Functional Expression, Characterization, and Purification of the Catalytic Domain of Human 11-β-Hydroxysteroid Dehydrogenase Type 1
2001; Elsevier BV; Volume: 276; Issue: 24 Linguagem: Inglês
10.1074/jbc.m011142200
ISSN1083-351X
AutoresElizabeth A. Walker, Anya M. Clark, Martin Hewison, Jon P. Ride, Paul M. Stewart,
Tópico(s)Coenzyme Q10 studies and effects
Resumo11-β-hydroxysteroid dehydrogenase type 1 catalyzes the conversion of cortisone to hormonally active cortisol and has been implicated in the pathogenesis of a number of disorders including insulin resistance and obesity. The enzyme is a glycosylated membrane-bound protein that has proved difficult to purify in an active state. Extracted enzyme typically loses the reductase properties seen in intact cells and shows principally dehydrogenase activity. The C-terminal catalytic domain is known to contain a disulfide bond and is located within the lumen of the endoplasmic reticulum, anchored to the membrane by a single N-terminal transmembrane domain. We report here the functional expression of the catalytic domain of the human enzyme, without the transmembrane domain and the extreme N terminus, inEscherichia coli. Moderate levels of soluble active protein were obtained using an N-terminal fusion with thioredoxin and a 6xHis tag. In contrast, the inclusion of a 6xHis tag at the C terminus adversely affected protein solubility and activity. However, the highest levels of active protein were obtained using a construct expressing the untagged catalytic domain. Nonreducing electrophoresis revealed the presence of both monomeric and dimeric disulfide bonded forms; however, mutation of a nonconserved cysteine residue resulted in a recombinant protein with no intermolecular disulfide bonds but full enzymatic activity. Using the optimal combination of plasmid construct and E. coli host strain, the recombinant protein was purified to apparent homogeneity by single step affinity chromatography. The purified protein possessed both dehydrogenase and reductase activities with a K m of 1.4 μm for cortisol and 9.5 μm for cortisone. This study indicates that glycosylation, the N-terminal region including the transmembrane helix, and intermolecular disulfide bonds are not essential for enzyme activity and that expression in bacteria can provide active recombinant protein for future structural and functional studies. 11-β-hydroxysteroid dehydrogenase type 1 catalyzes the conversion of cortisone to hormonally active cortisol and has been implicated in the pathogenesis of a number of disorders including insulin resistance and obesity. The enzyme is a glycosylated membrane-bound protein that has proved difficult to purify in an active state. Extracted enzyme typically loses the reductase properties seen in intact cells and shows principally dehydrogenase activity. The C-terminal catalytic domain is known to contain a disulfide bond and is located within the lumen of the endoplasmic reticulum, anchored to the membrane by a single N-terminal transmembrane domain. We report here the functional expression of the catalytic domain of the human enzyme, without the transmembrane domain and the extreme N terminus, inEscherichia coli. Moderate levels of soluble active protein were obtained using an N-terminal fusion with thioredoxin and a 6xHis tag. In contrast, the inclusion of a 6xHis tag at the C terminus adversely affected protein solubility and activity. However, the highest levels of active protein were obtained using a construct expressing the untagged catalytic domain. Nonreducing electrophoresis revealed the presence of both monomeric and dimeric disulfide bonded forms; however, mutation of a nonconserved cysteine residue resulted in a recombinant protein with no intermolecular disulfide bonds but full enzymatic activity. Using the optimal combination of plasmid construct and E. coli host strain, the recombinant protein was purified to apparent homogeneity by single step affinity chromatography. The purified protein possessed both dehydrogenase and reductase activities with a K m of 1.4 μm for cortisol and 9.5 μm for cortisone. This study indicates that glycosylation, the N-terminal region including the transmembrane helix, and intermolecular disulfide bonds are not essential for enzyme activity and that expression in bacteria can provide active recombinant protein for future structural and functional studies. 11-β-hydroxysteroid dehydrogenase short chain dehydrogenase/reductase endoplasmic reticulum isopropyl-β-d-thiogalactopyranoside polyacrylamide gel electrophoresis In mammalian tissues, two isozymes of 11-β-hydroxysteroid dehydrogenase (11β-HSD)1catalyze the interconversion of hormonally active C11-hydroxylated corticosteroids (cortisol, corticosterone) and their inactive C11-keto metabolites (cortisone, 11-dehydrocorticosterone). The 11β-HSD1 and 11β-HSD2 isozymes share only 14‥ identity and are separate gene products with different physiological roles, regulation, and tissue distribution (1Stewart P.M. Krozowski Z.S. Vitam. Horm. 1999; 57: 249-324Crossref PubMed Scopus (447) Google Scholar). 11β-HSD2 is a high affinity NAD-dependent dehydrogenase that protects the mineralocorticoid receptor from glucocorticoid excess; mutations in the HSD11B2 gene explain an inherited form of hypertension, the syndrome of apparent mineralocorticoid excess in which cortisol acts as a potent mineralocorticoid (2Stewart P.M. Krozowski Z.S. Gupta A. Milford D.V. Howie A.J. Sheppard M.C. Whorwood C.B. Lancet. 1996; 347: 88-91Abstract PubMed Scopus (191) Google Scholar). By contrast, 11β-HSD1 is a relatively low affinity NADP-dependent enzyme that acts predominantly as a reductase in vivo, although extracted enzyme typically shows predominant dehydrogenase activity (3Tannin G.M. Agarwal A.K. Monder C. New M.I. White P.C. J. Biol. Chem. 1991; 266: 16653-16658Abstract Full Text PDF PubMed Google Scholar). By converting cortisone to cortisol, 11β-HSD1 facilitates glucocorticoid hormone action in key target tissues such as liver and adipose tissue, and as such has been implicated in a number of disorders including insulin resistance and central obesity (4Kotelevtsev Y. Holmes M.C. Burchell A. Houston P.M. Schmoll D. Jamieson P. Best R. Brown R. Edwards C.R. Seckl J.R. Mullins J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 23: 14924-14929Crossref Scopus (806) Google Scholar, 5Bujalska I.J. Kumar S. Stewart P.M. Lancet. 1997; 349: 1210-1213Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar).11β-HSD1 is a member of the short chain alcohol dehydrogenase family, also known as the short chain dehydrogenase/reductases (SDRs). SDRs typically exhibit residue identities only at the 15–30‥ level, indicative of early duplicatory origins and extensive divergence (6Jornvall H. Persson B. Krook M. Atrian S. Gonzales-Duarte J.J. Gosh D. Biochemistry. 1995; 34: 6003-6013Crossref PubMed Scopus (1152) Google Scholar, 7Krozowski Z. J. Steroid Biochem. Mol. Biol. 1994; 51: 125-130Crossref PubMed Scopus (99) Google Scholar, 8Jornvall H. Hoog J.O. Persson B. FEBS Lett. 1999; 445: 261-264Crossref PubMed Scopus (171) Google Scholar). However, in contrast to other SDR members, 11β-HSD1 is unusual in possessing a single transmembrane helix at the N terminus. This is intrinsic to the endoplasmic reticulum (ER) membrane, with a short 5-amino acid N-terminal region on the cytosolic side and the main catalytic domain of the protein facing the lumen of the ER (9Ozols J. J. Biol. Chem. 1995; 270: 2305-2312Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. J. Biol. Chem. 1999; 274: 28762-28770Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). The importance of the transmembrane domain on 11β-HSD1 activity has been studied but with inconclusive results. An N-terminally truncated variant of rat 11β-HSD1 was expressed in COS cells and reported to be inactive (11Obeid J. Curnow K.M. Aisenberg J. White P.C. Mol. Endocrinol. 1993; 7: 154-160Crossref PubMed Scopus (0) Google Scholar, 12Mercer W. Obeyesekere V. Smith R. Krozowski Z. Mol. Cell. Endocrinol. 1993; 92: 247-251Crossref PubMed Scopus (41) Google Scholar). However, this construct encoded a protein that had lost more than just the transmembrane helix and therefore may have lost vital parts of the enzymatic domain. In addition, because the expression studies were performed in COS and Chinese hamster ovary cells, the truncated protein would have been targeted (because of the lack of signal sequence) to the cytosol and not the ER. The lumen of the ER promotes the formation of disulfide bonds, and studies have indicated that there are important intrachain disulfide bonds within the 11β-HSD1 protein (9Ozols J. J. Biol. Chem. 1995; 270: 2305-2312Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar).The catalytic domain is glycosylated (13Blum A. Martin H.-J. Maser E. Biochem. Biophys. Res. Commun. 2000; 276: 428-434Crossref PubMed Scopus (32) Google Scholar, 14Agarwal A.K. Tusie-Luna M.T. Monder C. White P.C. Mol. Endocrinol. 1990; 4: 1827-1832Crossref PubMed Scopus (164) Google Scholar, 15Agarwal A.K. Mune T. Monder C. White P.C. Biochim. Biophys. Acta. 1995; 1248: 70-74Crossref PubMed Scopus (45) Google Scholar), which is in agreement with a lumenal orientation. Experiments to resolve the importance of glycosylation have also yielded varying results. Enzymatic deglycosylation of rabbit (9Ozols J. J. Biol. Chem. 1995; 270: 2305-2312Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) and human (13Blum A. Martin H.-J. Maser E. Biochem. Biophys. Res. Commun. 2000; 276: 428-434Crossref PubMed Scopus (32) Google Scholar) 11β-HSD1 has indicated that glycosylation is not important for enzyme activity. However, partial inhibition of glycosylation of the rat enzyme by tunicamycin decreased dehydrogenase activity but not reductase activity (14Agarwal A.K. Tusie-Luna M.T. Monder C. White P.C. Mol. Endocrinol. 1990; 4: 1827-1832Crossref PubMed Scopus (164) Google Scholar), and mutation of the rat (15Agarwal A.K. Mune T. Monder C. White P.C. Biochim. Biophys. Acta. 1995; 1248: 70-74Crossref PubMed Scopus (45) Google Scholar) and human (13Blum A. Martin H.-J. Maser E. Biochem. Biophys. Res. Commun. 2000; 276: 428-434Crossref PubMed Scopus (32) Google Scholar) sequences at putativeN-glycosylation sites resulted in reduced or abolished activity.Expression of human (and squirrel monkey) clones of 11β-HSD1 has been achieved in COS cells (11Obeid J. Curnow K.M. Aisenberg J. White P.C. Mol. Endocrinol. 1993; 7: 154-160Crossref PubMed Scopus (0) Google Scholar, 12Mercer W. Obeyesekere V. Smith R. Krozowski Z. Mol. Cell. Endocrinol. 1993; 92: 247-251Crossref PubMed Scopus (41) Google Scholar), HEK cells (16Bujalska I. Shimojo M. Howie A. Stewart P.M. Steroids. 1997; 77: 77-82Crossref Scopus (95) Google Scholar), and the yeastPichia pastoris (13Blum A. Martin H.-J. Maser E. Biochem. Biophys. Res. Commun. 2000; 276: 428-434Crossref PubMed Scopus (32) Google Scholar, 17Blum A. Martin H. Maser E. Toxicology. 2000; 144: 113-120Crossref PubMed Scopus (33) Google Scholar) using a variety of vectors. This has led to ambiguous kinetic results with over 10-fold variation inK m values and often significant differences in activity between whole cells and lysates. These systems have not yielded large amounts of pure recombinant protein, and no structural information has come from them. Overexpression of 11β-HSD1 in bacterial cells has been reported (17Blum A. Martin H. Maser E. Toxicology. 2000; 144: 113-120Crossref PubMed Scopus (33) Google Scholar), but the resulting protein was inactive. Failure to obtain activity was attributed to either insolubility of the protein, and subsequent refolding problems, or a lack of glycosylation. In this study we sought to maximize the production of soluble recombinant human 11β-HSD1 withinEscherichia coli by varying the expression construct, the host strain, and the incubation conditions. In particular, because 11β-HSD1 is thought to contain disulfide bonds, we have assessed the value of E. coli strains that promote disulfide bond formation within the cytoplasm of the bacterium through mutations in the genes encoding thioredoxin reductase and/or glutathione reductase. We also tested the effect of thioredoxin fusions, histidine tags, glycosylation status, the presence of the transmembrane domain, and mutation of a nonconserved cysteine residue on the activity of human 11β-HSD1. Through these measures, we arrived at an optimal construct and E. coli host combination for producing sufficient protein for purification.DISCUSSION11-β-Hydroxysteroid dehydrogenase activity was first documented in the rat liver in the 1950s, but it was the studies of Tanninet al. (3Tannin G.M. Agarwal A.K. Monder C. New M.I. White P.C. J. Biol. Chem. 1991; 266: 16653-16658Abstract Full Text PDF PubMed Google Scholar) that resulted in the enzymatic characterization, purification, and cloning of the liver-type 11β-HSD isozyme. With the cloning of a second “kidney-type” 11β-HSD isozyme, the liver-type isozyme is now termed 11β-HSD1.The importance of these isozymes in the metabolism and clearance of glucocorticoids is well established; in addition, these enzymes are intricately involved in the pathogenesis of human diseases. For example, 11β-HSD2 is implicated in hypertension and fetal growth retardation (1Stewart P.M. Krozowski Z.S. Vitam. Horm. 1999; 57: 249-324Crossref PubMed Scopus (447) Google Scholar). Specifically, for 11β-HSD1, emerging data have highlighted the role of this enzyme in modulating insulin sensitivity and visceral adiposity. Thus mice lacking the HSD11B1gene are resistant to hyperglycemia of stress/feeding because of a failure to activate glucocorticoid within the liver and stimulate gluconeogenesis (4Kotelevtsev Y. Holmes M.C. Burchell A. Houston P.M. Schmoll D. Jamieson P. Best R. Brown R. Edwards C.R. Seckl J.R. Mullins J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 23: 14924-14929Crossref Scopus (806) Google Scholar). Improvements in insulin sensitivity in normal volunteers given the 11β-HSD1 inhibitor carbenoxolone support such a concept (27Walker B.R. Connacher A.A. Lindsay R.M. Webb D.J. Edwards C. J. Clin. Endocrinol. Metab. 1995; 80: 3155-3159Crossref PubMed Google Scholar). Similarly in visceral adipose tissue, 11β-HSD1 acts locally to generate active glucocorticoid concentrations, thereby stimulating adipogenesis (5Bujalska I.J. Kumar S. Stewart P.M. Lancet. 1997; 349: 1210-1213Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar, 28Bujalska I.J. Kumar S. Hewison M. Stewart P.M. Endocrinology. 1999; 140: 3188-3196Crossref PubMed Google Scholar). Defect in the activity of 11β-HSD1 is also thought to underpin an inherited form of polycystic ovary syndrome, the syndrome of apparent cortisone reductase deficiency (29Phillipov G. Palermo M. Shackleton C.H.L. J. Clin. Endocrinol. Metab. 1996; 81: 3855-3860Crossref PubMed Scopus (72) Google Scholar). Finally, further studies are investigating the role of the enzyme in central nervous system tissues and its relationship to neurodegenerative diseases (30Rajan V. Edwards C.R. Seckl J.R. J. Neurosci. 1996; 16: 65-70Crossref PubMed Google Scholar). In each case though, the exciting concept has emerged that modulation of 11β-HSD1 expression may represent a novel mechanism to modulate glucocorticoid action at the tissue level without changing circulating concentrations, thereby precipitating states of glucocorticoid excess or deficiency. Thus there is a clear clinical need to undertake a detailed characterization of the human 11β-HSD1 isozyme with a view to its purification.11β-HSD1 belongs to the SDR superfamily, as defined on the basis of an N-terminal nucleotide binding motif, a central active site, and consensus sequence data. Sophisticated analytical approaches suggest that there are over 1000 members of this superfamily (6Jornvall H. Persson B. Krook M. Atrian S. Gonzales-Duarte J.J. Gosh D. Biochemistry. 1995; 34: 6003-6013Crossref PubMed Scopus (1152) Google Scholar, 7Krozowski Z. J. Steroid Biochem. Mol. Biol. 1994; 51: 125-130Crossref PubMed Scopus (99) Google Scholar, 8Jornvall H. Hoog J.O. Persson B. FEBS Lett. 1999; 445: 261-264Crossref PubMed Scopus (171) Google Scholar), with only one residue (Tyr) being strictly conserved. A lysine 4 residues downstream and a serine 14 residues upstream are also largely conserved; all these residues are present in 11β-HSD1. A model for catalysis of SDRs has been proposed on the basis of these residues (31Tanabe T. Tanaka N. Uchikawa K. Kabashima T. Ito K. Nonaka T. Mitsui Y. Tsuru M. Yoshimoto T.J. Biochemistry. 1998; 124: 634-641Google Scholar). Binding of the coenzyme, NAD(H) or NADP(H), is in the N-terminal part of the molecule involving a common protein folding arrangement of α- and β-strands (“Rossmann” fold) associated with a common Gly(Xaa)3GlyXaaGly motif (also found in 11β-HSD1). The critically important tyrosine seems to maintain a fixed position relative to the scaffolding of the Rossmann fold and the cofactor position, whereas the substrate-binding pocket alters in such a way that the dehydrogenation/reduction reaction site is brought into bonding distance of the tyrosine hydroxyl group. The tyrosine therefore acts as a basic catalyst, the lysine binds to NAD/P(H) and lowers the pK a value of the tyrosine, and the serine plays a subsidiary role of stabilizing substrate binding (31Tanabe T. Tanaka N. Uchikawa K. Kabashima T. Ito K. Nonaka T. Mitsui Y. Tsuru M. Yoshimoto T.J. Biochemistry. 1998; 124: 634-641Google Scholar, 32Tanaka N. Nonaka T. Tanabe T. Yoshimoto T. Tsuru D. Mitsui Y. Biochemistry. 1996; 18: 7715-7730Crossref Scopus (215) Google Scholar).Several groups have evaluated the importance of the N-terminal domain of 11β-HSD1. Recently it has been shown that the orientation of the enzyme within the ER is determined by sequences close to the N terminus (10Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. J. Biol. Chem. 1999; 274: 28762-28770Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Chimeric proteins where the N-terminal regions, including the membrane anchors, of the 11β-HSD1 and 11β-HSD2 enzymes were exchanged adopted inverted orientations in the ER membrane (10Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. J. Biol. Chem. 1999; 274: 28762-28770Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Neither protein was catalytically active. However, mutation of a single lysine residue close to the N terminus of type 1 resulted in an inverted orientation without loss of activity. These results suggest that the N-terminal anchor is required for both activity and correct orientation, although it should be noted that the sequences exchanged in the chimeras included much more than just the transmembrane helix. Mercer et al. (12Mercer W. Obeyesekere V. Smith R. Krozowski Z. Mol. Cell. Endocrinol. 1993; 92: 247-251Crossref PubMed Scopus (41) Google Scholar) reported that expression of an N-terminally truncated 11β-HSD1 did not produce a soluble protein. However, these studies and others have employed mammalian expression systems where such constructs would not be appropriately targeted to the ER, and hence correct folding and disulfide bond formation may not have been facilitated.In this study, using a series of bacterial expression constructs, we have shown that the activity of human 11β-HSD1 does not depend on the N-terminal domain. Constructs where the N-terminal region had been removed (pET32CD and pET21CD) exhibited higher levels of expression and activity than constructs containing the entire 11β-HSD1 sequence. Moreover, inclusion of the transmembrane domain, either with or without the thioredoxin fusion partner, failed to produce soluble active protein. This is in agreement with a study carried out by Blum et al. (13Blum A. Martin H.-J. Maser E. Biochem. Biophys. Res. Commun. 2000; 276: 428-434Crossref PubMed Scopus (32) Google Scholar), in which the complete human 11β-HSD1 sequence was expressed in E. coli and resulted in a protein that was virtually insoluble, difficult to purify, and completely inactive.We also investigated expression systems in which thioredoxin, the product of the E. coli TrxA gene (25LaVallie E.R. DiBlasio E.A. Kovacic S. Grant K.L. Schendel P.F. McCoy J.M.A. Bio/Technology. 1993; 11: 187-193Crossref PubMed Scopus (825) Google Scholar), was a fusion partner. In many cases heterologous proteins produced as thioredoxin fusion proteins are correctly folded and display full biological activity (25LaVallie E.R. DiBlasio E.A. Kovacic S. Grant K.L. Schendel P.F. McCoy J.M.A. Bio/Technology. 1993; 11: 187-193Crossref PubMed Scopus (825) Google Scholar,33Morris J.C. Neben S. Bennett F. Finnerty H. Long A. Beier D.R. Kovacic S. McCoy J.M. DiBlasio-Smith E. LaVallie E.R. Caruso A. Calvetti J. Morris G. Weich N. Paul S.R. Crosier P.S. Turner K.J. Wood C.R. Exp. Hematol. 1996; 24: 1369-1376PubMed Google Scholar, 34Abdulaev N.G. Ngo T. Chen R. Lu Z. Ridge K.D. J. Biol. Chem. 2000; 275: 39354-39363Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 35Ribas A.V. Ho P.L. Tanizaki M.M. Raw I. Nascimento A.L. Biotechnol. Appl. Biochem. 2000; 31: 91-94Crossref PubMed Scopus (17) Google Scholar). This has been thought to be caused by the small, highly soluble nature of thioredoxin, which also has robust folding characteristics (36Homlgrem A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). However, in our study proteins produced as a fusion with thioredoxin at the N terminus (pET32CD and pET32FL) showed no overall increase in the levels of soluble protein when compared with nonfusion constructs (pET21CD and pET21CDH), indicating that such fusions are not always profitable. Similarly, fusion of a 6xHis tag at the C terminus, as a means to simplify purification, was also detrimental to the solubility, and particularly the activity, of the enzyme. Residues close to the C terminus of SDRs may frequently be important in substrate binding (37Duax W.L. Ghosh D. Pletnev V. Vitam. Horm. 2000; 58: 121-148Crossref PubMed Google Scholar), and modifications in this region may thus affect protein structure to the detriment of enzyme activity.This study also clearly resolves the issue of whether glycosylation is required for the activity of the human enzyme. Studies on the rat 11β-HSD1 enzyme indicated that partial inhibition of glycosylation with tunicamycin inhibited dehydrogenase activity by 50‥ but had no effect on reductase activity (14Agarwal A.K. Tusie-Luna M.T. Monder C. White P.C. Mol. Endocrinol. 1990; 4: 1827-1832Crossref PubMed Scopus (164) Google Scholar). Mutagenesis of the first of two potential N-glycosylation sites reduced dehydrogenase and reductase activities by 75 and 50‥, respectively, whereas mutagenesis of the second site completely abolished activity (15Agarwal A.K. Mune T. Monder C. White P.C. Biochim. Biophys. Acta. 1995; 1248: 70-74Crossref PubMed Scopus (45) Google Scholar). Conversely, studies carried out on the rabbit enzyme, which like the human homologue contains three potential glycosylation sites, suggest that glycosylation is not important for enzyme activity. No alteration in activity could be observed after complete deglycosylation of rabbit 11β-HSD1 (9Ozols J. J. Biol. Chem. 1995; 270: 2305-2312Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Conflicting studies on the human enzyme have also been reported. Recently, human 11β-HSD1 has been expressed in E. coli, where the biosynthesis of N-linked glycoproteins does not occur. This resulted in a recombinant protein that was completely devoid of enzyme activity (17Blum A. Martin H. Maser E. Toxicology. 2000; 144: 113-120Crossref PubMed Scopus (33) Google Scholar). The same group also investigated the effects of deglycosylation on human 11β-HSD1 purified from liver and recombinant protein produced by the yeastP. pastoris (13Blum A. Martin H.-J. Maser E. Biochem. Biophys. Res. Commun. 2000; 276: 428-434Crossref PubMed Scopus (32) Google Scholar). Site-directed mutagenesis of the three potential glycosylation sites yielded an inactive protein from yeast cells as assessed using metyrapone and metyrapol as the substrates. However, the enzyme purified from human liver, upon complete deglycosylation, remained fully active. The results here agree with the latter experiment and clearly show that nonglycosylated enzymatically active 11β-HSD1 can be generated within E. coli, with the recombinant enzyme possessing both reductase and dehydrogenase activities with similar kinetic properties to those reported previously from mammalian expression systems. Glycosylation is therefore not required for activity or protein folding, although it could still be important for protein stability within the endoplasmic reticulum.All the constructs used in this study gave only moderate levels of soluble protein but a high proportion of protein in an insoluble form. The lack of protein solubility in E. coli is a complex event with many contributing factors. Although fusion with heterologous proteins may sometimes help to redress many solubility problems, another factor that may be important is the inability of the recombinant protein to form key disulfide bonds in the reducing environment of the bacterial cytoplasm (38Derman A.I. Beckwith J. J. Bacteriol. 1991; 173: 7719-7722Crossref PubMed Scopus (160) Google Scholar). Rabbit 11β-HSD1 is known to contain an intrachain disulfide bond (9Ozols J. J. Biol. Chem. 1995; 270: 2305-2312Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), and therefore we investigated the expression levels and activity of our recombinant proteins in a variety of host E. coli strains, some of which have been developed to promote disulfide bond formation. WithinE. coli at least two systems are responsible for reducing disulfide bonds that form in the cytoplasm; the thioredoxin system that consists of thioredoxin reductase and thioredoxin and the glutaredoxin system that includes glutathione reductase, glutathione, and glutaredoxins. We evaluated this using three separate E. coli strains. It was anticipated that disulfide bonds, and therefore solubility and activity of the soluble protein, would be enhanced by the use of AD494(DE3) and particularly the Origami(DE3) strain. In effect, the reverse was observed with the highest levels of soluble protein and enzyme activity being observed using BL21(DE3) as the host strain. This result could imply that the intramolecular disulfide bond observed in the rabbit 11β-HSD1 protein (9Ozols J. J. Biol. Chem. 1995; 270: 2305-2312Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) is not present in the human enzyme, although this awaits experimental confirmation.We also tested for the presence of interchain disulfide bonds by probing Western blots of SDS-PAGE gels of bacterial lysates run under both reducing and nonreducing conditions. Both dimer and monomer bands were identified in the nonreducing lanes, suggesting that some of the recombinant protein exists in an interchain disulfide-bonded form. Examination of human liver extracts indicated that the native 11β-HSD1 enzyme existed in a similar combination of monomeric and dimeric forms, proving that the heterogeneity was not a consequence of expression in the bacterial system. However, this heterogeneity not only complicates the purification of 11β-HSD1, as has been noted previously for native enzyme (9Ozols J. J. Biol. Chem. 1995; 270: 2305-2312Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), but could also hinder crystallographic analysis because crystal growth requires pure protein in a homogeneous form. Tests on extracts of mouse liver, however, detected no intermolecular disulfide bridges. Because previous reports also suggested that rabbit 11β-HSD1 contains no intermolecular disulfide bonds (9Ozols J. J. Biol. Chem. 1995; 270: 2305-2312Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), we investigated the effect of mutating the additional cysteine (Cys-272), found only in the human sequence, to the corresponding residue from the most closely related 11β-HSD1 sequence (squirrel monkey). Expression of the resulting mutant (pET21CD-C272S) in the optimized bacterial expression system resulted in a protein with kinetic properties indistinguishable from the wild-type recombinant protein. However, nonreducing gels showed that the ability to form the interchain disulfide bonds had been abolished. Previous structural studies on other SDRs suggest that they exist naturally as nondisulfide-bonded dimers or tetramers (reviewed in Ref. 37Duax W.L. Ghosh D. Pletnev V. Vitam. Horm. 2000; 58: 121-148Crossref PubMed Google Scholar). The results here suggest that Cys-272 of human 11β-HSD1 may be involved in disulfide bonds between adjacent polypeptide chains of the enzyme, possibly stabilizing the dimeric form. However, this property does not seem to be vital to the activity of the enzyme.Using a modified expression construct that included an N-terminal 6xHis tag, we developed a simple purification protocol that allowed 157-fold purification of recombinant human 11β-HSD1 in one step from crude cell lysates. The purified protein demonstrated activity in both dehydrogenase and reductase directions with K m values of 1.4 μm for cortisol and 9.5 μmfor cortisone.In conclusion this study has shown that, despite reports to the contrary, bacterial expression systems have the potential to produce active soluble 11β-HSD1 protein. The results also demonstrate conclusively that the N-terminal region, containing the transmembrane domain, glycosylation, and interchain disulfide bonds are not essential for the activity of this enzyme. For the first time, active soluble 11β-HSD1 has been produced in vitro and purified to apparent homogeneity. This will now allow detailed functional analysis of the enzyme using E. coli-produced protein and facilitate future structure/crystallographic studies. In mammalian tissues, two isozymes of 11-β-hydroxysteroid dehydrogenase (11β-HSD)1catalyze the interconversi
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