From the
A full-length cDNA coding for a novel human serine hydrolase has
been cloned from a breast carcinoma cDNA library. Nucleotide sequence
analysis has shown that the isolated cDNA contains an open reading
frame coding for a polypeptide of 274 amino acids and a complete
Alu repetitive sequence within its 3`-untranslated region. The
predicted amino acid sequence contains the
Gly-X-Ser-X-Gly motif characteristic of serine
hydrolases and displays extensive similarity to several prokaryotic
hydrolases involved in the degradation of aromatic compounds. The
highest degree of identities was detected with four serine hydrolases
encoded by the bphD genes of different strains of
Pseudomonas with the ability to degrade biphenyl derivatives.
On the basis of these sequence similarities, this novel human enzyme
has been tentatively called Biphenyl hydrolase-related protein
(Bph-rp). The Bph-rp cDNA was expressed in Escherichia coli,
and after purification, the recombinant protein was able to degrade
p-nitrophenylbutyrate, a water-soluble substrate commonly used
for assaying serine hydrolases. This hydrolytic activity was abolished
by diisopropyl fluorophosphate, a covalent inhibitor of serine
hydrolases, providing additional evidence that the isolated cDNA
encodes a member of this protein superfamily. Northern blot analysis of
poly(A)
The serine hydrolases are defined as a functional class of
hydrolytic enzymes that contain a serine residue in their active site.
This enzyme class is composed of a large number of diverse members,
including serine proteinases, esterases, and lipases, which are able to
hydrolyze a wide variety of substrates, thus performing vastly
different biological roles. However, despite this marked variability,
the proteins belonging to this superfamily of serine-dependent
hydrolytic enzymes can be grouped into subfamilies with closely related
members in terms of substrate specificity or amino acid sequence
similarities (Derewenda and Derewenda, 1991; Cygler et al.,
1993). This is the case of a recently described group of serine
hydrolases isolated from diverse bacterial strains, which display
significant amino acid sequence similarities and whose common function
would be the degradation of aromatic compounds (Hofer et al.,
1993; Arand et al., 1994).
Over the last few years, the
employment of soil bacteria capable of utilizing a wide range of
aromatic compounds as sources of carbon and energy has raised
considerable interest in biodegradation processes, since many of these
compounds are widespread and persistent environmental pollutants
(Furukawa, 1982; Chaudhry and Chapalamadugu, 1991; Timmis et
al., 1994). These microorganisms employ a number of different
enzymes for the initial attack on the structurally diverse aromatic
substrates, but the catabolic pathways tend to converge on just a few
central intermediates such as catechol or substituted catechols. These
key intermediates are subsequently cleaved and processed by one of a
few central pathways to Krebs cycle intermediates. For example,
biphenyls and their polychlorinated derivatives, toluene, phenol, or
xylenes, are all metabolized to catechol by different enzymatic
reactions, but the conversion of catechol to Krebs cycle intermediates
is mediated by common enzymatic steps called the metacleavage pathway
(Harayama and Timmis, 1989). One of the key reactions of these aromatic
catabolic routes is catalyzed by a serine hydrolase with the ability to
perform the hydrolytic cleavage of carbon-carbon bonds, one of the
rarest of all enzyme reaction types (Duggleby and Williams, 1986). At
present, seven bacterial hydrolases catalyzing this type of reaction
have been characterized at the amino acid sequence level. Four of them,
named 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolases, are the
products of the respective bphD genes from four different
strains of Pseudomonas: Pseudomonas sp strain KKS102
(Kimbara et al., 1989), Pseudomonas putida KF715
(Hayase et al., 1990), Pseudomonas sp strain LB 400
(Hofer et al., 1993), and Pseudomonas testosteroni B-356 (GenBank
During the course of
studies directed to look for biochemical markers that could be of
interest in breast cancer (Freije et al., 1994;
López-Boado et al., 1994; Velasco et al.,
1994; Ura et al., 1994) we have cloned from a human breast
cancer cDNA library a novel human serine hydrolase with a high degree
of sequence similarity to the above mentioned prokaryotic proteins
involved in the degradation of aromatic compounds. In this work, we
present the molecular cloning and nucleotide sequence of this novel
enzyme named biphenyl hydrolase-related protein (Bph-rp).
Finally, and also in relation to the structural analysis of the
nucleotide sequence of the cDNA coding for Bph-rp, the occurrence of an
Alu repetitive sequence in the 3`-noncoding sequence of the
isolated cDNA clone is noteworthy. This Alu element is
oriented in the same transcriptional direction as the Bph-rp gene,
beginning at nucleotide 1207 and terminating at the poly(A)-rich region
of the 3`-end of the Bph-rp cDNA (Fig. 1). Computer analysis of
diagnostic nucleotides at specific positions in Alu sequences
(Jurka and Milosavljevic, 1991) revealed that this Alu repetitive sequence belongs to the Alu J subfamily, the
oldest Alu subfamily in the human genome, which is believed to
have been disseminated more than 55 million years ago (Labuda and
Striker, 1989). The occurrence of this ancient Alu sequence in
the 3`-flanking region of the mRNA for human Bph-rp is very unusual,
since despite the fact that the short interspersed elements constitute
about 5% of the human genome, most of them have been found in
intergenic DNA or in introns, being spliced out during processing to
the mature RNAs (Schmid and Jelinek, 1982). The biological significance
of the occurrence of this Alu repeat in the 3`-nontranslated
region of the human Bph-rp mRNA as well as in those equivalent cases
including mRNAs for human low density lipoprotein receptor (Yamamoto
et al., 1984), cytochrome P-450 4 (Quattrochi et al.,
1986), tumor supressor p53 (Matlashewski et al., 1984), acid
and alkaline phosphatases (Sharief et al., 1989; Henthorn
et al., 1986), or lysozyme (Chung et al., 1988)
remains to be determined. In this regard, it should be remembered that
Alu sequences have a high degree of similarity with the
elongation arrest domain of 7SL RNA, a component of the signal
recognition particle involved in protein secretion (Gundelfinger et
al., 1983; Walter et al., 1984). On the basis of this
homology, the Alu J element here detected within the Bph-rp
mRNA could be important in the translational modulation of Bph-rp
expression, as already proposed for Alu transcripts that are
transported to the cytoplasmic compartment (Siegel and Walter, 1986;
Chang and Maraia, 1993).
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Dr. S. Gascón for support; Drs. M.
Balbn, A. M. Pendás, G. Velasco, L. M. Sánchez, and V. de
Lorenzo for helpful comments; and Dr. S. G. Granda (Scientific Computer
Center, Universidad de Oviedo) for computer facilities.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
RNAs isolated from a variety of human tissues
revealed that Bph-rp is mainly expressed in liver and kidney, which was
also confirmed at the protein level by Western blot analysis with
antibodies raised against purified recombinant Bph-rp. According to
structural characteristics, hydrolytic activity and tissue distribution
of Bph-rp, a potential role of this enzyme in detoxification processes
is proposed.
accession number L34338). These
hydrolases are involved in the degradation of biphenyls and their
polychlorinated derivatives and show a high degree of amino acid
sequence similarity. These four enzymes are also significantly related
to dmpD, todF, and xylF, the equivalent hydrolases of
the metacleavage pathways responsible for the degradation of phenol and
methyl-substituted phenols (Nordlund and Shingler, 1990), toluene (Menn
et al., 1991), and xylene (Horn et al., 1991), by
different bacterial strains. All seven proteins share about 25%
identical residues, including the Gly-X-Ser-X-Gly
sequence encompassing the active site Ser of this enzyme class, which
suggests that despite their discrete substrate specificities, all of
them derive from a common ancestor, which was subsequently adapted to
degrade the different aromatic compounds.
(
)
We also analyze its expression in human tissues and perform
a functional analysis with the protein produced in Escherichia
coli. Finally, on the basis of the obtained results, we propose a
potential role of this enzyme in detoxification processes.
Materials
A human breast carcinoma cDNA library
constructed in gt11 and two Northern blots containing RNAs from
many different human tissues were purchased from Clontech (Palo Alto,
CA). Oligonucleotides were synthesized by the phosphoramidite method in
an Applied Biosystems DNA synthesizer, model 381 A, and used directly
after synthesis. Restriction endonucleases and other reagents used for
molecular cloning were from Boehringer Mannheim. Double-stranded DNA
probes were radiolabeled with [
P] dCTP (3000
Ci/mmol) from Amersham Corp. using a commercial random priming kit
purchased from Pharmacia Biotech Inc. p-Nitrophenylbutyrate
and diisopropyl fluorophosphate were purchased from Sigma. Reagents for
amino acid sequencing were from Applied Biosystems (Foster City, CA).
Screening of a Human Breast Carcinoma cDNA
Library
About 1 10
pfu of a human breast
carcinoma cDNA library were plated using E. coli Y1088 as host
and analyzed according to standard procedures (Maniatis et al., 1982) using as probe a 0.3-kilobase fragment corresponding to the
3`-end of a partial cDNA putatively encoding a human breast epithelial
mucin-associated antigen (Larocca et al., 1990). The probe was
generated by PCR amplification of DNA isolated from the human breast
carcinoma cDNA library with primers 5`-TGGAGTGAGAGAACAAG-3` and
5`-GCAAAACGCAAATGCAG-3`. Hybridization to the radiolabeled probe was
carried out at 55 °C for 18 h in 6
SSC (1
SSC
= 150 mM NaCl, 15 mM sodium citrate, pH 7.0),
5
Denhardt's solution (1
Denhardt's
solution = 0.02% bovine serum albumin, 0.02%
polyvinylpyrrolidone, 0.02% Ficoll), 0.1% SDS, and denatured herring
sperm DNA (100 µg/ml). The membranes were washed twice for 1 h at
60 °C in 1
SSC, 0.1% SDS and exposed to XAR-5 film (Kodak)
at -70 °C with intensifying screens. Following plaque
purification, the cloned insert was excised by EcoRI
digestion, and the resulting fragments were subcloned into the
EcoRI site of pEMBL19.
DNA Sequencing
Selected DNA fragments were
inserted in the polylinker region of phage vector M13 mp19 (Messing and
Vieira, 1982) and sequenced by the dideoxy chain termination method
(Sanger et al., 1977) using either M13 universal primer or
cDNA-specific primers and the Sequenase Version 2.0 kit (U. S.
Biochemical Corp.). All nucleotides were identified in both strands.
Sequence ambiguities were solved by substituting dITP for dGTP in the
sequencing reactions according to the instructions of the manufacturer
of the kit. Computer analysis of DNA and protein sequences was
performed with the software package of the University of Wisconsin
Genetics Computer Group (Devereux et al., 1984).
Expression in E. coli
A 995-bp DNA fragment
containing the complete coding sequence for Bph-rp was obtained by PCR
amplification of the isolated full-length cDNA with primers
5`-ATGCCCAGGAATCTGCT-3` and 5`-CTGAAGCTAGAGGCTGT-3`. The PCR reaction
was carried out for 30 cycles of denaturation (95 °C for 1 min),
annealing (48 °C for 1 min), and extension (72 °C for 1 min).
The PCR product was phosphorylated with T4 polynucleotide kinase and
ligated to the expression vector pET3c (Rosenberg et al.,
1987), previously treated with NdeI and nuclease S1. The
resulting plasmid, called pETX3, was transformed into E. coli strain BL21(DE3), and the transformed cells were grown in LB broth
containing 100 µg ampicillin/ml at 37 °C for about 16 h,
diluted 1:100 with the same medium, and grown to an A of 1.0. Then isopropyl-1-thio-
-D-galactopyranoside
was added to a final concentration of 1 mM, and the incubation
was continued for another 3 h. Cells were collected by centrifugation,
resuspended in volume of phosphate-buffered saline, lysed by using a
French press, and centrifuged at 20,000
g for 20 min
at 4 °C. The protein was purified by DEAE-chromatography in 50
mM Tris, pH 7.4, and the retained material was eluted with a
gradient of 50 mM Tris, pH 7.4, 0.5 M NaCl. Fractions
containing Bph-rp, as judged by SDS-polyacrylamide gel electrophoresis,
were pooled and gel chromatographed on a Superdex 75 HR 10/30 column
(Pharmacia Biotech Inc.) in 0.1 M ammonium acetate, pH 6.5, at
a flow rate of 0.1 ml/min and using a SMART system chromatograph
(Pharmacia Biotech Inc.). Fractions of 250 µl were collected, and
those containing pure recombinant Bph-rp were pooled and lyophilized.
Enzyme Activity Measurements
The enzyme activity
of purified Bph-rp was measured using 0.5 mMp-nitrophenylbutyrate as substrate and following the procedure
described by Shirai and Jackson(1982) with minor modifications. Assays
were performed at 37 °C, in 100 mM sodium phosphate
buffer, pH 7.0, 90 mM NaCl, and the activity was measured at
410 nm by a UV-visible recorder spectrophotometer UV-160 (Shimadzu).
For inhibition assays, the reaction mixture was preincubated with 0.1
mM diisopropyl fluorophosphate at 37 °C for 1 h, and the
remaining activity was determined using p-nitrophenylbutyrate
as above. Diisopropyl fluorophosphate interaction with the putative
serine residue of the Bph-rp active center was examined by incubating
15 µg of recombinant protein with 2 µl of
[1,3-H]diisopropyl fluorophosphate (10 Ci/mmol)
(Amersham), for 1 h at 37 °C. Then, 20 µl of 50 mM
unlabeled diisopropyl fluorophosphate was added, and the samples were
electrophoresed in 13% SDS-polyacrylamide gel. The gel was then fixed
with 20% trichloroacetic acid and washed for 30 min with water to
remove the excess trichloroacetic acid. Finally, the gel was incubated
in 1 M sodium salicylate, dried, and exposed to XAR-5 film
(Kodak) at -70 °C.
Amino Acid Sequence Analysis
Purified recombinant
Bph-rp or tryptic peptides generated from the purified protein were
subjected to automatic Edman degradation on an Applied Biosystems 477A
sequencer in the presence of Polybrene. The anilinothiazolinones were
converted to phenylthiohydantoin derivatives in the automatic
conversion flask of the sequencer and identified and quantified with an
on-line phenylthiohydantoin analyzer (model 120 A; Applied Biosystems).
The tryptic peptides were obtained after treatment of purified Bph-rp
with trypsin for 16 h at 37 °C at an enzyme:substrate ratio of 1:50
(w/w). The resulting fragments were fractionated by reverse-phase HPLC
on a µRPC C2/C18 column (Pharmacia Biotech Inc.) equilibrated with
0.1% trifluoroacetic acid. The runs were carried out in a SMART system
fast protein liquid chromatography at room temperature and at a flow
rate of 0.1 ml/min. Peptides were eluted with a linear gradient of
acetonitrile in 0.1% trifluoroacetic acid, and their absorbance was
monitored at 214 nm.
Northern Blot Hybridization
Northern blots
containing 2 µg of poly(A) RNA of different human
tissue specimens were prehybridized at 42 °C for 3 h in 50%
formamide, 5
SSPE (1
SSPE = 150 mM
NaCl, 10 mM NaH
PO
, 1 mM EDTA,
pH 7.4), 10
Denhardt's solution, 2% SDS, and 100
µg/ml of denatured herring sperm DNA and then hybridized with
radiolabeled Bph-rp cDNA (a 995-bp PCR fragment extending from bp 213
to 1207) for 20 h under the same conditions. Filters were washed with
0.1
SSC, 0.1% SDS for 2 h at 50 °C and exposed to
autoradiography. RNA integrity and equal loading was assessed by
hybridization with an actin probe as indicated by the manufacturer.
Antiserum Production and Western Blot Analysis
200
µg of purified Bph-rp was used to immunize a New Zealand White
rabbit according to the method described by Vaitukaitis(1981). The
rabbit was bled 6 weeks after the injection of the recombinant protein,
and IgGs were purified by anion exchange chromatography on a
DEAE-cellulose column (Whatman DE52), equilibrated and eluted with 20
mM phosphate buffer, pH 7.2. Finally, the obtained antibodies
(diluted 1:1000) were used for Western blot analysis as described
previously (Sánchez et al., 1992).
Isolation and Characterization of a cDNA Encoding Human
Biphenyl Hydrolase-related Protein
During the course of studies
directed to examine the possible relevance of human milk-fat globule
membrane proteins as tumor markers in breast cancer, we noted that a
partial cDNA sequence putatively encoding one of these proteins, with a
molecular mass of 70 kDa and sharing several properties with bovine
butyrophilin, was not related to the amino acid sequence of this
protein (Larocca et al., 1990; Jack and Mather, 1990). These
data together with recent findings indicating the occurrence in the
human genome of a homologue of bovine butyrophilin (Vernet et
al., 1993) prompted us to examine the possibility that the
reported nucleotide sequence could encode a different protein. To do
that, we first screened a human breast cancer cDNA library with a probe
generated by PCR using synthetic oligonucleotides derived from the
partial cDNA sequence proposed to encode the 70-kDa human milk-fat
globule protein (Larocca et al., 1990). Upon screening of
approximately 1 10
plaque-forming units, two
positive clones named x-14 and x-15 were identified. Nucleotide
sequence analysis of both clones revealed that the sequence
corresponding to x-15 was entirely contained within that obtained for
x-14, indicating that it was a partial cDNA clone. Further analysis of
the sequence of the largest clone showed an open reading frame 822 bp
long, starting with an ATG codon at position 213 and ending with a TGA
codon at position 1037 (Fig. 1). This open reading frame codes
for a protein of 274 amino acid residues with a calculated molecular
weight of 31,106.
Figure 1:
Nucleotide sequence
of BPH-rp cDNA from a human breast carcinoma. The deduced amino acid
sequence is shown below the nucleotide sequence. Restriction
sites relevant for subcloning in M13 are indicated. The Alu repetitive sequence identified in the 3`-untranslated region of
the isolated cDNA is underlined.
A comprehensive search of the EMBL and
GenBank nucleic acid data bases revealed that this human
sequence had significant homology with those from a group of bacterial
proteins involved in the degradation of aromatic compounds, including
biphenyl derivatives, phenol, toluene, and xylene (Hofer et
al., 1993; Arand et al., 1994). A more detailed search
for sequence similarities between the identified amino acid sequence
and those corresponding to these prokaryotic enzymes revealed that the
highest degree of identities (about 25%) was detected with four
different serine hydrolases (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate
hydrolases) encoded by the bphD genes of different strains of
Pseudomonas (Fig. 2). The percentage of identities of
the human protein with the remaining members of this family of
bacterial serine hydrolases was slightly lower, ranging from 20% with
dmpD to 22% with xylF, the equivalent hydrolases
(2-hydroxymuconic-semialdehyde hydrolases) of pathways responsible for
the degradation of phenol and xylene, respectively. However, in all
cases the sequence similarity extends throughout the complete amino
acid sequence of these proteins, being of special relevance to the
occurrence of some regions with a high degree of conservation in all of
them (Fig. 2). Thus, the human sequence contains at an equivalent
position the Gly-X-Ser-X-Gly consensus motif that is
a characteristic feature of serine hydrolases from different sources
(Brenner, 1988; Derewenda and Derewenda, 1991; Cygler et al.,
1993). The serine residue contained in this consensus sequence has been
proposed to be the essential catalytic residue in the above mentioned
hydrolases involved in the degradation of aromatic compounds.
Consequently, it is tempting to speculate that the Ser-122 of the
identified open reading frame would correspond to the catalytic serine
residue of this putative novel human serine hydrolase that, according
to its sequence similarity to bacterial proteins encoded by bph genes, has been tentatively called human Biphenyl
hydrolase-related protein (Bph-rp).
Figure 2:
Alignment of the amino acid sequence of
human Bph-rp with prokaryotic serine hydrolases. The amino acid
sequences of the bacterial proteins were extracted from the Swiss-Prot
data base, and the multiple alignment was performed with the PILEUP
program of the GCG package (Devereux et al., 1984). Residues
common to all sequences are marked with an asterisk. Gaps are
indicated by hyphens.
The amino acid sequence
alignment of Bph-rp with these prokaryotic serine hydrolases could also
be helpful to identify the histidyl and acidic residues that together
with Ser-122 could form the catalytic triad found to occur in proteins
belonging to the serine hydrolase superfamily. Thus, although at
present there is not experimental evidence on the identity of catalytic
residues in these prokaryotic hydrolases, Ollis et al.(1992)
have proposed that two conserved aspartic acid and histidyl residues
could be part of the catalytic triad in
2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase from
Pseudomonas sp KKS102 and in 2-hydroxymuconic-semialdehyde
hydrolase from P. putida. Both residues are perfectly
conserved in the human sequence (positions 227 and 255) as well as in
the remaining members of the bacterial protein family related to Bph-rp
( Fig. 2and data not shown). Consequently, these residues would
be the most likely candidates to form part of the putative catalytic
triad presumed to be essential for the enzymatic activity of both human
and microbial enzymes. The ongoing studies on the three-dimensional
structure of Bph-rp will contribute to confirm this prediction and to
elucidate whether this human enzyme belongs to the /
hydrolase fold family of proteins as already proposed for its bacterial
homologues (Ollis et al., 1992; Arand et al., 1994).
Production of Recombinant Biphenyl Hydrolase-related
Protein in E. coli
As a preliminary step to examine the
functional role of Bph-rp as well as to obtain specific antibodies
against the protein that could be useful for further studies of the
native protein in human tissues, we undertook the production of this
protein in E. coli. To do that, a 995-bp fragment containing
the entire Bph-rp open reading frame was first subcloned in the T7-RNA
polymerase-based expression vector pET3c developed by Rosenberg et
al. (1987). The resulting plasmid, named pETx3, was transformed
into E. coli strain BL21(DE3), and the transformed bacteria
were induced to produce the recombinant protein by treatment with
isopropyl-1-thio--D-galactopyranoside. Extracts were
prepared from the induced bacteria, and their protein composition was
examined by SDS-PAGE. As shown in Fig. 3, the soluble fraction of
the extract contained a major protein of about 30 kDa, which was absent
in the extracts from bacteria carrying the control plasmid. This
recombinant protein was isolated from the bacterial extracts using the
strategy outlined under ``Materials and Methods.'' Thus,
extracts containing about 50 mg of total protein were first
chromatographed on a DEAE anion exchange column. As judged by SDS-PAGE,
the recombinant Bph-rp was detected in the flow-through fraction of the
column, indicating that the protein did not bind to this
chromatographic support. The material containing the putative Bph-rp
was then fractionated by size exclusion fast protein liquid
chromatography and the eluate material analyzed by 280-nm absorption
and SDS-PAGE ( Fig. 3and 4A). The main peak of the
chromatogram corresponded to an isolated, single polypeptide chain
protein, with the expected molecular mass of 30 kDa. In order to
confirm the identity of this purified recombinant protein as human
Bph-rp, 25 µg of isolated protein were subjected to automatic Edman
degradation. However, no phenylthiohydantoins could be identified in
two separate runs, suggesting that the purified protein was blocked at
its N-terminal end. Then in order to obtain information regarding
internal amino acid sequences, the recombinant protein was treated with
trypsin, and the resulting peptides were separated by reverse-phase
HPLC in a C2/C18 column (Fig. 4B). Automatic Edman
degradation of a series of isolated tryptic peptides (T18,
Tyr-Pro-Ser-Tyr-Ile-His-Lys; T19, Asp-Val-Ser-Lys; T25,
Trp-Val-Asp-Gly-Ile-Arg) revealed that all of them had sequences that
could be matched with regions present in the amino acid sequence
predicted from the cDNA sequence determined for human Bph-rp
(Fig. 1). According to these data, we can conclude that the
30-kDa polypeptide produced in E. coli corresponds to human
Bph-rp.
Figure 3:
Characterization of recombinant Bph-rp
produced in E. coli BL21(DE3) and of Bph-rp from human liver.
A, SDS-PAGE analysis of soluble fractions of the bacterial
extracts (pET3c and pETX3), and of fractions obtained
after anion exchange chromatography (DEAE) and size exclusion
chromatography (Bph) of the extracts. The sizes in kilodaltons
of the molecular weight markers (MWM) are shown to the
left. B, human recombinant Bph-rp was incubated with
[1,3-H diisopropyl fluorophosphate]
(DIFP) and electrophoresed in 13% PAGE, and the gel was
exposed at -70 °C after treatment with sodium salicylate.
C, Western blot analysis of human liver protein extracts
(Clontech) separated by SDS-PAGE, transferred to polyvinylidene
difluoride membranes, and incubated with polyclonal antibodies (diluted
1:500) against purified recombinant Bph-rp.
Figure 4:
Isolation of recombinant Bph-rp by size
exclusion chromatography and separation of its tryptic peptides by
reverse-phase chromatography. A, flow-through fractions of
pETX3 bacterial extracts chromatographed on a DEAE column were pooled,
concentrated, and applied to a Superdex 75 HR 10/30 column (Pharmacia
Biotech Inc.) equilibrated and eluted with 0.1 M ammonium
acetate, pH 6.7. The flow rate was 100 µl/min, and 200-µl
fractions were collected. Fractions containing isolated recombinant
Bph-rp are indicated by the bar. B, tryptic peptides
from Bph-rp were fractionated by reverse-phase HPLC on a µRPC
C2/C18 column (Pharmacia Biotech Inc.), equilibrated with 0.1%
trifluoroacetic acid, and eluted with a gradient of acetonitrile in
0.1% trifluoroacetic acid. Peaks T18, T19, and T25 were directly
subjected to automatic Edman degradation.
In order to examine the enzymatic activity of this
recombinant protein, 2 µg of isolated Bph-rp was incubated with
p-nitrophenylbutyrate, which is a commonly used water-soluble
substrate for members of the serine hydrolase protein superfamily. In
addition, this aromatic ester may somewhat resemble the aromatic
compounds metabolized by the prokaryotic enzymes structurally related
to Bph-rp. The recombinant Bph-rp displayed an hydrolytic activity on
p-nitrophenylbutyrate (1.2 nmol/min/µg protein) that was
fully abolished by diisopropyl fluorophosphate, an inhibitor of serine
hydrolases. Furthermore, incubation of purified Bph-rp with radioactive
diisopropyl fluorophosphate followed by SDS-PAGE/fluorography analysis,
demonstrated that this active-site reagent was able to bind to the
recombinant protein (Fig. 3B). Taken together, these
results provide a strong indication that Bph-rp is an authentic serine
hydrolase. The degree of activity of Bph-rp on
p-nitrophenylbutyrate is similar to that reported for bovine
milk lipoprotein lipase against the same substrate (1.0 nmol/min/µg
protein) (Shirai and Jackson, 1982) but considerably lower than those
obtained for most serine hydrolases, including human milk bile-salt
activated lipase (200 nmol/min/µg protein) (Wang, 1991) or human
liver carboxylesterase (155 nmol/min/µg protein) (Kroetz et
al., 1993). These data suggest that the natural substrates of
Bph-rp are distantly related to p-nitrophenylbutyrate. In this
regard, it should also be mentioned that studies directed to examine
the possibility that some available biphenyl derivatives (kindly
provided by Dr. V. de Lorenzo) could be more appropriate substrates for
Bph-rp have failed to demonstrate significant levels of hydrolytic
activity, suggesting that despite its structural similarity to
polychlorinated biphenyl hydrolases from prokaryotic sources, this
novel human enzyme may have specific requirements in terms of
substrates or experimental conditions of analysis of its hydrolytic
activity. Nevertheless, it is also possible that the human Bph-rp
produced in bacteria may be only partially functional. Studies are in
progress to examine these possibilities as well as to try to identify
the natural substrates of this human enzyme and to precisely establish
the kinetics of hydrolysis of these substrates.
Expression Analysis of the Gene Encoding Biphenyl
Hydrolase-related Protein in Human Tissues
To examine the
expression of the Bph-rp gene in human tissues, two Northern blots
containing poly(A) RNAs obtained from different human
tissues including leukocytes, colon, small intestine, ovary, testis,
prostate, thymus, spleen, pancreas, kidney, skeletal muscle, liver,
lung, placenta, brain, and heart, were hybridized with a fragment of
the cDNA coding for Bph-rp, which lacked the 3`-flanking region
containing the Alu repetitive sequence. As shown in
Fig. 5
, a strong hybridizing band of about 1.8 kilobases was
detected in RNA from liver and kidney and at lower intensity in RNA
from other tissues including intestine, heart, and skeletal muscle. It
is also noteworthy that an additional hybridizing band of about 2.4
kilobases was recognized by the Bph-rp probe in some tissues,
especially in the lane corresponding to RNA from kidney. Interestingly,
this large band was absent in liver, the tissue in which the expression
of the 1.8-kilobase Bph-rp transcript was maximal. The origin of these
two transcripts is unclear, although it seems likely that they could be
the result of alternative splicing events or alternative utilization of
polyadenylation sites. However, the possibility of existence of
additional genes with a high degree of sequence similarity with Bph-rp
and differentially expressed in human tissues cannot be definitively
ruled out. The expression analysis of Bph-rp in human tissues was also
confirmed at the protein level by using antibodies raised in rabbits
against the recombinant protein produced in E. coli. As shown
in Fig. 3C, these antibodies recognize a protein in
human liver extracts that has virtually the same molecular mass as
recombinant Bph-rp. The close agreement between the molecular mass of
Bph-rp deduced from SDS-PAGE mobility and that calculated from the
amino acid sequence suggests that major posttranslational modifications
like proteolytic processing or extensive glycosylation events are not
occurring in Bph-rp. This last observation is also consistent with the
absence of N-linked glycosylation consensus sequences in the
amino acid sequence deduced in this work for human Bph-rp
(Fig. 1). These data, together with the absence of any
immunoreactive band of 70 kDa in the Western blot analysis of Bph-rp
production by human tissues, strongly suggest that Bph-rp is not
related to the 70-kDa mucin-associated antigen thought to be encoded by
the partial cDNA used herein for cloning Bph-rp. Since this partial
cDNA was isolated upon screening of a cDNA library with antibodies
raised against the 70-kDa component of human milk-fat globule membranes
(Larocca et al., 1990), it seems likely that the previously
isolated cDNA clone coding for the C-terminal end of Bph-rp was picked
up as a result of an immunological cross-reactivity event between these
two otherwise unrelated proteins.
Figure 5:
Northern blot analysis of Bph-rp mRNA in
human tissues. About 2 µg of poly(A) RNA from the
indicated tissues were analyzed by hybridization with a 995-bp fragment
of the cDNA (lacking the Alu repeat region) for human Bph-rp.
The positions of RNA size markers are shown. Filters were subsequently
hybridized to a human actin probe in order to ascertain the differences
in RNA loading among the different samples.
The high level expression of human
Bph-rp in liver and kidney, together with its structural relationship
to bacterial enzymes involved in biodegradative pathways of different
aromatic compounds, could be indicative of a potential role for this
protein in detoxification processes. This role may be somewhat parallel
to that proposed for other mammalian hydrolases, including nonspecific
carboxylesterases (Lee et al., 1986; Shibata et al.,
1993, Alexson et al., 1994) and epoxide hydrolases (Heinemann
and Ozols, 1984; Knehr et al., 1993; Grant et al.,
1993). These proteins are also produced at high levels in liver and
kidney, hydrolyze a variety of exogenous and endogenous substrates, and
are thought to function mainly in drug metabolism and detoxification of
many harmful substances (Satoh, 1987). Furthermore, Arand et
al.(1994) have recently described that some of these mammalian
hydrolases, especially epoxide hydrolases, display significant amino
acid sequence similarity with the diverse bacterial proteins involved
in the degradation of aromatic compounds that according to the present
results are most closely related to human Bph-rp. The availability of
the different reagents developed in the present work, including
recombinant Bph-rp, and specific antibodies and probes for this novel
human enzyme, will be useful for a better understanding of the
functional significance of the structural similarities between these
two sets of enzymes that diverged more than 1.5 billion years ago.
Finally, these reagents will also be of interest to examine the
significance of the production of Bph-rp by breast carcinomas and the
possibility that it could play some role in the metabolism of aromatic
endogenous substances like steroid hormones within the human mammary
gland.
/EMBL Data Bank with accession number(s) X81372.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.