(Received for publication, March 31, 1997, and in revised form, June 4, 1997)
From the Biotechnology Research Laboratories, Endoglycoceramidase (EGCase (EC 3.2.1.123)) is a
hydrolase that hydrolyzes the linkage between the oligosaccharide and
ceramide of various glycosphingolipids. This paper describes the
molecular cloning and expression of EGCase II, one of the isoforms of
EGCases. The gene encoding EGCase II was obtained by screening of a
genomic DNA library from Rhodococcus sp. strain M-777
constructed in pUC19 with oligonucleotide probes deduced from a partial
amino acid sequence of the enzyme protein. Recombinant
Escherichia coli cells in which the EGCase II gene was
expressed produced 14 units of the enzyme per liter of culture medium
but did not produce sphingomyelinase. Recombinant EGCase II was a
functioning enzyme with substrate specificity identical to that of the
wild-type enzyme. Sequence analysis showed the presence of an open
reading frame of 1470 base pairs encoding 490 amino acids. The
N-terminal region of the deduced amino acid sequence had the general
pattern of signal peptides of secreted prokaryotic proteins.
Interestingly, the consensus sequence in the active site region of the
endo-1,4- Glycosphingolipids are amphipathic compounds consisting of
oligosaccharides and ceramide moieties and are cell surface components of all vertebrates. Some glycosphingolipids are tumor-associated antigens and receptors of bacterial toxins and hormones and modulate cell growth and differentiation (1). Some of these biological functions
of glycosphingolipids have been elucidated in experiments in which
glycosphingolipids are added to cells. However, information on the
roles of endogenous glycosphingolipids in biological processes is
limited.
Endoglycoceramidase
(EGCase),1 found first in a
culture supernatant of Rhodococcus sp. strain G-74-2 (2),
cleaves the linkage between oligosaccharides and ceramides of various
glycosphingolipids. EGCases have been found in bacterial cells (3),
earthworms (4), leeches (5), rabbit mammary tissues (6), and clams (7),
as well. Three molecular species of the enzyme, EGCases I, II, and III,
each with different specificity, have been isolated from the culture
supernatant of Rhodococcus sp. strain M-750, a mutant of the
wild strain G-74-2 (8). EGCase II hydrolyzed globo-type
glycosphingolipids more slowly than did EGCase I. EGCase III
specifically hydrolyzed the galactosylceramide linkage of gala-type
glycosphingolipids, which were resistant to EGCase I and II. These
enzymes are useful in structural studies of glycosphingolipids (7,
9-14).
Protein activators of EGCase activity in the absence of detergents have
been purified from the culture supernatant of Rhodococcus sp. strain M-777, another mutant of strain G-74-2 (15). When an
activator is used, sugar chains of cell surface glycosphingolipids of
living cells are removed without cell viability being decreased (16,
17). It is thus possible to use EGCase II in conjunction with an
activator to elucidate the biological functions of glycosphingolipids (18-20). However, many steps of chromatographic separation are needed
to separate EGCase from contaminating enzymes, especially sphingomyelinases, before such use and so preparation of purified EGCase II in large amounts is still difficult.
We report here the isolation of an EGCase gene and its expression in
Escherichia coli. The protein sequence obtained from a clone
of EGCase II included the consensus sequence of an activity site of
endo-1,4- pUC19, pTV118N, EASYTRAP Version 2, a DNA
ligation kit, BcaBEST DNA polymerase, restriction and
DNA-modifying enzymes, a MEGALABEL DNA 5 Cells of Rhodococcus sp. strain
M-777 were lysed with lysozyme and proteinase K, and genomic DNA was
prepared as described elsewhere (21). Recombinant plasmid DNA from
E. coli clones was isolated by the alkaline lysis method
(22). Agarose gel electrophoresis, DNA restriction, and treatment with
alkaline phosphatase were done by standard procedures (22). DNA was
ligated with a DNA ligation kit as described by the manufacturer.
EGCase II from Rhodococcus
sp. strain M-777 was purified as described before (8). About 30 µg of
purified EGCase II was treated at 100 °C for 5 min with vapor from a
mixture of 4 µl of pyridine, 1 µl of 4-vinylpyridine, 1 µl of
tributylphosphin, and 5 µl of water and dried in a desiccator under
reduced pressure (23). The pyridylethylated EGCase II was digested with
8 pmol of lysylendopeptidase at 37 °C in 50 µl of 4 M
urea in 20 mM Tris-HCl, pH 9.0, for 16 h. The digest
was put on a reverse-phase column (µRPC C2/C18 SC 2.1/10, 2.1 × 100 mm; Pharmacia Biotech Inc.) and eluted with a linear gradient from
0 to 50% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of
0.1 ml/min by a SMART system for micropreparative liquid chromatography
(Pharmacia). The isolated peptides were numbered in the order of
elution. Amino acid sequences of the isolated peptides were identified
by automated Edman degradation (sequencer model 477A, Applied
Biosystems).
Genomic DNA (5 µg) was digested with
MluI, and the digest was fractionated by 0.7% agarose gel
electrophoresis by the standard method (22). DNA was transferred from
agarose gels to nylon membranes (Hybond N+, Amersham Corp.)
as described by the manufacturer. On the basis of the amino acid
sequence from residues 1 to 15 of peptide L1 (see Table I) obtained by
lysylendopeptidase digestion, and in the expectation that the residue
at position Table I.
Sequences of the peptides derived from lysylendopeptidase digestion of
EGCase II
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES
-glucanase family A was found in the amino acid sequence of
EGCase II.
-glucanase.
Materials
-end labeling kit, and a
BcaBEST random primer DNA labeling kit are products of
Takara Shuzo Co. (Kyoto, Japan). Radionucleotides were purchased from
Amersham Corp. The glycosphingolipids GT1b, GD1a, and GM2 were purchased from Bachem
California (Torrance, CA). GM1a, GM3,
asialo-GM1, and Forssman antigen were purchased from Iatron
Laboratories, Inc. (Tokyo, Japan). Globotetraosylceramide (Gb4-ceramide), sialosylparagloboside, and lysyl endopeptidase were
purchased from Wako Pure Chemicals (Osaka, Japan), and lactosylceramide was purchased from Sigma.
1 of peptide L1 must be lysine, a mixed oligonucleotide
probe,
AAGTC(G/C)GCCCCCGACGG(C/T)ATGCC(G/C)CAGTTCAC(G/C)GA(A/G)GCCGACCTCGC, was designed and designated probe 1. T4 polynucleotide kinase from the MEGALABEL kit was used to label the probe with
[
-32P]ATP. Hybridization was done in 5 × SSC
(1 × SSC = 0.15 M NaCl and 0.05 M
sodium citrate) containing 0.5% SDS, 5 × Denhardt's solution
(1 × Denhardt's solution = 0.02% (w/v) each of Ficoll 400, bovine serum albumin, and polyvinylpyrrolidone-40), and 0.1 mg/ml
denatured salmon sperm DNA at 65 °C for 16 h. After
hybridization, the membranes were washed for 30 min with 0.2 × SSC containing 0.1% SDS at 65 °C and used to expose an imaging
plate, which was examined later on an imaging analyzer (BAS2000, Fuji
Photo Film Co., Tokyo). Hybridization with probe 1 to Southern blots of
the MluI digest showed that only the 4.4-kbp fragment
contained the EGCase II gene. For cloning of this gene, a digest was
prepared with 20 µg of genomic DNA. Restriction fragments of genomic
DNA of Rhodococcus sp. strain M-777 were fractionated by
preparative 0.7% agarose gel electrophoresis. Fragments (4.4 kbp long)
were extracted from the gel by adsorption to glass beads from the
EASYTRAP Version 2 kit. A phosphorylated MluI linker,
pGACGCGTC, was inserted into the HincII site of pUC19, and
the resulting plasmid was designated pUC19M. The MluI
fragments from the fractionated DNA digest were ligated to the
MluI site of pUC19M. The recombinant plasmids obtained were
used to transform E. coli JM109, used in the preparation of
a gene library enriched with the EGCase II gene hybridized with probe
1. Colony hybridization was done by the standard procedure (22), and
hybridized clones were detected with probe 1 under the same conditions
as in Southern blotting. One clone was selected and the plasmid in the
clone was designated pEGCM36.
Peptide
Sequence
L1
SAPDGMPQFTEADLAREYADMGTNF
L2
IDDPRAGQQRIAYPPHLYPLPLDIG
L3
AWRAVADRFADNDAVVAYXLMNEPFGGSLQG
L4
VMLDMHQDVYSGAITPEGNSGNGAGAIGNG
L5
PYPRAVAGTPTEWSSTXDRLQ
L6
HPELVEHYAK
L7
DDDGRSLILRGFNTASSAK
A DNA probe was prepared by digestion of pEGCM36 with
HincII. The probe, which was the 3-end of the EGCase II
gene in pEGCM36, was 210 base pairs long. This probe 2 was random prime
labeled with [
-32P]dCTP using the BcaBEST
labeling kit. In the same way as when pEGCM36 was prepared, a gene
library enriched with the 3
-end of the EGCase II gene was constructed
from genomic DNA digested with BamHI with probe 2 used for
hybridization and detection. BamHI fragments (2.7 kbp long)
were ligated to the BamHI site of pUC19 and used to
transform E. coli JM109, used in the preparation of an
enriched gene library. The library was screened by colony hybridization
with probe 2.
The nucleotides were sequenced by the dideoxy chain termination method with BcaBest DNA polymerase and a DNA sequencer (Applied Biosystems, model 373A). Computer analysis including comparison of DNA sequences was done with DNASIS and GENEBRIGHT software (Hitachi Software Engineering, Tokyo). Frame analysis was done as described elsewhere (24).
Construction of Expression Plasmid with EGCase II GeneThe
vector pTV118N was treated with SalI, Klenow fragment, and
SphI. An insert that included the 5-end of the EGCase II
gene was prepared by digestion of clone pEGCM36 with AccIII,
blunting, digestion with MluI, and gel purification. An
insert that included the 3
-end of the EGCase II gene was prepared by
digestion of clone pEGC20 with MluI and SphI and
then gel purification. The inserts and vector were ligated and used to
transform E. coli JM109. The recombinant plasmid was
purified and designated pTEG3.
E. coli JM109 cells transformed with pTEG3 were
grown at 37 °C in Luria-Bertani medium containing 100 µg/ml
ampicillin upon reaching the optical density (absorbance at 600 nm) of
about 0.5. Then isopropylthio--D-galactopyranoside was
added to the final concentration of 1 mM to cause
transcription, and culture was continued at 37 °C for 4 h more.
Cells were harvested by centrifugation, suspended in extraction buffer
(10 mM Tris-HCl, pH 8.0, and 0.5 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride), and sonicated. Crude extracts were further purified by ion-exchange chromatography on Q-Sepharose.
EGCase activity was assayed with purified asialo-GM1 as the substrate in the presence of Triton X-100 as described before (8). One unit of enzyme was defined as the amount needed to catalyze the hydrolysis of 1 µmol of substrate/min. The substrate specificity was examined as reported elsewhere (8).
The N-terminal amino acid
of the EGCase II purified from Rhodococcus sp. strain M-777
was blocked, so we could not identify the N-terminal sequence. Seven
peptides, L1 to L7, were purified from a lysylendopeptidase digest and
sequenced (Table I). Southern blotting
with probe 1 showed a 4.4-kbp band in the hybridization pattern of the
genomic DNA digested with MluI. A clone, pEGCM36, containing
a 4.4-kbp insert was isolated from the gene library enriched with the
EGCase II gene hybridized with probe 1. Its nucleotides were sequenced,
and it was found to contain a putative open reading frame. The amino
acid sequences L1, L2, L3, L4, L6, and L7 were found in the deduced
amino acid sequence of the open reading frame, but sequence L5 and a
stop codon were not found so we screened the genomic library for the
missing sequences. A probe 2 was prepared from pEGCM36 for use in this
screening. Southern blotting with probe 2 of the BamHI
digest gave a 2.7-kbp band. A clone, pEGCB20, containing a 2.7-kbp
insert was isolated from the gene library enriched with the 3-end of
the EGCase II gene and sequenced. The deduced amino acid sequence
contained sequence L5 and a stop codon in the same frame. A partial
sequence of the 3
-end of the EGCase II gene in pEGCM36 was found in
pEGCB20. pEGCM36 and pEGCB20 overlapped (Fig.
1).
DNA and Amino Acid Sequence Analysis
We cloned and sequenced
2012 nucleotides of the two contiguous clones, pEGCM36 and pEGCB20,
including the coding regions of the EGCase II gene and found an open
reading frame at positions 1-1860. The initiation codon was not
identified because the N-terminal amino acid of native EGCase II was
blocked. In an attempt to tentatively identify the initiation codon and
open reading frame, we analyzed the nucleotide sequence by frame
analysis plotting, which shows codon position-specific differences in
the GC content. In organisms with a high GC content, bases of coding
regions have a high GC content at the third codon position, a low
content at the second position, and an intermediate content at the
first position (24). Frame analysis plotting of the 2012 nucleotides
that included the EGCase II gene suggested that there was a coding
region between the nucleotide positions of approximately 350 and 1850 (Fig. 2). A hydrophobic motif was found
in the deduced amino acid sequence (Fig.
3). This sequence motif, a putative
secretion signal peptide (25), had a positively charged N terminus
followed by a hydrophobic core and a string of polar residues. The
finding of a signal peptide sequence was in agreement with EGCase II
being secreted into the culture medium. The signal sequence was coded
at nucleotides 391-480, starting with GTG. A possible Shine-Dalgarno
ribosome binding sequence started 4 bases upstream from the GTG. These
results were in complete agreement with nucleotides 391-393 (GTG)
being initiation codons. The DNA sequence and deduced amino acid
sequence of the open reading frame of EGCase II are shown in Fig.
4. The open reading frame was 1470 base
pairs long with 490 codons. All of the peptide sequences shown in Table
I were in the deduced amino acid sequence.
Expression of EGCase II
The expression plasmid pTEG3 was
constructed by insertion of a fragment of the coding sequence at
nucleotide positions 482-1997, without a putative secretion signal
sequence, into plasmid pTV118N between the SalI and
SphI sites and in frame with the initiation codon of the
plasmid. In pTEG3, transcription of recombinant genes is controlled by
the promoter plac and can be induced by
isopropylthio--D-galactopyranoside. E. coli
JM109 cells transformed with pTEG3 were cultured in a medium containing
1 mM isopropylthio-
-D-galactopyranoside and separated from the medium by centrifugation. The EGCase II activity of
the cell lysate was assayed with asialo-GM1 as the
substrate. Recombinant E. coli cells produced 14 units of
EGCase II activity per liter of culture medium, but
Rhodococcus sp. strain M-777 produced 3 units of a mixed
enzyme activity of EGCase II and EGCase I (8). Extracts from the
negative control strain containing plasmid pTV118N without the EGCase
II gene had no EGCase II activity, so the enzyme activity found was due
entirely to expression of the cloned EGCase II gene. The specific
activity of purified recombinant EGCase II was 3.2 units/mg, the same
as native EGCase II with asialo GM1 as the substrate. The substrate
specificities of recombinant and native EGCase II were examined with
various glycosphingolipids. Recombinant EGCase II hydrolyzed various
glycosphingolipids at the same rates as native EGCase II under
conditions I and II in Table II. No
activity toward proteases, exoglycosidases, and sphingomyelinases by
the EGCase II purified from E. coli was found. No
sphingomyelinase activity was detected in the lysate of recombinant
E. coli cells. The protein activator of EGCase II
activity, activator II (15), was not detected in the lysate of
recombinant E. coli cells with polyclonal antibodies against
activator II purified from Rhodococcus sp. strain M-777.
|
The molecular weight of EGCase II isolated from Rhodococcus sp. strain M-777 was 58,900 by SDS-polyacrylamide gel electrophoresis. The molecular weight of the recombinant EGCase II was 57,500 by SDS-polyacrylamide gel electrophoresis, although the recombinant enzyme contained 18 additional amino acids from pTV118N. Native EGCase II might be cleaved within the signal sequence upstream from the putative cleavage sites, or its N terminus might be blocked with a bulky residue.
With various glycosphingolipids, we found that activator II increased the activities of both recombinant and native EGCase II. However, only native EGCase II hydrolyzed cell surface glycosphingolipids in the presence of activator II (15). That is, recombinant EGCase II did not hydrolyze the cell surface glycosphingolipids under the conditions we used (data not shown); the reason is not known. The N-terminal amino acid of native EGCase II was blocked (15). We speculate that the N-terminal structure of native EGCase II could be needed for the hydrolysis of cell surface glycosphingolipids.
In the putative amino acid sequence of EGCase II, those sequences of the N terminus of EGCase I and the sequence of activator II that have been isolated from a Rhodococcus sp. (15) were not found. These results suggested that the EGCase II gene is independent of the EGCase I and activator II genes.
Comparison with other nucleotide sequences published in computer data
bases did not show similarity. However, a search of protein sequence
data bases showed that an amino acid sequence of EGCase II was similar
to that of the active site region of bacterial endo-1,4--glucanases
(Fig. 5). Cellulase and
endo-1,4-
-glucanase genes can be grouped into families of related
enzymes on the basis of similarities of the amino acid sequence of
their putative catalytic domains (26, 27). All cellulase and
endo-1,4-
-glucanase sequences listed in Fig. 5 belong to family A. The sequence Asn-Glu-Pro, conserved in almost all cellulase and
endo-1,4-
-glucanase sequences of family A, was found in EGCase II at
amino acid positions 232-234. EGCase II hydrolyzes
D-glucose-
-1,1-ceramide glycosidic linkages but does not
hydrolyze D-galactose-
-1,1-ceramide (8). The Asn-Glu-Pro
sequence of EGCase II may recognize
-D-glucose residues and may have a catalytic role similar to that of
endo-1,4-
-glucanase. Hydrolysis by glycosyl hydrolases often
involves general acid catalysis, usually promoted by aspartic acid,
glutamic acid, or both. Results of site-directed mutagenesis of the
endo-1,4-
-glucanase sequence from Bacillus polymyxa,
Bacillus subtilis, and Ervinia chrysanthemi in
family A again suggest that the conserved glutamic acid residue in
Asn-Glu-Pro is part of the active site (28, 29). Active site residues
are usually highly conserved during evolution. EGCase II and
endo-1,4-
-glucanases may have evolved from the same ancestral
protein, although they have different substrate specificities.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U39554.
We thank Dr. T. Yamagata for encouragement throughout this work.