Lumenal Orientation and Post-translational Modifications of the Liver Microsomal 11
-Hydroxysteroid Dehydrogenase (*)
- From the Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06030
Abstract
The topology and post-translational modifications of microsomal 11β-hydroxysteroid dehydrogenase (11βDH) was investigated using the approaches of protein structure analysis. Sequence analysis of peptides generated by chemical and enzymatic cleavages revealed that carbohydrate is attached at Asn-122, −161, and −206. Enzymatic deglycosylation reactions of the protein identified the attached glycans as high mannose carbohydrates, implying that the bulk of the protein molecule is oriented on the lumenal side of the endoplasmic membrane. The carbohydrate moiety of native dehydrogenase was cleaved by endo-N-acetylglucosaminidase H without significantly affecting the 11β-DH activity. Chemical modification of cysteinyl residues, followed by amino acid sequence analysis, identified one disulfide bond linking Cys-77 and Cys-212. This disulfide bond was inaccessible to thiol reagents, unless the protein was denatured. Contrary to the partially purified 11β-DH preparations, the purified enzymatically active protein failed to bind to a 2,5′-ADP affinity column, suggesting that a conformational change has occurred in the enzyme during purification. The proposed model of the 11β-DH has a single trans-membrane segment at the N terminus, with the bulk of the polypeptide chain projecting into the lumen of endoplasmic reticulum. Limited proteolysis studies of 11β-DH concluded an absence of a flexible intradomain segment between the membranous and the lumenal domains. The lumenal localization of the 11β-DH requires a mechanism by which cortisol is transported to the endoplasmic reticulum of the lumen.
Footnotes
-
↵* This work was supported by National Institutes of Health, National Institute of General Medical Sciences Grant RO1 GM-26351. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The protein sequence reported in this study has been submitted to the Protein Identification Resource (PIR) National Biomedical Research Foundation with accession no. A44619.
-
↵1 The abbreviations used are:
- DH
-
hydroxysteroid dehydrogenase
- DTT
-
dithiothreitol
- HPLC
-
high performance liquid chromatography
- ER
-
endoplasmic reticulum
- PTH
-
phenylthiohydantoin
- PAGE
-
polyacrylamide gel electrophoresis
- endo H
-
endo-N-acetylglucosaminidase H.
-
- Received October 4, 1994.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
