Identification of the minimal lysosomal enzyme recognition domain in cathepsin D.
Specific recognition of lysosomal hydrolases by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the biosynthesis of mannose 6-phosphate residues, is governed by a common protein determinant. Previously, we generated a lysosomal enzyme recognition domain in the secretory protein glycopepsinogen by substituting in two regions (lysine 203 and amino acids 265-293 of the beta loop) from cathepsin D, a highly related lysosomal protease. Here we show that substitution of just two lysines (Lys-203 and Lys-267) stimulates mannose phosphorylation 116-fold. Substitution of additional residues in the beta loop, particularly lysines, increased phosphorylation 4-fold further, approaching the level obtained with intact cathepsin D. All the phosphorylation occurred at the carboxyl lobe glycan, indicating that additional elements are required for phosphorylation of the amino lobe glycan. These data support the proposal that as few as two lysines in the correct orientation to each other and to the glycan can serve as the minimal elements of the lysosomal enzyme recognition domain. However, our findings show that the spacing between lysines is flexible and other residues contribute to the recognition marker.
Steet
Richard
R
Department of Internal Medicine, Washington University School of Medicine, Saint Louis, Missouri 63110, USA.
Lee
Wang-Sik
WS
Kornfeld
Stuart
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002402:Cathepsin D / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002522:Chlorocebus aethiops; D016384:Consensus Sequence; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry; D008247:Lysosomes / Q000201:enzymology*; D008358:Mannose / Q000378:metabolism; D008360:Mannosephosphates / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis; D010766:Phosphorylation; D011134:Polysaccharides / Q000378:metabolism; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013462:Sulfur Radioisotopes; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism*; D014316:Tritium / Q000378:metabolism
D008360:Mannosephosphates; D009844:Oligosaccharides; D011134:Polysaccharides; D013462:Sulfur Radioisotopes; D014316:Tritium; C027693:mannose-6-phosphate; D017855:Transferases (Other Substituted Phosphate Groups); C030012:UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase; D002402:Cathepsin D; D008239:Lysine; D008358:Mannose