Human 11beta-hydroxysteroid dehydrogenase 1/carbonyl reductase: additional domains for membrane attachment?
11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) is a membrane integrated glycoprotein, which physiologically performs the interconversion of active and inactive glucocorticoid hormones and which also participates in xenobiotic carbonyl compound detoxification. Since 11beta-HSD 1 is fixed to the endoplasmic reticulum (ER) with a N-terminal membrane spanning domain, the enzyme is very difficult to purify in an active state. Upon expression experiments in Escherichia coli, 11beta-HSD 1 turns out to be hardly soluble without detergents. This study describes attempts to increase the solubility of 11beta-HSD 1 via mutagenesis experiments by generating several truncated forms expressed in E. coli and the yeast Pichia pastoris. Furthermore, we investigated if the codon for methionine 31 in human 11beta-HSD 1 could serve as an alternative start codon, thereby leading to a soluble form of the enzyme, which lacks the membrane spanning segment. Our results show that deletion of the hydrophobic membrane spanning domain did not alter the solubility of the enzyme. In contrast, the enzyme remained bound to the ER membrane even without the N-terminal membrane anchor. However, activity could not be found, neither with the truncated protein expressed in E. coli nor with that expressed in P. pastoris. Hydrophobicity plots proved the hydrophobic nature of 11beta-HSD 1 and indicated the existence of additional membrane attachment sites within its primary structure.
Blum
A
A
Department of Pharmacology and Toxicology, School of Medicine, Philipps-University of Marburg, Karl-von-Frisch-Strasse 1, D-35033, Marburg, Germany.
Raum
A
A
Martin
H
H
Maser
E
E
D043209:11-beta-Hydroxysteroid Dehydrogenase Type 2; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D008566:Membranes / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D008958:Models, Molecular; D010843:Pichia / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D017931:DNA Primers; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; D006913:Hydroxysteroid Dehydrogenases; D043209:11-beta-Hydroxysteroid Dehydrogenase Type 2; C468387:HSD11B2 protein, human