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 d eveloped to identify sequences in the propiece and amino lobe of cathe psin D which direct oligosaccharide phosphorylation by UDP-GlcNAc:lyso somal enzyme N-acetylglucosamine l-phosphotransferase. Propiece restri ction fragments and oligonucleotide cassettes corresponding to 13 regi ons of the cathepsin D and glyco pepsinogen amino lobes were randomly shuffled together to generate a large library of chimeric molecules, T he library was inserted into an expression vector encoding the carboxy l lobe of cathepsin D with a carboxyl-terminal myc epitope and a CD8 t ransmembrane extension. Transfected COS1 cells expressing the membrane -anchored forms of the cathepsin D/glycopepsinogen chimeras at the cel l surface were selected with solid phase mannose 6-phosphate receptor or an antibody to the myc epitope, Plasmids were rescued in Escherichi a coli and sequenced by hybridization to the original oligonucleotide cassettes. Two regions of the cathepsin D amino lobe (segments 7 and 1 2) were found to contribute to proper folding, surface expression, and selective phosphorylation of the carboxyl lobe oligosaccharide. Two d ifferent cathepsin D regions (the propiece and segment 5) cooperated w ith a previously identified recognition element in the carboxyl lobe t o allow efficient phosphorylation of both the amino and carboxyl lobe oligosaccharides. Three general models for extending the catalytic rea ch of N-acetylglucosamine l-phosphotransferase to widely spaced oligos accharides are presented.