Lukong K E, Elsliger M A, Mort J S, Potier M, Pshezhetsky A V
Service de Génétique Médicale, Hôpital Sainte-Justine, Université de Montréal, Québec, Canada.
Biochemistry. 1999 Jan 5;38(1):73-80. doi: 10.1021/bi981324r.
A key step in the targeting of soluble lysosomal enzymes is their recognition and phosphorylation by a 540 kDa multisubunit enzyme, UDP-N-acetylglucosamine-phosphotransferase (phosphotransferase). The molecular mechanism of recognition is still unknown, but previous experiments suggested that the phosphotransferase-binding sites on lysosomal proteins are represented by structurally conserved surface patches of amino acids. We identified four such regions on nonhomologous lysosomal enzymes, cathepsins A, B, and D, which were superimposed by rotating their structures around the Calpha atom of the glycosylated Asn residue. We proposed that these regions represent putative phosphotransferase-binding sites and tested synthetic peptides, derived from these regions on the basis of surface accessibility, for their ability to inhibit in vitro phosphorylation of purified cathepsins A, B, and D. Our results indicate that cathepsin A and cathepsin D have one closely related phosphotransferase recognition site represented by a structurally and topologically conserved beta-hairpin loop, similar to that previously identified in lysosomal beta-glucuronidase. The most potent inhibition of phosphorylation was demonstrated by homologous peptides derived from the regions located on cathepsin molecules opposite the oligosaccharide chains which are phosphorylated by the phosphotransferase. We propose that recognition and catalytic sites of the phosphotransferase are located on different subunits, therefore, providing an effective mechanism for binding and phosphorylation of lysosomal proteins of different molecular size.
可溶性溶酶体酶靶向的关键步骤是它们被一种540 kDa的多亚基酶UDP-N-乙酰葡糖胺磷酸转移酶（磷酸转移酶）识别并磷酸化。识别的分子机制仍然未知，但先前的实验表明，溶酶体蛋白上的磷酸转移酶结合位点由氨基酸的结构保守表面区域表示。我们在非同源溶酶体酶组织蛋白酶A、B和D上鉴定出四个这样的区域，通过围绕糖基化天冬酰胺残基的α碳原子旋转它们的结构使其重叠。我们提出这些区域代表推定的磷酸转移酶结合位点，并测试了基于表面可及性从这些区域衍生的合成肽抑制纯化的组织蛋白酶A、B和D体外磷酸化的能力。我们的结果表明，组织蛋白酶A和组织蛋白酶D有一个密切相关的磷酸转移酶识别位点，由一个结构和拓扑保守的β-发夹环表示，类似于先前在溶酶体β-葡糖醛酸酶中鉴定的位点。来自组织蛋白酶分子上与被磷酸转移酶磷酸化的寡糖链相对的区域的同源肽表现出最有效的磷酸化抑制作用。我们提出磷酸转移酶的识别和催化位点位于不同的亚基上，因此为不同分子大小的溶酶体蛋白的结合和磷酸化提供了一种有效机制。
