APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1990, p. 1103-1108 0099-2240/90/041103-06$02.00/0 Copyright C) 1990, American Society for Microbiology
Vol. 56, No. 4
Cross-Reactive and Specific Monoclonal Antibodies against Cellobiohydrolases I and II and Endoglucanases I and II of Trichoderma reesei R. A. NIEVES,'* M. E. HIMMEL,2 R. J. TODD,' AND R. P. ELLIS' Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523,1 and Applied Biological Sciences Section, Biotechnology Research Branch, Biofuels Research Division, Solar Energy Research Institute, Golden, Colorado 804012 Received 18 September 1989/Accepted 11 January 1990
Splenocytes derived from mice inoculated with a commercial cellulase preparation or purified cellulases were fused with a stable myeloma cell line (SP2/0). Specific monoclonal antibodies to cellobiohydrolases I and II and endoglucanases I and II were established. In addition to specific monoclonal antibodies, we were also able to establish stable hybridoma cell lines which produced monoclonal antibodies that recognized similar epitopes possessed by two or more of the above cellulases. By obtaining monospecific antibodies for all four individual celiulases, the role and function of the individual cellulases can thus be studied in greater detail.
The cellulase system of Trichoderma reesei has been studied extensively since the 1950s (1, 4-6, 14, 16, 23, 26). The hydrolysis of cellulose to small oligosaccharides and glucose is accomplished by synergism among cellobiohydrolases (EC 3.2.1.91; 1,4-p-D-glucan cellobiohydrolase), endoglucanases (EC 3.2.1.4; 1,4-,B-D-glucan 4-glucanohydrolase), and cellobiase (EC 3.2.1.21; ,-D-glucoside glucohydrolase). In general, the endoglucanases hydrolyze the cellulose chain randomly, whereas the cellobiohydrolases cleave cellobiose units from the nonreducing end of the chain (2, 8). Immunological and immunochemical approaches have been applied to the detection and differentiation of the various cellulase components. In 1979, Fagerstam and Pettersson described immunological distinctions between the endoglucanase and cellobiohydrolase of T. reesei QM 9414 (3). Later, Nummi and colleagues were able to identify immunological differences among the different enzymes of the cellulase complex of T. reesei by immunoelectrophoresis
which include CBH I and II and endoglucanases I and II (EG I and II). Also, we were able to generate hybridoma cell lines which secrete MAbs which recognize shared epitopes on two or more of these cellulases. MATERIALS AND METHODS Crude cellulase preparation and purified celiulase component enzymes. The preparation of crude T. reesei cellulases used in this study was cellulase 150L from Genencor, Inc., South San Francisco, Calif. Samples of purified T. reesei CBH I and II and EG I and II were obtained as a gift from Sharon Shoemaker. These enzymes were purified to homogeneity from the commercial cellulase 150L preparation, using sequential chromatographic methods following the procedures in Shoemaker et al. (22). MAb production. Six- to 8-week-old female BALB/c mice were immunized subcutaneously with approximately 100 ,ug of the commercial cellulase preparation emulsified in 0.5 ml of Freund complete adjuvant (Difco Laboratories, Detroit, Mich.) and 0.5 ml of sterile phosphate-buffered saline, pH 7.0. After 2 weeks, the mice were boosted intraperitoneally with 100 ,ug of the commercial cellulase emulsified with 0.5 ml of incomplete Freund adjuvant and 0.5 ml of sterile phosphate-buffered saline, pH 7.0. Ten days later, mice were bled and antibody titers were measured by an indirect enzyme-linked immunosorbent assay (ELISA). Three days prior to fusion, the mouse to be used for fusion was inoculated intraperitoneally with 100 ,ug of the cellulase in phosphate-buffered saline, pH 7.0. On the day of fusion, the mouse was sacrificed and the spleen was removed. The splenocytes were collected and fused with the myeloma cell line SP2/0 (20). Hybridoma fusions with the commercial cellulase yielded MAbs specific for CBH I, but all such fusions yielded cross-reactive MAbs for EG I, EG II, and CBH II. To obtain MAbs specific for EG I, EG II, and CBH II, mice inoculated with these purified enzymes were used for fusions as described above. Fused cells were suspended in RPMI culture medium supplemented with 50 IU of penicillin per ml, 50 ,ug of streptomycin per ml, 2 mM L-glutamine, 1 mM sodium pyruvate (Sigma Chemical Co., St. Louis, Mo.), and 10%
(13).
By inoculating rabbits with purified enzymes, Shoemaker et al. reported antigenic differences between various cellulase components of T. reesei L27 (22). Although these prior reports seemed conclusive, the antibodies used for such studies were of a polyvalent nature.
Recently, monoclonal antibodies (MAbs) generated by modifications of the Kohler and Milstein technique (7) have been applied to studies of the different cellulase components. Riske and co-workers reported the production of MAbs which recognized the cellobiohydrolase I (CBH I) of T. reesei and not the other cellulase components (17). Also in 1987, McHale reported the production of a MAb capable of recognizing an endoglucanase of T. emersonii (10). More recently, Mischak and colleagues reported the production of a mouse MAb specific to CBH I and CBH II from T. reesei. With the use of these MAbs, they were able to define the specific immunological recognition sites for the enzymes (11). In this study, we report the first production of MAbs specific for all individual cellulase components of T. reesei, *
Corresponding author. 1103
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NIEVES ET AL.
fetal bovine serum (Irvine Scientific, Irvine, Calif.). They were then transferred into sterile 96-well plates (Nunclon, Denmark) and cultivated at 37°C in a humidified incubator containing 5% Co2. Hybridomas were selected by the addition of hypoxanthine-aminopterin-thymidine-RPMI medium for 5 consecutive days. After 2 weeks, all wells were assayed by an indirect ELISA. Fusions of spleens derived from mice inoculated with purified cellulases were also performed as described above. Indirect ELISA. To detect hybridomas producing MAb against the commercial cellulase or purified enzymes, an indirect ELISA was performed on all cultured plates. Wells on polyvinyl chloride plates (Becton Dickinson Labware, Oxnard, Calif.) were coated with 250 ng of commercial cellulase or 100 ng of the purified cellulase of interest. The plates were incubated at 37°C overnight. The following day, the ELISA plates were washed and blocked with 1% polyvinylpyrrolidone-phosphate-buffered saline, pH 7.0 (Sigma), for 30 min. All incubation times were 30 min. Culture supernatants were added to the plates, and plates were incubated at 25°C and washed. Subsequently, 50 RI of horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G, diluted 1:1,000, was added. The plates were washed, and 50 RI of o-phenylenediamine in 80 mM citratephosphate solution containing H202 was added as substrate. Selected positive clones were dilution cloned twice, using BALB/c mice thymocytes as feeder cells. SDS-PAGE and Western blotting. Two weeks after dilution cloning, single-colony clones were assayed with a miniblotter system (Immunetics, Cambridge, Mass.) for antibody against the commercial cellulase or the purified enzyme of interest. A 38.5:1 acrylamide-bisacrylamide stock solution (Eastman Kodak Co., Rochester, N.Y.) was prepared and filtered for casting of the gels. Sodium dodecyl sulfate (SDS)-polyacrylamide gels consisted of a 6% stacking gel and a 12.5% resolving gel. Gels were cast for SDS-polyacrylamide gel electrophoresis (PAGE) and conventional Western blots (immunoblots). The gels used for the miniblot system were different in that the comb inserted into the stacking gel was reversed so as to give one large lane. The pretreated sample of commercial cellulase or purified enzyme was applied across the length of the large lane and electrophoresed. Proteins on the gel were transferred to nitrocellulose (NC) paper (Bio-Rad Laboratories, Richmond, Calif.) overnight. When molecular weight estimates were required, two lanes were charged with 10-jig samples of low- and high-molecular-weight standard proteins (Bio-Rad). The following day, the NC was blocked for 1 h with a 10% powdered milk-TEN buffer (pH 7.4) solution. The paper was then washed extensively with 0.05% Tween 20 in TEN buffer (pH 7.4) and placed on the miniblot apparatus. Approximately 75 jil of supernatants from the single-colony clones being tested was placed into the corresponding slots, and the apparatus was left standing for 1 h at 25°C. The NC was then extensively washed with TEN-Tween buffer. A 75-,ul portion of goat anti-mouse horseradish peroxidase-conjugated antibody, diluted 1:1,000, was added to each slot, and wells were incubated as described above. Reactive MAbs were detected by the addition of substrate, which consisted of diaminobenzidene, H202, and 0.05 M ammonium acetate, pH 5.0. To verify the specificity of the MAb, conventional Western blots were performed with the four purified enzymes. For conventional Western blots, 4.3 to 6 jig of purified enzymes was applied to individual wells of a 12.5% poly-
APPL. ENVIRON. MICROBIOL.
00w0
94,000
0
am
67,000
-sto
43,000
30,000 9~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
20,000 14,400
_ * -
FIG. 1. SDS-PAGE demonstrating the four purified cellulases used to assay specificity of the MAbs.
acrylamide gel; then wells were subjected to SDS-PAGE and transferred to NC paper as described above. Two NC sheets were obtained per gel. The NC sheets were blocked and washed, as stated above, in separate glass containers. A 100-jil amount of an antibody recognizing all four enzymes was diluted 1:1,000 in 13 ml of a 10% milk-TEN buffer solution and added to one of the NC sheets. The culture supernatant of the clone being assayed was diluted accordingly and added to the other NC sheet. Both sheets were gently stirred for 1 h at 25°C. The NC was then washed and 13 ml of diluted goat anti-mouse horseradish peroxidaseconjugated antibody was added as described for the miniblotter system. The NC was washed and the substrate was added.
RESULTS MAb against CBH I and cross-reactive MAbs. Figure 1 demonstrates the four purified enzymes used for specificity assays by Western blots. These preparations appeared to be homogeneous by SDS-PAGE and molecular weights ranged from 59,000 to 70,000, which is consistent with earlier reports (22). Initial fusions of splenocytes from mice immunized with the commercial cellulase preparation yielded, almost exclusively, MAbs which reacted with epitopes shared by two or more of the purified enzymes. These MAbs are referred to as cross-reactive MAbs. Figure 2 illustrates a MAb which recognized an epitope shared among all four purified enzymes as observed by a conventional Western blot. Another blot shows a MAb obtained in a similar fashion which recognized EG I and CBH I (Fig. 3). The lighter bands detected in lanes 1 and 4 are speculated to be a slight CBH I contamination in the EG I and CBH II preparations since they migrate similarly to CBH I (lane 3). After various fusions of splenocytes from mice immunized with the commercial cellulase, it was possible to obtain two different MAbs which reacted exclusively with CBH I. Figure 4 demonstrates the specificity of one of these clones.
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MAbs AGAINST T. REESEI CBH I AND II AND EG I AND II
VOL. 56, 1990
94,000 67,000
43,000
FIG. all four II;
2.
Conventional Western
purified
lane 4,
blot of
a
MAb which
enzymes. Lane 1, CBH II; lane
2, CBH
recognized
I; lane
3, EG
EG
Specificity of MAb CI-P1-4G-5H-12B to CBH I obtained splenocytes of a mouse immunized with a commercial cellulase preparation. FIG. 4.
from was designated CI-P1-4G-5H-12B. Production of EG and EG specific for CBH proved to be difficult with fusions obtained with splenocytes from mice 4~~~~~~~~~~~~0 inoculated with the commercial cellulase preparation. For this reason, purified cellulases were used to inoculate mice
This clone MAb
II,
for fusions. In addition, nizes
a
I,
cross-reactive MAb which recog-
and CBH I was obtained by EG from the fusion of splenocytes from a mouse immunized with purified EG (Fig. 5). EG I-specific mouse MAb production. A fusion of splenocytes from a mouse inoculated with purified EG I initially produced 31 wells with supernatant fluids positive by an
ELISA.
epitope
Five
shared
of these
clones
were
selected for dilution
cloning, and the rest were subsequently frozen in liquid N2. After cloning twice, only clone El-Pl-6H-11D-11F was found to react positively by the miniblot assay. Further testing of this stable clone demonstrated it to be specific for EG I (Fig. 6a). MAb proCBH HI-specific MAb production. For CBH duction, 5.95 x 108 spleen cells were fused with SP2/0
FIG. 3. Western blot illustrating specificity of a MAb to EG I and CBH I. Lane 1, EG I; lane 2, EG II; lane 3, CBH I; lane 4, CBH II.
wells from eight plates positive by indirect ELISA. Thirteen clones were tested against purified CBH using the miniblotter. Seven hybridomas demonstrated reactivity to After dilution cloning, two different hybridomas CBH
myeloma cells.
were
Two weeks later,
observed to be
II,
showed
specificity to CBH
CII-P5-9G-12B-8G of
clone EG
and
CII-P5-9G-12B-8G
I1-specific
mouse
These
clones
were
CII-P5-12F-10E-11C. MAb
is demonstrated
The in
designated specificity
Fig.
6b.
production. After three fusions
splenocytes from mice immunized with purified EG II, 15 clones from the third fusion were found to be ELISA
of
FIG. 5. Evidence showing that a MAb obtained from splenocytes of a mouse inoculated with purified EG II recognized EG II and CBH I exclusively.
6b _
a
amW _ := _
_
am: i3_ d
Mi
0
0
fts4 ,
i
6
400
43,000
0
43,000
FIG. 6. Western blots illustrating the specificity of MAbs for EG I, CBH II, and EG II. Each of the clones was obtained by using the respective purified enzyme as antigen. (a) Recognition of EG I by clone EI-P1-6H-11D-11F is demonstrated by this Western blot. (b and c) From each SDS-PAGE, it was possible to obtain two nitrocellulose sheets. A MAb which recognized all four enzymes was used as a positive control (left blot), and MAb specificity is illustrated on the right blot. (b) Recognition of CBH II by clone CII-P5-9G-12B-8G. (c) Recognition of EG II by clone EII-P3-2A-11B-8E. 1106
MAbs AGAINST T. REESEI CBH I AND II AND EG I AND II
VOL. 56, 1990
positive. When these were tested by the miniblot, only five recognized EG II. These five clones were subsequently frozen and cloned twice. Conventional Western blots demonstrated clone EII-P3-2A-11B-8E to be specific against EG II (Fig. 6c). DISCUSSION
The initial purpose of our studies was to produce individual cellulase-specific MAbs against the four major cellulases of T. reesei by utilizing a commercial cellulase preparation. When this commercial cellulase was used for inoculating mice, the MAbs produced mainly recognized shared epitopes among the individual cellulases. This is not unexpected, since it has been shown that small conserved amino acid regions on all four cellulases are present (15, 19, 24). At this time, we can only speculate whether these regions are responsible for cellulase degradation or binding. A crossreactive MAb which recognizes a shared epitope which participates in the hydrolysis of cellulose could be utilized to study these enzymatic reactions. It is believed that T. reesei cellulases pertain to a related gene family due to their highly conserved regions (24). From our studies it is apparent that epitopes exist which are not shared by these enzymes. Whether these regions are functional or whether they are structural epitopes is not known. It is possible that divergence of these cellulases from the ancestral gene has led to the elimination of unproductive genes in some of the enzymes while in others they are retained. Since cellulase protein in culture broth is mainly composed of CBH I (12), it was possible to obtain a MAb specific for CBH I from mice immunized with the commercial cellulase. Production of MAbs to the other three enzymes proved to be more difficult, possibly because these are present in lesser amounts in the cellulase preparation. Even though some lymphocytes may have been stimulated by the presence of these proteins, the probability of isolating such a cell from the vast array of stimulated lymphocytes is minimal. For this reason it was necessary to obtain specific MAbs for CBH II, EG I, and EG II by inoculating mice with purified enzymes. This procedure proved to be more successful as specific MAbs were produced. We do not know to which particular epitopes these MAbs are directed, but further studies should clarify this. The use of such MAbs is varied and extensive. Riske et al. have used a specific MAb to purify CBH I by affinity chromatography (18). Similarly, we have recently reported the purification of EG I from a diafiltered crude culture broth of T. reesei (R. A. Nieves, M. E. Himmel, and R. P. Ellis, Appl. Biochem. Biotechnol., in press). By utilizing these antibodies, purification of the other individual cellulases can also be achieved by simplified procedures as opposed to conventional purification techniques. Also, conventional purification methods can be greatly simplified by using a specific MAb to assay purification steps (i.e., chromatography fractions), replacing the tedious cellulase assays. The cloning and expression of T. reesei cellulase genes into eucaryotes and procaryotic microorganisms have been reported recently (21, 24, 25). Since one of the major setbacks in the utilization of cellulases as a potential producer of utilizable substrates is the cost of enzyme production (9), it is foreseeable that recombinant techniques and structurefunction studies of these cellulases will be further emphasized. For such applications, labeled MAbs directed against these enzymes could be used as probes for detecting transformed cells expressing cellulase gene products.
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ACKNOWLEDGMENTS We thank Sharon Shoemaker for the purified T. reesei cellulase enzymes. This work was funded through the Biofuels Program Office at the Solar Energy Research Institute by the Biochemical Conversion Program at the Department of Energy Biofuels and Municipal Waste Technology Division. LITERATURE CITED
1. Berghem, L. E. R., L. G. Pettersson, and U. B. Axio-Fredriksson. 1976. The mechanism of enzymatic cellulose degradation. Eur. J. Biochem. 61:621-630. 2. Coughlan, M. P. 1985. The properties of fungal and bacterial cellulase with comment on their production and application. Biotechnol. Genet. Eng. Rev. 31:39-109. 3. Fagerstam, L. G., and L. G. Pettersson. 1979. The cellulytic complex of Trichoderma reesei QM 9414. FEBS Lett. 98: 363-367. 4. Fagerstam, L. G., and L. G. Pettersson. 1980. The 1,4-betaglucan cellobiohydrolases of Trichoderma reesei QM 9414. FEBS Lett. 119:97-100. 5. Henrissat, B., H. Driguez, C. Viet, and M. Schulein. 1985. Synergism of cellulases from Trichoderma reesei in the degradation of cellulose. Bio/Technology 3:722-726. 6. Johansson, G., J. Stahlberg, G. Lindeberg, A. Engstrom, and G. Pettersson. 1989. Isolated fungal cellulase terminal domains and a synthetic minimum analogue bind to cellulose. FEBS Lett. 243:389-393. 7. Kohler, G., and C. Milstein. 1975. Continuous cultures of fixed cells secreting antibody of pre-defined specificity. Nature (London) 256:495-497. 8. Mandels, M. 1982. Cellulases, p. 35-78. In Annual reports on fermentation processes, vol. 5. Academic Press, Inc., New York. 9. Mandels, M. 1985. Applications of cellulases. Biochem. Soc. Trans. 13:414-416. 10. McHale, A. P. 1987. Production and characterization of monoclonal antibodies to the cellulases produced by Talaromyces emersonii CBS 814.70. Biochim. Biophys. Acta 924:147-153. 11. Mischak, H., F. Hofer, R. Messner, E. Weissinger, M. Hayn, P. Tomme, H. Esterbauer, E. Kuchler, M. Claeyssens, and C. P. Kubicek. 1989. Monoclonal antibodies against different domains of cellobiohydrolase I and II from Trichoderma reesei. Biochim. Biophys. Acta 990:1-7. 12. Natick Research and Development Command, U.S. Army. 1981. Enzymatic hydrolysis of cellulose to glucose. A report on the Natick Programme. Natick Research and Development Command, U.S. Army, Natick, Mass. 13. Nummi, M., M. L. Niku-Paavola, T. M. Enari, and V. Raunio. 1980. Immunoelectrophoretic detection of cellulases. FEBS Lett. 113:164-166. 14. Okada, G. 1976. Enzymatic studies on a cellulase system of Trichoderma viride. J. Biochem. 80:913-922. 15. Penttila, M., P. Lehtovaara, H. Nevalainen, R. Bhikhabhai, and J. Knowles. 1986. Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene. Gene 45:253-263. 16. Reese, E. T., R. G. H. Siu, and H. S. Levinson. 1950. The biological degradation of soluble cellulose derivatives and its relationship to the mechanism of cellulose hydrolysis. J. Bacteriol. 59:485-497. 17. Riske, F., I. Labudova, L. Miller, J. D. Macmillan, and D. E. Eveleigh. 1987. Monoclonal antibodies against cellobiohydrolase from Trichoderma reesei, p. 167-176. In M. Moo-Young (ed.), Biomass Conversion technology. Principles and practice. Pergamon Press, New York. 18. Riske, F. J., D. E. Eveleigh, and J. D. Macmillan. 1986. Purification of cellobiohydrolase I from Trichoderma reesei using monoclonal antibodies in an immunomatrix. J. Ind. Microbiol. 1:259-264. 19. Saloheimo, M., P. Lehtovaara, M. Penttila, T. T. Teeri, J. Stahlberg, G. Johansson, G. Pettersson, M. Claeyssens, P. Tomme, and J. K. C. Knowles. 1988. EG III, a new endogluca-
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22.
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nase from Trichoderma reesei: the characterization of both gene and enzyme. Gene 63:11-21. Schulman, M., C. D. Wilde, and G. Kohler. 1978. A better cell line for making hybridomas secreting specific antibodies. Nature (London) 276:269-270. Shoemaker, S., V. Schweickart, M. Ladner, D. Gelfand, S. Kwok, K. Myambo, and M. Innis. 1983. Molecular cloning of exocellobiohydrolase I derived from Trichoderma reesei strain L27. Bio/Technology 1:691-696. Shoemaker, S., K. Watt, G. Tsitovsky, and R. Cox. 1983. Characterization and properties of cellulases purified from Trichoderma reesei strain L27. Bio/Technology 1:687-690. Shoemaker, S. P., and R. D. Brown, Jr. 1978. Characterization
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of endo-1,4-beta-D-glucanases purified from Trichoderma viride. Biochim. Biophys. Acta 523:147-161. 24. Teeri, T. T., P. Lehtovaara, S. Kauppinen, I. Salovuori, and J. Knowles. 1987. Homologous domains in Trichoderma reesei cellulolytic enzymes: gene sequence and expression of cellobiohydrolase II. Gene 51:43-52. 25. Van Arsdell, J. N., S. Kwok, V. L. Schweickart, M. B. Ladner, D. H. Gelfand, and M. A. Innis. 1987. Cloning, characterization, and expression in Saccharomyces cerevisiae of endoglucanase I from Trichoderma reesei. Bio/Technology 5:60-64. 26. Woodward, J., M. K. Hayes, and N. E. Lee. 1988. Hydrolysis of cellulose by saturating and non-saturating concentrations of cellulase: implications for synergism. Bio/Technology 6:301-304.
Vol. 56, No. 4
Cross-Reactive and Specific Monoclonal Antibodies against Cellobiohydrolases I and II and Endoglucanases I and II of Trichoderma reesei R. A. NIEVES,'* M. E. HIMMEL,2 R. J. TODD,' AND R. P. ELLIS' Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523,1 and Applied Biological Sciences Section, Biotechnology Research Branch, Biofuels Research Division, Solar Energy Research Institute, Golden, Colorado 804012 Received 18 September 1989/Accepted 11 January 1990
Splenocytes derived from mice inoculated with a commercial cellulase preparation or purified cellulases were fused with a stable myeloma cell line (SP2/0). Specific monoclonal antibodies to cellobiohydrolases I and II and endoglucanases I and II were established. In addition to specific monoclonal antibodies, we were also able to establish stable hybridoma cell lines which produced monoclonal antibodies that recognized similar epitopes possessed by two or more of the above cellulases. By obtaining monospecific antibodies for all four individual celiulases, the role and function of the individual cellulases can thus be studied in greater detail.
The cellulase system of Trichoderma reesei has been studied extensively since the 1950s (1, 4-6, 14, 16, 23, 26). The hydrolysis of cellulose to small oligosaccharides and glucose is accomplished by synergism among cellobiohydrolases (EC 3.2.1.91; 1,4-p-D-glucan cellobiohydrolase), endoglucanases (EC 3.2.1.4; 1,4-,B-D-glucan 4-glucanohydrolase), and cellobiase (EC 3.2.1.21; ,-D-glucoside glucohydrolase). In general, the endoglucanases hydrolyze the cellulose chain randomly, whereas the cellobiohydrolases cleave cellobiose units from the nonreducing end of the chain (2, 8). Immunological and immunochemical approaches have been applied to the detection and differentiation of the various cellulase components. In 1979, Fagerstam and Pettersson described immunological distinctions between the endoglucanase and cellobiohydrolase of T. reesei QM 9414 (3). Later, Nummi and colleagues were able to identify immunological differences among the different enzymes of the cellulase complex of T. reesei by immunoelectrophoresis
which include CBH I and II and endoglucanases I and II (EG I and II). Also, we were able to generate hybridoma cell lines which secrete MAbs which recognize shared epitopes on two or more of these cellulases. MATERIALS AND METHODS Crude cellulase preparation and purified celiulase component enzymes. The preparation of crude T. reesei cellulases used in this study was cellulase 150L from Genencor, Inc., South San Francisco, Calif. Samples of purified T. reesei CBH I and II and EG I and II were obtained as a gift from Sharon Shoemaker. These enzymes were purified to homogeneity from the commercial cellulase 150L preparation, using sequential chromatographic methods following the procedures in Shoemaker et al. (22). MAb production. Six- to 8-week-old female BALB/c mice were immunized subcutaneously with approximately 100 ,ug of the commercial cellulase preparation emulsified in 0.5 ml of Freund complete adjuvant (Difco Laboratories, Detroit, Mich.) and 0.5 ml of sterile phosphate-buffered saline, pH 7.0. After 2 weeks, the mice were boosted intraperitoneally with 100 ,ug of the commercial cellulase emulsified with 0.5 ml of incomplete Freund adjuvant and 0.5 ml of sterile phosphate-buffered saline, pH 7.0. Ten days later, mice were bled and antibody titers were measured by an indirect enzyme-linked immunosorbent assay (ELISA). Three days prior to fusion, the mouse to be used for fusion was inoculated intraperitoneally with 100 ,ug of the cellulase in phosphate-buffered saline, pH 7.0. On the day of fusion, the mouse was sacrificed and the spleen was removed. The splenocytes were collected and fused with the myeloma cell line SP2/0 (20). Hybridoma fusions with the commercial cellulase yielded MAbs specific for CBH I, but all such fusions yielded cross-reactive MAbs for EG I, EG II, and CBH II. To obtain MAbs specific for EG I, EG II, and CBH II, mice inoculated with these purified enzymes were used for fusions as described above. Fused cells were suspended in RPMI culture medium supplemented with 50 IU of penicillin per ml, 50 ,ug of streptomycin per ml, 2 mM L-glutamine, 1 mM sodium pyruvate (Sigma Chemical Co., St. Louis, Mo.), and 10%
(13).
By inoculating rabbits with purified enzymes, Shoemaker et al. reported antigenic differences between various cellulase components of T. reesei L27 (22). Although these prior reports seemed conclusive, the antibodies used for such studies were of a polyvalent nature.
Recently, monoclonal antibodies (MAbs) generated by modifications of the Kohler and Milstein technique (7) have been applied to studies of the different cellulase components. Riske and co-workers reported the production of MAbs which recognized the cellobiohydrolase I (CBH I) of T. reesei and not the other cellulase components (17). Also in 1987, McHale reported the production of a MAb capable of recognizing an endoglucanase of T. emersonii (10). More recently, Mischak and colleagues reported the production of a mouse MAb specific to CBH I and CBH II from T. reesei. With the use of these MAbs, they were able to define the specific immunological recognition sites for the enzymes (11). In this study, we report the first production of MAbs specific for all individual cellulase components of T. reesei, *
Corresponding author. 1103
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fetal bovine serum (Irvine Scientific, Irvine, Calif.). They were then transferred into sterile 96-well plates (Nunclon, Denmark) and cultivated at 37°C in a humidified incubator containing 5% Co2. Hybridomas were selected by the addition of hypoxanthine-aminopterin-thymidine-RPMI medium for 5 consecutive days. After 2 weeks, all wells were assayed by an indirect ELISA. Fusions of spleens derived from mice inoculated with purified cellulases were also performed as described above. Indirect ELISA. To detect hybridomas producing MAb against the commercial cellulase or purified enzymes, an indirect ELISA was performed on all cultured plates. Wells on polyvinyl chloride plates (Becton Dickinson Labware, Oxnard, Calif.) were coated with 250 ng of commercial cellulase or 100 ng of the purified cellulase of interest. The plates were incubated at 37°C overnight. The following day, the ELISA plates were washed and blocked with 1% polyvinylpyrrolidone-phosphate-buffered saline, pH 7.0 (Sigma), for 30 min. All incubation times were 30 min. Culture supernatants were added to the plates, and plates were incubated at 25°C and washed. Subsequently, 50 RI of horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G, diluted 1:1,000, was added. The plates were washed, and 50 RI of o-phenylenediamine in 80 mM citratephosphate solution containing H202 was added as substrate. Selected positive clones were dilution cloned twice, using BALB/c mice thymocytes as feeder cells. SDS-PAGE and Western blotting. Two weeks after dilution cloning, single-colony clones were assayed with a miniblotter system (Immunetics, Cambridge, Mass.) for antibody against the commercial cellulase or the purified enzyme of interest. A 38.5:1 acrylamide-bisacrylamide stock solution (Eastman Kodak Co., Rochester, N.Y.) was prepared and filtered for casting of the gels. Sodium dodecyl sulfate (SDS)-polyacrylamide gels consisted of a 6% stacking gel and a 12.5% resolving gel. Gels were cast for SDS-polyacrylamide gel electrophoresis (PAGE) and conventional Western blots (immunoblots). The gels used for the miniblot system were different in that the comb inserted into the stacking gel was reversed so as to give one large lane. The pretreated sample of commercial cellulase or purified enzyme was applied across the length of the large lane and electrophoresed. Proteins on the gel were transferred to nitrocellulose (NC) paper (Bio-Rad Laboratories, Richmond, Calif.) overnight. When molecular weight estimates were required, two lanes were charged with 10-jig samples of low- and high-molecular-weight standard proteins (Bio-Rad). The following day, the NC was blocked for 1 h with a 10% powdered milk-TEN buffer (pH 7.4) solution. The paper was then washed extensively with 0.05% Tween 20 in TEN buffer (pH 7.4) and placed on the miniblot apparatus. Approximately 75 jil of supernatants from the single-colony clones being tested was placed into the corresponding slots, and the apparatus was left standing for 1 h at 25°C. The NC was then extensively washed with TEN-Tween buffer. A 75-,ul portion of goat anti-mouse horseradish peroxidase-conjugated antibody, diluted 1:1,000, was added to each slot, and wells were incubated as described above. Reactive MAbs were detected by the addition of substrate, which consisted of diaminobenzidene, H202, and 0.05 M ammonium acetate, pH 5.0. To verify the specificity of the MAb, conventional Western blots were performed with the four purified enzymes. For conventional Western blots, 4.3 to 6 jig of purified enzymes was applied to individual wells of a 12.5% poly-
APPL. ENVIRON. MICROBIOL.
00w0
94,000
0
am
67,000
-sto
43,000
30,000 9~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
20,000 14,400
_ * -
FIG. 1. SDS-PAGE demonstrating the four purified cellulases used to assay specificity of the MAbs.
acrylamide gel; then wells were subjected to SDS-PAGE and transferred to NC paper as described above. Two NC sheets were obtained per gel. The NC sheets were blocked and washed, as stated above, in separate glass containers. A 100-jil amount of an antibody recognizing all four enzymes was diluted 1:1,000 in 13 ml of a 10% milk-TEN buffer solution and added to one of the NC sheets. The culture supernatant of the clone being assayed was diluted accordingly and added to the other NC sheet. Both sheets were gently stirred for 1 h at 25°C. The NC was then washed and 13 ml of diluted goat anti-mouse horseradish peroxidaseconjugated antibody was added as described for the miniblotter system. The NC was washed and the substrate was added.
RESULTS MAb against CBH I and cross-reactive MAbs. Figure 1 demonstrates the four purified enzymes used for specificity assays by Western blots. These preparations appeared to be homogeneous by SDS-PAGE and molecular weights ranged from 59,000 to 70,000, which is consistent with earlier reports (22). Initial fusions of splenocytes from mice immunized with the commercial cellulase preparation yielded, almost exclusively, MAbs which reacted with epitopes shared by two or more of the purified enzymes. These MAbs are referred to as cross-reactive MAbs. Figure 2 illustrates a MAb which recognized an epitope shared among all four purified enzymes as observed by a conventional Western blot. Another blot shows a MAb obtained in a similar fashion which recognized EG I and CBH I (Fig. 3). The lighter bands detected in lanes 1 and 4 are speculated to be a slight CBH I contamination in the EG I and CBH II preparations since they migrate similarly to CBH I (lane 3). After various fusions of splenocytes from mice immunized with the commercial cellulase, it was possible to obtain two different MAbs which reacted exclusively with CBH I. Figure 4 demonstrates the specificity of one of these clones.
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MAbs AGAINST T. REESEI CBH I AND II AND EG I AND II
VOL. 56, 1990
94,000 67,000
43,000
FIG. all four II;
2.
Conventional Western
purified
lane 4,
blot of
a
MAb which
enzymes. Lane 1, CBH II; lane
2, CBH
recognized
I; lane
3, EG
EG
Specificity of MAb CI-P1-4G-5H-12B to CBH I obtained splenocytes of a mouse immunized with a commercial cellulase preparation. FIG. 4.
from was designated CI-P1-4G-5H-12B. Production of EG and EG specific for CBH proved to be difficult with fusions obtained with splenocytes from mice 4~~~~~~~~~~~~0 inoculated with the commercial cellulase preparation. For this reason, purified cellulases were used to inoculate mice
This clone MAb
II,
for fusions. In addition, nizes
a
I,
cross-reactive MAb which recog-
and CBH I was obtained by EG from the fusion of splenocytes from a mouse immunized with purified EG (Fig. 5). EG I-specific mouse MAb production. A fusion of splenocytes from a mouse inoculated with purified EG I initially produced 31 wells with supernatant fluids positive by an
ELISA.
epitope
Five
shared
of these
clones
were
selected for dilution
cloning, and the rest were subsequently frozen in liquid N2. After cloning twice, only clone El-Pl-6H-11D-11F was found to react positively by the miniblot assay. Further testing of this stable clone demonstrated it to be specific for EG I (Fig. 6a). MAb proCBH HI-specific MAb production. For CBH duction, 5.95 x 108 spleen cells were fused with SP2/0
FIG. 3. Western blot illustrating specificity of a MAb to EG I and CBH I. Lane 1, EG I; lane 2, EG II; lane 3, CBH I; lane 4, CBH II.
wells from eight plates positive by indirect ELISA. Thirteen clones were tested against purified CBH using the miniblotter. Seven hybridomas demonstrated reactivity to After dilution cloning, two different hybridomas CBH
myeloma cells.
were
Two weeks later,
observed to be
II,
showed
specificity to CBH
CII-P5-9G-12B-8G of
clone EG
and
CII-P5-9G-12B-8G
I1-specific
mouse
These
clones
were
CII-P5-12F-10E-11C. MAb
is demonstrated
The in
designated specificity
Fig.
6b.
production. After three fusions
splenocytes from mice immunized with purified EG II, 15 clones from the third fusion were found to be ELISA
of
FIG. 5. Evidence showing that a MAb obtained from splenocytes of a mouse inoculated with purified EG II recognized EG II and CBH I exclusively.
6b _
a
amW _ := _
_
am: i3_ d
Mi
0
0
fts4 ,
i
6
400
43,000
0
43,000
FIG. 6. Western blots illustrating the specificity of MAbs for EG I, CBH II, and EG II. Each of the clones was obtained by using the respective purified enzyme as antigen. (a) Recognition of EG I by clone EI-P1-6H-11D-11F is demonstrated by this Western blot. (b and c) From each SDS-PAGE, it was possible to obtain two nitrocellulose sheets. A MAb which recognized all four enzymes was used as a positive control (left blot), and MAb specificity is illustrated on the right blot. (b) Recognition of CBH II by clone CII-P5-9G-12B-8G. (c) Recognition of EG II by clone EII-P3-2A-11B-8E. 1106
MAbs AGAINST T. REESEI CBH I AND II AND EG I AND II
VOL. 56, 1990
positive. When these were tested by the miniblot, only five recognized EG II. These five clones were subsequently frozen and cloned twice. Conventional Western blots demonstrated clone EII-P3-2A-11B-8E to be specific against EG II (Fig. 6c). DISCUSSION
The initial purpose of our studies was to produce individual cellulase-specific MAbs against the four major cellulases of T. reesei by utilizing a commercial cellulase preparation. When this commercial cellulase was used for inoculating mice, the MAbs produced mainly recognized shared epitopes among the individual cellulases. This is not unexpected, since it has been shown that small conserved amino acid regions on all four cellulases are present (15, 19, 24). At this time, we can only speculate whether these regions are responsible for cellulase degradation or binding. A crossreactive MAb which recognizes a shared epitope which participates in the hydrolysis of cellulose could be utilized to study these enzymatic reactions. It is believed that T. reesei cellulases pertain to a related gene family due to their highly conserved regions (24). From our studies it is apparent that epitopes exist which are not shared by these enzymes. Whether these regions are functional or whether they are structural epitopes is not known. It is possible that divergence of these cellulases from the ancestral gene has led to the elimination of unproductive genes in some of the enzymes while in others they are retained. Since cellulase protein in culture broth is mainly composed of CBH I (12), it was possible to obtain a MAb specific for CBH I from mice immunized with the commercial cellulase. Production of MAbs to the other three enzymes proved to be more difficult, possibly because these are present in lesser amounts in the cellulase preparation. Even though some lymphocytes may have been stimulated by the presence of these proteins, the probability of isolating such a cell from the vast array of stimulated lymphocytes is minimal. For this reason it was necessary to obtain specific MAbs for CBH II, EG I, and EG II by inoculating mice with purified enzymes. This procedure proved to be more successful as specific MAbs were produced. We do not know to which particular epitopes these MAbs are directed, but further studies should clarify this. The use of such MAbs is varied and extensive. Riske et al. have used a specific MAb to purify CBH I by affinity chromatography (18). Similarly, we have recently reported the purification of EG I from a diafiltered crude culture broth of T. reesei (R. A. Nieves, M. E. Himmel, and R. P. Ellis, Appl. Biochem. Biotechnol., in press). By utilizing these antibodies, purification of the other individual cellulases can also be achieved by simplified procedures as opposed to conventional purification techniques. Also, conventional purification methods can be greatly simplified by using a specific MAb to assay purification steps (i.e., chromatography fractions), replacing the tedious cellulase assays. The cloning and expression of T. reesei cellulase genes into eucaryotes and procaryotic microorganisms have been reported recently (21, 24, 25). Since one of the major setbacks in the utilization of cellulases as a potential producer of utilizable substrates is the cost of enzyme production (9), it is foreseeable that recombinant techniques and structurefunction studies of these cellulases will be further emphasized. For such applications, labeled MAbs directed against these enzymes could be used as probes for detecting transformed cells expressing cellulase gene products.
1107
ACKNOWLEDGMENTS We thank Sharon Shoemaker for the purified T. reesei cellulase enzymes. This work was funded through the Biofuels Program Office at the Solar Energy Research Institute by the Biochemical Conversion Program at the Department of Energy Biofuels and Municipal Waste Technology Division. LITERATURE CITED
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of endo-1,4-beta-D-glucanases purified from Trichoderma viride. Biochim. Biophys. Acta 523:147-161. 24. Teeri, T. T., P. Lehtovaara, S. Kauppinen, I. Salovuori, and J. Knowles. 1987. Homologous domains in Trichoderma reesei cellulolytic enzymes: gene sequence and expression of cellobiohydrolase II. Gene 51:43-52. 25. Van Arsdell, J. N., S. Kwok, V. L. Schweickart, M. B. Ladner, D. H. Gelfand, and M. A. Innis. 1987. Cloning, characterization, and expression in Saccharomyces cerevisiae of endoglucanase I from Trichoderma reesei. Bio/Technology 5:60-64. 26. Woodward, J., M. K. Hayes, and N. E. Lee. 1988. Hydrolysis of cellulose by saturating and non-saturating concentrations of cellulase: implications for synergism. Bio/Technology 6:301-304.
