Cuozzo J W, Tao K, Wu Q L, Young W, Sahagian G G
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
J Biol Chem. 1995 Jun 30;270(26):15611-9. doi: 10.1074/jbc.270.26.15611.
The recognition of lysosomal enzymes by UDP-GlcNAc: lysosomal-enzyme GlcNAc-1-phosphotransferase (phosphotransferase) is mediated by a protein structure on lysosomal enzymes. It has been previously demonstrated that lysine residues are required for phosphorylation of procathepsin L and are a common feature of the site on many lysosomal proteins. In this work, the procathepsin L recognition structure was further defined by identification of the region of the protein containing the structure and the critical lysine residues involved. Removal of the cathepsin L propeptide by low pH-induced autocatalytic processing abolished phosphorylation. The addition of either the purified propeptide or a glutathione S-transferase-propeptide fusion protein to the processed protein restored phosphorylation. Mutagenesis of individual lysine residues demonstrated that two propeptide lysine residues (Lys-54 and Lys-99) were required for efficient phosphorylation of procathepsin L. By comparison of the phosphorylation rates of procathepsin L, lysine-modified procathepsin L, and the procathepsin L oligosaccharide, lysine residues were shown to account for most, if not all, of the protein-dependent interaction. On this basis, it is concluded that the proregion lysine residues are the major elements of the procathepsin L recognition site. In addition, lysine residues in cathepsin D were shown to be as important for phosphorylation as those in procathepsin L, supporting a general model of the recognition site as a specific three-dimensional arrangement of lysine residues exposed on the surface of lysosomal proteins.
UDP-GlcNAc对溶酶体酶的识别：溶酶体酶GlcNAc-1-磷酸转移酶（磷酸转移酶）对溶酶体酶的识别是由溶酶体酶上的一种蛋白质结构介导的。先前已经证明，赖氨酸残基是组织蛋白酶L原磷酸化所必需的，并且是许多溶酶体蛋白上该位点的共同特征。在这项工作中，通过鉴定含有该结构的蛋白质区域和涉及的关键赖氨酸残基，进一步确定了组织蛋白酶L原的识别结构。低pH诱导的自催化加工去除组织蛋白酶L原肽后，磷酸化作用消失。将纯化的原肽或谷胱甘肽S-转移酶-原肽融合蛋白添加到加工后的蛋白中可恢复磷酸化。对单个赖氨酸残基进行诱变表明，两个原肽赖氨酸残基（Lys-54和Lys-99）是组织蛋白酶L原有效磷酸化所必需的。通过比较组织蛋白酶L原、赖氨酸修饰的组织蛋白酶L原和组织蛋白酶L原寡糖的磷酸化速率，发现赖氨酸残基即使不是全部，也是大部分蛋白质依赖性相互作用的原因。在此基础上，可以得出结论，原结构域赖氨酸残基是组织蛋白酶L原识别位点的主要元素。此外，组织蛋白酶D中的赖氨酸残基对磷酸化的重要性与组织蛋白酶L原中的赖氨酸残基相同，这支持了识别位点是溶酶体蛋白表面暴露的赖氨酸残基的特定三维排列的一般模型。
