The Phosphorylation of Bovine DNase I Asn-linked Oligosaccharides Is Dependent on Specific Lysine and Arginine Residues

Abstract

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...

The DMAP interaction domain of UDP-GlcNAc:lysosomal enzyme N -acetylglucosamine-1-phosphotransferase is a substrate recognition module

“…A number of studies from our laboratory and others have shown that the protein recognition domain of acid hydrolases is a complex conformation-dependent determinant that includes a number of critical lysine residues (16)(17)(18)(19)(20)(21)(22)(23). On this basis, it seems likely that the interaction of an acid hydrolase with GlcNAc-1-phosphotransferase would involve multiple contacts between the surfaces of the two proteins.…”

Role of N-Linked Oligosaccharide Flexibility in Mannose Phosphorylation of Lysosomal Enzyme Cathepsin L

“…for lysine in some contexts (15)(16)(17). Other residues in the vicinity of critical lysine residues may affect the accessibility or properties of the lysine residues.…”

“…The chimeric studies of cathepsin D, as the authors note, can be interpreted by having two independent phosphorylation signals, instead of having a single extended signal (9). The existence of two or more signals on cathepsin D explains why mutation of individual lysine residues affects phosphorylation of the two cathepsin D oligosaccharides differently and why localized phosphorylation of engineered glycosylation sites was not observed for this protein (12,15).…”

“…On the basis of these results, a relatively simple model involving lysine residues was proposed as a general phosphorylation signal for lysosomal proteins. Similar involvement of lysine residues (15,16) has since been demonstrated for other proteins, and it is now widely accepted that lysine residues are the primary determinants for mannose phosphorylation of a wide range of proteins by the transferase.…”

Recognition of Arylsulfatase A and B by the UDP-N-acetylglucosamine:lysosomal Enzyme N-Acetylglucosamine-phosphotransferase

“…1 The abbreviations used are: ER, endoplasmic reticulum; M6P, mannose-6-phosphate; ASA, arylsulfatase A; ASB, Arylsulfatase B; ASE, arylsulfatase E; ASU, sea urchin arylsulfatase; BHK, baby hamster kidney; HPLC, high-performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. enzymes (8,11,14), suggesting that the spacing of surface lysines is critical for phosphotransferase recognition (8).…”