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 Lys(27) and Lys(74), 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 (Tyr(54), Lys(124), and Ser(190)) along with the presence of a valine at position 23 that is absent in the bovine species. We show that Val(23) inhibits phosphorylation at the Asn(18) glycosylation site, whereas Tyr(54), Lys(124), and Ser(190) enhance phosphorylation at the Asn(106) glycosylation site. Tyr(54) and Ser(190) are widely separated from each other and from Asn(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.