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J. Biol. Chem., Vol. 276, Issue 41, 37929-37933, October 12, 2001

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Molecular Cloning and Expression of Human Bile Acid -Glucosidase^*

Heidrun Matern, Henrike Boermans, Friedrich Lottspeich^§, and Siegfried Matern

From the Department of Internal Medicine III, Rheinisch-Westfälische Technische Hochschule Aachen, 52057 Aachen, Germany and ^§ Genzentrum Martinsried, 82152 Martinsried, Germany

Received for publication, May 11, 2001, and in revised form, July 26, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A novel microsomal -glucosidase was recently purified and characterized from human liver that catalyzes the hydrolysis of bile acid 3-O-glucosides as endogenous compounds. The primary structure of this bile acid -glucosidase was deduced by cDNA cloning on the basis of the amino acid sequences of peptides obtained from the purified enzyme by proteinase digestion. The isolated cDNA comprises 3639 base pairs containing 524 nucleotides of 5'-untranslated and 334 nucleotides of 3'-untranslated sequences including the poly(A) tail. The open reading frame predicts a 927-amino acid protein with a calculated M_r of 104,648 containing one putative transmembrane domain. Data base searches revealed no homology with any known glycosyl hydrolase or other functionally identified protein. The cDNA sequence was found with significant identity in the human chromosome 9 clone RP11-112J3 of the human genome project. The recombinant enzyme was expressed in a tagged form in COS-7 cells where it displayed bile acid -glucosidase activity. Northern blot analysis of various human tissues revealed high levels of expression of the bile acid -glucosidase mRNA (3.6-kilobase message) in brain, heart, skeletal muscle, kidney, and placenta and lower levels of expression in the liver and other organs.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A microsomal -glucosidase has been previously purified to apparent homogeneity from human liver (1) and characterized as a novel enzyme that is not identical with the known -glucosidases described in human tissues, the lysosomal enzyme glucosylceramidase (EC 3.2.1.45, Refs. 2 and 3), the membrane-bound lactase-phlorizin hydrolase found in the brush-border of the small intestine (EC 3.2.1.62/108, Ref. 4), and a cytosolic broad specificity -glucosidase (5). The purified human liver microsomal -glucosidase preparation exhibited a 100-kDa protein band in SDS-polyacrylamide gel electrophoresis and catalyzed the hydrolysis of bile acid 3-O-glucosides (1). Bile acid 3-O-glucosides had been shown to be synthesized in human liver microsomes by a sugar nucleotide-independent mechanism (6) and had been identified in humans as naturally occurring bile acid conjugates (7). Since these compounds are at present the only known natural substrates of the human liver microsomal -glucosidase the enzyme was named bile acid -glucosidase. To provide a basis for the understanding of the physiological role of this enzyme attempts have been undertaken to define its primary structure. The present report describes the molecular cloning and expression of a cDNA encoding human bile acid -glucosidase.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein Purification and Sequencing-- Bile acid -glucosidase was isolated from human liver microsomes as described previously (1). The purified protein was run on a 7.5% SDS-polyacrylamide gel under reducing conditions. After staining with Coomassie Blue R250 the 100-kDa band was cut out. Following digestion of the protein with endoproteinase Lys-C directly in the gel, peptides were separated by reverse phase high performance liquid chromatography. Amino acid sequences of the purified peptides were determined by automated Edman degradation as described previously (8).

cDNA Synthesis-- A BLAST sequence similarity search of a human EST data base with peptides from human bile acid -glucosidase resulted in two EST clones (GenBank^TM accession numbers T09191 and AA063366). From the cDNA sequence of these clones two oligonucleotide primers were derived representing determined sequences of purified peptides (P3 and P4, Fig. 1). With these primers a 894-base pair cDNA fragment was amplified by PCR¹ using Marathon-Ready human liver cDNA (CLONTECH) as the template. Based on the sequence of the first PCR product, the 5'-sequence was extended with five rounds of PCR using 5'-RACE with Marathon-Ready cDNA from human liver. Final extension of the 5'-end was performed with a SMART RACE cDNA amplification kit (CLONTECH) using poly(A) RNA (1 µg) prepared from HepG2 cells, which show endogenous expression of bile acid -glucosidase activity. SMART RACE-PCR amplifications gave optimal results in the presence of 5 M GC Melt (CLONTECH) due to the high percentage of GC residues including stretches of multiple GC repeats in this region. The sequence of the 3'-untranslated region was determined by 3'-RACE experiments using Marathon-Ready human liver cDNA as the template. A full-length -glucosidase cDNA was generated between base pair positions 348-3305 (Fig. 1) by PCR amplification using reverse-transcribed total RNA from HepG2 cells or Marathon-Ready human liver cDNA as the templates, which yielded the same cDNA products. The PCR products were subcloned into pBluescript II KS. The nucleotide sequence was obtained for both strands using the automated fluorescent dye terminator technique (PerkinElmer ABI model 373A). Each PCR product was analyzed by sequencing at least 10 separate clones.

Expression of -Glucosidase-- -Glucosidase-coding cDNA from pBluescript II containing 177 bases upstream from the translation initiation site was inserted into the NotI/EcoRI sites of the eukaryotic expression vector pcDNA3.1() (Invitrogen). To produce the -glucosidase as a fusion protein containing at its carboxyl-terminal end a Strep-tag II peptide (WSHPQFEK), the tag sequence (IBA, Göttingen, Germany) was inserted in-frame into the EcoRI/AflII sites of the pcDNA3.1--glucosidase construct to yield a recombinant vector coding for the Strep-tagged -glucosidase.

Plasmid DNA (10 µg) was transiently transfected into COS-7 cells (1.5 × 10⁷ cells/100-mm dish) by the DEAE-dextran method in the presence of chloroquine. Cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 100 mg/liter streptomycin, and 60 mg/liter penicillin. At 24 h post-transfection cells were split 1:2, and after an additional 24 h in culture medium, cells were washed with ice-cold phosphate-buffered saline and lysed at 4 °C in this buffer by brief sonication in the presence of proteinase inhibitors (complete mixture tablets, Roche Molecular Biochemicals). After centrifugation at 100,000 × g for 60 min the pellet was taken up in 0.25 M sucrose containing 5 mM Tris-HCl, pH 7.4, and proteinase inhibitors (400 µl of buffer/100-mm dish of COS-7 cells). Expression of -glucosidase was monitored by immunoblotting and determination of enzyme activity in fractions obtained from cell lysates.

General Methods and Analyses-- Standard methods for molecular biology were used (9). Determination of -glucosidase activity toward the bile acid lithocholic acid glucoside as substrate, SDS-polyacrylamide gel electrophoresis, and determination of protein were performed as described previously (1).

For Northern blot analysis a human multiple tissue Northern blot (CLONTECH) was used representing 1 µg of poly(A) RNA from each of several human tissues. Hybridization was performed with an antisense RNA probe synthesized in vitro and labeled with digoxigenin using a DIG RNA labeling kit (Roche Molecular Biochemicals). The template for the RNA probe was a cDNA fragment of nucleotides 2411-3305 of the -glucosidase cDNA (Fig. 1) subcloned into pBluescript II KS. Prehybridization and hybridization were performed at 68 °C using DIG Easy Hyb (Roche Molecular Biochemicals). The blots were washed at high stringency conditions as suggested by the manufacturer (0.1× SSC (15 mM sodium chloride, 1.5 mM sodium citrate, pH 7.0) and 0.1% SDS at 68 °C for the two final washes). The hybridized probe was detected by a chemiluminescent technique with 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1^3,7]decan]-4-yl)phenyl phosphate (CSPD, Roche Molecular Biochemicals) according to the instructions of the manufacturer. The same blot was then stripped and hybridized with a digoxigenin-labeled control human cDNA -actin probe.

Immunoblotting was performed after transfer of proteins from SDS-polyacrylamide gels to nitrocellulose membranes. Blots were blocked, treated with a 1:2000 dilution of a Strep-tag II-specific antiserum from rabbits (IBA), and washed as suggested by the supplier. The bound antibodies were visualized using an alkaline phosphatase-conjugated goat anti-rabbit secondary antibody, 5-bromo-4-chloro-3-indolyl phosphate, and nitro blue tetrazolium.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

cDNA Cloning, Nucleotide Sequence, and Predicted Amino Acid Sequence of -Glucosidase-- The 100-kDa protein of the purified bile acid -glucosidase (1) was subjected to microsequencing. The inability to generate amino-terminal sequence data by Edman degradation suggested that the amino terminus was blocked. Therefore, protein sequences were obtained from four peptide fragments (P1-P4) derived after digestion of the purified -glucosidase with endoproteinase Lys-C. The position of peptides P1-P4 within the cDNA and the amino acid sequences are indicated in Fig. 1. Based on the amino acid sequences of peptides P3 and P4 (Fig. 1) a cDNA fragment of 894 base pairs was initially amplified by PCR as described under "Experimental Procedures." Additional 5'- and 3'-sequences were obtained with the use of RACE-PCR. After five rounds of 5'-RACE-PCR a cumulative sequence of about 3400 base pairs was obtained including the poly(A) tail. No additional 5'-sequence could be amplified with this method in the presence or absence of conditions that lead to disruption of strong secondary structure. To further analyze the 5'-end, SMART RACE-PCR was performed. This procedure generated overlapping cDNA products that gave an additional 5'-extension of ~240 base pairs. Most of the products ended with an identical 5'-sequence. Due to the high GC content in this stretch (80%, Fig. 1) this additional sequence information could only be obtained under conditions that lead to disruption of strong secondary structure.

View larger version (61K): [in this window] [in a new window]   Fig. 1.   Nucleotide sequence and deduced amino acid sequence of human bile acid -glucosidase. Shadowed amino acid sequences are those corresponding to the sequenced peptides P1-P4 of purified bile acid -glucosidase spanning the amino acid sequence at the following positions: P1, 39-46; P2, 619-623; P3, 624-636; P4, 919-927. Potential asparagine-linked glycosylation sites are at amino acid positions 105 and 245. The polyadenylation signal is underlined.

The cDNA sequence and the deduced amino acid sequence of bile acid -glucosidase are shown in Fig. 1. The total cDNA consists of 3639 nucleotides. There are two potential initiation sites at positions 525 and 549, both of which agree well with the Kozak consensus sequence (10). Based on the scanning model for translation (10) the initiation site was assumed to start with the first ATG at position 525, which was preceded by an in-frame termination codon at position 387. Consequently the open reading frame extends over 2781 nucleotides, thus predicting a polypeptide of 927 amino acids with an M_r value of 104,648. The deduced amino acid sequence contains all of the four peptide sequences, which were experimentally determined from the purified protein (Fig. 1). Within the 3'-untranslated region a potential poly(A) signal was identified 14 bases upstream of a stretch of A residues at position 3591, suggesting the presence of a full-length cDNA.

Computer-aided analysis of the cloned -glucosidase revealed an acidic protein with a theoretical isoelectric point of 5.6. Polar and nonpolar amino acids constitute 42.3 and 57.7 residues per 100 total residues, respectively. The hydropathy plot for the -glucosidase open reading frame indicated the presence of a highly hydrophobic sequence at residues 689-708 (Fig. 2), which is predicted to be -helical (not shown) and of sufficient length to span the membrane. No amino-terminal signal sequence could be identified. Two potential N-linked glycosylation sites were found in the amino acid sequence (Fig. 1). However, no evidence for the existence of glycosyl residues was obtained for the purified -glucosidase from human liver microsomes or the COS-7 cell-expressed enzyme since the electrophoretic mobility of these proteins was not affected by digestion with N-glycosidase F or endoglycosidase H (not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Hydrophobicity profile of the bile acid -glucosidase polypeptide. The plot was evaluated according to Kyte and Doolittle (11) with a window size of 15 amino acids (hydrophobic, positive values; hydrophilic, negative values).

A search of the available data bases with the deduced amino acid sequence of bile acid -glucosidase revealed no significant sequence similarity with any known glycosyl hydrolase or other functionally identified protein. With regard to unidentified gene products showing significant similarity to the deduced -glucosidase sequence, various cDNA or DNA sequences with significant homology to the cDNA sequence reported herein were released from GenBank^TM during the preparation of this manuscript. The genomic sequence from the human chromosome 9 clone RP11-112J3 (GenBank^TM accession number AL133410) contained the -glucosidase sequence on an ~12-kilobase stretch. One base pair present in position 1435 of the -glucosidase sequence was deleted in the genomic sequence.

Compared with the cDNA sequence of the present study the human cDNA sequences released from GenBank^TM (accession numbers XM 048516, XM 048517, XM 048518, AK027884, AB046825 (12), and AF258662) were all incomplete at the 5'-ends starting at the following positions of the present -glucosidase sequence: 11 for clones AB046825 and XM 048518, and 338 for clones XM 048517 and AK027884. The cDNA sequence of clones AF258662 and XM 048516 started with a 139-base pair stretch apparently derived from an intron followed by sequence with strong homology to the present -glucosidase cDNA sequence beginning at position 1092. Compared with the cDNA sequence of the present study, most of the cDNA sequences revealed insertions or deletions of single base pairs in the open reading frame (accession numbers XM 048516, XM 048517, XM 048518, and AF258662). Two cDNA sequences (accession numbers AB046825 and XM 048518) showed a 157-base pair insertion within the open reading frame apparently derived from another intron as compared with the present -glucosidase sequence and with the sequence of the genomic clone RP11-112J3 containing the -glucosidase sequence. Two cDNA sequences (accession numbers AF258662 and XM 048516) were truncated at the 3'-end with regard to the present -glucosidase sequence, ending within the open reading frame at position 2865 of the -glucosidase cDNA.

The proteins predicted from the cDNA sequences of all clones mentioned above could not be identified or classified into a functional category. Most of the deduced amino acid sequences were truncated as compared with the amino acid sequence reported herein containing the following number of amino acids: 367 for clones AF258662 and XM 048516, 654 for clones XM 048517 and XM 048518, 877 for clone AB046825, and 927 for clone AK027884. The deduced amino acid sequence from clone AK027884 corresponds in length to the amino acid sequence of the -glucosidase reported herein. However, within the amino acid sequence deduced from clone AK027884, three residues were discrepant in comparison to the -glucosidase sequence. Two cysteine residues in the -glucosidase sequence were exchanged in the deduced amino acid sequence of clone AK027884 for tyrosine (position 60) or arginine (position 222); asparagine was replaced by lysine (position 482). Since amino acid sequences deduced from other cDNA clones did not exhibit these discrepancies in comparison to the present -glucosidase sequence, e.g. clones AB046825 or XM 048517, the amino acid replacements observed in the deduced amino acid sequence of clone AK027884 appear to be artifacts of sequencing or PCR.

With regard to unidentified proteins from other organisms the deduced amino acid sequence of the present study shared marked sequence homology (34-48%) with hypothetical proteins from Arabidopsis thaliana (BAA97011), Caenorhabditis elegans (AAB54243), Drosophila melanogaster (AAF44865), Oryza sativa (AAG16864), or a Synechocystis species (BAA17664; all numbers given as GenBank^TM accession numbers). The strongest sequence homologies with these gene products were observed within the COOH-terminal ~500-886 amino acids of the deduced -glucosidase sequence. These apparently conserved regions may contain important domains for structure and function of the enzyme.

Screening EST data bases with the cDNA sequence of the -glucosidase revealed that more than 100 EST sequences from various human sources have been generated up to the present time that exhibit significant homology to the sequence of the present study. The EST fragments were spread over the whole -glucosidase cDNA sequence. One of these fragments reached the 5'-end until position 22 (GenBank^TM accession number BG393407). Furthermore, EST sequences showing significant homology to parts of the -glucosidase cDNA sequence were detectable from other vertebrates, e.g. EST clones from cow, mouse, pig, or rat with the following examples of GenBank^TM accession numbers, respectively: BE752928, BG293326, BG834883, or BF543949.

Expression of -Glucosidase Activity in COS-7 Cells-- Two days after transient transfection of COS-7 cells with the recombinant plasmid coding for the Strep-tagged -glucosidase (see "Experimental Procedures") or with the plasmid as control, -glucosidase assays were conducted on fractions from cell lysates. As shown in Table I, 100,000 × g pellets from COS-7 cells transfected with the recombinant vector showed high overexpression of -glucosidase activity compared with pellets from nontransfected cells or cells transfected with the expression vector without -glucosidase cDNA. In contrast to the 100,000 × g pellets, the 100,000 × g supernatants exhibited little bile acid -glucosidase activity amounting to about 20 and 5% of the activities estimated in the pellets from cells transfected with and without the tagged -glucosidase cDNA, respectively (not shown). Like the enzyme from human liver microsomes (13) the COS-7 cell-expressed tagged -glucosidase was sensitive to inhibition by the glucosidase inhibitor 1-deoxynojirimycin, which produced about 50% inhibition of enzyme activity at a concentration of 0.01 mM of the inhibitor (not shown).

View this table: [in this window] [in a new window]   Table I Overexpression of -glucosidase in COS-7 cells COS-7 cells were transfected, and -glucosidase activity toward lithocholic acid glucoside was determined from the 100,000 × g pellets of COS-7 cells as described under "Experimental Procedures."

Western blot analysis of fractions from lysates of transfected cells using a polyclonal antibody directed against the Strep-tag II peptide revealed a polypeptide of about 110 kDa only in cells transfected with the tagged -glucosidase cDNA (Fig. 3). The molecular mass of this protein is identical within experimental error with that of the purified human liver bile acid -glucosidase (1) and with that deduced from the open reading frame of the -glucosidase cDNA including the Strep-tag II of about 1 kDa.

View larger version (53K): [in this window] [in a new window]   Fig. 3.   Western blot analysis of the expressed human bile acid -glucosidase fusion protein in COS-7 cells. Pellet fractions after cell lysis of plasmid-transfected control cells (lane 2) and cells transfected with the recombinant plasmid containing the tagged -glucosidase cDNA (lane 3) were analyzed by immunoblotting as described under "Experimental Procedures." Molecular mass markers (prestained, lane 1) are indicated on the left. Bands around 60 kDa in lanes 2 and 3 may result from reducing agent (dithiothreitol) in the sample buffer, which has been shown to produce irrelevant bands after immunoblotting (14).

Tissue Distribution of -Glucosidase mRNA-- To determine the distribution of the -glucosidase mRNA Northern blot analyses were performed with poly(A) RNA from different human tissues. Using a probe that contained sequences for two isolated peptides of the -glucosidase (P3 and P4, Fig. 1) and high stringency conditions, a signal at about 3.6 kilobases corresponding to the -glucosidase cDNA was observed in the tissues examined (Fig. 4). The 3.6-kilobase message was mainly expressed in brain, heart, skeletal muscle, kidney, and placenta and at minor levels in liver, spleen, small intestine, and lung. Very low levels were detectable in colon (without mucosa), thymus, and peripheral blood leukocytes. A human -actin probe served as a control and gave a positive signal in all lanes (Fig. 4). Identical results were obtained using two different lot numbers of human multiple tissue poly(A) RNA membranes.

View larger version (71K): [in this window] [in a new window]   Fig. 4.   Northern blot analysis of bile acid -glucosidase expression in human tissues. The Northern blot with poly(A) RNA from brain (lane 1), heart (lane 2), skeletal muscle (lane 3), colon without mucosa (lane 4), thymus (lane 5), spleen (lane 6), kidney (lane 7), liver (lane 8), small intestine (lane 9), placenta (lane 10), lung (lane 11), and peripheral blood leukocytes (lane 12) was hybridized as described under "Experimental Procedures." A kilobase (kb) marker is indicated at the left.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A full-length cDNA clone of human bile acid -glucosidase was isolated by 5'- and 3'-RACE-PCRs on the basis of the amino acid sequences of four peptides obtained from the purified human liver enzyme by proteinase digestion. Several lines of evidence confirm that the cDNA is indeed encoding the bile acid -glucosidase.

First, the amino acid sequences of the four peptides of the purified protein are contained in the amino acid sequence deduced from the open reading frame (Fig. 1). Second, the molecular mass of the protein of 104.6 kDa calculated from the open reading frame is in agreement with the value of 100 kDa that was estimated by SDS-polyacrylamide gel electrophoresis of the purified enzyme (1) and with the molecular mass of the 110-kDa protein resulting from expression of the Strep-tagged -glucosidase cDNA in COS-7 cells (Fig. 3). Third, high overexpression of bile acid -glucosidase activity was observed in COS-7 cells upon transfection with -glucosidase cDNA linked to the Strep-tag II sequence (Table I).

The isolated cDNA contains a long 5'-untranslated segment of 524 nucleotides. No larger product could be generated in repeated RACE experiments using different methods, suggesting that the 5'-end of the cDNA is at or near the transcription start site. This site, however, has to be characterized by further analyses.

Screening a human sequence-tagged site data base with the amino acid sequence of the protein revealed that a partial sequence as contained in peptides P2 and P3 (Fig. 1) is represented in sequence-tagged site clone WI-17107, which was assigned to human chromosome 9. The apparent location of the human -glucosidase gene on chromosome 9 was also confirmed by the human chromosome 9 clone RP11-112J3 of the human genome project that contained the -glucosidase cDNA with significant identity.

Previous studies on the subcellular location of bile acid -glucosidase in human liver showed that the enzyme was mainly enriched in the microsomal fraction where it appeared to be confined to the smooth endoplasmic reticulum after subfractionation of microsomes by isopycnic centrifugation (13, 15). In agreement with a microsomal location, the purified -glucosidase showed characteristics of a membrane protein (1). For purification from human liver microsomes bile acid -glucosidase had to be solubilized and stabilized by the addition of detergents, and the catalytic activity of the purified enzyme was phospholipid-dependent as has been described for other microsomal enzymes (16). Furthermore, a transmembrane segment was predicted from the amino acid sequence of bile acid -glucosidase at residues 689-708 (Fig. 2), and the COS-7 cell-expressed enzyme was mainly associated with the 100,000 × g fraction of the cell lysate. However, no ER retrieval or retention motif could be identified within the deduced amino acid sequence of the protein. Therefore, further studies are needed on the subcellular location of the bile acid -glucosidase, e.g. by immunohistochemical methods when an antibody against the protein will be available. However, the enzyme may be retained in the ER by an as yet unknown mechanism as has been discussed by other authors on the ER location of a Man₉-mannosidase, which also lacked an ER recognition motif but was shown to be ER-resident by immunofluorescence microscopy (17).

Judging from nucleic acid and protein data base searches it appears that the nucleotide sequence of the -glucosidase codes for a unique protein. No significant amino acid sequence similarities with other glycosyl hydrolases were detectable using conventional sequence comparison methods. The absence of a molecular relation with other -glucosidases is not unexpected in view of the fact that bile acid -glucosidase is quite distinct in its substrate specificity as well as response to inhibitors and divalent metal ions (1) from other -glucosidases (18). The isolation of the cDNA encoding the human bile acid -glucosidase will provide the basis for future studies on the physiological role of this enzyme.

	ACKNOWLEDGEMENTS

We thank Gabriele Czeczor, Stephanie Erschfeld, and Melanie Flecken for excellent technical assistance and Dr. Iris Behrmann, Institute of Biochemistry, Rheinisch-Westfälische Technische Hochschule Aachen, for help in the initial stages of this investigation.

	FOOTNOTES

^* This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AJ309567.

To whom correspondence should be addressed: Medizinische Klinik III, RWTH Aachen, Pauwelsstrasse 30, 52057 Aachen, Germany. Tel.: 49-241-8088590; Fax: 49-241-8888455; E-mail: MK3@post.klinikum.rwth-aachen.de.

Published, JBC Papers in Press, August 6, 2001, DOI 10.1074/jbc.M104290200

	ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; SMART, switching mechanism at 5'-end of RNA transcript; EST, expressed sequence tag; ER, endoplasmic reticulum; lithocholic acid, 3-hydroxy-5-cholanoic acid; lithocholic acid glucoside, 3-glucosido-lithocholic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 276/41/37929    most recent M104290200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Matern, H. Articles by Matern, S. Search for Related Content PubMed PubMed Citation Articles by Matern, H. Articles by Matern, S. Social Bookmarking                      What's this?