Varlamov O, Leiter E H, Fricker L
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Biol Chem. 1996 Jun 14;271(24):13981-6. doi: 10.1074/jbc.271.24.13981.
Carboxypeptidase E (CPE) is involved in peptide processing in the brain and various neuroendocrine tissues. In mice homozygous for the Cpefat mutation, the virtual absence of CPE activity in islets of Langerhans and pituitary was associated with a missense mutation effecting a Ser202 to Pro shift (Naggert, J. K., Fricker, L. D., Varlamov, O., Nishina, P. M., Rouille, Y., Steiner, D. F., Carroll, R. J., Paigen, B. J., and Leiter, E. H. (1995) Nat. Genet. 10, 135-142). To examine the importance of Ser202 in CPE function, several mutations in this position were generated (Pro202, Ala202, Gly202, and Phe202). When the mutant proteins were expressed in a Baculovirus system, both Phe202 and Pro202CPE were enzymatically inactive, were unable to bind to a substrate affinity column, and were not secreted from Sf9 cells. In contrast, Ala202CPE or Gly202CPE exhibited enzymatic properties similar to those of wild-type CPE and were secreted from Sf9 cells. When expressed in AtT-20 cells, a mouse pituitary-derived cell line, CPE with Pro202 and Phe202 were not secreted. Pulse-chase analysis with [35S]Met indicated that Pro202CPE was degraded in AtT-20 cells within several hours. This degradative process was blocked by incubation at 15 degrees C but not by brefeldin A or by lysosomotrophic drugs. Pulse-chase analysis using dispersed pituitary cells from C57BLKS/Lt-Cpefat/Cpefat mutant mice shows similar results; Pro202-CPE produced in these cells was not secreted but rather was degraded within 5 h. Immunofluorescence analysis of epitope-tagged CPE revealed Ser202CPE to be present primarily in secretory vesicles, whereas Pro202CPE was localized to the endoplasmic reticulum and not the secretory vesicle-like structures. These results support the previous finding that Cpefat/Cpefat mice are defective in CPE activity because of the point mutation producing the Ser202 to Pro substitution. Furthermore, these results are consistent with a model that Ser202 is important for the intracellular folding of CPE.
羧肽酶E（CPE）参与大脑和各种神经内分泌组织中的肽加工过程。在Cpefat突变纯合的小鼠中，胰岛和垂体中几乎没有CPE活性，这与一个错义突变有关，该突变导致了Ser202到Pro的转变（Naggert, J. K., Fricker, L. D., Varlamov, O., Nishina, P. M., Rouille, Y., Steiner, D. F., Carroll, R. J., Paigen, B. J., and Leiter, E. H. (1995) Nat. Genet. 10, 135 - 142）。为了研究Ser202在CPE功能中的重要性，在该位置产生了几个突变（Pro202、Ala202、Gly202和Phe202）。当突变蛋白在杆状病毒系统中表达时，Phe202和Pro202 CPE均无酶活性，无法与底物亲和柱结合，也不能从Sf9细胞中分泌出来。相比之下，Ala202 CPE或Gly202 CPE表现出与野生型CPE相似的酶学性质，并从Sf9细胞中分泌出来。当在小鼠垂体来源的AtT - 20细胞中表达时，含有Pro202和Phe202的CPE不被分泌。用[35S]Met进行脉冲追踪分析表明，Pro202 CPE在AtT - 20细胞中数小时内就被降解。在15摄氏度下孵育可阻断这种降解过程，但布雷菲德菌素A或溶酶体营养药物不能阻断。使用来自C57BLKS/Lt - Cpefat/Cpefat突变小鼠的分散垂体细胞进行脉冲追踪分析显示了类似的结果；这些细胞中产生的Pro202 - CPE不被分泌，而是在5小时内被降解。对抗表位标记的CPE进行免疫荧光分析发现，Ser202 CPE主要存在于分泌小泡中，而Pro202 CPE定位于内质网，而非分泌小泡样结构。这些结果支持了之前的发现，即Cpefat/Cpefat小鼠由于产生Ser202到Pro替代的点突变而在CPE活性方面存在缺陷。此外，这些结果与Ser202对CPE细胞内折叠很重要的模型一致。
