|Budget Amount *help
¥2,700,000 (Direct Cost : ¥2,700,000)
Fiscal Year 1998 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 1997 : ¥1,400,000 (Direct Cost : ¥1,400,000)
We have reported that bovine DNase I, a secretary glycoprotein, acquires mannose 6-phosphate residues on 12.6% of its Asn-linked oligosaceharides when expressed in COS-1 cells and that the extent of phosphorylation increase to 79.2% when lysine are replaced at positions at 27 and 74 of the mature protein (Nishikawa, et al. (1997) J.Biol. Chem. 272, 19408-19412). We now demonstrate that muiine DNase I, which contains Lys^<27> and Lys74, is only phosphorylated 20.9% when expressed in the same COS-1 cell system. The difference is mostly due to the presence of three residues in murine sequence (Val^<23>, Lys^<117>, and Pro^<190>) that inhibit phosphorylation. Replacement of these residues with ahnines resulted in a strong stimulation of phosphorylation. In addition, substitution oh two other residues in the mouse sequence with the equivalent residues present in bovine DNase I (Glu57Tyr and Glul24Lys), but not replacement of these residues with an alanine, also resulted in enhanced phosphorylation, suggesting that these bovine residues have a positive stimulatory effect. The quadruple mutant (Lys117Ala-Prol 9OAla-Glu54Tyr-Val23Ala) was 65% phosphorylated, almost equivalent to the level obtained with bovine DNase I containing Lys^<27> and Lys^<74>.
These results indicate that the conformation-dependent recognition domain on murine DNase I that serves as the binding site for UDP-GIcNAc : Lysosomal enzyme NU-acetylglucosamine- l -phosphotransferase is suppressed by several inhibitory amino acid. In addition, murme DNase I lacks two of the stimulatory amino acids present in bovine DNase I, inducing a critical tyrosine residues.