Generation of a lysosomal enzyme targeting signal in the secretory protein pepsinogen.
Lysosomal enzymes contain a common protein determinant that is recognized by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the formation of mannose 6-phosphate residues. To identify this protein determinant, we constructed chimeric molecules between two aspartyl proteases: cathepsin D, a lysosomal enzyme, and pepsinogen, a secretory protein. When expressed in Xenopus oocytes, the oligosaccharides of cathepsin D were efficiently phosphorylated, whereas the oligosaccharides of a glycosylated form of pepsinogen were not phosphorylated. The combined substitution of two noncontinuous sequences of cathepsin D (lysine 203 and amino acids 265-292) into the analogous positions of glycopepsinogen resulted in phosphorylation of the oligosaccharides of the expressed chimeric molecule. These two sequences are in direct apposition on the surface of the molecule, indicating that amino acids from different regions come together in three-dimensional space to form this recognition domain. Other regions of cathepsin D were identified that may be components of a more extensive recognition marker.
Baranski
T J
TJ
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Faust
P L
PL
Kornfeld
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002402:Cathepsin D / Q000235:genetics; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D006801:Humans; D008239:Lysine; D008247:Lysosomes / Q000201:enzymology*; D008360:Mannosephosphates / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D009844:Oligosaccharides / Q000302:isolation & purification; D009865:Oocytes / Q000378:metabolism; D010435:Pepsinogens / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D011487:Protein Conformation; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D017855:Transferases (Other Substituted Phosphate Groups); D014982:Xenopus laevis
D008360:Mannosephosphates; D015345:Oligonucleotide Probes; D009844:Oligosaccharides; D010435:Pepsinogens; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C030012:UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase; D002402:Cathepsin D; D008239:Lysine