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(Received for publication, June
27, 1995; and in revised form, September 6, 1995) From the
A clone encoding glyoxalase II has been isolated from a human
adult liver cDNA library. The sequence of 1011 base pairs consists of a
full-length coding region of 780 base pairs, corresponding to a protein
with a calculated molecular mass of 28,861 daltons. Identities
(50-60%) were found to partial 5` and 3` cDNA sequences from Arabidopsis thaliana as well as within a limited region of
glutathione transferase I cDNA from corn. A vector was constructed for
heterologous expression of glyoxalase II in Escherichia coli.
For optimal yield of enzyme, silent random mutations were introduced in
the 5` coding region of the cDNA. A yield of 25 mg of glyoxalase II per
liter of culture medium was obtained after affinity purification with
immobilized glutathione. The recombinant enzyme had full catalytic
activity and kinetic parameters indistinguishable from those of the
native enzyme purified from human erythrocytes.
The glyoxalase system (1, 2, 3) consists of two distinct enzymes,
glyoxalase I (EC 4.4.1.5., lactoylglutathione lyase) and glyoxalase II
(EC 3.1.2.6., hydroxyacylglutathione hydrolase). Glyoxalase I (4) catalyzes the isomerization of the hemimercaptal adduct,
formed spontaneously from methylglyoxal and glutathione, to S-D-lactoylglutathione. This product is hydrolyzed by
glyoxalase II into D-lactic acid and glutathione. The
biological substrate methylglyoxal is produced mainly from
dihydroxyacetone phosphate and glyceraldehyde 3-phosphate in
glycolysis, but can derive also from aminoacetone and hydroxyacetone
formed in the catabolism of threonine and acetone,
respectively(3, 5) . Glyoxalases I and II have been
found in most tissues of mammals, as well as in other species such as
bacteria and plants. The enzymes have broad substrate specificities for
2-oxoaldehydes and their corresponding S-2-hydroxyacylglutathione derivatives, respectively. Although
the glyoxalase system has been studied for a long time, the biological
role of these ubiquitous enzymes is still unclear. They are probably
involved in detoxication of 2-oxoaldehydes, which can be formed from
both xenobiotics and endogenous compounds(4) . Research areas
of current interest include diabetes (6) and cancer
therapy(7) . cDNA encoding glyoxalase I has been isolated from
human colon and U937 cells (8, 9) and a corresponding
DNA sequence has been identified in Pseudomonas
putida(10) . Glyoxalase II activity has been found in
the cytosol fraction and in the mitochondria (11) of higher
eukaryotes. The enzyme is a monomer with a molecular mass of 29 kDa. It
is a basic protein with an isoelectric point of
8.4(12, 13) . Human glyoxalase II has been reported to
be essentially monomorphic, but a rare variant has also been observed
in certain populations(14, 15) . In this paper we
describe the cloning of a cDNA coding for glyoxalase II from human
liver. This is the first DNA sequence reported for the enzyme from any
species. A high level expression clone was constructed for heterologous
expression in Escherichia coli. The recombinant enzyme was
characterized and showed a kinetic behavior indistinguishable from that
of the native enzyme.
For PCR
amplification, 5 µl of the liver The amplified DNA was digested with restriction enzymes
at sites introduced via the PCR primers, and ligated to the vector
pGEM-3Zf(+). After transformation to E. coli XL-1, clones
harboring the DNA fragments were sequenced (23) on both
strands. The clone was called pGHGII.
Figure 1:
Nucleotide sequence and deduced amino
acid sequence of cDNA encoding human glyoxalase II. The termination
codon is marked ``END.'' The dotted lines correspond
to amino acid sequences determined for the peptides from glyoxalase II
from erythrocytes. The altered bases in the 5`-region of the cDNA used
for protein expression are indicated. The regions covered by the
primers used in the PCR for the expression construct are marked with dashes.
PCR was performed as described (above) using the liver cDNA library
as DNA template. After PCR, the fragments were digested with the
restriction endonucleases EcoRI and SalI and ligated
into the expression vector pKK-D. The resulting library of variant cDNA
sequences for expression was transformed to E. coli XL-1. For identification of clones expressing glyoxalase II, antiserum
against the rat erythrocyte protein was used for immunoscreening on
nitrocellulose filters(25) . The clone selected, pKHGII, was
transformed into E. coli JM 109 for large scale expression.
Isoelectric focusing was performed using a
model 8101 column (110 ml) with 1% (w/v) Ampholine, pH 7.0-9.0,
at 5 °C and 450 V for 48 h (Pharmacia Biotech); 300 ng of
recombinant glyoxalase II was analyzed and measurements were made to
monitor active fractions in which pH was determined. The six first
residues of the N terminus of the recombinant protein were determined
after electroblotting to polyvinylidene difluoride membranes.
The kinetic determinations were carried out at 37 °C in 1 ml of
100 mM MOPS, pH 7.2. The amount of enzyme in the assay ranged
from 7.4 to 150 ng/ml. The concentrations of S-D-lactoylglutathione and S-D-mandeloylglutathione were in the intervals
17.4-1840 µM and 0.52-520 µM,
respectively.
In a similar
manner, primers number 2 (5`-ATAC GAATTC TTY TAY GAR GGN ACN GCN GAY
GAR ATG-3`) and number 3 (5`-TCAA CTGCAG RTT NCC NGG YTC NAC RTG-3`),
corresponding to peptides f and h, respectively, were
designed as primers directed downstream and upstream, respectively. A
primary PCR was performed with number 1 and oligo-dT. A second PCR
followed to increase the specificity, using the primers number 2 and
oligo-dT with the first PCR product as a template. Finally, a third PCR
was carried out with primers numbers 2 and 3. This final combination of
nested primers yielded a DNA fragment of 163 bp, which was digested
with EcoRI and PstI, cloned, and sequenced. The
sequence corresponded to positions between 430 and 570 in the finally
determined cDNA sequence. The 96 bp between the primers contained
codons corresponding to the amino acid sequence of peptide g. The partial sequence cloned was used for design of primer number 4
(5`-ACTC GTCGAC TTG AGG TTG TTG ATG GTG TA-3`), directed upstream (Fig. 2), which in combination with number 1, allowed the
isolation of the first 542 bp of the 5` part of the coding sequence.
For the 5`-noncoding region, primer number 5 (5`-TCAA GAATTC GTCGAC CGG
ATC CAC AAT GGC AGC-3`), directed upstream and located close to the 5`
part of the coding region, was used together with two nested primers
against the
Figure 2:
Isolation of cDNA via nested PCR.
Localization of the primers designed from peptide sequences in Table 1. Three consecutive nested PCR yielded a 167-bp fragment (A). The sequence information of fragment A was used for the
isolation of the 5`-coding region (B). The 5`-coding and
noncoding region (C) was isolated with a primer close to the
start codon and nested vector specific primers. A fragment (D)
of 450 bp which corresponded to the 3`-coding and noncoding region was
isolated with a specific primer, number 6, and nested vector specific
primers. For both the 5` end and 3` regions two consecutive nested PCR
were performed.
The remaining 3` part of the cDNA was
isolated using primer number 6 (5`-ATAC GAATTC GTCGAC TAC ACC ATC AAC
AAC CTC AA-3`) (Fig. 2) and primers directed to the The isolated
cDNA contained 1011 bp (Fig. 1) including a coding region of 780
bp. The 5`-noncoding region consisted of 36 bp and the 3`-noncoding
region of 195 bp. The cDNA encodes a protein of 260 amino acid
residues. The calculated molecular mass of the protein is 28,861 Da.
Two partial 5` and 3` cDNA sequences from Arabidopsis thaliana,( The human glyoxalase II shares some
sequence similarity with corn glutathione transferase I (30) in
an overlap of 178 bp (including gaps, data not shown). Nucleotides
200-373 in glyoxalase II and 394-565 in glutathione
transferase I are 56% identical.
Figure 3:
Silver-stained SDS-PAGE. From left to
right: recombinant glyoxalase II, mixture of recombinant enzyme, and
enzyme purified from erythrocytes, glyoxalase II from
erythrocytes.
N-terminal sequence analysis of the
purified recombinant glyoxalase II revealed a sequence MKVEVL identical
to that determined for the protein prepared from erythrocytes and to
the amino acid sequence deduced from the cDNA. The Nterminal methionine
was 100% retained in the recombinant protein.
The sequence of the cDNA encoding human glyoxalase II
reported here provides the primary structure of a new member of the
large group of glutathione-linked enzymes(31) . The nature of
the protein as revealed by the nucleotide sequence is unequivocally
glyoxalase II. This was further confirmed by peptide analysis of the
purified protein and by the heterologous expression of a protein with
full glyoxalase II activity. The cDNA sequence encodes a 260-amino acid
residue protein with a calculated molecular mass of 28,861 Da, which is
in accordance with the mass estimated in earlier studies(32) .
The protein is identified as the major variant of glyoxalase II (14, 15) based on its isoelectric point (8.5) and the
finding that several cDNA isolates had the same sequence. No evidence
for a second variant was found in the cDNA library studied. The
optimized expression clone for glyoxalase II was found to have six
alterations in the 5`-coding region in comparison with the wild-type
sequence (Fig. 1). Sequence analysis of the entire coding region
demonstrated that no additional mutations were present in the
expression clone. Thus, the overall change in the new cDNA template
made it compatible with the requirements for expression of the protein
in E. coli without altering the amino acid sequence of the
translation product. The original ``wild-type'' cDNA did not
produce any detectable amount of enzyme in E. coli (data not
shown). The yield of recombinant glyoxalase II (70 mg/3-liter culture)
is approximately 100-fold higher than that from human erythrocytes (0.3
mg/liter hemolysate, cf. (32) ). The relative
migration in SDS-PAGE of the recombinant glyoxalase II and the protein
purified from erythrocytes further confirmed the expected molecular
mass of the enzyme. Isoelectric focusing of the purified recombinant
protein was carried out to confirm that no mutations or
post-translational modifications influencing the isoelectric point were
present. In addition, direct N-terminal sequence analysis demonstrated
the presence of the first six amino acid residues deduced from the cDNA
sequence. Many recombinant proteins have their N-terminal methionine
removed when expressed in a prokaryotic host. In the case of glyoxalase
II, the initiator methionine is present to 100%. This might be due to
the penultimate residue lysine, which does not promote removal of
methionine in bacteria(33) . The catalytic properties of the
recombinant glyoxalase II are of obvious importance for further
studies. Table 2shows that the kinetic constants for glyoxalase
II with the standard substrate, S-D-lactoylglutathione, and for the more hydrophobic S-D-mandeloylglutathione were in good agreement with
those obtained for the enzyme purified from human
erythrocytes(32) . Thus, the protein appears properly folded
and fully functional as required for more incisive mechanistic studies
in the future. Data base homology searches showed that two groups
independently have determined the 5` and 3` regions of human cDNA
sequences. Interestingly, also two partial cDNA sequences from A. thaliana are structurally similar to the 5` and 3` ends of the
glyoxalase II cDNA and overlap each other. They were isolated by two
groups independently, but not related to any known protein. From an
evolutionary perspective, it is interesting to note that not only
mammalian and plant glyoxalase II sequences show extensive sequence
similarities, but also that the maize glutathione transferase I appears
to have a substantial, but spatially restricted, sequence similarity
with glyoxalase II. At the DNA level, glutathione transferase I from
corn (30) shares sequence similarity with human glyoxalase II
in an overlap of 178 bp. Among glutathione transferases, the enzymes
from plants have primary structures that differ strongly from those of
the mammalian enzymes(34) . However, certain residues of
importance for glutathione binding are fairly well conserved between
mammalian and corn sequences and include residues 65-70,
represented by QSNAIL in several mammalian enzymes(34) . Although the glyoxalase system has been studied for a long time, its
biological function remains unclear. Cloning of the cDNA encoding human
glyoxalase II and expression of the protein in large amounts will
facilitate studies of structural and functional aspects of the enzyme
as well as the transcriptional regulation of its gene.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
X90999[GenBank].
Volume 271,
Number 1,
Issue of January 5, 1996 pp. 319-323
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Materials
A human adult liver cDNA library was
obtained from Clontech Laboratories, Inc. Oligonucleotides were custom
synthesized by Operon Technologies Inc. (Alameda, CA). Enzymes used for
PCR (
)and cloning were purchased from Boehringer Mannheim
(Mannheim, Germany). ProBlott membranes were obtained from Applied
Biosystems Inc. LambdaSorb and the vector pGEM-3Zf(+) were bought
from Promega Corp. Sequenase version 2.1 and
[
-P]dCTP were purchased from Amersham
International (Amersham, Buckinghamshire, United Kingdom). The
expression vector pKK223-3 was obtained from Pharmacia Biotech
(Uppsala, Sweden). The vector was modified by digestion with AccI to eliminate the second restriction site for SalI and called pKK-D(16) . Affi-Gel 10 and protein
assay reagent were bought from Bio-Rad.
5,5`-Dithio-bis(2-nitrobenzoate), glutathione, and other chemicals were
purchased from Sigma. Thiol esters of glutathione were enzymatically
synthesized (17) and S-p-nitrocarbobenzoxyglutathione chemically
synthesized and purified as described(18) . Antiserum against
rat glyoxalase II was prepared as described(19) .
Purification of Glyoxalase II from Human
Erythrocytes
About 2 liters of red blood cells (from a blood
bank) were treated with 6 liters of cold acetone (4 °C). The
mixture was kept in the cold room overnight and then centrifuged at
5,000 
g for 15 min at 4 °C. The sediment (about
1.2 liter) was recovered and 3 volumes of 10 mM MOPS buffer,
pH 7.1, containing 100 µM phenylmethanesulfonyl fluoride
were added. The mixture was kept in the cold room with vigorous
stirring for 2 h and then centrifuged at 15,000 g for
15 min at 4 °C. The sediment was discarded and the supernatant
(about 3 liters) was added to 20 ml of Affi-Gel 10 with immobilized
glutathione(20) . The mixture was kept overnight in the cold
room under shaking and then filtered under vacuum. The affinity matrix
was resuspended in 10 mM MOPS buffer, pH 7.1, and poured into
a column. After extensive washing with 10 mM MOPS buffer, pH
7.1, glyoxalase II activity was eluted with the same buffer containing
3 M NaCl. The active fractions were pooled (about 50 ml),
diluted 1:6 (v/v) with 10 mM MOPS buffer, pH 7.1, and filtered
through a column of hydroxyapatite (8 ml). Under these conditions,
glyoxalase II activity is not bound to the column (less than 5%). The
filtrate (hemeoglobin-free) was diluted 1:4 (v/v) and rechromatographed
on Affi-Gel 10 with immobilized glutathione. Glyoxalase II activity was
eluted with 2 mMN,S-di-Fmoc-glutathione (N-(9-fluorenyl)methoxycarbonyl) in 10 mM MOPS
buffer; this thiol ester analog is a more effective eluant than is free
glutathione.Determination of Amino Acid Sequences
The protein
sample was reduced with dithiothreitol and alkylated with
4-vinylpyridine. After SDS-PAGE and Coomassie Brilliant Blue staining
on a 12% (w/v) gel, the band corresponding to glyoxalase II was
excised, and the protein in the gel was subjected to in situ tryptic digestion(21) . Briefly, the gel was washed twice
with 0.2 M ammonium bicarbonate, 50% (v/v) acetonitrile, then
completely dried and finally rehydrated with a buffered solution (21) containing modified trypsin, sequence grade (Promega
Corp.). After incubation overnight at 30 °C, the fragments
generated were extracted with 0.1% trifluoroacetic acid, 60%
acetonitrile and subsequently separated by reverse-phase liquid
chromatography on a µRPC C2/C18 SC 2.1/10 column, operated in the
SMART System (Pharmacia Biotech). A 160-min
gradient (0-40%) of acetonitrile in 0.06% trifluoroacetic acid
was developed at a flow rate of 100 µl/min. Several fractions were
selected for automated sequence analysis in an Applied Biosystems Model
470A sequencer. Phenylthiohydantoin derivatives from the degradations
were analyzed by reverse-phase high performance liquid chromatography
as described(22) .
Purification of
For the purification of cDNA Library and Nested
PCR
cDNA, 10 ml (5.5
10
plaque-forming units) of phage lysate of the human
liver library was used. The purification was carried out with
LambdaSorb phage adsorbent in accord with the manufacturer's
instructions. The DNA was dissolved in 500 µl of water.
cDNA library was used in a
100-µl reaction mixture with 10 mM Tris-HCl, pH 8.3, 1.5
mM MgCl
, 50 mM KCl, 0.2 mM of
each dNTP, and 0.8 µM 5` and 3` primers. The mixture was
overlaid with 100 µl of mineral oil. Incubation at 95 °C for 10
min was followed by a decrease to 70 °C and 2.5 units of Taq DNA polymerase was added. Amplification with specific primers
involved 30 cycles at 95 °C for 1 min, 55 °C for 2 min, and 72
°C for 2 min. For degenerate primers, 3 cycles of denaturation at
95 °C for 1 min, annealing at 35-45 °C for 3 min, and
extension at 72 °C for 2 min were performed, followed by 30 cycles
with the annealing temperature increased to 45-55 °C. The
-specific primers V1 and V2 had the following sequences 5`-CG
GAATTC GAG CTC ACA CCA GAC CAA CTG GTA ATG-3` and 5`-CTC GAATTC ACC AAC
TGG TAA TGG TAG CG-3`, respectively. The underlined sequence is the
endonuclease EcoRI restriction site used for cloning of the
PCR product.
Vector Construction and Screening for High Level
Expression in E. coli
The primer GII15Ex (5`-T CTA GAATTC ATG
AAR GTD GAR GTD CTB CCD GCN CTB ACY GAY AAC TAY ATG TAY CTG GTC ATT GAT
GAT-3`, including restriction sites for EcoRI, underlined) was
designed for optimal expression in E. coli(24) by
randomizing the silent positions in some of the triplets coding for the
first 14 amino acid residues following the initiator methionine (Fig. 1). In the 3` end of the coding region, two stop codons
(TAA) in tandem were introduced via the primer GIIstop (5`-T
AAGCTTGTCGAC TTA TTA GTC CCG GGG CAT CTT GA-3`) which included
restriction sites for HindIII and SalI (underlined).
Purification
A 3-liter bacterial culture (grown in
2% (w/v) tryptone, 1.5% (w/v) yeast extract, 0.5% (w/v) NaCl, and 1%
(w/v) glycerol) was induced with 0.2 mM
isopropyl-
-D-thiogalactoside at OD 0.25 and
cultured overnight at 37 °C. The bacteria were harvested by
centrifugation, resuspended in 10 mM MOPS, pH 7.2, 0.1
mM phenylmethanesulfonyl fluoride containing 10 mg of lysozyme
and incubated on ice for 30 min. Sonication was performed three times
for 30 s followed by centrifugation. The bacterial lysate was applied
to a column of Affi-Gel 10 with immobilized glutathione. After washing
with 10 mM MOPS, pH 7.2, the enzyme was eluted with 3 M NaCl in the same buffer. Fractions with glyoxalase II activity
were pooled. Protein concentration was estimated using the method of
Bradford(26) .
Characterization of Purified Recombinant Glyoxalase
II
Recombinant glyoxalase II, the erythrocyte enzyme, and a
mixture of the two were analyzed by SDS-PAGE in a 12% (w/v) acrylamide
gel(27) . The protein bands were stained with
silver(28) .Assay for Glyoxalase II Activity and Kinetic
Measurements
Specific activity was measured at 37 °C in a
1-ml reaction volume containing 900 µMS-D-lactoylglutathione, and 200 µM 5,5`-dithiobis(2-nitrobenzoate) in 100 mM MOPS, pH 7.2.
The formation of glutathione in the presence of
5,5`-dithiobis(2-nitrobenzoate) (29) was monitored
spectrophotometrically at 412 nm ( = 13.6
mM
cm
). As an
alternative, the glutathione thiol ester hydrolysis was followed
spectrophotometrically at 240 nm (
= 3.1
mM
cm
)(1) .
Peptide Sequence Analysis
The fragments
generated by tryptic digestion of glyoxalase II were separated by
reverse-phase liquid chromatography. Edman degradation revealed the 12
peptide sequences shown in Table 1. The corresponding positions
in the cDNA encoding glyoxalase II subsequently isolated are indicated
in Fig. 1. The fragment sequences were used for primer design in
the PCR reactions and, subsequently, for identification of the PCR
products.
Cloning of cDNA Encoding Human Glyoxalase II
The
N-terminal sequence of peptide a (Table 1) was used for
design of primer number 1 (5`-ATAC GAATTC GTCGAC ATG AAR GTN GAR GTN
YTN CCN GCN YTN ACN AC-3`), in which all the possible mRNA triplets
coding for the amino acids were realized by randomizing some of the
``wobble base'' positions in the primer.gt11 vector.
vector. A fragment of 500 bp was cloned and sequenced.
Comparison with Other DNA Sequences
A search for
homology with other DNA sequences in the GenBank/EMBL Data Bank
revealed 91-99% identity to 5` and
3`()(
)(
)(
)(
)partial human cDNA sequences. These sequences cover about
one-third of the glyoxalase II cDNA from each end and contain a few
undetermined nucleotides.
)(
)were shown to
overlap each other and revealed 57% identity to human glyoxalase II.
The deduced amino acid sequences were about 51% identical and 68%
similar. Some regions of the primary structure showed significantly
higher degree of identity (100% for residues 50-65, and 82% for
residues 128-149).Expression and Purification
Ten clones were
isolated after screening the library of expression clones with antisera
against rat glyoxalase II. In all cases the expressed protein had
activity with S-D-lactoylglutathione. The clone
giving the highest activity with S-D-lactoylglutathione, pKHGII, was chosen for large
scale purification involving affinity chromatography with immobilized
glutathione. From a 3-liter culture, 70 mg of enzyme was recovered in
pure form. The specific activity of the purified enzyme was 1,400
µmol/min/mg of protein as determined with S-D-lactoylglutathione.Characterization of the Recombinant Protein
The
apparent molecular mass of recombinant glyoxalase II and the enzyme
prepared from human erythrocytes were the same as judged from SDS-PAGE (Fig. 3). The silver-stained gel showed a single protein band of
29 kDa for both preparations (Fig. 3), indicating no major
post-translational modifications in the enzyme from the natural source.
The isoelectric point of the recombinant enzyme was determined as 8.5
by use of isoelectric focusing.
Kinetic Studies
Kinetic parameters for the
recombinant enzyme were determined with S-D-lactoylglutathione and S-D-mandeloylglutathione (Table 2). For S-D-lactoylglutathione, the K
value was 187 µM and the k value 780 s
. For the more hydrophobic S-D-mandeloylglutathione, the K
value was 29 µM and the k value 201 s
. These values are in good
agreement with those estimated for glyoxalase II purified from
erythrocytes (Table 2).
Although there are some ambiguities in
the deposited sequences, they resemble the first one-third of the human
glyoxalase II cDNA sequence in the 5` end and the last one-third in the
3` end. These cDNA sequences have not been assigned to any protein.
The overlapping cDNA sequences of 762 bp show 57% identity with
that of human glyoxalase II. The deduced amino acid sequences share 51%
identity and are 68% similar. Some regions of the sequences are partly
ambiguous, but the cDNA sequences from A. thaliana are clearly
related and most probably represent glyoxalase II.
))))))))
We thank Drs. Ulf Landegren and Maria
Lagerström-Fermer, Uppsala University, Uppsala, for
valuable advice and Dr. Mikael Widersten of our laboratory for kind
help with computer searches. We also thank Dr. Helena Danielson for
helpful advice.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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