Characterization of a membrane-associated receptor from bovine liver that binds phosphomannosyl residues of bovine testicular beta-galactosidase.

Abstract

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 ...

Demonstration of tumor suppression by mannose 6-phosphate/insulin-like growth factor 2 receptor

“…While initially characterized with respect to its role in lysosomal enzyme trafficking (Sahagian et al, 1981) and IGF-2 endocytosis (Tong et al, 1988), the receptor has been reported to be involved in a number of other growth-related processes including TGF-b activation (Dennis and Rifkin, 1991), growth suppression by retinoic acid (Kang et al, 1999), granzyme B-mediated cytotoxicity (Motyka et al, 2000), and uPA receptor turnover (Nykjaer et al, 1998). With the exception of granzyme B-mediated cytotoxicity, which is ruled out by the SCID mouse experiment described here, any or all of these functions could potentially be involved in tumor suppression.…”

“…The Man-6-P/IGF-2 receptor is a multifunctional protein originally characterized with respect to its role in trafficking mannose phosphorylated hydrolases to lysosomes in mammalian cells and tissues (Sahagian et al, 1981). Cloning and sequencing of the high-affinity receptor for IGF-2 led to the realization that the two receptors were the same protein (Morgan et al, 1987) that had distinct binding sites for the two ligands (Braulke et al, 1988;Kiess et al, 1988;MacDonald et al, 1988).…”

Synthesis of beta-hexosaminidase in cell-free translation and in intact fibroblasts: an insoluble precursor alpha chain in a rare form of Tay-Sachs disease.

“…12 and 13). Enzymes destined for lysosomes are then equipped with mannose 6-phosphate recognition markers in the Golgi apparatus (14)(15)(16)(17), bound to phosphomannosyl receptors (18)(19)(20), and transported to lysosomes. A portion of the newly formed phosphorylated lysosomal enzymes may be secreted, and a fraction thereof may be recaptured by the phosphomannosyl receptors on the cell surface.…”

Trafficking of lysosomal enzymes in normal and disease states.

“…In fibroblasts, this secretion-recapture mechanism functions as a salvage pathway, which accounts for the delivery of5-10% oftotal lysosomal enzymes to lysosomes (23). A M-6-P receptor has been purified from a variety of tissues and appears to be a single glycoprotein with an apparent molecular weight of 215,000 (24)(25), although there is one report indicating that the receptor may consist ofsmaller subunits (26). This receptor binds acid hydrolases independent ofdivalent cations.…”