Induced and Spontaneous Mutations at Ser202 of Carboxypeptidase E

doi:10.1074/jbc.271.24.13981

Induced and Spontaneous Mutations at Ser²⁰² of Carboxypeptidase E

EFFECT ON ENZYME EXPRESSION, ACTIVITY, AND INTRACELLULAR ROUTING*

From the Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461 and
^‡ The Jackson Laboratory, Bar Harbor, Maine 04609

^§ To whom correspondence should be addressed:
Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461.
Tel.: 718-430-4225; Fax: 718-430-8922; E-mail: fricker{at}aecom.yu.edu

Abstract

Carboxypeptidase E (CPE) is involved in peptide processing in the brain and various neuroendocrine tissues. In mice homozygous for the Cpe^fat 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°C but not by brefeldin A or by lysosomotrophic drugs. Pulse-chase analysis using dispersed pituitary cells from C57BLKS/Lt-Cpe^fat/Cpe^fat 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 Cpe^fat/Cpe^fat 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.

Footnotes

↵* This work was supported in part by National Institute on Drug Abuse Grant DA-04494, National Institute on Drug Abuse Research Scientist Development Award DA-00194, a grant from the Juvenile Diabetes Foundation (to L. D. F.), and a grant from the American Diabetes Association (to E. H. L.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

CPE

carboxypeptidase E

CPA

carboxypeptidase A

DMEM

Dulbecco's modified Eagle's medium

PBS

phosphate-buffered saline.
↵² G. Nagle, personal communication.
↵³ O. Varlamov, X. Xin, and L. Fricker, unpublished observation.
↵⁴ S. Almo, personal communication.
↵⁵ L. Fricker and E. H. Leiter, manuscript in preparation.
↵⁶ L. Song and L. Fricker, unpublished observations.
- Received February 12, 1996.
- Revision received March 25, 1996.