Identification of UDP-N-acetylglucosamine-phosphotransferase-binding sites on the lysosomal proteases, cathepsins A, B, and D.
A key step in the targeting of soluble lysosomal enzymes is their recognition and phosphorylation by a 540 kDa multisubunit enzyme, UDP-N-acetylglucosamine-phosphotransferase (phosphotransferase). The molecular mechanism of recognition is still unknown, but previous experiments suggested that the phosphotransferase-binding sites on lysosomal proteins are represented by structurally conserved surface patches of amino acids. We identified four such regions on nonhomologous lysosomal enzymes, cathepsins A, B, and D, which were superimposed by rotating their structures around the Calpha atom of the glycosylated Asn residue. We proposed that these regions represent putative phosphotransferase-binding sites and tested synthetic peptides, derived from these regions on the basis of surface accessibility, for their ability to inhibit in vitro phosphorylation of purified cathepsins A, B, and D. Our results indicate that cathepsin A and cathepsin D have one closely related phosphotransferase recognition site represented by a structurally and topologically conserved beta-hairpin loop, similar to that previously identified in lysosomal beta-glucuronidase. The most potent inhibition of phosphorylation was demonstrated by homologous peptides derived from the regions located on cathepsin molecules opposite the oligosaccharide chains which are phosphorylated by the phosphotransferase. We propose that recognition and catalytic sites of the phosphotransferase are located on different subunits, therefore, providing an effective mechanism for binding and phosphorylation of lysosomal proteins of different molecular size.
Lukong
K E
KE
Service de Génétique Médicale, Hôpital Sainte-Justine, Université de Montréal, Québec, Canada.
Elsliger
M A
MA
Mort
J S
JS
Potier
M
M
Pshezhetsky
A V
AV
D000818:Animals; D001665:Binding Sites; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D043402:Cathepsin A; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002402:Cathepsin D / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D008958:Models, Molecular; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D051381:Rats; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000378:metabolism*
D010455:Peptides; D017855:Transferases (Other Substituted Phosphate Groups); C030012:UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase; D002268:Carboxypeptidases; C468629:CTSA protein, human; D043402:Cathepsin A; D002401:Cathepsin B; D002402:Cathepsin D