Mannose phosphorylat10n on secretory glycoproteins

UDP-GlcNAc:Lysosornal enzyme N-acetylglucosaminylphosphotransferase recognizes a conformation-dependent protein determinant that is present in acid hydrolases but absent m most secretory glycoprotems. However, a few secretory glycoproteins have been reported tc contain Man-6-P moieties, including DNase 1 which is secreted by the pancreas. To pursue the basis for this, bovine and mouse DNase i cDNAs were transfected into COS cells and the resultant protem analyzed for Man-6-P content. The oligosaccharides of these DNase l were phosphorylated only 1 3 and 1 6%, respectively. By analyzing a number of mutant and chimeric constructs, we determined that the low phosphorylation of bovine DNase I is due to the lack of two critical lysmes present m the mouse sequence whereas the poor phosphorylation of mouse DNase 1 is due to the presence of "inhibitory" ammo acids that block interaction with phosphotransferase. Insertion of the critical lysines into bovme DNase I or removal of the inhibitory am.no acids from mouse DNase I increases the extent of phosphorylation to 40-90%, equivalent to that observed with authentic acid hydrolases. Thus, mouse DNase I has a potent recognition domain that is masked by inhibitory amino acids. These results extend previous studies implicating lysine residues as essential components cf the phosphotransferase recognition domain and reveal a second level of regulation, mediated by " inhibitory" ar-.mo acids that prevent mannose phosphorylatior.