Analysis of Lysosomal Protein Targeting Signals.

我们已经报道，牛DNase I（一种分泌糖蛋白）在COS-1细胞中表达时，12.6%的asn连接的寡糖糖苷上获得甘露糖6-磷酸残基，当赖氨酸在成熟蛋白的27和74位被取代时，磷酸化程度增加到79.2% （Nishikawa等人（1997）J.Biol）。化学272,19408-19412)。我们现在证明，在相同的COS-1细胞系统中表达的muiine DNase I，包含lysys ^<27>和Lys74，仅磷酸化20.9%。这种差异主要是由于小鼠序列中存在三个抑制磷酸化的残基（Val^<23>, Lys^<117>和Pro^<190>）。用氨基酸取代这些残基导致磷酸化的强烈刺激。此外，将小鼠序列中的另外两个残基（Glu57Tyr和Glul24Lys）与牛dna酶I中的等效残基（glul57tyr和Glul24Lys）替换，而不是用丙氨酸替换这些残基，也会导致磷酸化增强，这表明这些牛残基具有积极的刺激作用。四重突变体（Lys117Ala-Prol 9oala - glu54tyrr - val23ala）被65%磷酸化，几乎相当于含有Lys^<27>和Lys^<74>的牛dna酶I的磷酸化水平。这些结果表明，作为UDP-GIcNAc：溶酶体酶nu -乙酰氨基葡萄糖- 1 -磷酸转移酶结合位点的小鼠dna酶I的构象依赖性识别结构域被几种抑制氨基酸抑制。此外，奶牛dna酶I缺乏牛dna酶I中存在的两种刺激性氨基酸，诱导了一个关键的酪氨酸残基。

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.