A NOVEL MUTAGENESIS STRATEGY IDENTIFIES DISTANTLY SPACED AMINO-ACID-SEQUENCES THAT ARE REQUIRED FOR THE PHOSPHORYLATION OF BOTH THE OLIGOSACCHARIDES OF PROCATHEPSIN-D BY N-ACETYLGLUCOSAMINE 1-PHOSPHOTRANSFERASE

A novel combinatorial mutagenesis strategy (shuffle mutagenesis) was developed to identify sequences in the propiece and amino lobe of cathepsin D which direct oligosaccharide phosphorylation by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine l-phosphotransferase. Propiece restriction fragments and oligonucleotide cassettes corresponding to 13 regions of the cathepsin D and glyco pepsinogen amino lobes were randomly shuffled together to generate a large library of chimeric molecules, The library was inserted into an expression vector encoding the carboxyl lobe of cathepsin D with a carboxyl-terminal myc epitope and a CD8 transmembrane extension. Transfected COS1 cells expressing the membrane-anchored forms of the cathepsin D/glycopepsinogen chimeras at the cell surface were selected with solid phase mannose 6-phosphate receptor or an antibody to the myc epitope, Plasmids were rescued in Escherichia coli and sequenced by hybridization to the original oligonucleotide cassettes. Two regions of the cathepsin D amino lobe (segments 7 and 12) were found to contribute to proper folding, surface expression, and selective phosphorylation of the carboxyl lobe oligosaccharide. Two different cathepsin D regions (the propiece and segment 5) cooperated with a previously identified recognition element in the carboxyl lobe to allow efficient phosphorylation of both the amino and carboxyl lobe oligosaccharides. Three general models for extending the catalytic reach of N-acetylglucosamine l-phosphotransferase to widely spaced oligosaccharides are presented.