J Biol Chem, Vol. 274, Issue 27, 19309-19315, July 2, 1999

Identification of Amino Acids That Modulate Mannose Phosphorylation of Mouse DNase I, a Secretory Glycoprotein

Atsushi Nishikawa, Akash Nanda, Walter Gregory, John Frenz^§, and Stuart Kornfeld

From the Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, the  Department of Biochemistry, Okayama University of Science, Okayama 700, Japan, and ^§ Genentech, Inc., South San Francisco, California 94080

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 Val²³ inhibits phosphorylation at the Asn¹⁸ glycosylation site, whereas Tyr⁵⁴, Lys¹²⁴, and Ser¹⁹⁰ enhance 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 enzyme N-acetylglucosamine-1-phosphotransferase, which is responsible for the formation of mannose 6-phosphate residues on lysosomal enzymes.