Short Communication

Isolation of a Multi-functional
Endogenous Cellulase Gene from Mollusc, Ampullaria crossean

WANG Ji, DING Ming, LI Yan-Hong, CHEN Qing-Xi, XU
Gen-Jun, ZHAO Fu-Kun*

( Key Laboratory of Proteomics, Institute of
Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the
Chinese Academy of Sciences, Shanghai 200031, China )

Abstract        The
cellulase genes of some animals, most coding for endo-β-1,4-glucanases, were
found and cloned. There has been no reports about genes encoding
exo-β-1,4-glucanase or endo-β-1,4-xylanase from animal. Here we cloned the cDNA
of a cellulase designated as EGX from mollusc, Ampullaria crossean, and
expressed it in Pichia pastoris for the first time. The cellulase EGX is a
multi-functional β cellulase with the activities of exo-β-1,4-glucanase,
endo-β-1,4-glucanase and endo-β-1,4-xylanase. The opening reading frame of EGX cDNA is 1185 bp and
encodes 395 amino acids. The EGX gene can also be amplificated from the genomic
DNA by PCR, which verified the endogenous origin of this gene. This EGX gene
was the first multi-functional cellulase gene that was directly isolated from
animals.

Key
words     multi-functional
cellulase; EGX; gene isolation; Ampullaria crossean

Cellulose is one
of the most abundant renewable sources in the world. Many researches showed
that it had been technically feasible to obtain fermentable carbohydrate by the
enzymatic degradation of plant biomass for ethanol production[1]. The key
enzymes of this process are cellulases and xylanases. Cellulases include exo-β-1,4-glucanase
(E.C.3.2.9.11), endo-β-1,4-glucanase (E.C.3.2.1.4) and β-glucosidase
(E.C.3.2.1.21)[2]. Xylanases include endo-β-1,4-xylanase (E.C.3.2.1.8),
β-xylosidase (E.C.3.2.1.37) and some side-chain cleaving enzymes[3]. Till now,
cellulases and xylanases applied for practical purpose are mainly from
microorganisms, there are great needs to discover new cellulases and xylanases
with novel characteristics.

According to the
traditional view, animals do not produce cellulase themselves and live in
symbiosis with cellulytic microorganisms[4]. However, since the discovery of
endogenous endo-β-1,4-glucanase in termite[5] and nematode[6], this knowledge
should be corrected. By now, about 20 animal endo-β-1,4-glucanase and
β-glucosidase genes have been cloned, but there was no report about cloning of
exo-β-1,4-glucanase and xylanase gene from animal. Furthermore, among those
cloned cellulase genes, few have been successfully expressed[7,8], which
hindered the further study on animal cellulases.

Is the
endogenous cellulase system in animal incomplete? Does this animal cellulase
system include only endo-β-1,4-glucanase and β-glucosidase as plant cellulase
system? Could animal bodies degrade xylan? To explore above questions, we
purified an endogenous cellulase designated EGX from the mollusc, Ampullaria
crossean[9]. The cellulase EGX was characterized as a multi-functional
enzyme with the activity of exo-β-1,4-glucanase, endo-β-1,4-glucanase and
endo-β-1,4-xylanase[9].

In this paper, we report the cloning and
expression of the animal cellulase gene EGX. Our data demonstrates that
cellulase EGX is an endogenous cellulase and suggests that a complete cellulase
system might also exist in animals.

1    Materials and Methods

1.1   Materials

The mollusc, Ampullaria
crossean, were collected in Xiamen, China.
The thermal cycle was according to the Single BlockTM system (Ericomp Inc., San
Diego, CA, USA).
The Multi-copy Pichia expression kit was purchased from Invitrogen (San
Diego, CA, USA).

1.2   Protein N-terminal sequencing

The stomach
juice was isolated from Ampullaria crossean to purify EGX[9].

The purified
tissue enzyme was subjected to SDS-PAGE and electroblotted on a polyvinylidene
fluoride membrane. Blotted protein bands were stained with Ponceau S and cut
out for protein sequencing. To obtain the internal sequence of EGX, the
purified EGX was digested by Endo Glu C protease (Promega, Madison,
WI, USA).
The fragments were subjected to SDS-PAGE and electroblotted on a polyvinylidene
fluoride membrane. A band of about 31 kD was cut out for protein sequencing.

1.3   RNA isolation

Total RNA was
isolated from the stomach of the mollusc, Ampullaria crossean. The
stomach tissue, which was freshly cut, was dissected and homogenized in a
sterile glass homogenizer to isolate total RNA using Trizol (Life Technologies, Cergy Pontoise, France).

1.4   cDNA
synthesis and DNA amplification

The first strand
cDNA was primed with oligo(dT)12－18 on total RNA isolated from stomach tissue of Ampullaria
crossean. Amplification of the central region of EGX cDNA was performed
using the two degenerate primers, EGX01f and EGX01r (Table 1). To obtain the
3-terminal sequence of the gene, PCR was performed using the gene specific
primer EGX04f and oligo(dT)18. In the hemi-nested PCR, the gene specific primer
EGX03f and oligo(dT)18 were employed. Amplification of the 5-terminal sequence
was performed according to the protocol of the 5′ RACE system for rapid amplification of cDNA ends version 2.0 (Life
Technologies, Cergy Pontoise, France). The first strand cDNA was primed with
the primer EGX05f. The first PCR was performed with the gene specific primer
EGX06f and the anchor primer. Then, in the nested PCR, the gene specific primer
EGX07f and the universal amplification primer (UAP) were employed. To obtain
the opening reading frame, a 1.3 kb DNA fragment was amplified with the primer
EGX10f and EGX10r. The 1.3 kb DNA fragment was cloned and sequenced. Plasmids
from six clones were sequenced to avoid possible errors caused by DNA
amplification.

1.5   Genomic DNA PCR amplification

Genomic DNA was
isolated from ovary tissue of Ampullaria crossean using the 3S Spin
cell/tissue genomic DNA isolation kit (Shenergy Biocolor Biological Science
& Technology Company, Shanghai, China).
The TaKaRa Ex Taq (TaKaRa, Dalian, China)
was employed in DNA amplification. PCR amplification was carried out on 45 μg
of Ampullaria crossean genomic DNA template using the cDNA specific
primer EGX11f (containing the start codon) and EGX11r (just before the stop
codon) (Table 1). The PCR was performed with an annealing temperature of 62 ℃ for 30 cycles. The resulted PCR
product was cloned and sequenced.

1.6   Recombinant
expression of EGX in Pichia pastoris

The opening
reading frame of EGX was amplified with the primer Wt-11f and Wt-10r. After
digested with EcoRI and NotI, the fragment was ligated into the P. pastoris
expression vector pPIC9K (Invitrogen, De schlep, Netherlands).
The P. pastoris EGX expression clone was constructed according to the Pichia
User Manual (Invitrogen, De schlep, Netherlands).
P. pastoris GS115 strain was selected to be used as a host strain and the
restriction enzyme Bpu1102I was chosen to linearize the constructed vector
before being tranformed to the host cell. A P. pastoris GS115 control clone
transformed with the Bpu1102I-linearized pPIC9K plasmid was also constructed.

To detect the endo-β-1,4-xylanase activity
of expressed EGX, the EGX expression clone and the control clone were dotted on
a the BMMY plate containing 1 g/L xylan from birchwood, 20 mmol/L NaCl, 100
mmol/L phosphate buffer, pH 5.8. After a 6-day culture in 29 ℃, the plate was stained with 1 g/L
Congo red and destained with 1 mol/L NaCl to visualize the hydrolysed zone[10].

To detect the
endo-β-1,4-glucanase activity, the P. pastoris expression clone and the control
clone were cultured and induced in the liquid medium according to the Pichia
User Manual (Invitrogen, De schlep, Netherlands) except the BMGY and BMMY
contain 20 mmol/L NaCl and were buffered with 100 mmol/L phosphate buffer, pH
6.0. After a 6-day induction at 28 ℃, the culture supernatants were collected for activity assay. The
culture supernatants were added on agarose plate containing 0.2 g/L
carboxylmethyl cellulose sodium (CMC-Na), 100 mmol/L NaCl and 100 mmol/L
acetate buffer, pH 5.2. After incubation in 30 ℃ for 48 h, the endo-β-1,4-glucanase activity on the plate was
visualized by being stained with 1 g/L Congo red and destained with 1 mol/L
NaCl[10].

The detection of
exo-β-1,4-glucanase activity was similar with endo-β-1,4-glucanase activity
except that 0.2 g/L carboxylmethyl cellulose sodium (CMC-Na) was substituted by
4.32 mmol/L p-nitrophenyl cellobioside (pNPC) and 5.61 mmol/L D-gluconic acid
lactone and 2% Na2CO3 was added to visualized the activity[11].

2    Results and Discussion

2.1   cDNA sequence

The N-terminal
and internal sequences of EGX were determined to isolate cDNA of EGX (Table 1).

The sequence of EGX cDNA reveals an opening
reading frame of 1185 bp(Fig.1). The putative ATG start codon is found to be at
162 bp from the 5′ end of the
cDNA. The 3′ untranslated
region contains a putative polyadenylation signal (AATAAA), 24 nucleotides
upstream from the poly(A) tail, suggesting that the 3′ untranslated region was complete.

Table 1   Partial amino acid sequences of purified
EGX and oligonucleotide primers used to synthesizing the first strand cDNA
synthesis, PCR amplification and EGX cloning

^a Degenerative
primers derived from the N-terminal and the internal amino acid sequence of
EGX.

The opening reading
frame is conceptually translated into 395 amino acids. The N-terminus of the
purified EGX starts at nucleotide 13. There are only four amino acids absent in
the N-terminus of the purified EGX. After being deleted the four amino acids,
the calculated molecular weight of EGX is 44.12 kD, which is similar to the
apparent molecular weight of 41.5 kD of the purified EGX as estimated by
SDS-PAGE[9]. On the other hand, the software of SignalP V2.0 failed to predict
the potential signal peptide cleavage site in the N-terminal sequence of EGX.
There is no evidence for a typical N-terminal signal peptide in the putative
amino acid sequence. Similar to EGX, it has also been reported the
endoglucanase CelC from anaerobic bacterium Clostridium thermocellum does not
display a signal peptide but could be secreted to the growth medium with an
unknown mechanism[12]. The mechanism of EGX secretion in animal cell remains to
be further identified.

Many fungal
cellulases with hydrolytic activity to crystalline cellulose substrates, such
as Sigma cell type 101, consist of not only catalytic domains, but also
cellulose binding domains and Pro/Thr/Ser-rich sequence linkers hinging
catalytic domains and cellulose binding domains[13]. The putative amino acid
sequence of EGX does not contain Pro/Thr/Ser-richsequence. In addition, as
analyzed with the Conserved domain database search, EGX does not show
meaningful similarity towards any type of cellulose binding domain. Thus, it is
considered that EGX does not contain a structurally well defined cellulose
binding domain, which is similar to the single domain endoglucanase NtEG from
termite, Nasutitermes takasagoensis[14].

Fig.1       The
cDNA sequence and predicted EGX amino acid sequence

The putative start codon and
stop codon are in bold. The polyadenylation signal (AATAAA) is underlined. The
sequenced fragments of purified EGX are boxed.

2.2   Sequence comparison

As shown in Fig.2,
sequence comparison has revealed that the putative amino acid sequence of EGX
is similar to the members of the glycosyl hydrolase family 10 (GHF10)[15]. The
residues that may serves as catalytic nucleophile and proton donor are
conserved. The nuleophilic residue is deduced to Glu268, which is in the
consensus sequence, Thr-Glu-Leu-Asp[16]. The putative acid/base catalyst,
Glu167, is in the highly conserved region, Trp-Asp-Val-Asn-Asn-Glu[17]. The
amino acid sequence Asn-Asp-Tyr-Asn, which is a consensus region in most
members of glycosyl hydrolase family 10, is also conserved in EGX. The putative
amino acid sequence of EGX displays 29% identity to the xylanase from
Penicillium simplicissimum (GenBank Accession No: AF070417), 28% identity to
the thermostable xylanase from Thermoascus aurantiacus (AF127529), and 28%
identity to xylanase from Streptomyces lividans (M64551). To the
exo-β-1,4-glucanase/endo-β-1,4-xylanase Cex from Cellulomonas fimi (M15824),
EGX displays 29% identity.

Fig.2       Sequence
comparison of EGX with other members of glycoside hydrolase family 10

“C”, the exo-β-1,4-glucanase/ endo-β-1,4-xylanase
cex from Cellulomonas fimi (GenBank Acession No: M15824); “P.”, the xylanase
from Penicillium simplicissimum (AF070417); “S.”, the xylanase from
Streptomyces lividans (M64551); “T.”, the thermostable xylanase from
Thermoascus aurantiacus (AF127529).

2.3   Endogenous origin of the EGX gene

Symbiosis
microorganisms are traditionally considered to provide the ability of cellulose
hydrolysis to animals. To verify the endogenous origin of the EGX gene, the
genomic fragment encoding EGX was amplified on the genomic DNA prepared from
ovary of mollusc, Ampullaria crossean, to avoid the possibility of
gut-microbial contamination.

Amplification of
the EGX gene on the genomic DNA was carried out using the specific primers
EGX11f and EGX11r (Table 1). The PCR fragment of 4892 bp contains nine exons
and eight introns (Fig.3). The eight introns are located after nucleotide 74,
122, 280, 336, 473, 646, 753 and 972 of opening reading frame. The lengths of
the eight introns are 205 bp, 1032 bp, 371 bp, 366 bp, 269 bp, 279 bp, 234 bp
and 960 bp, respectively. The intron 1, 5, 7 and 8 have typical eukaryotic
splice site (GU-AG). The amplification of the EGX gene on the genomic DNA
confirms the endogenous origin of the EGX gene.

Ampullaria
crossean is a kind of phytophagous animal[18]. By
Western blot assay using the anti-EGX sera[9], EGX was detected to exist in its
stomach and liver, both belonging to the digestive system of the mollusc, so
EGX is believed to be its digestive enzyme. In plant, xylan hinders the
hydrolysis of cellulase towards plant cell wall by locating between the
cellulose fibres[19]. The endo-β-1,4-xylanase activity of EGX might be helpful
to the hydrolytic reaction. However, whether the mollusc could utilize xylan as
one of its carbon sources remained to be investigated.

Fig.3       Organization
of the genomic fragment of EGX

The genomic fragment is 4892 bp in
length and contains 9 exons and 8 introns. The eight introns are located after
nucleotide 74, 122, 280, 336, 473, 646, 753 and 972 of opening reading frame.
The lengths of the eight introns are 205 bp, 1032 bp, 371 bp, 366 bp, 269 bp,
279 bp, 234 bp and 960 bp, respectively.

2.4   Expression of EGX by Pichia
pastoris

The eukaryotic expression of
methylotrophic yeast Pichia pastoris was used to express EGX. The pPIC9K vector
was selected to permit expression of the EGX gene using the alcohol oxidase
promotor 1 (AOX1). Fusion of the EGX gene to the α-factor signal peptide from
Saccharomyces cerevisiae could direct the efficient secretion of the target
protein to the culture medium. As shown in Fig.4, the P. pastoris EGX
expression clone has the activities of exo-β-1,4-glucanase, endo-β-1,4-glucanase
and endo-β-1,4-xylanase, suggesting the multi-functional cellulase EGX was
expressed with all of its hydrolytic activities.

Fig.4       Detection
of recombinant EGX activities expressed by P. pastoris

(A), (B) and (C) are the control clone
without EGX gene; (B), (D) and (F) were the expression clone with EGX gene. (B)
shows the endo-β-1,4-xylanase activity; (D) shows the endo-β-1,4-glucanase
activity; (F) shows the exo-β-1,4-glucanase activity.

2.5 Conclusion

Since 1998,
there have been about 20 animal cellulase genes isolated, including
endo-β-1,4-glucanase gene and β-glucosidase gene[13,20]. The cloning of EGX
gene in this paper confirms the presence of endogenous animal
exo-β-1,4-glucanase, which suggests that the cellulase system in animal might
be a complete system. Additionally, cloning of EGX gene also proves the being
of endogenous animal xylanase. These bring us new insight into the cellulase
and hemi-cellulase system from animals. What’s more, the recombinant expression of EGX gene enable us to further
study the hydrolysis mechanism of this multi-functional cellulase.

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_____________________________________________

Received: July
9, 2003   Accepted: August
9, 2003

This work was supported by the grants from
the Key Science and Technology Project of the Chinese Academy of Sciences (No. KY
951-JI-310), the Creation Foundation from Shanghai
Institutes for Life Sciences and the Shenglongda Science Foundation

*Corresponding author: Tel, 86-21-54921155;
Fax, 86-21-54921011; e-mail, [email protected]

Updated at: 2003-10-09

福寿螺(Ampullaria
crossean)内源性多功能纤维素酶基因的克隆

王骥    丁明    李燕红     陈清西     许根俊     赵辅昆*

（ 中国科学院上海生命科学研究院生物化学与细胞生物学研究所蛋白质组学重点实验室，
上海 200031 ）

摘要       迄今为止已克隆的动物纤维素酶基因都是编码内切-β-1,4-葡聚糖酶或β-葡萄糖苷酶的， 还没有有关动物外切-β-1,4-葡聚糖酶基因的报道。 并且， 动物是否对植物细胞壁的另一主要成分木聚糖具有水解能力也依然是个疑问。
已经由草食性软体动物福寿螺的胃液中纯化得到一种纤维素酶， 该酶同时具有外切β-1, 4-葡聚糖酶、 内切β-1, 4-葡聚糖酶和内切β-1, 4-木聚糖酶等三种活性， 是一种多功能纤维素酶(命名为EGX)。 在此基础上， 从福寿螺胃组织中克隆得到的EGX cDNA开放阅读框全长为1185 bp， 共编码395个氨基酸。 由氨基酸顺序同源和活性部位的保守顺序的比较分析， EGX 可归属于糖苷水解酶第10家族。 该多功能纤维素酶cDNA在毕赤酵母(Pichia
pastoris)中表达的产物具有水解外切β-1,
4-葡聚糖酶底物(pNPC)， 羧甲基纤维素(内切β-1, 4-葡聚糖酶的底物)和木聚糖(木聚糖酶的底物)的活力。 另外也从福寿螺卵巢组织中获得编码福寿螺多功能纤维素酶EGX基因， 长度为4892 bp， 含有9个外显子和8个内含子。 8个内含子分别位于开放阅读框的74 bp、 122 bp、 280 bp、 336 bp、 473 bp、 646 bp、 753 bp和972 bp处。 以上结果证明了多功能纤维素酶EGX是福寿螺自身产生的, 同时也证明了福寿螺具有与微生物相似的纤维素酶系, 即也含有内源性的外切β-1, 4-葡聚糖酶活力组分。

关键词   多功能纤维素酶； EGX； 基因克隆；
福寿螺(Ampullaria crossean)