A novel cellulase gene from the mulberry longicorn beetle, Apriona germari: gene structure, expression, and enzymatic activity

Comparative Biochemistry and Physiology, Part B 140 (2005) 551 – 560 www.elsevier.com/locate/cbpb

A novel cellulase gene from the mulberry longicorn beetle, Apriona germari: Gene structure, expression, and enzymatic activity Seong Jin Leea,1, Kwang Sik Leea,1, Seong Ryul Kima, Zhong Zheng Guia, Yong Soon Kimb, Hyung Joo Yoonb, Iksoo Kimb, Pil Don Kangb, Hung Dae Sohna, Byung Rae Jina,* b

a College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea Department of Agricultural Biology, National Institute of Agricultural Science and Technology, Suwon 441-100, Korea

Received 17 September 2004; received in revised form 26 November 2004; accepted 8 December 2004

Abstract We have previously cloned a cellulase [h-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Ag-EGase I) belonging to glycoside hydrolase family (GHF) 45 from the mulberry longicorn beetle, Apriona germari. We report here the gene structure, expression and enzyme activity of an additional celluase (Ag-EGase II) from A. germari and also described the gene structure of Ag-EGase I. The AgEGase II gene spans 1033 bp and consisted of two introns and three exons coding for 239 amino acid residues. The 2713-bp-long genomic DNA of Ag-EGase I also consisted of two introns and three exons. The Ag-EGase II showed 61% protein sequence identity to Ag-EGase I and 51% to another beetle, Phaedon cochleariae, cellulase, belonging to GHF 45. The catalytic sites of GHF 45 are conserved in Ag-EGase II. The Ag-EGase II has 14 conserved cysteine residues and three putative N-glycosylation sites. Northern blot analysis confirmed midgut-specific expression of Ag-EGase II, suggesting that the midgut is the prime site for cellulase synthesis in A. germari larvae. The cDNA encoding Ag-EGase II was expressed as a 36-kDa polypeptide in baculovirus-infected insect Sf9 cells and the enzyme activity of the purified recombinant Ag-EGase II was approximately 812 U/mg of recombinant Ag-EGase II. The enzymatic properties of the purified recombinant Ag-EGase II showed the highest activity at 50 8C and pH 6.0, and were stable at 60 8C at least for 10 min. D 2004 Elsevier Inc. All rights reserved. Keywords: Apriona germari; Baculovirus; cDNA cloning; Cellulase; Endoglucanase; Enzyme; Genomic structure; Insect cells; Mulberry longicorn beetle

1. Introduction Cellulose, the most abundant carbohydrate polymer on earth, is composed of repeating glucose units linked by h1,4-glycosidic bonds. Cellulose, which is used as a food source by a wide variety of organisms, is mainly produced by terrestrial plants. However, the exploitation of cellulose as a food source is restricted by cellulose-degrading ability. Cellulases are multicomponent complexes which are composed of endoglucanases (EC 3.2.1.4), exoglucanases (EC 3.2.1.91) and cellobiases (EC 3.2.1.21), and cellulose is * Corresponding author. Tel./fax: +82 51 200 7594. E-mail address: [email protected] (B.R. Jin). 1 These authors contributed equally to this paper. 1096-4959/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2004.12.003

efficiently hydrolyzed through the synergistic action of three major types of cellulolytic enzymes (Henrissat et al., 1985; Beguin and Aubert, 1994). Along with an extensive interest in cellulose as a major source for renewable energy and raw materials, the cellulolytic enzyme also has been subjected to investigation. To exploit the energy and carbon available from cellulose, organisms such as fungi and bacteria produce mixtures of synergistically acting cellulases (Beguin and Aubert, 1994; Teeri, 1997). Among the cellulolytic fungi, Trichoderma reesei has very strong cellulose-degrading activity, and its cellulase has been widely investigated (Penttila¨ et al., 1986; Shoemaker et al., 1983; Tomme et al., 1988; Van Arsdell et al., 1987; Chen et al., 1987; Henrissat et al., 1985; Linder et al., 1995).

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Although cellulose is a major food source for many insect species, most insects depend on symbiotic microorganisms, which produce cellulolytic enzymes responsible for cellulose hydrolysis. From this view of cellulase activity, it was believed that cellulose digestion in insects was mediated by microbial cellulase activity in their gut. However, recent works confirmed the production of cellulases from insect for themselves (Watanabe et al., 1998; Girard and Jouanin, 1999; Tokuda et al., 1999; Nakashima et al., 2002, Sugimura et al., 2003; Lee et al., 2004), along with reports on the production from symbiotic organisms harboring in the insect gut (Ohtoko et al., 2000) and both (Breznak and Brune, 1994; Watanabe et al., 1998; Ohtoko et al., 2000; Scharf et al., 2003). Known cellulases of insect origin belong to three glycosyl hydrolase families (GHFs): GHF 45 (Phaedon cochleariae beetle), GHF 5 (Psacothea hilaris beetle) and GHF 9 (termites and cockroaches). These three GHFs are structurally unrelated and their evolutionary origins are likely to be independent (Sugimura et al., 2003). We have previously reported a cellulase [h-1,4-endoglucanase (EGase), Ag-EGase I] belonging to GHF 45 from the mulberry longicorn beetle, Apriona germari (Coleoptera: Cerambycidae), that feed on mulberry tree, tunneling inside the stem and ingesting the living wood (Lee et al., 2004). In this paper, we report the gene structure, expression and enzyme activity of an additional cellulase (Ag-EGase II) belonging to GHF 45 from A. germari. The Ag-EGase II cDNA from A. germari was expressed functionally in baculovirus-infected insect cells and the purified recombinant Ag-EGase II was assayed for some enzymatic properties. We also describe here the gene structure of Ag-EGase I.

2. Materials and methods 2.1. Animals The mulberry longicorn beetle, A. germari (Coleoptera: Cerambycidae), was reared on an artificial diet as described previously (Yoon and Mah, 1999).

systems, Foster City, CA). The sequences were compared using the DNASIS and BLAST programs provided by the NCBI (http://www.ncbi.nlm.nih.gov/BLAST). GenBank, EMBL and SwissProt databases were searched for sequence homology using a BLAST algorithm program. MacVector (ver. 6.5, Oxford Molecular) was used to align the amino acid sequences of cellulase. Phylogenetic analysis was performed using PAUP (Phylogenetic Analysis Using Parsimony) version 4.0 (Swofford, 2000). The accession numbers of the sequences in GenBank are as follows: A. germari Ag-EGase II (AY451326; this study), A. germari Ag-EGase I (AY162317), P. cochleariae cellulase (CAA76931), representative clones 2–6, 7–50, 7–10, and 45–6 from the protists of the termite Reticulitermes speratus (BAA98036, BAA98041, BAA98043, and BAA98048, respectively), R. speratus endoglucanase2 (BAA34050), and R. speratus cellulase (BAA31326). 2.3. Genomic DNA isolation and PCR of cellulase genes in A. germari Genomic DNA was extracted from the fat body tissues of A. germari using a Wizardk Genomic DNA Purification Kit, according to the manufacturer’s instructions (Promega). The primers used for amplification of a genomic DNA encoding the Ag-EGase I were 5V-ATGAAGGTGTTCGTAGCAATCCTCGCTG-3V for the translational start sequence region and 5V-TTAATAATTGCATCCAGTAATGGAAACCAAC-3V for the 3V coding region, based on the Ag-EGase I cDNA (Lee et al., 2004). In addition, the primers used for amplification of Ag-EGase II genomic DNA were 5V-ATGAAGGTATTGTTGGCAGTCGTCGCTGTGC-3V for the translational start sequence region and 5V-TTATGAATAATTGCATCCGGAAGCTG-3V for the 3V coding region, based on the Ag-EGase II cDNA cloned in this study. After a 35-cycle amplification (94 8C for 30 s; 48 8C for 40 s; 72 8C for 2 min), PCR products were ethanol precipitated, centrifugated at 10,000g for 15 min, and rinsed with 70% ethanol. These DNAs were analyzed with 1.0% agarose gel electrophoresis. The PCR products for sequencing were cloned into pGem-T vector (Promega). The construct was transformed into Escherichia coli TOP10FV cells (Invitrogen).

2.2. cDNA library screening, nucleotide sequencing and data analysis

2.4. RNA isolation and Northern blot analysis

A cDNA library constructed using whole bodies of A. germari larvae was used as previously described (Kim et al., 2001). The clones harboring cDNA inserts were randomly selected and sequenced to generate the expressed sequence tags (ESTs) (Kim et al., 2003). The plasmid DNA from the clones harboring cDNA inserts was extracted by Wizard mini-preparation kit (Promega, Madison, WI). The nucleotide sequence was determined by using a BigDyeTerminator cycle sequencing kit and an automated DNA sequencer (model 310 Genetic Analyzer; Perkin-Elmer Applied Bio-

The larvae of A. germari were dissected under a Stereomicroscope (Zeiss, Jena, Germany). Individual samples such as midgut, fat body, and epidermis were harvested, and washed twice with PBS (140 mM NaCl, 27 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4). Total RNA was isolated from the whole body, midgut, fat body, and epidermis of A. germari larvae by using the Total RNA Extraction Kit (Promega). Total RNA (10 ~g/lane) from A. germari was denatured by glyoxalation (McMaster and Carmichael, 1977), transferred onto a nylon blotting

S.J. Lee et al. / Comparative Biochemistry and Physiology, Part B 140 (2005) 551–560

membrane (Schleicher and Schuell, Dassel, Germany) and hybridized at 42 8C with a probe in a hybridization buffer containing 5d SSC, 5d Denhardt’s solution, 0.5% SDS, and 100-Ag/ml denatured salmon sperm DNA. The 834-bp AgEGase II cDNA clone was labeled with [a-32P] dCTP (Amersham, Arlington Heights, IL) using the Prime-It II Random Primer Labeling Kit (Stratagene) for use as a probe for hybridization. After hybridization, the membrane filter was washed three times for 30 min each in 0.1% SDS and 0.2d SSC (1d SSC is 0.15 M NaCl and 0.015 M sodium citrate) at 65 8C and exposed to autoradiography film. For rehybridization, the membrane was washed for 20 min at room temperature in sterile millipore water. Then, the membrane was washed overnight at 65 8C in 50 mM Tris–HCl (pH 8.0), 50% dimethylformamide and 1% SDS in order to remove the hybridized probe. The membrane was then rehybridized to [a-32P] dCTP-labeled 60S rRNA probe (Kim et al., 2001). The 60S rRNA gene was used as an internal loading control. 2.5. Cell culture and virus Insect Sf9 cells (Vaughn et al., 1977) were maintained at 27 8C in TC100 medium (GIBCO BRL LIFE Technologies, Gaithersburg, MD), supplemented with 10% fetal bovine serum (FBS; GIBCO BRL LIFE Technologies) as described by standard methods (O’Reilly et al., 1992). Wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) and recombinant AcNPV were propagated in Sf9 cells. The titer was expressed as plaque forming units (PFU) per ml (O’Reilly et al., 1992). 2.6. Construction of transfer vector and recombinant virus The 834-bp Ag-EGase II cDNA from pBlueScriptAgEGase II was subcloned between SacI and KpnI sites of pBacPAK9 (Clontech, Palo Alto, CA) to produce transfer vector pBacPAK9-AgEGase II. In the transfer vector, the Ag-EGase II cDNA is under the control of the AcNPV polyhedrin promoter. One microgram of BacPAK6 viral DNA (Clontech), five Ag of pBacPAK9-AgEGase II in 20 mM HEPES buffer and sterile water to make a total volume of 50 Al were mixed in a polystyrene tube. Fifty microliters of 100 Ag/ml Lipofectink (GIBCO BRL LIFE Technologies) was gently mixed with the DNA solution and the mixture was incubated at room temperature for 30 min. The Lipofectin–DNA complexes were added dropwise to the medium covering the cells (1.0–1.5d 106 cells per 35-mm cell culture dish). After incubation at 27 8C for 5 h, TC100 medium containing antibiotics and 10% FBS was added to each dish and incubation at 27 8C was continued. At 5 days of postinfection (p.i.), the supernatant was harvested, clarified by centrifugation at 2000 rpm for 5 min, and stored at 4 8C. Recombinant AcNPV was plaque purified on 6-well plates seeded with 1.5d 106 Sf9 cells as described by O’Reilly et al. (1992).

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2.7. SDS-polyacrylamide gel electrophoresis (PAGE) Insect Sf9 cells were mock-infected or infected with the wild-type AcNPV and recombinant AcNPV in a 35-mm diameter dish (1d 106 cells) at an multiplicity of infection (MOI) of 5 PFU per cell. After incubation at 27 8C, cells were harvested at 1, 2, and 3 days p.i. For SDS-PAGE (Laemmli, 1970) of cell lysates, uninfected Sf9 cells and cells infected with virus were washed twice with PBS and mixed with protein sample buffer and boiled. The total cellular lysates were subjected to 10% SDS-PAGE. After electrophoresis, gels were fixed and stained with 0.1% Coomassie brilliant blue R-250. 2.8. Purification of recombinant cellulase Insect Sf9 cells were infected with the recombinant AcNPV expressing Ag-EGase II at a MOI of 5 PFU per cell. The culture supernatant was harvested at 5 days p.i. and clarified by centrifugation (10,000g) at 4 8C for 10 min. The culture supernatant was supplemented with 1 M ammonium sulphate and subjected to gel permeation chromatography using a Superdex 200 HR 10/30 column (Pharmacia LKB) in 20 mM Tris–HCl buffer (pH 8.0) containing 0.1 mM PMSF with a flow rate of 0.5 ml/min. Fractions containing recombinant Ag-EGase II were respectively assayed for enzyme activity. The recombinant Ag-EGase II-enriched fractions, identified by enzyme activity, were subjected to anion exchange chromatography on a MonoQ HR 5/5 column (Pharmacia LKB) in 20 mM Tris–HCl buffer (pH 8.0) containing 0.1 mM PMSF with a flow rate of 1 ml/min. Enzymes were eluted in a linear segment sodium chloride gradient (0–0.6 M). Fractions containing purified recombinant Ag-EGase II were respectively identified by SDS-PAGE and enzyme activity. 2.9. N-terminal sequencing Purified recombinant Ag-EGase II was subjected to 10% SDS-PAGE and electroblotted on to a polyvinylidene difluoride (PVDF) membrane (ProBlottk, Applied Biosystems) in 10 mM CAPS (3-[cyclohexylamino]-1-propanesulfonic acid) buffer and 10% methanol at 50 V for 45 min at room temperature. Protein sample on PVDF membrane was detected with staining by amido black staining solution (0.1% amido black 10B in 1% acetic acid/40% methanol). The Ag-EGase II band was excised from the membrane and then subjected to automated Edman degradation using an Applied Biosystems sequencer (Perkin-Elmer Applied Biosystems). 2.10. Determination of enzyme activity The recombinant Ag-EGase II mixed in substrate solution [2% (w/v) carboxymethyl cellulose (CMC; Sigma)

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for 1 h and the remaining activities were measured. The buffers used were acetate (pH 2 and 3), citrate (pH 4 to 6), phosphate (pH 7), Tris–HCl (pH 8 and 9), glycine–NaOH (pH 10), and KCl–NaOH solution (pH 11 and 12). The amount of reducing sugars produced by these reactions was measured by a dinitrosalicylic (DNS) reagent method (Miller, 1959). One unit of the enzyme was defined as the activity producing 1 Amol of reducing sugars in glucose equivalents per min.

in 0.1 M acetate buffer (pH 6.0)] was incubated at various temperatures ranging from 15 8C to 85 8C with 5 8C intervals for 1 h to determine the optimal temperature, then the remaining activities were measured. The recombinant Ag-EGase II for thermal stability was preincubated at various temperatures ranging from 15 8C to 85 8C with 5 8C intervals for 10 min without substrate and then assayed at 20 8C. For the optimal pH, the recombinant Ag-EGase II was treated at various pH ranges (pH 2.0–pH 12.0) at 50 8C

A. germari EGase II A. germari EGase I P. cochlearie R. speratus symbiont

1 1 1 1

A. germari EGase II A. germari EGase I P. cochlearie R. speratus symbiont

MK V L MK V F MQ V I M L

L V V L

A A L L

V I P I

V L L L

A A V S

V V F V

L F L V

C C A A

T T T T

F F F V

E E A L

A V T G

S S S L

26 26 31 16

P K P -

L V V -

V P D -

G Y G -

G G G -

V I L -

S S S -

G G G -

T S Y D

G G G G

K T T R

T T T T

T T T T

R R R R

Y Y Y Y

W W W W

D D D D

C C C C

A. germari EGase II A. germari EGase I P. cochlearie R. speratus symbiont

56 56 61 38

N T -

K K P -

S E T V

G G M S

K T T K

P P P P

V V V V

E A Q D

A T T T

C C C C

A S A A

A A I K

D D D D

G G G G

K S N T

T T T T

V T V R

A. germari EGase II A. germari EGase I P. cochlearie R. speratus symbiont

86 85 91 66

G T

A S S A

Y Y Y Y

M M M M

C C C C

S S S Y

D N N D

Q Q Q Q

Q Q Q T

P P A P

K K F R

V S V A

V V V V

N N N N

S S S D

T T T S

A. germari EGase II A. germari EGase I P. cochlearie R. speratus symbiont

116 114 120 96

D D D K

V T N -

N N N A

M Y L A

C C C C

C C C C

A A S T

C C C C

L I M Y

R K L E

L L L L

K T T T

F F F F

Q Q Q T

G D G S

A. germari EGase II A. germari EGase I P. cochlearie R. speratus symbiont

145 143 149 125

G G G G

S G G G

D D D D

L L L L

G G G G

S S S S

N N T N

Q Q S Q

F F S F

D D I D

I A I A W P I A

I I F I

P P P P

A. germari EGase II A. germari EGase I P. cochlearie R. speratus symbiont

175 173 179 154

T T P A

P P R P

S S G A

N N A D

GW GW A G GW

G G G G

D D D S

Q Q Q R

Y Y Y Y

G W G G

G W G G

V V V V

A. germari EGase II A. germari EGase I P. cochlearie R. speratus symbiont

204 203 208 183

C C C C

K K R Q

F F F W

R R R R

F F F F

F F F W

M M L F

K E E Q

S G N N

V A V A

S S S D

N N N N

A. germari EGase II A. germari EGase I P. cochlearie R. speratus symbiont

234 233 238 213

S T S T

G G N N

N N A R

Y S 239 237 Y 242 L R Q 218

G -

S -

S S D -

Q K A -

D V S -

Y Y P -

H N E -

V L I -

T N V -

25 25 30 15

C C C C

S G A G

WK WV WK WD

A E E G

N N N K

L L I A

K A N S

55 55 60 37

T V V A

K K Q K

S S S S

A S G A

C C C C

E V I D

E G G S

G G G G

A T S G

85 84 90 65

Y F F F

V V A A

A A A A

A A G A

S S S A

F F F V

T T T S

G G G G

G G G G

I A V E

115 113 119 95

G G G G

K K K K

Q T Q K

M I MV F L MT

V V V V

Q Q Q Q

V V I V

T T T T

N N N N

T T T T

144 142 148 124

I I I L

F F F Y

T T T N

K D Q -

G G G G

C C C C

S S H T

S S D S

QW QW QW Q S

G G T G

174 172 178 153

C C C C

S A S S

Q Q D Q

L L L L

P P P P

S S E S

S D V G

L L L L

R Q Q Q

E E P A

G G G G

203 202 207 182

Q Q Q E

V V V V

S D Q S

C C C C

P P P P

S S A G

E E E D

I L I L

V V V T

S S A S

A I I K

233 232 237 212

L -

A -

A -

L L P -

C C C C

K K K K

P P P G

S S S S

V V V V

K A N A

E A A T

S S S S

F F L Y

A A A A

L L F I

G G G G

D A Q G

L L L P

S Q A V

N

G G G G

G G G G

G G G G

V V V V

G G G G

S W Y S

G -

S S S S

E E V R

S A E S

Q D Q E

P P P P

A G Q S

T T S T

F F F F

E E Q N

**

*

*

*

*

C C C C

*

**

T E E D

*

*

*

V V V I

*

*

Fig. 1. Alignment of the amino acid sequence of Ag-EGase II with known cellulases. The identical residues are shown in solid boxes. Dots represent gaps introduced to preserve alignment. Vertical arrow indicates the end of the signal peptides. The conserved catalytic sites in cellulase residues are shown in dotted box. The cysteine residues are marked with asterisks. The putative N-glycosylation sites are bold-lined above the alignment. The Ag-EGase II cDNA sequence has been deposited in GenBank under accession number AY451326.

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3. Results 3.1. Cloning, sequencing and phylogenetic analysis of AgEGase II cDNA In a search of A. germari ESTs (expressed sequence tags), we identified a cDNA showing homology with previously reported cellulases. The cDNA clone including the full-length open reading frame (ORF) was sequenced and characterized. The Ag-EGase II cDNA is an ORF of 834 bp encoding 239 amino acid residues (Fig. 1; GenBank Accession No. AY451326). The deduced amino acid sequence of the Ag-EGase II cDNA contains three putative N-glycosylation sites at positions, 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S). The calculated molecular mass and estimated pI of Ag-EGase II are 25.0 kDa and 8.62, respectively. A multiple sequence alignment of the deduced protein sequence of Ag-EGase II cDNA with available cellulase sequences is shown in Fig. 1. Alignment with the deduced amino acid sequence indicated that Ag-EGase II sequence

A

555

was closely related to Ag-EGase I (Lee et al., 2004) and another beetle, P. cochleariae (Girard and Jouanin, 1999), cellulase, those belonging to GHF 45. The catalytic sites of GHF 45 (Girard and Jouanin, 1999; Bourne and Henrissat, 2001; Li et al., 2003; Lee et al., 2004) in Ag-EGase II are well conserved at positions 36–48 (K-T-T-R-Y-W-D-C-C-KP-S-C). But T36 in catalytic sites of Ag-EGase II was replaced by K (T36K). The Ag-EGase II alignment shows 14 conserved cysteine residues and three putative N-glycosylation sites. Among the three N-glycosylation sites of AgEGase II, a putative N-glycosylation site (Asn99–Ser100– Thr101) is commonly present in both Ag-EGase I and P. cochleariae beetle cellulase. A phylogenetic analysis using the deduced amino acid sequences of known GHF 45 EGases derived from beetle and symbiont, and two different endogenous termite cellulases of GHF 9 family revealed that the cellulases are divided into two groups (Fig. 2A). The deduced amino acid sequences of the EGase genes from two beetle species such as A. germari and P. cochleariae, and termite symbiont together formed a subgroup, excluding two endogenous

Species 71

A. germari EGaseII A. germari EGaseI

99

Beetle

P. cochlearie 80

GHF 45

R. speratus protist(45-6) 99

R. speratus protist(7-50) 62

Symbiont 59

R. speratus protist(2-6) R. speratus protist(7-10)

100

R. speratus endoglucanase2 Termite

GHF 9

R. speratus cellulase

B

Percent similarity

Species

GenBank No.

1. A. germari EGase II

AY451326

1

2

3

4

5

6

7

8

9

74

66

61

58

58

58

18

18

58

57

58

58

17

16

50

51

49

52

15

15

88

84

83

19

19

84

89

18

18

87

21

21

19

19

2. A. germari EGaseI

AY162317

64

3. P. cochlearie

CAA76931

52

51

4. R. speratus symbiont(45-6) BAA98048

46

43

5. R. speratus symbiont(7-50) BAA98041

44

45

40

78

BAA98036

44

45

38

77

7. R. speratus symbiont(7-10) BAA98043

44

43

39

75

82

82

8. R. speratus endoglucanase2 BAA34050

8

10

7

12

11

14

11

BAA31326

8

9

7

12

11

14

11

6. R. speratus symbiont(2-6)

9. R. speratus cellulase

62 37

79

98 98

Percent identity Fig. 2. Relationships among amino acid sequences of the Ag-EGase II and the known cellulases. (A) A maximum parsimony analysis for the amino acid sequences of Ag-EGase II and known cellulases derived from insects and symbiont. The sequence sources are described in Materials and methods. The tree was obtained by bootstrap analysis with the option of heuristic search and the numbers on the branches represent bootstrap values for 1000 replicates. (B) Pairwise similarities and identities of the deduced amino acid sequence of Ag-EGase II among cellulase sequences.

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w

ho le m bod id gu y fa t tb od ep y id er m is

termite cellulases of GHF 9 family. Similarly, the deduced protein sequence of the Ag-EGase II showed 64% protein sequence identity to Ag-EGase I, 52% to another beetle P. cochleariae cellulase and 44–46% to R. speratus protist cellulases, while the protein sequence identity to the two endogenous termite cellulases of GHF 9 family (8%) was low (Fig. 2B).

EtBr

Ag-EGase II

3.2. Genomic structure of A. germari EGase genes To identify the genomic structure of A. germari EGase genes, we designed primer sets based on the sequences of the Ag-EGase I (Lee et al., 2004) and Ag-EGase II cDNAs. A band was amplified from A. germari genomic DNA for each primer set of Ag-EGase I or Ag-EGase II. The PCR product was cloned and sequenced. Genomic PCR product sequences showed 100% identity to Ag-EGase I and AgEGase II cDNAs. The organization of the gene is illustrated in Fig. 3. Comparison of the genomic sequence with the sequence of the cDNA revealed the presence of three exons and two introns in both the Ag-EGase I and Ag-EGase II. The sequences at the exon–intron boundaries conformed to the typical eukaryotic splice sites, including an invariant GT at the intron 5V boundary and an invariant AG at its 3V boundary (Fig. 3B). The genomic DNA size from translation start codon to stop codon was 2713 bp for Ag-EGase I and 1033 bp for Ag-EGase II, respectively (Fig. 3A). Substantial length difference between the genomic DNAs of Ag-EGase I and Ag-EGase II was detected in intron 1. Interestingly, the length of intron 1 (1885 bp) in Ag-EGase I

A

Northern blot rRNA 1

2

3

4

Fig. 4. Northern blot analysis of Ag-EGase II. Total RNA was isolated from whole body (lane 1), midgut (lane 2), fat body (lane 3) and epidermis (lane 4) of the seventh instar A. germari larvae. The RNA was separated by 1.0% formaldehyde agarose gel electrophoresis (upper panel), transferred on to a nylon membrane, and hybridized with radiolabelled 834 bp Ag-EGase II cDNA (middle panel). The 60S rRNA gene was used as an internal control (lower panel). Transcripts are indicated on the right side of the panel by arrow.

was approximately 8.6-fold longer than that of intron 1 (218 bp) in Ag-EGase II. 3.3. Tissue-specific expression of Ag-EGase II To confirm the expression of Ag-EGase II gene at transcriptional level, Northern blot analysis was performed

140

1925

2286 2408

2713(bp)

Ag-EGase I

Intron 1

Exon 1

Exon 2

Intron 2

Exon 3

Ag-EGase II 140

258

625 725

1033(bp)

B Cellulase type

Ag-EGase I

Ag-EGase II

Exon

Length of exon(bp) Position in gene

1

40

1-40

2

362

1925-2286

3

306

2408-2713

1

40

1-40

2

368

258-625

3

309

725-1033

Sequence at exon-intron junction M F

K E

V V

F S

V

F

C

T

Q

G

K

T Y Stop

M

V

V Q

G

C

N

M

K

V

L

L

C

T

F

E

A

S

G

K

Q

I

V

N

M

Y

S Stop

Fig. 3. Genomic organization of Ag-EGase I and Ag-EGase II. (A) Schematic drawing of genomic structure of Ag-EGase I and Ag-EGase II. Solid and open boxes represent exons and introns, respectively. Numbers indicate the position in the genomic sequences. The GenBank accession numbers of the genomic sequences are AY741064 for Ag-EGase I and AY451326 for Ag-EGase II, respectively. (B) Lengths of exons and exon/intron boundaries.

S.J. Lee et al. / Comparative Biochemistry and Physiology, Part B 140 (2005) 551–560

M ar M ker oc w k t-A cN PV A cN PV

-A

gE

G as

eI

I

A

(kDa)

1

2

3 (day p.i.)

4

5

6

66 45

31

1

2

3

have termed AcNPV-AgEGase II, was produced in insect Sf9 cells. To examine the expression of Ag-EGase II by recombinant virus in insect cells, the protein synthesis in Sf9 cells infected with the recombinant virus was analyzed by SDS-PAGE (Fig. 5A). The recombinant Ag-EGase II expressed by the Ag-EGase II cDNA was present as a single band of about 36-kDa polypeptide in the cells infected with the recombinant virus, but not in the cells infected with the wild-type AcNPV or mock-infected cells. Furthermore, the activity of the recombinant Ag-EGase II was clearly detected in culture supernatant from the recombinant virus-infected insect cells (Fig. 5B), whereas mock- or wild-type AcNPV-infected cell supernatants did not. 3.5. Purification and characterization of the recombinant Ag-EGase II

1

A

cN t-A w

M

oc

k

PV

cN

PV

-A

gE

G as

eI

I

B

557

2

1

2

3

3

4

5

(day p.i.)

Fig. 5. Expression of the Ag-EGase II in baculovirus-infected insect cells. (A) SDS-PAGE analysis of the Ag-EGase expressed in recombinant baculovirus (AcNPV-AgEGase II)-infected insect cells. Sf9 cells were mock-infected (lane 2) or infected with the wild-type AcNPV (lane 3) and the recombinant AcNPV (lanes 4, 5 and 6) at an MOI of 5 PFU per cell. Cells were collected at 1 (lane 4), 2 (lanes 3 and 5) and 3 (lane 6) days p.i. Total cellular lysates were subjected to 10% SDSPAGE. The arrow on the right of the panel indicates the 36-kDa recombinant Ag-EGase II polypeptide. Molecular weight standards were used as size marker (lane 1). (B) Enzyme activity assay of Ag-EGase II expressed in recombinant baculovirus-infected insect cells. Sf9 cells were mock-infected (lane 1) or infected with the wild-type AcNPV (lane 2) and the recombinant AcNPV (lanes 3, 4 and 5) at an MOI of 5 PFU per cell. The culture supernatants were collected at 1 (lane 3), 2 (lanes 2 and 4) and 3 (lane 5) days p.i. The Ag-EGase II activity assay was performed with the culture supernatants by using CMC agar plate (Beguin, 1983). The Ag-EGase activity was detected by yellow halo zone.

using mRNA prepared from midgut, fat body and epidermis, respectively. Hybridization signal was detected as a single band in mRNA from whole body as a positive control and midgut, evidencing the midgut as a specific site for AgEGase II synthesis (Fig. 4). 3.4. Expression of Ag-EGase II cDNA in baculovirusinfected insect cells To assess Ag-EGase II gene, the 834 bp for Ag-EGase II cDNA was inserted into baculovirus transfer vector. Recombinant AcNPV expressing Ag-EGase II, which we

In order to characterize the recombinant Ag-EGase II expressed in baculovirus-infected insect cells, the recombinant Ag-EGase II with a molecular mass of approximately 36 kDa was purified from the culture supernatant using FPLC techniques. Fractions showing enzyme activity from Superdex 200 HR column were applied to anion exchange chromatography. Four peaks from MonoQ HR column were eluted and one protein peak corresponded to the activity peak. The purified recombinant Ag-EGase II was identified as a single band of 36 kDa by SDS-PAGE and the enzymatic activity of the purified recombinant Ag-EGase II was approximately 812 U/mg of recombinant Ag-EGase II (Table 1). To verify removal of the signal peptide of Ag-EGase II, furthermore, N-terminal amino acid sequencing was performed for the purified recombinant Ag-EGase II by the Edmann degradation. Cleavage of the signal peptide occurred between Leu18 and Ser19 in Ag-EGase II, as represented in Fig. 1. The mature Ag-EGase II is predicted to be 221 amino acid residues. The molecular mass and pI of the mature Ag-EGase II were deduced to be 22.8 kDa and 8.92, respectively.

Table 1 Purification of the recombinant Ag-EGase II expressed in baculovirusinfected insect cells Step

Total protein (mg)

Specific activity (Ua/mg)

Total activity (Ua)

Recovery (%)

Supernatant Ammonium sulfate D-52 Sephadex Gel-filtration Ion-exchange

525.40 86.95

2.25 9.12

1182.15 792.98

100 67.08

1.0 4.1

7.257

18.70

135.71

11.48

8.3

0.741 0.0293

49.20 812.00

36.46 23.79

3.08 2.01

21.9 360.9

Purification fold

a One unit of cellulase activity is defined as the amount of enzyme that releases 1 Amol of glucose min 1 at 50 8C.

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S.J. Lee et al. / Comparative Biochemistry and Physiology, Part B 140 (2005) 551–560

A Absorbance A 280

1.2 0.9 0.6 0.3

20

30

40

50

60

70

Temperature(°C)

80

B Absorbance A 280

1.0 0.8 0.6 0.4 0.2 3

5

7

9

11

pH Fig. 6. Enzymatic properties of the recombinant Ag-EGase II expressed in baculovirus-infected insect cells. (A) Optimum temperature and thermal stability of the recombinant Ag-EGase II. Enzyme activity for optimum temperature was determined by incubating the recombinant Ag-EGase II in acetate buffer (0.05 M, pH 6.0) containing 1% (w/v) CMC at the temperature ranges from 15 8C to 85 8C with 5 8C increment and by assaying the reducing sugars released for DNS reagent method (solid diamond). Thermostability of the recombinant Ag-EGase II was determined by measurement of residual activity after incubation in acetate buffer (0.05 M, pH 6.0) at each temperature for 10 min (shaded square). Error bars represent the standard deviation. (B) Optimal pH of the recombinant AgEGase II. Activities at different pHs are shown with shaded diamond. The recombinant Ag-EGase II was incubated in 0.05 M buffer for 1 h at 50 8C prior to measuring the residual EGase activity under standard assay conditions. Error bars represent the standard deviation.

3.6. Enzymatic properties of the recombinant Ag-EGase II The activity of the purified recombinant Ag-EGase II was determined at various temperatures, ranging from 15 8C to 85 8C at pH 6.0. The optimum temperature of the recombinant Ag-EGase II activity was 50 8C (Fig. 6A). Thermal stability of the recombinant Ag-EGase II was determined by measurement of residual activity after incubation without substrate at various temperatures for 10 min. The recombinant Ag-EGase II was stable up to 60 8C for at least 10 min (Fig. 6A). The optimum pH of the recombinant Ag-EGase II was determined in a pH range of 2.0–12.0 by incubating the enzyme at 50 8C for 1 h, and pH 6.0 was optimal (Fig. 6B).

4. Discussion We have previously reported an endogenous cellulase (Ag-EGase I) belonging to GHF 45 from the mulberry

longicorn beetle, A. germari (Lee et al., 2004). We report here the gene structure, expression and enzyme activity of a novel cellulase (Ag-EGase II) of insect origin from A. germari. The 834-bp Ag-EGase II cDNA encodes an EGase protein of 239 amino acid residues. The Ag-EGase II is proposed to belong to GHF 45 and the conserved catalytic site of GHF 45 (Girard and Jouanin, 1999; Bourne and Henrissat, 2001; Li et al., 2003; Lee et al., 2004) was found in the amino acid sequence. It is noted that Ag-EGase II appears to consist only of a single catalytic domain and lacks both a cellulose-binding domain and a spacer sequence, like the amino acid sequences from the AgEGase I (Lee et al., 2004), P. cochleariae (Girard and Jouanin, 1999), R. speratus hindgut protists (Ohtoko et al., 2000) and M. darwiniensis hindgut flagellates (Li et al., 2003). Among the known cellulase sequences, Ag-EGase II was closest to that of Ag-EGase I (64% protein sequence identity) (Lee et al., 2004) and next to another beetle P. cochleariae (52% protein sequence identity) cellulase (Girard and Jouanin, 1999). In addition, still relatively high amino acid sequence identity was observed compared with several R. speratus hindgut protists (44–46% protein sequence identity) (Ohtoko et al., 2000), but it was low to the two endogenous termite cellulases (8%) (Scharf et al., 2003). The amino acid sequence identity was further reflected to the phylogenetic relationships. The genomic organization of the EGase genes in A. germari analyzed by PCR amplification based on the sequences of the Ag-EGase I (Lee et al., 2004) and AgEGase II cDNAs showed that both Ag-EGase I and AgEGase II contain three exons and two introns, indicating that the two EGase genes from A. germari are endogenous. This is the first report about intron/exon structure of endogenous insect EGase gene belonging to GHF 45. The noteworthy structural property in A. germari EGase genes is that the intron 1 of Ag-EGase I possesses more than two-thirds in the length of entire Ag-EGase I gene, indicating that the length of intron 1 in Ag-EGase I was approximately 8.6-fold longer than that of intron 1 in Ag-EGase II. Girard and Jouanin (1999) suggested that in Southern blot analysis, another beetle, P. cochleariae, has at least 3 cellulase genes, which probably encode divergent enzymes. Furthermore, an EGase cDNA from P. cochleariae was previously cloned (Girard and Jouanin, 1999), but the genomic DNA structure was not reported yet. Intron/exon structure from endogenous cellulase genes of insect origin is reported from a different family of glycosyl hydrolases (GHF 9), showing that the EGase gene of the termite, Nasutitermes takasagoensis, consists of 10 exons interrupted by 9 introns (Tokuda et al., 1999). The expression of Ag-EGase II performed by Northern blot analysis suggested that the Ag-EGase II is specifically expressed in the midgut, as is true for Ag-EGase I (Lee et al., 2004). It is likely that the midgut in A. germari is a site where large quantities of EGase are synthesized for the

S.J. Lee et al. / Comparative Biochemistry and Physiology, Part B 140 (2005) 551–560

degradation of the absorbed cellulose from the diet. The midgut-specific expression of EGases in A. germari is in good agreement with the previous findings that the cellulase genes of beetle origin of P. cochleariae and P. hilaris are expressed only in the midgut (Girard and Jouanin, 1999; Sugimura et al., 2003). The Ag-EGase II cDNA was expressed in baculovirusinfected insect Sf9 cells and the recombinant Ag-EGase II protein was detected as a band of about 36 kDa by SDSPAGE. The calculated molecular mass of mature Ag-EGase II, without the signal peptide, is 22.8 kDa. However, the apparent molecular mass of the purified recombinant AgEGase II was estimated to be 36 kDa from its mobility on SDS-PAGE. The difference between the calculated and observed molecular mass on SDS-PAGE was 13.2 kDa. This inconsistency may be explained by a possible posttranslational modification at three potential N-glycosylation sites, 56–58 (N–K–S), 99–101 (N–S–T) and 237–239 (N– Y–S), in the amino acid sequence of Ag-EGase II (Sugimura et al., 2003). Similarly, slower migration than predicted from the calculations in SDS-PAGE also was reported for P. hilaris cellulase (Sugimura et al., 2003). The enzymatic properties of the Ag-EGase II were assayed in terms of optimum temperature, thermal stability and optimal pH for the Ag-EGase II expressed in baculovirus-infected insect Sf9 cells. The purified recombinant Ag-EGase II showed activity of approximately 812 U/ mg. The enzymatic properties of the recombinant AgEGase II expressed in Sf9 cells showed that the Ag-EGase II activity was most effective at 50 8C and pH 6.0, and was stable at 60 8C for at least 10 min. This result is similar to the previous report of enzymatic properties on optimum temperature and optimum pH of the Ag-EGase I from A. germari (Lee et al., 2004). However, comparison of enzymatic properties of two EGases from A. germari suggested that the Ag-EGase II (with thermal stability at 60 8C for at least 10 min) was more thermo-stable than Ag-EGase I (with thermal stability at 55 8C for at least 10 min), whereas the enzyme activity of Ag-EGase II (812 U/ mg of recombinant EGase) was approximately 18% less than that of Ag-EGase I (992 U/mg of recombinant EGase). It is interesting that thermal stability and enzyme activity of Ag-EGase I and Ag-EGase II are slightly different from each other, although optimum temperature and optimum pH of these EGases are identical to each other. Our result suggests that synergism between these cellulases would be needed in the efficient hydrolysis of cellulose by A. germari larva. In conclusion, we have cloned a novel EGase gene belonging to GHF 45 from the beetle A. germari. The two endogenous EGase genes from A. germari both have three exons and two introns. We also have compared the enzyme properties of two EGases of recombinant Ag-EGase I and Ag-EGase II. Our result may help to further our understanding on gene structure and enzyme activity of insect cellulases.

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Acknowledgements This work was supported by a grant from BioGreen21 program, Rural Development Administration, Republic of Korea. We thank Dr. Hirofumi Watanabe (National Institute of Agrobiological Sciences, Tsukuba 305-8634, Japan) for critical reading of the manuscript.

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