Confirmation of non N-glycan Linked Mannose Glycosylation in Chitinase 42 kDa Secreted by Trichoderma harzianum BIO10671
T. Nor Farizan,
Chitinase 42 kDa produced by Trichoderma harzianum
has been proven as a prime compound to be excreted onto the hyphae of
the pathogen causing localised cell wall lysis at the point of interaction.
This finally initiate the process of the host cell becomes empty of cytoplasm,
disintegrates and shows a rapid collapse. This study investigates the
existence of N-glycan linked mannose in chitinase 42 kDa produced by the
Malaysian T. harzianum strain BIO10671. The chitinase 42 kDa from
T. harzianum BIO10671 was initially purified using anion exchange
chromatography prior to a series of experiments such as immunoblotting
against the chitinase 42 kDa antibody, lectin staining for detecting any
terminal linked mannose and galactofuranose detection to determine the
presence of galatofuranose components in glycoproteins. The enzyme purification
harvested about 12-fold of chitinase 42 kDa from T. harzianum BIO10671
with strong indication of the presence chitinase 42 kDa presence on SDS-Page.
This was confirmed by immunoblotting with a strong response around 42
kDa after overnight incubation in chitinase 42 kDa antibody suggesting
that the gene for chitinase 42 kDa was greatly expressed in this strain.
There are no intervation of galatofuranose on any of the terminal mannose
in chitinase 42 kDa as shown by negative results on samples treated with
or without endoglycosidase-H and lectin staining. Therefore, it can be
concluded that glycosylation occurred in the chitinase 42 kDa from T.
harzianum 42 kDa was not in the form of N-glycan linked mannose as
The chitinolytic system of T. harzianum is complex with several
types of chitinase working together simultaneously to fulfil all the niche
of Trichoderma sp. One of the components in the chitinolytic system
is the chitinase 42 kDa which has a molecular weight of around 42 kDa.
Filamentous fungi such as T. harzianum are able to secrete high
levels of protein into the culture medium and this advantage has been
used widely in biotechnology industry to produce heterologous proteins
(Archer and Peberdy, 1997). Chitinase 42 kDa by itself is capable of degrading
fungal cell walls and its antifungal activity can be enhanced by adding
chitinase 33 kDa or combined with gliotoxin (Carsolio et al., 1994).
Chitinase 42 kDa from different strains display different physical characteristics
such as pI values and substrate specificity (Gakul et al., 2000).
Due to its high potential as biocontrol and biotechnology compounds,
we are interested to study one of the interesting characteristics of chitinase
42 kDa such as glycosylation, post-translational modification of the protein
to strengthen the protein conformation, stability and biological activity
in order to produce a high-end protein. One of the problems in producing
recombinant glycoprotein is the correct intracellular environment at the
moment of glycosylation. Combination of correct intracellular environment,
genetic combination, mannosyltransferase activity and the substrate
specificity have an effect on the formation on N-linked or O-linked
glycans as shown in of the Saccharomyces cerevisiae (Moo et
al., 2006) and both types of glycosylation can effect the function
of the final recombinant protein produced (Yan et al., 1999). However,
the glycosylation process is required before glycoprotein production can
truly benefit the industry.
Glycosylation is the process of saccharides addition to proteins and
lipids using two types of linkage which are N-linked and O-linked glycosylations.
For N-linked oligosaccharides, a 14-sugar precursor contains three glucose,
nine mannose and two N-acetylglucosamine molecules is first added to the
asparagine in the polypeptide chain of the target protein. Then a complex
set of reactions attaches this branched chain to a carrier molecule called
dolichol bwfore benn translocated into the ER lumen. While O-linked glycosylation
occurs at a later stage during protein processing, probably in the Golgi
apparatus which involve the addition of N-acetyl-galactosamine to serine
or threonine residues by the enzyme UDP-N-acetyl-D-galactosamine: Polypeptide
N-acetylgalactosaminyltransferase, followed by other carbohydrates such
as galactose and sialic acid (Shoda, 2001). The aim and objectives of
the present study were to obtain the evidence and to confirm the non-N-linked
mannose glycosylation in chitinase 42 kDa produced by a Malaysian T.
harzianum strain BIO10671.
MATERIALS AND METHODS
Trichoderma harzianum strain BIO10671 obtained from personal
collection, Department of Biology, Faculty of Science was used in the
present study. The culture was maintained on Potato Dextrose Agar (PDA)
at 28 °C and subcultured every fortnight.
Extracellular Chitinase Purification
The spore suspensions of T. harzianum BIO10671 in approximately
1x107 spores mL-1 were added to 25 mL of Trichoderma
Complete Medium (pH 5.5; 0.5% w/v glucose) to produce seed cultures. Seed
cultures were shaken at 180 rpm at 28 °C for 24 h before being filtered
through a sterile Whatman No. 1 paper. Then were washed three times with
sterile distilled water and transferred into 25 mL of Trichoderma Minimal
Medium (pH 5.5; 1.0% w/v Pleurotus sajor-caju mycelium). Culture
filtrates were harvested, filtered through a Whatman No. 1 filter paper,
centrifuged at 6000 x g for 10 min before being dialysed against the distilled
water for at least 24 h at 4 °C.
The purification was carried out using anion-exchange method by Lima
et al. (1997). Crude culture containing extracellular chitinase was
precipitated with ice-cold 80% acetone and incubated at -20 °C for
30 min. The precipitate was recovered by centrifugation at 28000 x g for
10 min at 4 °C, re-dissolved in distilled water and dialysed against
distilled water for another 24 h at 4 °C.
Ninety microliter of buffer A (50 mM Tris-HCL pH 7.5) and 20 μL
1 M Tris pH 7.5 were added to the dialysed crude culture sample and the
pH was adjusted to pH 7.0. The sample was centrifuged at 12000 x g for
10 min. Meanwhile, an anion exchange Neobar AQ column was washed with
10 column volumes of Buffer B (50 mM Tris-HCL pH 7.5; 1 M NaCl) followed
by 10 column volumes of Buffer A. The resulting supernatant was loaded
onto the column and eluted at a flow rate of 1 mL min-1 and
the bound protein was eluted with a 0-1 mM NaCl gradient. The fractions
with high β-1,3-glucanase activity were pooled before the dialysis
against distilled water for 24 h at 4 °C.
Dialysed fraction was collected and assayed for chitinase activity using
the chitinase assay method (Reissig et al., 1955) which involved
the estimation of N-acetlyglucosamine (GlcNAc) released from chito-oligosaccharides
by the β-N-acetylhexosaminidase. Discontinuous sodium dodecyl sulphate-polyacrylamide
gel electrophoresis (SDS-PAGE) was carried out on collected fraction in
10% acrylamide gels and stained with Coomasie R-250 brilliant blue (Sigma)
according to the method of Laemmli (1970). Low molecular mass standard
proteins were used for molecular mass determination.
Determination of N-Linked Glycosylation
Lyophilised sample of purified chitinase 42 kDa was resuspended in
90 μL of nanopure water followed by adding 40 μL 4xbuffer (100
mM sodium acetate phosphorus, pH 5.5). The sample was heated for 10 min
at 100 °C to denature the sample and any contaminating proteases.
The sample was then divided into two portions; (i) a control sample i.e.
without endoglycosidase-H enzyme (Oxford GlycoSystems) and (ii) test sample
with 2 μ unit of endoglycosidase-H enzyme added. Finally, 5 μL
of toluene was added to prevent any fungal or bacteria growth during incubation
at 37 °C overnight. Four types of assay, namely immunoblotting, lactin
staining, galactofuranose detection and general glycan detection were
carried out using both of the prepared samples.
Treated purified chitinases was tested against chitinase 42 kDA antibody
(CHIT42), kindly supplied by Benitez, University of Seville, Spain. Fifty
microliter lyophilised CHIT42 antibody was diluted 1:2000 in 0.1% (v/v)
TBS buffer. This antibody was raised in rabbits against the CHIT42 from
T. longibrachiatum. Immunoblotting was carried out according to
Wallis et al. (1999).
Lectin Staining: Detection of Terminal Linked Mannose
The detection of terminal linked mannose in purified chitinase 42
kDA from T. harzianum BIO10671 was done by staining the membrane
blots of the chitinase pre and post-endoglycosidase-H enzyme treatments
with Galanthus nivalis agglutinin (GNA) lectin (Boehringer Mannheim)
according to the manufacturer`s recommendation.
An investigation for the presence of galatofuranose components of
glycoproteins was also carried out by blotting against EBA2 antibody.
The EBA2 is the antibody raised against the immunogenic polysaccharide
from Aspergillus fumigatus, which recognises glycoproteins containing
β-linked galactofuranose residues. The membrane was incubated overnight
in EBA2 antibody (2.5 μL in 5 mL 0.1% (v/v) TBS-Tween) at 4 °C
(Wallis et al., 1999).
RESULTS AND DISCUSSION
Although chitinase-producing microorganisms are considered an effective
biological control, the role of chitinase in the antagonistic process,
literatures concerning the purification, molecular properties and molecular
structure of extracellular chitinase from mycoparasitic fungi are still
limited (Gakul et al., 2000; Nampoothiri et al., 2004).
The crude enzyme from T. harzianum BIO10671 contained 26.22 μmoles
μL-1 of chitinase activity was used at the beginning of
the anion exchange. The presumed mixture of chitinase was purified from
T. harzianum using acetone precipitation. The elution pattern of
anion exchange chromatography of this crude enzyme fraction is shown in
Fig. 1 with two peaks for chitinase activities in T.
harzianum BIO10671 arbitrarily BIO (C1) and BIO (C2) for fraction
4-6 and 24-39, respectively. Only 75.6% (21.33 μmoles μL-1)
of chitinase activity was recovered after the anion exchange, which consist
of 0.18 μmoles μL-1 in BIO (C1) and 21.15 μmoles
μL-1 in BIO
||Purification of chitinase by anion exchange chromatography.
Bound protein was eluted with a 0 to 0.5M NaCl gradient. Elution profile
of T. harzianum BIO10671 for protein on Neobar AQ exchanger
column with peaks at fraction 4-6 (BIO C1) and fraction 24-39 (BIO
||Purification of chitinase by anion exchange chromatography.
Bound protein was eluted with a 0 to 0.5 M NaCl gradient. SDS-PAGE
(10%) of protein from pooled peaks and stained with Coomassie blue.
Lane 1: BIO C1 (fraction 4-6); Lane 2: BIO C2 (fraction 24-39); Lane
3: Low range standard molecular weight marker
(C2). Since the BIO (C1) contained only a small percentage of chitinase
activity; it was not used for further analysis. The analysis of both pooled
fractions by SDS-PAGE (Fig. 2) showed several major
proteins bands in BIO (C1) while only one band was observed in BIO (C2)
at approximately 42 kDa.
Three chitinases purified by De La Cruz et al. (1992) showed
molecular masses of 42, 37 and 33 kDa. Recently, several other chitinases
have been purified and tested for their antifungal
||Immunoblot of BIO C2 (fraction 2-39) incubated in chitinase
42 kDa antibody (CHIT 42) and detected using secondary anti-rabbit
antibody for 1 h (1:1000 in 10 mL TBS-Tween)
activity (Viterbo et al., 2001; De Las Mercedes et al.,
2001). The antibodies raised against the two higher molecular mass enzymes
reacted specifically and did not cross react suggesting that each protein
is encoded by a different gene. The protein size revealed by SDS-PAGE
for T. harzianum BIO10671 was 42 kDA which was larger than the
purified chitinase collected by Deane et al. (1998) from T.
harzianum T198, but similar to the range of endochitinases reported
previously by Haran et al. (1995) and Matsumiya et al. (2001).
The molecular weight of purified chitinase may differ between species
and also within species (Pitson et al., 1993) and it is not known
whether they are differently processed gene products from the same gene
or from separate genes. Sometimes the type of growth substrate used can
also influence the number of bands and molecular weight on SDS-PAGE (Vazquez-Garciduenas
et al., 1998). Matsuzawa et al. (1996) concluded that although
the same type of chitinase was purified, characterisation of the purified
form will be different from each other and it depends on the species,
the type of reaction (exo-or endo-) and the method of purification.
Since Trichoderma chitinases are extracellular enzymes, they are
expected to be glycosylated. In order to determine whether or not the
purified chitinase from T. harzianum BIO10671 was N-linked glycosylated,
a series of tests were carried out including immunoblotting with chitinase
42 kDa antibody, lactin staining, galactofuranose detection and general
glycan detection. Immunoblotting process by incubating the membrane with
CHIT42 antibody with the pooled fraction of BIO (C2) revealed four bands
within the range of 31to 45 kDa molecular weight (Fig. 2).
A strong response around 42 kDa after overnight incubation in CHIT42 antibody
suggested that there was a high level of chitinase 42 kDa activity in
this strain. In other words, this result indicated that the gene expression
for chitinase 42 kDa was greater than the other types of chitinase and
similar finding was also reported by Peréz-Martínez et
al. (2007) and Carsolio et al. (1994). The studies by Ike et
al. (2006) proved the ability of chitinase 42 kDa to be expressed
its activity greater than other enzyme in Escherichia coli but
still influence by substrate specificity.
Another positive reaction was also seen on the membrane from 21 to 97
kDa although only a single chitinase band was detected in SDS-PAGE after
the anion purification (Fig. 3). This would
||Lectin staining membrane for mannose detection using
Galanthus nivalis agglutinin (GNA). Lane 1: BIO C2 (fraction
24-39) incubated with endoglycosidase-H; Lane 2: BIO C2 (fraction
24-39) incubated without endoglycosidase-H
||Galactofuranose detection of BIO C2 (fraction 24-39)
against 2.5 μL EBA2 antibody in 5 mL 1% (v/v) TBS-Tween at 4
indicate the existence of chitinase 42 kDa isoform in this strain. However,
this result is in contrast to the findings described by Lima et al.
(1997) who found only one band in immunoblotting of chitinase 42 kDa
from T. harzianum T6. The differences in the number of chitinase
42 kDa form in this study and previous report might due to the difference
in type of strain, carbon source and culture used in the experiment. This
was explained by Steyaert et al. (2004) which described that regulation
and expression of mycoparasitism`s genes in/among Trichoderma species
is highly variable and might be regulated by a metabolic pathway since
mycoparasitism is an alternative means of carbon assimilation, characterized
by catabolite repression and induction during carbon starvation.
Lectin staining using GNA is an effective method to detect any mannose
in the glycan component since most glycosylated enzymes in fungi contain
mannose (Neethling and Nevalainen, 1995). Prior to this step, purified
chitinases from T. harzianum BIO10671 was incubated in 37 °C
in endoglycosidase-H overnight in order to remove any N-linked oligosaccharides
from the polypeptide so that mannose branches can be easily detected by
GNA. There was no positive response for sample treated with or without
endoglycosidase-H as shown in Fig. 4, This can be seen
as an evidence of the non existence of N-glycan linked in chitinase 42
kDA as expected. The test also revealed that no lectin stained was aroused
at chitinase 42 kDa`s position. This showed no mannose branches formation
in this glycoprotein structure.
The negative of response to endoglycosidase-H might imply a possibility
that the terminal mannose was capped by galatofuranose as these residues
were not detected by GNA. To investigate this possibility, a galactofuranose
binding antibody was employed. However no galatofuranose could be detected
in the chitinase 42 kDa area (Fig. 5), thus indicating
that galatofuranose was not present in this glycoprotein structure to
conceal mannose branches.
In conclusion, all the results obtained in this experiment through three
experiments suggested that chitinase 42 kDa did not exist in the form
of N-glycan linked mannose glycosylation.
The authors are indebted to Universiti Putra Malaysia (UPM) for their
technical assistance and Ministry of Science, Technology and Innovation
for the research funding under EA project (09-02-04-0667-EA001) for IRPA
1: Archer, D.B. and J.F. Peberdy, 1997. The molecular biology of secreted enzyme production by fungi. Crit. Rev. Biotechnol., 17: 273-306.
Direct Link |
2: Carsolio, C., A. Gutierrez, B. Jimenez, M. Van Montagu and A. Herrera-Estrella, 1994. Characterization of ech42, a Trichoderma harzianum endochitinase gene expressed during mycoparasitism. Microbiology, 91: 10903-10907.
3: De La Cruz, J., A. Hidalgo-Gallego, J.M. Lora, T. Benitez, J.A. Pintor-Taro and A. Llobell, 1992. Isolation and characterization of three chitinase in Trichoderma harzianum. Eur. J. Biochem., 206: 859-867.
4: De Las Mercedes, D.M., M.C. Limon, R. Mejias, R.L. Mach, T. Benitez, J.A. Pinto-Toro and C.P. Kubicek, 2001. Regulation of chitinase 33 (chit33) gene expression in Trichoderma harzianum. Curr. Gen., 6: 335-342.
5: Deane, E.E., J.M. Whipps, J.M. Lynch and J.F. Peberdy, 1998. The purification and characterization of a Trichoderma harzianum exochitinase. Biochim. Biophys. Acta, 1383: 101-110.
6: Gakul, B., J.H. Lee, K.B. Song, S.K. Rhee, C. Kim and T. Panda, 2000. Characterization and applications of chitinase from Trichoderma harzianum-A review. Bioprocess Biosyst. Eng., 23: 691-694.
7: Haran, S., H. Schickler, A. Oppenheim and I. Chet, 1995. New components of the chitinolytic system of Trichoderma harzianum. Mycol. Res., 99: 441-446.
CrossRef | Direct Link |
8: Ike, M., K. Nagamatsu, A. Shioya, M. Nogawa, W. Ogasawara, H. Okada and Y. Morikawa, 2006. Purification, characterization and gene cloning of 46 kDa chitinase (Chi46) from Trichoderma reesei PC-3-7 and its expression in Escherichia coli. Applied Microbiol. Biotechnol., 71: 294-303.
9: Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685.
CrossRef | Direct Link |
10: Lima, L.H.C., C.J. Ulhoa, A.P. Fernandes and C.R. Felix, 1997. Purification of a chitinase from Trichoderma sp. and its action on Sclerotium rofsii and Rhzioctonia solani cell walls. J. Gen. Applied Microbiol., 43: 31-37.
11: Matsumiya, M., K. Miyauchi and A. Mochizuki, 2001. Characterization of 38 and 42 kDa chitinase isozymes from the liver of Japanese common squid Todarodes pacificus. Fish. Sci., 68: 603-609.
12: Matsuzawa, T., Y. Amano, M. Kubo and T. Kanda, 1996. Mode of action of exo-β-1, 3-glucanase from Trichoderma pseudokoningii TM37 on various β-D-glucans. J. Soc. Ferment. Bioeng., 74: 261-267.
Direct Link |
13: Moo, W.K., J.K. Eun, J.Y. Kim, J.S. Park and D.B. Oh et al., 2006. Functional characterization of the Hansenula polymorpha HOC1, OCH1 and OCR1 genes as members of the yeast OCH1 mannosyltrans ferase family involved in protein glycosylation. J. Biol. Chem., 281: 6261-6272.
Direct Link |
14: Nampoothiri, K.M., T.V. Baiju, C. Sandhya, A. Sabu, G. Szakacs and A. Pandey, 2004. Process optimization for antifungal chitinase production by Trichoderma harzianum. Process Biochem., 39: 1583-1590.
15: Neethling, D. and H. Nevalainen, 1995. Mycoparasitic species of Trichoderma harzianum produce lectins. Can. J. Microbiol., 42: 141-146.
16: Peréz-Martínez, A.S., A. De León-Rodr•uez, L.J. Harris, A. Herrera-Estrella and A.P. Barba de la Rosa, 2007. Overexpression, purification and characterization of the Trichoderma atroviride endochitinase, Ech42, in Pichia pastoris. Protein Exp. Purif., 55: 183-188.
17: Pitson, S.M., R.J. Sevior and B.M. Dougal, 1993. Non-celluloytic fungal β-glucanase: their physiology and regulation. Enzyme Microbiol. Technol., 15: 178-192.
Direct Link |
18: Reissig, J.L., J.L. Strominger and L.P. Lelor, 1955. A modified calorimetric method for the estimation of N-acetylamino sugar. J. Biol. Chem., 217: 959-966.
19: Shoda, S., 2001. Enzymatic Glycosylation. In: Glycoscience Chemistry and Chemical Biology, Fraser, B., Reid, K. Tatsuta and J. Thiem (Eds.). Springer, Berlin, pp: 1465-1496
20: Steyaert, J.M., A. Stewart, M.V. Jaspers, M. Carpenter and H.J. Ridgway, 2004. Co-expression of two genes, a chitinase (chit42) and proteinase (prb1), implicated in mycoparasitism by Trichoderma hamatum. Mycologia, 96: 1245-1252.
Direct Link |
21: Vazquez-Garciduenas, A., C.A.L. Morales and A.H. Estrella, 1998. Analysis of the β-1, 3-glucanolytic system of the biocontrol agent Trichoderma harzianum. Applied Environ. Microbiol., 64: 1442-1446.
PubMed | Direct Link |
22: Viterbo, A., S. Haran, D. Friesem, O. Ramot and I. Chet, 2001. Antifungal activity of a novel endochitinase gene (chit36) from Trichoderma harzianum Rifai TM. FEMS Microbiol. Lett., 200: 169-174.
CrossRef | PubMed |
23: Wallis, G.L.F., R.J. Swift, F.W. Hemming, A.P. Trinci and J.F. Peberdy, 1999. Glucoamylase overexpression and secretion in Aspergillus niger: Analysis of glycosyation. Biochim. Biophys. Acta, 1472: 576-586.
CrossRef | PubMed |
24: Yan, B., W. Zhang, J Ding and P. Goa, 1999. Sequence pattern for the occurance of N-glycosylation in protein. J. Protein Chem., 15: 511-521.