Walter Gregory

Endoplasmic reticulum-associated degradation of mammalian glycoproteins involves sugar chain trimming to Man6-5GlcNAc2.

Endoplasmic reticulum-associated degradation of misfolded or misprocessed glycoproteins in mammalian cells is prevented by inhibitors of class I α-mannosidases implicating mannose trimming from the precursor oligosaccharide Glc3Man9GlcNAc2 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 Man6GlcNAc2 and Man5GlcNAc2. These structures lack the mannose residue that is the acceptor of glucose transferred by UDP-Glc: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.

The rate of internalization of the mannose 6-phosphate/insulin-like growth factor II receptor is enhanced by multivalent ligand binding.

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, β-glucuronidase (a homotetramer bearing multiple Man-6-P moieties) and IGF-II. We found that β-glucuronidase entered the cell ∼3–4-fold faster than IGF-II. Unlabeled β-glucuronidase stimulated the rate of internalization of125I-IGF-II to equal that of125I-β-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 β-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 β-glucuronidase generated a complex composed of two receptors and one β-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 Phosphorylation of Bovine DNase I Asn-linked Oligosaccharides Is Dependent on Specific Lysine and Arginine Residues

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 enzymeN-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.

Identification of amino acids that modulate mannose phosphorylation of mouse DNase I, a secretory glycoprotein.

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). We now demonstrate that murine DNase I, which contains Lys27 and Lys74, is phosphorylated only 20.9% when expressed in the same COS-1 cell system. This difference is mostly due to the absence of three residues present in bovine DNase I (Tyr54, Lys124, and Ser190) along with the presence of a valine at position 23 that is absent in the bovine species. We show that Val23inhibits phosphorylation at the Asn18 glycosylation site, whereas Tyr54, Lys124, and Ser190enhance phosphorylation at the Asn106 glycosylation site. Tyr54 and Ser190 are widely separated from each other and from Asn106 on the surface of DNase I, indicating that residues present over a broad area influence the interaction with UDP-GlcNAc:lysosomal enzymeN-acetylglucosamine-1-phosphotransferase, which is responsible for the formation of mannose 6-phosphate residues on lysosomal enzymes.

The identification and partial characterization of lysosomes in human reticulocytes

Abstract The activities of several lysosomal enzymes have been determined in erythrocytes from human donors with and without spleens. The activities of the lysosomal enzymes β - N -acetylglucosaminidase (EC 3.2.1.30) and β -galactosidase (EC 3.2.1.23) are strikingly increased in erythrocytes from splenectomized donors, especially those with high reticulocyte counts. In contrast, erythrocytes from normal subjects have low levels of these enzymes regardless of the reticulocyte count. The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49), a soluble enzyme, was the same in erythrocytes from these two groups of subjects as was the total activity of acid β - glycerophosphate phosphohydrolase (EC 3.1.3.2), a characteristic lysosomal enzyme. This discrepancy was resolved by gel filtration studies which showed that erythrocytes from normal subjects contain two acid phosphatases while erythrocytes from splenectomized patients contain three different acid phosphatases. The third acid phosphatases is localized to membrane-bounded particles. Each of the three acid phosphotases has a different molecular weight and responds differently to various substrates and inhibitors, Mg 2+ , pH, and temperature. These studies provide biochemical evidence for the existence of lysosomes in ery erythrocytes and demonstrate that the spleen determines the level of lysosomes in these cells.