137

Biochimica et Biophysica Acta, 1087 (1990) 137-141 Elsevier

BBAEXP 92166

The conserved terminal region of Trichoderma reesei cellulases forms a strong antigenic epitope for polyclonal antibodies Sirpa Aho and Marja Paloheimo Research Laboratories, Alko Ltd, Helsinki (Finland)

(Received 8 February 1990) (Revised manuscript received 28 May 1990)

Key words: Cellulase cDNA deletion; Cellulolytic yeast; Cellulase antibody; (Saccharomyces cerevisiae, T. reesei)

The specificity of polyclonal antibodies (Pab) raised against Triehoderma reesei eellulases has been studied, cDNAs lacking regions coding for certain functional domains were produced by preparing series of 3'-end deletions from the cDNAs for two cellobiohydrolases, CBH I and CBH II, and an endoglucanase, EG I. The proteins coded by the full length cDNAs and the truncated proteins coded by the deleted cDNAs were expressed in yeast Saccharomyces cerevisiae, under the control of the ADC1 promoter. Each polydonal antiserum showed cross-reactivity with other cellulases. Pabs for CBH I and CBH II both recognized EG I. Pab for EG I strongly recognized both CBH I and CBH II. By analyzing the truncated proteins, we found that these antibodies were almost entirely directed against the conserved tail of the cellulase enzymes.

Introduction The filamentous fungus Trichoderma reesei produces a set of extracellular cellulolytic enzymes. Cellulose is formed from chains of/~-l,4-1inked glucose units. Enzymes degrading cellulose are traditionally divided according to their way of action into three groups, exoglucanases or cellobiohydrolases (EC 3.2.1.9t), endoglucanases (EC 3.2.1.4) and fl-glucosidases (EC 3.2.1.21). There are multiple enzyme proteins in each of these groups. Genes for two cellobiohydrolases, CBH I [1,2] and CBH II [3], and for two endoglucanases, EG I [4,5] and EG III [6], have been isolated and sequenced. The detailed examination of the deduced protein sequences has revealed an interesting domain structure. Each enzyme is composed of a core region, beating the active site, and 'tail' with the Ser-Thr-rich B-region and highly conserved A-region [7-9]. For example CBH I and EG I share 45% sequence identity [4]. CBH I and EG I have the tail domain at the COOH-terminal end, whereas in CBH II and EG III it is found at the NHz-terminal end. The region showing most similarity is the 30 amino acids long A-region, where 70% of the amino acids are identical in all four Trichoderma cellulases [3]. The

Correspondence: S. Aho, Research Laboratories, Alko Ltd., POB 350, SF-00101 Helsinki, Finland.

putatively O-glycosylated Ser-Thr-rich B-region also shows considerable sequence similarity. The yeast Saccharomyces cerevisiae is an excellent expression host for the cellulase cDNAs, because it does not produce endogenous fl-l,4-glucanases. First, we expressed each cDNA, coding for CBH I, CBH II, EG I and EG III, separately in yeast and used the secreted proteins to study the specificity of polyclonal antibodies, raised against native T. reesei CBH I, CBH II and EG I. Second, truncated proteins coded by cDNAs with deletions of various lengths from their 3'-ends were used to study the distribution of the antigenic epitopes on intact cellulases. Materials and Methods Production of polyclonal antibodies The CBH I, CBH II and EG I proteins were purified from the culture liquid of T. reesei VTT-D-80133 as described earlier [10-12] and were a kind gift from Dr. Marja-Liisa Niku-Paavola (Technical Research Centre of Finland, Espoo, Finland). The rabbit polyclonal antisera were prepared at the National Public Health Institute (Helsinki, Finland), using their standard immunization procedure. Preparation of deleted cDNAs cDNA clones for T. reesei VTT-D-80133 celtulases CBH I, CBH II and EG I were made by Dr. Tuula

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138 Teeri [13]. cDNA inserts were separated from the plasmids pTTc01 and pTTc09 [14] and plasmid p T T c l l [15] and transfered into the multiple cloning site of plBI 76 (International Biotechnologies). To create the 3'-end deletions the plasmids were opened at the 3'-end of the cDNA insert and treated with exonuclease III [16]. Samples were drawn at different time points, treated with mung bean nuclease (Pharmacia) and purified by phenol extraction and ethanol precipitation. Plasmids were recircularized by T4 DNA ligase and transformed into Escherichia coli DH5a [17]. The exact length of each deleted cDNA was studied by DNA sequencing, using the dideoxy-chain termination method [18]. A 17-nucleotide-long oligonucleotide was synthesized using the DNA synthesizer (model 381A, Applied Biosystems) according to the T7 promoter sequence in plBI 76 and used as the sequencing primer. The deleted cDNAs were separated from the plBI 76 and ligated into the HindlIl cloning site in the pAAH5 yeast expression vector [19] after filling in the 3'-ends with the Klenow fragment of DNA polymerase I.

Expression of cellulases in yeast Plasmids (pALK220, pALK221, pALK222 and pALK223, respectively) containing the full length cDNAs for CBH I, CBH II, EG I and EG III in pAAH5 and plasmids (see above) containing the selected, deleted cDNAs coding for truncated CBH I, CBH II and EG I were transformed [20] into S. cerevisiae strain YF135 (MATs, leu2-3,112, his3-11,15). Yeast strains were grown on agar plates containing per liter 20 g glucose and 6.7 g yeast nitrogen base without amino acids (Difco) and supplemented with amino acids except leucine. For filter immunodetection yeast strains were streaked onto an agar plate, covered with a nitrocellu-

lose filter (Hybond C, Amersham) and grown at 30 ° C for 2-4 days. Filters were lifted from the plates, the cell mass was washed away with 20 mM Tris-HC1 (pH 7.5) 500 mM NaC1 and the proteins on the filter were visualized using the antibodies and the Protoblot lmmunoblotting System (Promega Biotec) according to the manufacturer's instructions. For dot blot assays yeast cells were inoculated at 2-106 cells/ml into medium containing per liter 20 g glucose and 6.7 g yeast nitrogen base with amino acids except leucine and grown in shake flasks at 30 o C. The growth was monitored by optical density measurements. The samples were collected when cells had reached the stationary phase (2.5.10 "1 cetls/ml). Cells were separated by centrifugation for 15 rain at 5000 × g, 4 ° C, the supernatants were collected, blotted on the nitrocellulose filter and detected with antibodies as described above. Results

Polyclonal antibodies were raised against non-denatured proteins purified from T. reesei culture medium. Two rabbits were immunized with each antigen. Polyclonal antisera KH1050 and KH1051 against CBH I, KH1052 and KH1053 against CBH lI and KH1057 and KH1058 against EG I were obtained. The two antisera against each enzyme gave qualitatively identical results. Therefore, only the results obtained with one set of antibodies, KH1050, KH1052 and KH1057, are presented here. The recognition of Trichoderma cellulases expressed in yeast, by these polyclonal antibodies is shown in Fig. 1. Each antiserum strongly recognized the enzyme against which it had been raised. In addition, antisera against CBH I faintly recognized EG I, antisera against CBH II strongly recognized EG I, and finally, antisera

A

3

2 4

5

Fig. 1. Specificityof polyclonal antibodies against cellulasesproduced by yeast. Yeast strains containingplasmids pALK220 (1), pALK221 (2), pALK222 (3), pALK223 (4), expressingproteins CBH I, CBH II, EG I and EG III, respectively,and cloningvector pAAH5 (5) were grown for 2 days on the nutrient agar plates covered with nitrocellulosefilter. Proteins bound to nitrocellulosewere detected with antiserum KHI050 against CBH I, diluted 1 : 10000 (A); KH1052 againstCBH II, diluted 1 : 5000 (B); KH1057 against EG I, diluted 1 : 10000 (C).

139 against EG I strongly recognized CBH I and CBH II. None of these antisera recognized EG III. Under the conditions used, polyclonal antibodies against CBH I did not recognize CBH II and polyclonal antibodies against CBH II did not recognize CBH I. Pre-immune serum from each rabbit was tested against the cellulase producing yeast strains. They all gave the same background as the polyclonal antibodies in Fig. 1 with the control yeast strain containing the plain cloning vector. Because the proteins secreted by the yeast cells growing on nutrient agar plates covered with nitrocellulose filters accumulated on the filter beyond the linear response range of antibody detection, yeast strains containing cellulase cDNAs were grown in liquid culture. The medium was collected from a fixed amount of ceils and serial 1 : 2 dilutions from each medium were made and applied to the nitrocellulose filter as dot blots (Fig. 2). The culture medium from 1 . 1 0 7 tO 7 . 8 " 10 4 cells containing the CBH I cDNA, gave a linear response with CBH I antiserum. To detect CBH II and EG I with CBH I antiserum, 50- and 100-fold more culture medium was needed from yeast strains containing CBH II and EG I cDNAs, respectively. C B H I protein was not detectable with CBH II antiserum under the conditions used and 100-fold more EG I culture medium was needed than CBH II culture medium. In contrast, EG I antiserum detected CBH I and CBH II 10-fold better than it detected EG I (Fig. 2). To find out which parts of the enzyme molecules are responsible for these cross-reactions, we prepared series of 3'-end deletions from CBH I, CBH II and EG I c D N A (Fig. 3). Each deleted c D N A was expressed in yeast under the control of yeast ADC1 promoter and terminator sequences. Fig. 4A shows that CBH I Pab strongly recognized the protein coded by the d22 but only faintly the protein coded by d26, which suggested that the antibodies were directed against the BA region of the CBH I enzyme. EG I Pab recognized only the intact CBH I molecule (Fig. 4B). CBH II Pab strongly recognized all deleted CBH II proteins tested here (Fig. 4C). Also EG I Pab recognized them (Fig. 4D). As shown in Fig. 3, the AB region is located at the NHz-terminal end of 1

2

3

4

1

2

3

4

O

~

1

2

3

4

PAAH5 EG III EG I

eO

CBH II CBN I P a b - CBHI

P a b - CBH II

Pab-

EG I

Fig. 2. Reaction of antibodies against ceUulases secreted into the culture medium by 5-106 (1), 2.5.106 (2), 1.2.106 (3) and 0.6.106 (4) yeast cells, containing the cDNA indicated and detected with 1 : 10,000 dilution of each antiserum.

CBHI ATG

TAA B AI Kx-x,v~4x\\\~

~/A I

I

1

,

I

d28

d26

d22

i

d2

dl

CBH]] ATG

A

TAA

B

t

1

II

1

o&2 d32

a29

d46 d16

EGI ATG B

TAG AI

11 , 1

[

d l O d9

] d7

l d6

I d'4

d3 d2

d5

d8

Fig. 3. The structure of T. reesei CBH I, CBH II and EG I cDNAs and the location of 3'-end deletions. Hatched, sloping left, signal sequence, cross-hatched, Ser-Thr-rich B-region, hatched, stoping fight, highly conserved A-region, and the start (ATG) and stop (TAA; TAG) of translation as well as the cDNAs after deletions are indi cated.

CBH II and was not removed by COOH-terminal deletions. Most likely it provided the strongest antigenic determinants. Finally, also EG I Pab recognized the intact EG I molecule (Fig. 4E). The deleted proteins coded by d5 and d8 show that some antibodies are directed also against other epitopes. Within the EG I protein, the C B H II Pab recognized only the AB-region. Discussion

We raised the antibodies against the non-denatured Trichoderma cellulases in the hope of getting antibodies against the native functional domains of these enzymes. Such antibodies might be used to identify and quantitate cellulases with or without certain functional domains. Polyclonal antibodies have been reported to show some cross-reactivity between related cellulases [21,22]. Due to the known homology between different cellulases, we could expect some cross-reactivity between our antibodies and different cellulases, but the strong cross-reactivity shown in Fig. 1 was unexpected. When yeast cells are grown on the agar plates covered with nitrocellulose filters, the secreted proteins accumulate

i

140

A

CBH I

V

CBHI

B

P a b - EG I

V

428

d22 ....

• z.

al

C

~

C . . . .

Fig. 4. Reaction of antibodies with the truncated proteins coded by deleted cDNAs. Yeast strains containing CBH 1 cDNA and deletions thereof (A, B), CBH II cDNA and deletions thereof (C, D), and EG I cDNA and deletions thereof (E, F), were grown on the agar plates covered with nitrocellulose filters. Antibodies used for each filter were KHI050 against CBH I (A), KH1052 against CBH II (C, F) and KH1057 against EG I (B, D, E). On each filter V denotes the yeast strain containing the cloning vector pAAH5 and C for the yeast strata containing the full length cDNA.

on the filter. Also proteases secreted by yeast cells are bound onto the filter and thus do not have free access to other secreted proteins. The amount of proteins accumulated on the filter in the immediate surroundings of the yeast cells is far above the linear range of staining, when detected with antibodies. The dot blots showed that CBH I Pab detects CBH I protein and CBH II Pab detects CBH II protein 50- to 100-fold better than the other cellulases, but EG I Pab detected CBH I and CBH II 10-fold better than EG I. These antibodies cannot be used for identification or quantitation of any single cellulase in, for example, the 7". reesei culture medium, where different amounts of several cellulases are present. This leads to the situation that

when the amount of one enzyme is optimized for detection on the Western blot, the other cellulases are either under-represented or over-represented. This stresses the importance of acquiring antibodies of higher specificity, probably monoclonal antibodies. Because the cellulases are glycoproteins and have very similar Ser-Thr-rich O-glycosylated B-regions, some cross-reactivity of antibodies was expected to be due to the shared antigenic epitopes formed by the carbohydrate chains [23]. However, EG I Pab recognized only the full length CBH 1 and CBH II Pab only the full length EG I, which suggests that the common antigenic site is located within the highly conserved A-region. The finding that, even though these antibodies are polyclonal, all their main epitopes seem to be in a limited region of the cellulases, emphasises the caution needed when attempting quantitative immunological assays of genetically engineered proteins. By hydrophobic cluster analysis EG III has been shown to belong to a different family of cellulases [24]. Consistent with this it did not show any cross reactivity with the antibodies tested here. The latest model of the structure of CBH I A-region [25] suggests that the last 13 C-terminal amino acids form a distinct loop structure. Our CBH I deletion 22, which is lacking the last 12 amino acids, was recognized by CBH I Pab but not by EG I Pab. Therefore, the loop formed by the most conserved terminal amino acids forms the strongest antigenic epitope on the 7: reesei celhilases and also the antigenic determinant common to the T. reesei cellulases studied here.

Acknowledgements The excellent technical assistance by Outi Nikkil~i and Taina Jalava is gratefully acknowledged. We thank Marja-Liisa Niku-Paavola (Technical Research Centre of Finland) for providing purified proteins and the personnel of National Public Health Institute for the pleasant collaboration during the immunization procedure. We thank Rolf Bfihler for critical reading of this manuscript.

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The conserved terminal region of Trichoderma reesei cellulases forms a strong antigenic epitope for polyclonal antibodies.

The specificity of polyclonal antibodies (Pab) raised against Trichoderma reesei cellulases has been studied. cDNAs lacking regions coding for certain...
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