The cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) undergoes constitutive endocytosis, mediating the internalization of two unrelated classes of ligands, mannose 6-phosphate (Man-6-P)-containing acid hydrolases and insulin-like growth factor II (IGF-II). To determine the role of ligand valency in M6P/IGF-II receptor-mediated endocytosis, we measured the internalization rates of two ligands, ␤-glucuronidase (a homotetramer bearing multiple Man-6-P moieties) and IGF-II. We found that ␤-glucuronidase entered the cell ϳ3-4-fold faster than IGF-II. Unlabeled ␤-glucuronidase stimulated the rate of internalization of 125 I-IGF-II to equal that of 125 I-␤-glucuronidase, but a bivalent synthetic tripeptide capable of occupying both Man-6-P-binding sites on the M6P/IGF-II receptor simultaneously did not. A mutant receptor with one of the two Man-6-P-binding sites inactivated retained the ability to internalize ␤-glucuronidase faster than IGF-II. Thus, the increased rate of internalization required a multivalent ligand and a single Man-6-P-binding site on the receptor. M6P/IGF-II receptor solubilized and purified in Triton X-100 was present as a monomer, but association with ␤-glucuronidase generated a complex composed of two receptors and one ␤-glucuronidase. Neither IGF-II nor the synthetic peptide induced receptor dimerization. These results indicate that intermolecular cross-linking of the M6P/IGF-II receptor occurs upon binding of a multivalent ligand, resulting in an increased rate of internalization.The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) 1 is a type I transmembrane glycoprotein that cycles through the Golgi, endosomes, and the plasma membrane to carry out its role in the biogenesis of lysosomes and in the clearance of the polypeptide insulin-like growth factor II (IGF-II) (1, 2). In the Golgi, the receptor binds newly synthesized acid hydrolases modified with mannose 6-phosphate (Man-6-P) residues on their asparagine-linked oligosaccharides and transports them to endosomes via clathrincoated vesicles (3-5). The acid hydrolases are released in the acidified endosome and then packaged into lysosomes while the receptor either returns to the Golgi to bind another ligand or moves to the plasma membrane (6, 7). At the plasma membrane, the M6P/IGF-II receptor mediates internalization of Man-6-P-containing ligands and IGF-II (3,5,8).The interactions of IGF-II and Man-6-P-containing ligands with the M6P/IGF-II receptor have been characterized in several studies (8 -12). The extracellular portion of the M6P/ IGF-II receptor contains 15 homologous repeating domains of ϳ147 amino acids each (13). Domains 3 and 9 (numbering from the amino terminus) each bind 1 mol of Man-6-P, and the single IGF-II-binding site has been mapped to domain 11 in the extracellular region (14 -16). Man-6-P residues do not inhibit binding of IGF-II to the receptor, verifying that the two ligandbinding sites are distinct. However, proteins containing Man-6...

Endoplasmic reticulum-associated degradation of misfolded or misprocessed glycoproteins in mammalian cells is prevented by inhibitors of class I ␣-mannosidases implicating mannose trimming from the precursor oligosaccharide Glc 3 Man 9 GlcNAc 2 as an essential step in this pathway. However, the extent of mannose removal has not been determined. We show here that glycoproteins subject to endoplasmic reticulum-associated degradation undergo reglucosylation, deglucosylation, and mannose trimming to yield Man 6 GlcNAc 2 and Man 5 GlcNAc 2 . These structures lack the mannose residue that is the acceptor of glucose transferred by UDPGlc:glycoprotein glucosyltransferase. This could serve as a mechanism for removal of the glycoproteins from folding attempts catalyzed by cycles of reglucosylation and calnexin/calreticulin binding and result in targeting of these molecules for proteasomal degradation.Trimming of the asparagine-linked precursor oligosaccharide Glc 3 Man 9 GlcNAc 2 in the endoplasmic reticulum (ER) 1 yields Glc 1 Man 9 -7 GlcNAc 2 (G1M9 -7) that associates with the ER chaperones/lectins calnexin (CNX) and calreticulin (CRT) (1). A folding and quality control process is then initiated that consists of cycles of deglucosylation by glucosidase II, release from the chaperone, reglucosylation by the folding sensor enzyme UDP-Glc:glycoprotein glucosyltransferase (GT), and reassociation with CNX and CRT (2). Glycoproteins that achieve proper folding are no longer recognized by GT and leave this cycle.Trimming of mannose (Man) residues is involved in the disposal of ER-retained glycoproteins that cannot be driven to their native forms during the cycles of binding to CNX/CRT (3). It was proposed that Man trimming may provide a timer that allows these folding attempts before degradation starts (3, 4). Thus, inhibitors that prevent Man trimming were found to inhibit the ERAD of many defective glycoproteins (4 -8). In the absence of these inhibitors, the glycoproteins are ubiquitinated and delivered to the cytosolic proteasomes for degradation (9). Prior to dislocation to the cytosol, the substrates are transported to a specialized compartment termed the quality control (QC) compartment together with CNX and CRT (10). This compartment is pericentriolar, located near (but not overlapping) the Golgi and the ER-Golgi intermediate compartment (ERGIC), and is dependent on microtubules.Whereas the inhibitor studies provide strong evidence that Man trimming is involved in ERAD, the structures of the N-linked oligosaccharides that result from trimming have not been determined in mammalian cells. The inhibitors that prevent Man trimming leading to ERAD (such as 1-deoxymannojirimycin (dMNJ) and kifunensine) are blockers of class I mannosidases. Therefore, the major ER class I enzyme, ER mannosidase I, has been proposed to be responsible for this pathway. Consequently, the product of cleavage by this enzyme, an M8 isomer lacking the middle branch terminal Man (M8B), has been suggested as the signal that marks the glycoprotein for...

The secretory glycoprotein DNase I acquires mannose 6-phosphate moieties on its Asn-linked oligosaccharides, indicating that it is a substrate for UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (phosphotransferase) (Cacia, J., Quan, C., and Frenz, J. (1995) Glycobiology 4, 99). Phosphotransferase recognizes a conformation-dependent protein determinant that is present in lysosomal hydrolases, but absent in most secretory glycoproteins. To identify the amino acid residues of DNase I that are required for interaction with phosphotransferase, wild-type and mutant forms of bovine DNase I were expressed in COS-1 cells and the extent of oligosaccharide phosphorylation determined. Phosphorylation of DNase I oligosaccharides decreased from 12.6% to 2.3% when Lys-50, Lys-124, and Arg-27 were mutated to alanines, indicating that these residues are required for the basal level of phosphorylation. Mutation of lysines at other positions did not impair phosphorylation, demonstrating the selectivity of this process. When Arg-27 was replaced with a lysine, phosphorylation increased to 54%, showing that phosphotransferase prefers lysine residues to arginines. Mutation of Asn-74 to a lysine also increased phosphorylation to 50.3%, and the double mutant (R27K/ N74K) was phosphorylated 79%, equivalent to the values obtained with lysosomal hydrolases. Interestingly, Lys-27 and Lys-74 caused selective phosphorylation of the neighboring Asn-linked oligosaccharide. Finally, mutation of Lys-117 to an alanine stimulated phosphorylation, demonstrating that some residues may be negative regulators of this process. We conclude that selected lysine and arginine residues on the surface of DNase I constitute the major elements of the phosphotransferase recognition domain present on this secretory glycoprotein.In many cell types, the sorting of newly synthesized acid hydrolases from secreted proteins is mediated by the phosphomannosyl recognition system (1, 2). The specificity of this pathway is determined by the enzyme UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (phosphotransferase) 1 which recognizes a conformation-dependent protein determinant that is present in lysosomal hydrolases but absent in most secretory glycoproteins (3). Using a variety of approaches, evidence has been obtained that the recognition determinant involves a broad surface patch that includes critical lysine residues (4 -10). The interaction of phosphotransferase with its acid hydrolase substrates results in the transfer of GlcNAc-P to mannose residues on the Asn-linked high mannose oligosaccharides of the lysosomal hydrolases. The GlcNAc residues are then removed by N-acetylglucosamine-1-phosphodiester ␣-N-acetylglucosaminidase to generate phosphomannosyl residues which mediate binding of the hydrolases to mannose 6-phosphate (Man-6-P) receptors present in the Golgi. These complexes are subsequently translocated via clathrincoated vesicles to endosomes where the hydrolases are discharged for packaging into lysosomes.While ph...

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We have reported that bovine DNase I, a secretory glycoprotein, acquires mannose 6-phosphate residues on 12.6% of its Asn-linked oligosaccharides when expressed in COS-1 cells and that the extent of phosphorylation increases to 79.2% when lysines are placed at positions 27 and 74 of the mature protein (Nishikawa, A., Gregory, W., Frenz, J., Cacia, J., and Kornfeld, S. (1997) J. Biol. Chem. 272, 19408 -19412 106 on the surface of DNase I, indicating that residues present over a broad area influence the interaction with UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, which is responsible for the formation of mannose 6-phosphate residues on lysosomal enzymes.In a previous report, we demonstrated that bovine DNase I, a secretory glycoprotein of the pancreas and the salivary gland, acquires mannose 6-phosphate moieties on its Asn-linked oligosaccharides (1), confirming the findings of Cacia et al. (2) with human DNase I. However, the level of oligosaccharide phosphorylation (12.6%) was considerably less than that observed with lysosomal hydrolases (usually 50% or greater). This indicated that DNase I is a weak substrate for UDPGlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (phosphotransferase), 1 the enzyme that synthesizes the Man-6-P recognition marker for targeting to lysosomes. Phosphotransferase is known to recognize a conformation-dependent protein determinant that is present in lysosomal hydrolases, but absent in most secretory glycoproteins (3). This determinant has been shown to involve a broad surface patch that includes critical lysine residues (4 -11). The binding of lysosomal hydrolases to phosphotransferase is followed by the transfer of GlcNAc-P from UDP-GlcNAc to selected mannose residues on the Asn-linked high mannose oligosaccharides of the hydrolases. The N-acetylglucosamine is then removed by N-acetylglucosamine-1-phosphodiester-␣-Nacetylglucosaminidase to generate the phosphomannosyl recognition determinant that allows binding to the mannose 6-phosphate receptors in the Golgi and subsequent targeting to lysosomes (12). The basal level of phosphorylation of bovine DNase I was shown to be dependent on three residues (Lys 50 , Lys 124 , and Arg 27 ), consistent with the finding that lysine residues are important components of the phosphotransferase recognition determinant on lysosomal hydrolases (1). When Arg 27 was replaced with a lysine, oligosaccharide phosphorylation increased to 54%, demonstrating that phosphotransferase prefers lysine residues over arginines. Furthermore, mutation of Asn 74 to a lysine also increased phosphorylation to 50.3%, and the double mutant (R27K/N74K) was phosphorylated 79.2%, equivalent to the values observed with authentic lysosomal hydrolases. Interestingly, the lysine at position 27 specifically stimulated phosphorylation of the oligosaccharide at Asn 18 , whereas the lysine at position 74 selectively stimulated phosphorylation of the oligosaccharide at Asn 106 . The R27K and N74K bovine constructs were initially prepared bec...

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