Synthesis of phosphorylated oligosaccharides in lysozyme is enhanced by fusion to cathepsin D.
Chinese hamster ovary cells transfected with human lysozyme cDNA encoding Asn instead of Gly22 synthesize a mutant lysozyme, [Asn22]lysozyme, with about 60% of the molecules bearing carbohydrate. This carbohydrate is predominantly of the complex type and contains a varied number of lactosamine repeats. In this study we show that the glycosylation of [Asn22] lysozyme fused to human cathepsin D is altered relative to [Asn22]lysozyme alone. The fusion protein is synthesized as a 66-kDa precursor that is cleaved to enzymatically active and antigenically positive cathepsin D and lysozyme. As compared with [Asn22]lysozyme the lysozyme moiety of the fusion protein shows an increased N-glycosylation and a decreased synthesis of lactosamine repeats. Cleavage of the precursor with cathepsin L has revealed that the lysozyme portion of the secreted fusion protein bears a complex type carbohydrate. The intracellularly released lysozyme portion of the fusion protein contains trimmed oligosaccharides. In the presence of NH4Cl the lysosomal targeting of the fusion protein is inhibited. The secreted protein is then enriched in molecules bearing phosphorylated high mannose oligosaccharides in their lysozyme moiety. Our results indicate that carbohydrate processing in [Asn22]lysozyme, including the synthesis of mannose 6-phosphate residues and of lactosamine repeats, is altered by the attached cathepsin D. The phosphorylation of the carbohydrate on the lysozyme portion results in a very efficient lysosomal targeting of the concerned fusion protein molecules.
Horst
M
M
Institute of Physiological Chemistry, Westfälische Wilhelms Universität, Münster, Federal Republic of Germany.
Mares
M
M
Zabe
M
M
Hummel
M
M
Wiederanders
B
B
Kirschke
H
H
Hasilik
A
A
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002402:Cathepsin D / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009113:Muramidase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D009844:Oligosaccharides / Q000096:biosynthesis*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
D006023:Glycoproteins; D009838:Oligodeoxyribonucleotides; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D009113:Muramidase; D002402:Cathepsin D