Carbohy drate Research, 152 (1986) 183-194 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands LYSOSOMAL-ENZYME TARGETING: THE PHOSPHORYLATION OF SYNTHETIC D-MANNOSYL SACCHARIDES BY UDP-N-ACETYL- GLUCOSAMINE:LYSOSOMAL-ENZYME N-ACETYLGLUCOSAMINE- PHOSPHOTRANSFERASE FROM RAT-LIVER MICROSOMES AND FIBROBLASTS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA RAGUPATHY MADIYALAKAN. MANJIT S. CHOWDHARY, SURIIT S. RANA. AND KHUSHI L. MATTA* Department of Gynecologic Oncology, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, NY 14263 (U.S.A.) (Received September 4th, 1984; accepted for publication in revised form, November 22nd, 1985) ABSTRACT Phosphorylation of the D-mannose residues of lysosomal enzymes is essential for the uptake and intracellular transport of these enzymes to lysosomes. The GlcNAc-P-transferase which is involved in the phosphorylation reaction seems to recognize a signal, probably a protein conformation, common to many lysosomal enzymes. To evaluate the role of the carbohydrate portion of the enzyme in these phosphorylation reactions, the acceptor specificity of GlcNAc-P-transferase from rat-liver microsomes and fibroblasts was examined with the aid of synthetic D- mannosyl disaccharides and derivatives that are closely related to the high-mannose type of oligosaccharides. Four methyl D-mannobiosides were synthesized, and their structures were established by i3C-n.m.r. spectroscopy. Of all the o-mannosyl sac- charides tested, cY-D-Man-(1+2)-a-D-Man-(l+OMe) was found to be the best ac- ceptor, thereby suggesting that oligosaccharide structure may also have a role to play in recognition by this enzyme. INTRODUCTION The phospho-o-mannopyranosyl recognition-marker of lysosomal enzymes that is considered zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA to be responsible for the targeting of newly synthesized, lysosomal enzymes to 1ysosomesiJ is generated in two steps. First, N-acetyl-o- glucosamine l-phosphate is transferred to the 6-hydroxyl group situated on a D- mannose residue of high-mannose type oligosaccharides in lysosomal enzymes. This reaction is catalyzed3 by a specific UDP-GlcNAc-lysosomal-enzyme N-acetyl- glucosaminephosphotransferase (EC 2.7.8.17; “GlcNAc-P-transferase”). In the next step, the outer N-acetyl-D-glucosamine residues are removed4 by the action of an N-acetylglucosamine-l-phosphodiester N-acetylglucosaminidase (EC 3.1.4.45). *To whom correspondence should be addressed. 0008-6215/86/$03.50 @ 19% Elsevier Science Publishers B.V.