Expression of the yeast aspartyl protease, proteinase A. Phosphorylation and binding to the mannose 6-phosphate receptor are altered by addition of cathepsin D sequences.
Proteinase A, a yeast aspartyl protease that is highly homologous to the mammalian lysosomal aspartyl protease, cathepsin D, was expressed in Xenopus oocytes and its biosynthesis and post-translational modifications were characterized. While 29-45% of the proteinase A was secreted from oocytes, approximately 37% of the cell-associated proteinase A underwent proteolytic cleavage, characteristic of delivery to a lysosomal organelle. Although proteinase A is not targeted to the yeast vacuole by a mannose 6-phosphate receptor-dependent pathway, 2-5% of the proteinase A molecules expressed in oocytes bound to a Man-6-P receptor column. However, analysis of its [2-3H]mannose-labeled oligosaccharides revealed that 14-23% of these units contain phosphomannosyl residues. A hybrid molecule (H6), in which the propiece and first 12 amino acids of proteinase A were changed to the cathepsin D sequence, was also expressed in oocytes. The binding of H6 to the Man-6-P receptor was approximately 12-fold greater than observed for proteinase A. This increased level of receptor binding could be accounted for by three factors: 1) a small increase in the total amount of phosphorylated oligosaccharides, 2) an increase in the number of oligosaccharides which acquire two phosphomonoesters, and 3) the presence of a greater percentage of oligosaccharides with one phosphomonoester which exhibit high affinity binding to the Man-6-P receptor. These results demonstrate that proteinase A is recognized by UDP-GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase. However, this interaction is altered by the addition of cathepsin D sequences, resulting in the generation of a higher affinity ligand for binding to the Man-6-P receptor.
Faust
P L
PL
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Kornfeld
S
S
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D002352:Carrier Proteins / Q000378:metabolism*; D002402:Cathepsin D / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006801:Humans; D008360:Mannosephosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009865:Oocytes / Q000201:enzymology*; D010766:Phosphorylation; D017527:Receptor, IGF Type 2; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013552:Swine; D014981:Xenopus
D002352:Carrier Proteins; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D029701:Saccharomyces cerevisiae Proteins; D010450:Endopeptidases; C022325:aspartic proteinase A; C499267:PEP4 protein, S cerevisiae; D016282:Aspartic Acid Endopeptidases; D002402:Cathepsin D