide are highly conserved in Dictyostelium. Thus, it is very likely that the Dictyostelium genes share considerable homology with the mammalian genes. In this proposal, the Dictyostelium genes will be identified using a novel insertional mutagenesis approach. In this method, a selectable vector/plasmid randomly integrates into the genome and disrupts the gene. Transformants are screened using the mammalian Man6P receptor to isolate those that cannot bind to receptor because either of the two genes for Man6P biosynthesis has been disrupted. Enzyme activity assays and structural analysis of the oligosaccharides will distinguish which enzyme is missing in the transformants. Reisolation of the disrupting plasmid together with flanking regions of the disrupted gene will be used to target the vector/plasmid specifically into a new set of untransformed cells. Homologous recombination through the flanking regions of the target gene now drives the specific gene mutations. Loss of enzyme activity and attendant oligosaccharide structure confirms that the same gene was disrupted. The entire gene is sequenced and protein is expressed to make antibodies that can precipitate one of the enzyme activities to prove that the disruption occurred in the structural gene for that enzyme. Later, the sequenced Dictyostelium gene may be useful as a probe under reduced stringency to screen a mammalian cDNA library and identify the homologous mammalian genes.
