Induced and Spontaneous Mutations at Ser202 of Carboxypeptidase E: EFFECT ON ENZYME EXPRESSION, ACTIVITY, AND INTRACELLULAR ROUTING

Oleg Varlamov,Edward H Leiter,Lloyd Fricker

doi:10.1074/jbc.271.24.13981

Oleg Varlamov, Edward H Leiter + Show 1 more

Open Access

https://doi.org/10.1074/jbc.271.24.13981

Copy DOI

Abstract

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.

Highlights

Carboxypeptidase E (CPE) is involved in peptide processing in the brain and various neuroendocrine tissues
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
On expression in Sf9 insect cells using the Baculovirus system, CPE with Ser202, Gly202, Ala202, Phe202, or Pro202 was expressed at relatively high levels in the cells (Fig. 1, left)

Summary

Introduction

Carboxypeptidase E (CPE) is involved in peptide processing in the brain and various neuroendocrine tissues. Immunofluorescence analysis of epitopetagged 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. We examined CPE with a Pro202 in further detail The results of this analysis indicate that Ser202 is not essential for the production of enzymatically active CPE since Ala or Gly can replace the Ser. Analysis of the fate of the Pro202CPE in AtT-20 cells and in Cpefat/Cpefat mice is consistent with the hypothesis that this mutant form of CPE is misfolded and subsequently degraded within the cell

Methods

Results

Conclusion