Lumenal orientation and post-translational modifications of the liver microsomal 11 beta-hydroxysteroid dehydrogenase.
The topology and post-translational modifications of microsomal 11 beta-hydroxysteroid dehydrogenase (11 beta-DH) 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 11 beta-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 11 beta-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 11 beta-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 11 beta-DH concluded an absence of a flexible intradomain segment between the membranous and the lumenal domains. The lumenal localization of the 11 beta-DH requires a mechanism by which cortisol is transported to the endoplasmic reticulum of the lumen.
Ozols
J
J
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030.
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000595:Amino Acid Sequence; D000818:Animals; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006596:Hexosaminidases / Q000494:pharmacology; D006913:Hydroxysteroid Dehydrogenases / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D008562:Membrane Glycoproteins; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; D006596:Hexosaminidases