Oshima A, Nolan C M, Kyle J W, Grubb J H, Sly W S
E.A. Doisy Department of Biochemistry, St. Louis University School of Medicine, Missouri 63104.
J Biol Chem. 1988 Feb 15;263(5):2553-62.
We have cloned and sequenced the full-length cDNA for the human cation-independent mannose 6-phosphate receptor from four overlapping clones. The 9104-nucleotide sequence contains 7473 nucleotides which encode a protein of 2491 amino acids. The amino acid sequence includes a putative signal sequence of 40 amino acids, an extracytoplasmic domain consisting of 15 homologous repeat sequences of 134-167 amino acids, a transmembrane region of 23 amino acids, and a cytoplasmic domain of 164 amino acids. The predicted molecular size is greater than 270 kDa. Repeats 7-15 of the extracytoplasmic domain of the human receptor are highly homologous with the sequence recently reported for the partial cDNA for the bovine receptor (Lobel, P., Dahms, N. M., Breitmeyer, J., Chirgwin, J. M., and Kornfeld, S. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2233-2237). The nucleotide sequence for the full-length cDNA and the deduced amino acid sequence for the cation-independent mannose 6-phosphate receptor, which are reported here, are strikingly similar (99.8% identical at the nucleotide level and 99.4% identical at the amino acid level) to those recently reported for the human insulin-like growth factor II receptor from HepG2 hepatoma cells (Morgan, D. O., Edman, J.D., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature 329, 301-307). These findings support the suggestion that the cation-independent mannose 6-phosphate receptor for lysosomal enzymes is a multifunctional binding protein which is identical with the insulin-like growth factor II receptor. A cDNA construct containing the full coding sequence for the cation-independent mannose 6-phosphate receptor in the expression vector pSVL was used to transfect COS cells. Expression of the cDNA in transfected COS cells produced a cell-surface protein which co-migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with authentic human receptor, bound to an affinity column and was specifically eluted with mannose 6-phosphate, mediated cell-surface binding and endocytosis of beta-glucuronidase, and targeted the endocytosed enzyme to lysosomes.
我们从四个重叠克隆中克隆并测序了人阳离子非依赖性甘露糖6-磷酸受体的全长cDNA。9104个核苷酸的序列包含7473个核苷酸，编码一个2491个氨基酸的蛋白质。氨基酸序列包括一个40个氨基酸的推定信号序列、一个由15个134 - 167个氨基酸的同源重复序列组成的胞外结构域、一个23个氨基酸的跨膜区域和一个164个氨基酸的胞质结构域。预测的分子大小大于270 kDa。人受体胞外结构域的第7 - 15个重复序列与最近报道的牛受体部分cDNA序列高度同源（Lobel, P., Dahms, N. M., Breitmeyer, J., Chirgwin, J. M., and Kornfeld, S. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2233 - 2237）。这里报道的阳离子非依赖性甘露糖6-磷酸受体全长cDNA的核苷酸序列和推导的氨基酸序列与最近报道的来自HepG2肝癌细胞的人胰岛素样生长因子II受体的序列惊人地相似（在核苷酸水平上99.8%相同，在氨基酸水平上99.4%相同）（Morgan, D. O., Edman, J.D., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature 329, 301 - 307）。这些发现支持了这样的观点，即溶酶体酶的阳离子非依赖性甘露糖6-磷酸受体是一种多功能结合蛋白，与胰岛素样生长因子II受体相同。一个在表达载体pSVL中包含阳离子非依赖性甘露糖6-磷酸受体完整编码序列的cDNA构建体被用于转染COS细胞。cDNA在转染的COS细胞中的表达产生了一种细胞表面蛋白，该蛋白在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上与真实的人受体共迁移，与亲和柱结合并被甘露糖6-磷酸特异性洗脱，介导β-葡萄糖醛酸酶的细胞表面结合和内吞作用，并将内吞的酶靶向溶酶体。
