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 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.
Nishikawa
A
A
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Gregory
W
W
Frenz
J
J
Cacia
J
J
Kornfeld
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D019556:COS Cells; D002417:Cattle; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D008239:Lysine; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism*; D010766:Phosphorylation
D009844:Oligosaccharides; D001120:Arginine; D003850:Deoxyribonuclease I; D008239:Lysine