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A receptor that binds the phosphomannosyl recognition marker of bovine testicular P-galactosidase (13-D-galactoside galactohydrolase, EC 3.2.1.23) was isolated from bovine liver membranes. The receptor was extracted from crude plasma membrane preparations with Triton X-100 and immunoprecipitated as a receptor-f-galactosidase complex with anti-,B-galactosidase. The receptor was dissociated from the precipitate with mannose 6-phosphate, labeled with`RI, and purified on a (3-galactosidase-Sepharose 4B affinity matrix. A quantitative binding assay employing anti-(-galactosidase and IgGsorb (formalin-fixed Staphylococcus aureus) was devised to study the binding of`5I-labeled receptor to (3-galactosidase. Maximal binding of receptor to enzyme occurred at pH values between 5.7 and 6.5. Divalent cations were not required for binding. The values of the dissociation constant obtained for 83-galactosidase varied between 200 nM observed with "lower uptake" forms and 20 nM for "higher uptake" forms of the enzyme. A number of phosphorylated monosaccharides were tested as inhibitors of binding of enzyme to receptor; mannose 6-phosphate and fructose 1-phosphate served as inhibitors and exhibited 1I values of 0.064 mM and 0.24 mM, respectively. The receptor has a subunit molecular weight of 215,000. Similar receptors were also demonstrated in Triton X-100 extracts of human skin fibroblasts, Chinese hamster ovary cells, and rat hepatocytes. These cell types are known to assimilate lysosomal enzymes containing covalently bound mannose 6-phosphate residues.Certain lysosomal enzymes are selectively and efficiently taken up by cultured human fibroblasts (1). This process is thought to be mediated by a specific cell surface receptor that recognizes phosphomannosyl residues on oligosaccharide chains of the enzymes (2-5). Recognition of lysosomal enzymes by a phosphomannosyl receptor has been proposed as an essential step for the delivery of newly synthesized lysosomal enzymes to lysosomes (6-8). Direct evidence for the existence of phosphomannosyl receptors has been obtained by demonstration of the reversible binding of a-L-iduronidase to the cell surface ofhuman skin fibroblasts (9) and by the binding of P3-glucuronidase to fibroblast cell membranes (10).Phosphomannosyl receptors also occur in other mammalian cell types and tissues. Phosphomannosyl-dependent uptake or binding of lysosomal enzymes has been observed in Chinese hamster ovary cells (11), normal rat kidney cells (12), a rat liver epithelial cell line (13), rat hepatocytes (12,14), and Swarm rat chondrosarcoma (15). Binding studies using f-hexosaminidase suggest the presence of the phosphomannosyl receptor in all major tissues of the rat and in several rat liver subcellular fractions (16).In previous studies we demonstrated the presence of mannose 6-phosphate residues in f3-galactosidase (P-D-galactoside galactohydrolase, EC 3.2.1.23) (17,18) and showed that the enzyme is subject to endocytosis by the phosphomannosyl uptake system in human skin fibroblasts (5). We ...

Observations that extracellular lysosomal enzymes are assimilated by fibroblasts and serve to correct genetic lysosomal enzyme deficiencies has stimulated interest in the mechanisms by which the enzyme assimilation occurs. It has been proposed that the assimilation (absorptive endocytosis or uptake) of lysosomal enzymes by fibroblasts is initiated by cellular "recognition" of a carbohydrate-containing "marker" bound to the lysosomal enzymes (1). Studies in this laboratory implicated enzyme-bound mannosyl residues in the assimilation of bovine testicular f-galactosidase (f-D-galactoside galactohydrolase, EC 3.2.1.23) (2). Recently, Kaplan et al. (3) suggested that phosphate, probably as mannose 6-phosphate, participated in the assimilation of platelet 3-glucuronidase by fibroblasts. This conclusion was based on the strong inhibition of the assimilation of (-glucuronidase by free mannose 6-phosphate and the lack of assimilation of 3-glucuronidase when the enzyme was pretreated with alkaline phosphatase. Subsequently, similar experiments implicated mannose phosphate in the assimilation of other lysosomal enzymes by fibroblasts. These enzymes included highly purified a-L-iduronidase (4) and 3-galactosidase (5) as well as several other partially purified lysosomal enzymes from platelets (6) and urine (7). These observations strongly suggested that mannose 6-phosphate was a normal constituent of many lysosomal enzymes. However, direct evidence of the occurrence of mannose 6-phosphate in lysosomal enzymes has not been presented. Furthermore, the type of carbohydrate unit of these glycoproteins that may contain mannose 6-phosphate and constitute the "recognition marker" was not apparent.Elucidation of the structural features that characterize the recognition marker has been restricted by the limited amounts of highly purified enzymes. Fortunately, glycoproteins prepared from urine (4), platelets (6), and bovine testes (2, 5) furnish abundant materials which, based on inhibition studies, bear recognition markers similar to those of lysosomal enzymes. * These glycoprotein inhibitors were found to contain several lysosomal enzymes that may compete for the same cell receptors (4, 5). MATERIALS AND METHODSDiphenylcarbamyl chloride-treated (DCC) trypsin, soybean trypsin inhibitor, jack bean ca-mannosidase, and mannose 6-phosphatet were obtained from Sigma. Potato acid phosphatase, glycoaldehyde phosphate diethylacetal, and DL-glyceraldehyde-3-phosphate diethylacetal were obtained from Calbiochem; acetals were hydrolyzed as recommended by the supplier. Concanavalin A (Con A)-Sepharose was obtained from Pharmacia. Sodium borol3H]hydride [specific activity, 422 mCi/mmol (1 Ci = 3.7 X 1010 becquerels)] was obtained from Amersham. Radioactivity was measured by liquid scintillation spectrometry on squares of Whatman 3 MM paper immersed in a toluene liquid scintillation cocktail. Methyl 3-mannopyranoside was prepared from methyl tetra-O-acetyl-3-mannopyranoside. The latter compound and synthetic a-mannopyranosyl-(1l-2)-m...

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