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J. Biol. Chem., Vol. 279, Issue 30, 31131-31138, July 23, 2004

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Appropriate Function of 11-Hydroxysteroid Dehydrogenase Type 1 in the Endoplasmic Reticulum Lumen Is Dependent on Its N-terminal Region Sharing Similar Topological Determinants with 50-kDa Esterase^*

Christoph Frick, Atanas G. Atanasov, Peter Arnold, Juris Ozols, and Alex Odermatt¶

From the Division of Nephrology and Hypertension, Department of Clinical Research, University of Berne, 3010 Berne, Switzerland and the Biochemistry Department, University of Connecticut Health Center, Farmington, Connecticut 06030

By interconverting glucocorticoids, 11-hydroxysteroid dehydrogenase type 1 (11-HSD1) exerts an important pre-receptor function and is currently considered a promising therapeutic target. In addition, 11-HSD1 plays a potential role in 7-ketocholesterol metabolism. Here we investigated the role of the N-terminal region on enzymatic activity and addressed the relevance of 11-HSD1 orientation into the endoplasmic reticulum (ER) lumen. Previous studies revealed that the luminal orientation of 11-HSD1 and 50-kDa esterase/arylacetamide deacetylase (E3) is determined by their highly similar N-terminal transmembrane domains. Substitution of Lys⁵ by Ser in 11-HSD1, but not of the analogous Lys⁴ by Ile in E3, led to an inverted topology in the ER membrane, indicating the existence of a second topological determinant. Here we identified Glu²⁵/Glu²⁶ in 11-HSD1 and Asp²⁵ in E3 as the second determinant for luminal orientation. Our results suggest that the exact location of specific residues rather than net charge distribution on either side of the helix is critical for membrane topology. Analysis of charged residues in the N-terminal domain revealed an essential role of Lys³⁵/Lys³⁶ and Glu²⁵/Glu²⁶ on enzymatic activity, suggesting that these residues are responsible for the observed stabilizing effect of the N-terminal membrane anchor on the catalytic domain of 11-HSD1. Moreover, activity measurements in intact cells expressing wild-type 11-HSD1, facing the ER lumen, or mutant K5S/K6S, facing the cytoplasm, revealed that the luminal orientation is essential for efficient oxidation of cortisol. Furthermore, we demonstrate that 11-HSD1, but not mutant K5S/K6S with cytoplasmic orientation, catalyzes the oxoreduction of 7-ketocholesterol. 11-HSD1 and E3 constructs with cytosolic orientation of their catalytic moiety should prove useful in future studies addressing the physiological function of these proteins.

Received for publication, December 15, 2003 , and in revised form, April 28, 2004.

^* This work was supported by grants from the Cloëtta Research Foundation and Swiss National Science Foundation Grant 3100AO-100060. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Division of Nephrology and Hypertension, Dept. of Clinical Research, University of Berne, Freiburgstrasse 15, 3010 Berne, Switzerland. Tel.: 41-31-632-9438; Fax: 41-31-632-9444; E-mail: alex.odermatt{at}dkf1.unibe.ch.

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