Identification of UDP-N-acetylglucosamine-phosphotransferase sites on the lysosomal proteases, cathepsins A, B, and D

A key step in the targeting of soluble lysosomal enzymes is their recogniti on and phosphorylation by a 540 kDa multisubunit enzyme, UDP-N-acetylglucos amine-phosphotransferase (phosphotransferase). The molecular mechanism of r ecognition is still unknown, but previous experiments suggested that the ph osphotransferase-binding sites on lysosomal proteins are represented by str ucturally 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 Ca atom of the gl ycosylated Asn residue. We proposed that these regions represent putative p hosphotransferase-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 re sults indicate that cathepsin A and cathepsin D have one closely related ph osphotransferase recognition site represented by a structurally and topolog ically conserved beta-hairpin loop, similar to that previously identified i n lysosomal beta-glucuronidase. The most potent inhibition of phosphorylati on was demonstrated by homologous peptides derived from the regions located on cathepsin molecules opposite the oligosaccharide chains which are phosp horylated by the phosphotransferase. We propose that recognition and cataly tic sites of the phosphotransferase are located on different subunits, ther efore, providing an effective mechanism for binding and phosphorylation of lysosomal proteins of different molecular size.