(Received for publication, December 14, 1994; and in revised form, December 29, 1994)
From the
In vitro single point mutagenesis, inositol
phospholipid hydrolysis, and substrate protection experiments were used
to identify catalytic residues of human phosphatidylinositide-specific
phospholipase C1 (PLC
1) isolated from a human aorta cDNA
library. Invariant amino acid residues containing a functional side
chain in the highly conserved X region were changed by in vitro mutagenesis. Most of the mutant enzymes were still able to
hydrolyze inositol phospholipid with activity ranging from 10 to 100%
of levels in the wild type enzyme. Exceptions were mutants with the
conversion of Arg
to Leu (R338L), Glu
to
Gly (E341G), or His
to Leu (H356L), which made the enzyme
severely defective in hydrolyzing inositol phospholipid. Phospholipid
vesicle binding experiments showed that these three cleavage-defective
mutant forms of PLC
1 could specifically bind to
phosphatidylinositol 4,5-bisphosphate (PIP
) with an
affinity similar to that of wild type enzyme. Western blotting analysis
of trypsin-treated enzyme-PIP
complexes revealed that a
67-kDa major protein fragment survived trypsin digestion if the wild
type enzyme, E341G, or H356L mutant PLC
1 was preincubated with 7.5
µM PIP
, whereas if it was preincubated with 80
µM PIP
, the size of major protein surviving
was comparable to that of intact enzyme. However, mutant enzyme R338L
was not protected from trypsin degradation by PIP
binding.
These observations suggest that PLC
1 can recognize PIP
through a high affinity and a low affinity binding site and that
residues Glu
and His
are not involved in
either high affinity or low affinity PIP
binding but rather
are essential for the Ca
-dependent cleavage activity
of PLC.
Phospholipase C hydrolyzes inositol phospholipids into
diacylglycerol and inositol 1,4,5-trisphosphate (IP), (
)a process that constitutes a major pathway for
receptor-coupled signaling at the plasma membrane of most eukaryotic
cells. Both diacylglycerol and IP
function as important
second messengers that activate protein kinase C and mobilize
intracellular Ca
, respectively, thus triggering
multiple enzymatic cascades to regulate cellular functions including
cell growth and neuronal activity(1) . Phospholipase C exists
as isoenzymes (PLC
, PLC
, and PLC
)(2) . PLC
is activated following phosphorylation by nonreceptor or receptor
protein tyrosine kinase activities(3, 4) , whereas
PLC
s are regulated by
subunits of G proteins (Gq family) (5, 6, 7) or by
subunits(8, 9, 10) . How PLC
1 is
regulated still remains to be determined.
Although PLC isozymes
differ in the way they are regulated, they have similar enzymatic
properties(2) . All three members of the PI-PLC family are able
to recognize phosphatidylinositol (PI), phosphatidylinositol
4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate
(PIP) and to carry out the Ca
-dependent
hydrolysis of these inositol phospholipids. Comparison of the amino
acid sequences of all three isoforms reveals that PI-PLCs are highly
conserved in two distinct regions designated X and Y(2) .
Structural integrity of the highly conserved X and Y region is
essential for a functional catalytic core, as partial deletion of
either the X or Y region sequence in PLC
1 (11, 12) or PLC
(13, 14) inactivates the enzyme. The intervening
peptide connecting the X and Y regions is not essential for the
hydrolytic properties of either PLC
or PLC
1, as partial
deletion of these sequences in PLC
1 (14) or PLC
2 (13) or trypsin cleavage of this sequence in PLC
1 (11) does not inactivate the truncated enzymes. The first 60
NH
-terminal residues of the PLC
1 sequence are not
essential for Ca
-dependent catalysis, but are
required for the enzyme to hydrolyze PIP
in a processive
manner (15) . A subfamily-specific sequence of 400 amino acid
residues in the
family is located between the X and Y regions and
is characterized by src homology domains (SH2 and SH3), which
are essential for the activation of PLC
by tyrosine protein
kinases(3) . Partial deletion of either the NH
terminus or COOH terminus of PLC
does not affect its
catalytic activity, but the enzyme loses its ability to be activated by
G proteins(10, 16) . These observations strongly
indicate that residues essential for specific substrate recognition and
Ca
-dependent cleavage can be identified among those
conserved residues located in the X and Y regions.
To identify
specific amino acid residues involved in catalysis, we isolated cDNA of
the smallest PLC from human aorta, expressed it in Escherichia
coli, and purified the protein. To minimize structural disturbance
of the enzyme and to facilitate genetic analysis of the role of
PLC1 in cellular function, we used single amino acid residue
substitution mutagenesis to evaluate the contribution of each conserved
residue in PI-PLC to its catalytic function. To narrow down the number
of potential residues involved in catalysis, only those residues
containing a functional side chain and which are invariant in both
prokaryotic and eukaryotic PI-PLC were subjected to base substitution
mutagenesis. The mutant enzymes were assayed for their abilities to
hydrolyze PI and PIP
, to bind inositol phospholipid, and to
form trypsin-resistant enzyme-substrate complexes. We demonstrate that
His
and Glu
, located in the X conserved
region of PLC
1, are essential for the
Ca
-dependent hydrolysis of PIP
rather
than for substrate binding.
Figure 6:
Schematic presentation of two-stage PCR
method to construct point mutations in PLC1. The first stage of
mutagenesis consists of two independent primary PCRs. One reaction uses
the mismatch primer (mutagenic primer) paired with one of the external
primers flanking one of the unique restriction sites; the other uses
the internal primer paired with another external primer flanking the
other unique restriction site. Both reactions used pRSETAplc plasmid as
template, and the PCRs were carried out as described under
``Experimental Procedures.'' The primary PCR products were
isolated and purified, mixed, and reannealed. The hybrid template was
first extended with Taq polymerase and followed by using the
same pair of external primers for the secondary stage PCR as described
under ``Experimental Procedures.'' The final mutagenic DNA
fragment was cleaved with a pair of unique restriction endonuclease (SphI/SacII or SphI/EcoRI) and used
to replace the corresponding fragment in wild type
pRSETAplc.
Figure 1:
Nucleotide and deduced amino acid
sequence of human PLC1 cDNA. Panel A, the structure of
human PLC
1 cDNA (2.6 kb) is schematically shown with the coding
region (open box) flanked by untranslated sequences (solid
line). The 2040- and 591-base pair (bp) EcoRI
fragments from the phage clone, corresponding to the sequence coding
for the NH
-terminal and COOH-terminal region of PLC
1,
respectively, were subcloned further into the EcoRI site of
pTZ19R to generate pTZ19R5`plc and pTZ19R3`plc. Panel B, the
nucleotide sequence of the 2.6-kb full-length human phospholipase
C
1 cDNA was determined as described under ``Experimental
Procedures.'' The nucleotide residue numbers are given to the right of each line. The deduced amino acid sequence encoded by
the longest open reading frame (beginning at nucleotide 95 ATG) is
shown using the single letter amino acid code. Deduced amino acid
residues are numbered beginning with the initiation methionine, and the
residue numbers are shown to the left of each line. Amino acid
residues different from those of rat enzyme are shown. Conserved X and
Y regions of PI-PLC are underlined.
Fig. 2compares the deduced
amino acid sequence of the most conserved X and Y regions of human
PLC1 with those of the previously described PI-specific PLCs.
Within these regions, human PLC
1 has 40 and 38% sequence identity
to rat PLC
1, 35 and 43% sequence identity to rat PLC
, 40 and
38% sequence identity to the Drosophila NorpA gene, and 38 and
32% sequence identity to yeast PLC1. PI-specific PLC from Bacillus
cereus only contains the X domain, with which the human clone
shares 30% similarity.
Figure 2:
Comparison of the primary structures of
the conserved regions of PLC isoenzymes from rat, human, Drosophila, yeast, and bacillus. Amino acid sequence
comparison of human PLC1 with the conserved X and Y regions of rat
PLC
1(17) , PLC
3(42) , PLC
1(43) ,
PLC
2(13) , human PLC
2 (44) , yeast
PLC
(45) , Drosophila norpA (Dro
PLC
)(46) , and the X region of bacillus
PI-PLC(41) . Organisms, PI-PLC isoenzyme classes, and the
starting position in each protein sequence of the residues shown are
indicated on the far left. The boxed areas denote
positions at which amino acids in seven or more sequences are identical
or represent conservative substitutions grouped as follows: A and G; T
and S; I, L, M and V; K, H, and R; W, Y, and F; D and E; N and Q. Gaps
introduced to optimize the alignment are indicated by hyphens.
Identical residues containing functional side chain in the X region of
PI-PLC from human to bacillus are indicated by an asterisk and
were subjected to base substitution mutagenesis
study.
Figure 3:
Northern analysis of RNA from various
human tissues. Human multiple tissue Northern blot obtained from
CLONTECH was hybridized with full-length human PLC1 cDNA washed
and exposed as suggested by the supplier. Lane 1, pancreas; lane 2, kidney; lane 3, skeletal muscle; lane
4, liver; lane 5, lung; lane 6, placenta; lane 7, brain; and lane 8,
heart.
Figure 4:
SDS-polyacrylamide gel electrophoresis and
immunoblots of human PLC1 expressed from E. coli BL21. Panel A, 100 µg of crude extracts from E. coli BL21 carrying pRSETAplc (lane a), vector pRSET (lane
b), or 0.1 µg of heparin-Sepharose column
chromatography-purified PLC
1 (lane c) were separated on
10% SDS-polyacrylamide gels and further detected with mixed monoclonal
anti-PLC
1 antibodies. Panel B, 5 µg of
heparin-Sepharose column chromatography-purified PLC
1 (lane
a), 100 µg of crude extracts from E. coli BL21
carrying pRSETAplc (lane b), and 10 µg of
Ni
-NAT agarose column-purified PLC
1 (lane
c) were separated on 10% SDS-polyacrylamide gels and stained with
Coomassie Blue.
Figure 5:
Ca dependence of
PLC
1. The PIP
hydrolysis activity of purified
PLC
1 expressed from E. coli BL21 was assayed as described
under ``Experimental Procedures,'' and the free calcium
concentrations were calculated according to Fabiato and
Fabiato(47) . The values are expressed relative to the activity
at a free Ca
concentration of 95
nM.
Figure 7:
Centrifugation assay of PLC1 binding
to lipid vesicles. Panel A, binding of wild type PLC
1 and
cleavage-defective mutants to phospholipid vesicles of different lipid
content: 400 µM phospholipid vesicles with a PE/PC molar
ratio of 4:1 (
), 600 µM phospholipid vesicles of
PE/PC/PI with a molar ratio of 4:1:2.5 (&cjs2108;), and 420 µM (
) phospholipid vesicles of PE/PC/PIP
with a
molar ratio of 4:1:0.25 (
). Panel B, dose-dependent
binding of PE/PC/PIP
(4:1:0.25) lipid vesicles to wild type
and cleavage-defective mutants. The concentration of PIP
(µM) was a fraction of phospholipid vesicle
containing PE/PS/PIP
with a molar ratio of 4:1:0.25. All of
the centrifugation assays were carried out in a 0.2-ml total volume
using a Beckman TL-100 table top ultracentrifuge and TLA-100 rotor (see
``Experimental Procedures''). The unbound enzyme fractions
(supernatant) were quantified by PI hydrolysis assay and immunoblotting
using mixed monoclonal antibodies (for cleavage-defective mutant). The
bound enzyme fractions (pellets) were dissolved in 0.05 ml of
phosphate-buffered saline buffer then quantified by Western blotting
analysis.
Figure 8:
Effect of PIP on the trypsin
cleavage of PLC
1. Panel A, Western blotting analysis of
PLC
1 (0.2 µg) digested with 0 (lane 1), 0.005 (lane 2), 0.01 (lane 3), 0.05 (lane 4), and
0.1 µg (lane 5) of trypsin for 5 min in the presence of
400 µM PE. Panel B, kinetic analysis of PLC
1
inactivation by the proteolytic action of trypsin. 0.2 µg of the
PLC
1 was preincubated with PE/PIP
micelles containing
200 µM PIP
and 400 µM PE
(
); PE micelles containing 600 µM PE (
) or in
the absence of phospholipid (
). At each indicated time point,
an aliquot of incubation mixture was diluted and used to determine the
residual PI hydrolysis activity. Panel C, the residual
activity of PLC
1 after a 5-min trypsin digestion in the presence
of PE/PIP
micelles containing 400 µM PE and
the indicated concentration of PIP
(
) or PE only
(
).
The ability of
PIP to protect PLC
1 against trypsin-inflicted loss of
PI hydrolysis activity was correlated to the extent of degradation of
PLC
1 by trypsin. As shown in Fig. 9A, incubating
PLC
1 with PE/PIP
micelles prior to trypsin digestion
markedly reduced the rate of trypsin cleavage; at least 50% of the
PLC
1 protein still remained intact after a 5-min trypsin
digestion, and this was reduced to less than 10% as the
enzyme-PIP
complexes were digested further with trypsin for
a total period of 15 min. However, 90% of the enzyme was degraded by
trypsin within 5 min when the enzyme was preincubated with lipid
micelles containing PE only. The pattern and extent of digestion of
PLC
1
PIP
complexes by trypsin were highly
dependent on the concentration of PIP
used to form the
specific complexes. As the concentration of PIP
was
increased to 7.5 and 35 µM, the major protein species
surviving trypsin cleavage was a 67-kDa fragment. As the PIP
concentration increased to 200 µM, the size of
protein surviving trypsin cleavage was similar to that of the intact
enzyme. The proportion of the 67-kDa fragment and intact protein
surviving the action of trypsin was dependent on the concentration of
PIP
. Trypsin digestion of PLC
1 preincubated with low
concentrations of PIP
yielded a major 67-kDa proteolytic
fragment. As the concentration of PIP
increased, the size
of the major protein species surviving the trypsin digestion was 89
kDa. We were not able to detect the 80-kDa fragment, which was the
major proteolytic product when free PLC
1 was digested with the
reduced level of trypsin, suggesting that both the sensitivity and
specificity of the enzyme to trypsin cleavage were changed as a result
of specific binding of PIP
. This observation shows that the
site of enzyme-PIP
complex and its sensitivity to trypsin
cleavage were highly dependent on the concentration of PIP
used to form the specific enzyme-PIP
complex.
Figure 9:
Western blotting analysis of trypsin
cleavage of PLC1. Panel A, trypsin (0.1 µg) cleavage
of PLC
1 for 0, 5, and 15 min in the presence of 200 µM or absence of PIP
(control). Panel B, trypsin
(0.1 µg) cleavage of PLC
1 (0.2 µg) for 5 min in the
presence of PE/PIP
micelles containing the indicated
concentration of PIP
. All of the trypsin cleavage reactions
were carried out at 37 °C in 40 µl of 12.5 mM sodium
phosphate, 37.5 mM HEPES, pH 7.0, 5 mM EGTA, 3.75
mM EDTA, 75 mM NaCl, 25 mM KCl, 0.025% Tween
20, 0.075% sodium cholate, 375 µg/ml BSA containing 400 µM PE and the indicated concentration of
PIP
.
Figure 10:
Analysis of the trypsin sensitivity of
mutant forms of PLC1 in the presence or absence of
PIP
. Panel A, trypsin (0.1 µg) cleavage of 0.2
µg of mutant PLC
1 E341G, H356L, and R338L proteins in the
presence of 200 µM PIP
or in the absence of
PIP
(control) for 0, 5, 10, and 15 min. Panel B,
trypsin (0.1 µg) cleavage of 0.2 µg of mutant PLC
1 E341G,
H356L, and R338L proteins for 5 min in the presence of the indicated
concentration of PIP
. All of the trypsin cleavage reactions
were carried out in a buffer (40 µl) containing 12.5 mM sodium phosphate, 37.5 mM HEPES, pH 7.0, 5 mM EGTA, 3.75 mM EDTA, 75 mM NaCl, 25 mM KCl, 0.025% Tween 20, 0.075% sodium cholate, 375 µg/ml BSA
containing 400 µM PE and the indicated concentrations of
PIP
. At the indicated time point, the reactions were
stopped and analyzed by Western blotting as described under
``Experimental Procedures.''
The
cleavage site(s) of preformed substrate-enzyme complexes by trypsin was
also highly dependent on the PIP concentration. For mutants
H356L and E341G, enzyme-substrate complexes formed at a low
concentration of PIP
(7.5 µM) were easily
cleaved to a 67-kDa fragment by trypsin, whereas enzyme-substrate
complexes formed at a saturating concentration of PIP
(200
µM) were relatively resistant to trypsin cleavage (Fig. 10B). Preincubating mutant R338L with increasing
concentrations of PIP
from 7.5 to 200 µM did
not protect it from trypsin cleavage (Fig. 10B). The
saturation concentration of PIP
required to protect H356L
and E341G from trypsin digestion was estimated to be 100
µM, and the half-maximal protection concentration of
PIP
was estimated to be 35 µM. This result
demonstrated that according to their sensitivity to trypsin cleavage,
mutant E341G and H356L are able to form at least two types of complex
with PIP
in a way similar to that of the wild type enzyme.
This indicates that Glu
and His
may play a
role in the step of cleavage rather than in the steps of substrate
binding.
At least four isoforms of PLC have been identified and
cloned:
1,
2,
3(23, 24) , and
4(25) . Judged by the slight deviation of its amino acid
sequence from those of rat and bovine
isoforms, the present PLC
isoform we isolated from a human aorta library is classified as
1
type. Patterns of expression of PLC
1 from various tissues of rats
and bovines examined by immunoanalysis (26) and Northern
analysis (27) reveal that although the level of expression is
relatively lower than for other isoforms of PLC, PLC
1 is
widespread among most tissues. We found that transcripts of PLC
1
are also present in almost all human tissues, implying that the enzyme
may play a role in some fundamental cellular process. Several lines of
evidence agree with this implication. First, in an attempt to select
mutants from mutagenized Chinese hamster lung fibroblasts defective in
thrombin-induced mitogenesis, several of these mutants were later shown
to lack PLC
1 protein(28) . Second, disruption of a
PLC
-like (PLC1) gene in yeast resulted in slow growth or
death of the cells, which could be rescued by exogenous expression from
a cloned rat PLC
1 cDNA(29) . Third, a mutation in
PLC
1 leading to an 8-fold increase in specific activity has been
identified in the spontaneous hypertensive
rat(30, 31) . Finally, our laboratory has recently
found that suppression of PLC
1 in a rat cell line arrests cell
growth and diminishes mitogen-induced intracellular calcium
mobilization. (
)
In at least two structural aspects, the
present recombinant version of PLC1 differs from purified forms.
First, a 34-amino acid region including 6 consecutive histidine
residues was fused to the NH
terminus of the recombinant
enzyme. Second, post-translational modification of PLC
1 expressed
in E. coli may be quite different from that of PLC
1
expressed in mammalian cells. Since we have demonstrated here that
recombinant human PLC
1 expressed from E. coli displays PI
and PIP
hydrolyzing activity similar to that of the
purified forms(7, 32) , this version of recombinant
human PLC
1 should be suitable for in vitro structure-function analyses.
Centrifugation binding assays show
that recombinant human PLC1 expressed from E. coli can
form specific complexes with PIP
vesicles, with an affinity
comparable to that of the purified form(22, 33) . The
structure of the enzyme-substrate complex differs from that of the free
enzyme, as the enzyme-PIP
complex is much more resistant to
trypsin cleavage than its free form. Moreover, the site and sensitivity
of the enzyme-PIP
complex to trypsin cleavage are highly
dependent on the concentrations of PIP
used to bind
PLC
1. One of the cleavage-defective mutants PLC
1 (R338L)
bound PIP
vesicles as tightly as the wild type enzyme did,
but its complex was cleaved by trypsin as readily as the free protein.
Therefore, these changes in the sites and sensitivities to trypsin
cleavage are not caused by hindrance of the phospholipid moiety but
rather reflect a temporal rearrangement of potential trypsin-sensitive
sites during binding.
At least two types of specific
PLC1
PIP
complexes were demonstrated in these
experiments; complexes formed at a low concentration of PIP
were readily degraded to a 67-kDa fragment, whereas specific
complexes formed at a higher concentration of PIP
micelles
were relatively resistant to trypsin cleavage. The simplest
interpretation is that there are at least two types of PIP
binding sites in PLC
1: a high affinity site and a low
affinity site. When the PIP
substrate concentration is low,
PLC
1 will bind to PIP
through the high affinity site
to form a complex that is cleaved readily by trypsin to give a 67-kDa
fragment. As the concentration of PIP
increases, another
binding site with lower affinity will be saturated and cause formation
of a complex much more resistant to trypsin digestion. Because the
67-kDa fragment was retained in a Ni
-NAT gel (data
not shown), the present results also suggest that the COOH-terminal
sequences of PLC
1, although required for
catalysis(11, 12) , are not involved in the high
affinity binding of PIP
. Two lines of evidence are
consistent with this interpretation that at least two PIP
binding sites exist in PLC
1. Deletion of the first 60 amino
acids of bovine PLC
1 does not eliminate its PIP
hydrolysis activity but dramatically reduces the binding of the
truncated enzyme to PIP
lipid vesicles(15) .
Furthermore, a peptide corresponding to residues 30-43 of
PLC
1 binds to IP
(12) . These observations
indicate that in addition to the PIP
cleavage site,
PLC
1 can bind to PIP
via a noncatalytic PIP
binding site in the NH
terminus of the protein.
Indeed, residues 16-134 in the NH
terminus of
PLC
1 have been found to share homologous sequences with pleckstrin
(PH domain)(34, 35, 36) , which can
specifically bind to PIP
(37) . The binding of
PIP
to the noncleavage site of PLC
1 can either allow
the enzyme to hydrolyze the phospholipid in a processive manner (15) or may be required for other regulatory
process(38, 39) .
All the eukaryotic PI-PLC
identified so far can be structurally divided into conserved X and Y
regions(2, 17, 40) . Deletion mutagenesis
studies have shown that both X and Y region sequences are essential for
the catalytic
activity(11, 12, 13, 14) .
Consistent with this observation, conversion of several highly
conserved amino acid residues in the Y region causes the enzyme to lose
its catalytic functions (data not shown). In particular, conversion of
Arg to Gly (R549G) makes the enzyme selectively defective
in the cleavage of PIP
, whereas this mutant enzyme can
retain 20% of the ability to hydrolyze PI. We are investigating further
the contribution of these conserved residues to the catalytic function
of PLC
1.
Prokaryotic PI-PLC does not contain a Y
conserved region(41) . Its catalytic activity is independent of
Ca and does not recognize PIP or PIP
.
However, structural comparison of the X region sequences between
eukaryotic and prokaryotic PI-PLC reveals a high degree of similarity.
This supports the hypothesis that catalytic sites of PLCs might reside
in the X region. Within this region, 9 amino acid residues are
invariant from prokaryotic to human PLCs. Our preliminary in vitro mutagenesis and enzymatic characterization of the mutant human
PLC
1 show that side chain switching of most of these residues does
not cause significant loss of catalytic activity of the mutant enzyme.
It is unlikely that switching their functional side chains does not
alter catalytic function of PLC
1. We would rather believe that
these mutant enzymes might be defective in other functions not detected
in the present in vitro enzymatic assay.
However, in
agreement with the hypothesis that catalytic residues could be
localized in the X region, we found that conversion of Glu to Gly, His
to Leu, or Arg
to Leu
totally eliminated the catalytic activity of PLC
1. Although these
three cleavage-defective mutant enzymes can bind to PIP
vesicles as tightly as the wild type enzyme does, our substrate
protection experiments demonstrated that the structures of specific
PIP
-protein complexes of these three mutants are not quite
the same. Both the sites of and sensitivity to trypsin digestion of the
specific PIP
-protein complexes of H356L and E341G are
similar to those of the specific complexes of the wild type enzyme,
whereas binding of R338L to PIP
does not affect its site or
sensitivity to trypsin digestion. The simplest interpretation is that
upon sequential binding of PIP
, both mutant PLC
1 E341G
and H356L can adopt structural adjustments similar to those of the wild
type enzyme, thus Glu
and His
are not
involved in either high affinity or low affinity interactions with
PIP
. Although R338L is still able to bind tightly to
PIP
vesicles, its structural adjustment upon binding to
PIP
is distinct from that of the wild type enzyme.
In
conclusion, this study shows that Glu and His
are not involved in specific interaction with PIP
.
Since Ca
is not essential for the PIP
binding activity of PLC but is required for cleavage, this
implies that Glu
and His
may be involved
either in the binding of Ca
or in the cleavage step.
However, His
and Glu
are also invariant in
prokaryotic PI-PLC, whose activity is independent of
Ca
. Therefore, these two residues are most likely
involved in the hydrolysis of PIP
once the specific
substrate-enzyme complex has been formed.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U09117[GenBank].