SYNTHESIS OF PHOSPHORYLATED OLIGOSACCHARIDES IN LYSOZYME IS ENHANCED BY FUSION TO CATHEPSIN-D

Chinese hamster ovary cells transfected with human lysozyme cDNA encod ing Asn instead of Gly22 synthesize a mutant lysozyme, [Asn22]lysozyme , with about 60% of the molecules bearing carbohydrate. This carbohydr ate is predominantly of the complex type and contains a varied number of lactosamine repeats. In this study we show that the glycosylation o f [Asn22] lysozyme fused to human cathepsin D is altered relative to [ Asn22]lysozyme alone. The fusion protein is synthesized as a 66-kDa pr ecursor that is cleaved to enzymatically active and antigenically posi tive cathepsin D and lysozyme. As compared with [Asn22]lysozyme the ly sozyme moiety of the fusion protein shows an increased N-glycosylation and a decreased synthesis of lactosamine repeats. Cleavage of the pre cursor with cathepsin L has revealed that the lysozyme portion of the secreted fusion protein bears a complex type carbohydrate. The intrace llularly released lysozyme portion of the fusion protein contains trim med oligosaccharides. In the presence of NH4Cl the lysosomal targeting of the fusion protein is inhibited. The secreted protein is then enri ched in molecules bearing phosphorylated high mannose oligosaccharides in their lysozyme moiety. Our results indicate that carbohydrate proc essing in [Asn22]lysozyme, including the synthesis of mannose 6-phosph ate residues and of lactosamine repeats, is altered by the attached ca thepsin D. The phosphorylation of the carbohydrate on the lysozyme por tion results in a very efficient lysosomal targeting of the concerned fusion protein molecules.