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- Materials and methods
A β-1,4-endoglucanase named MI-ENG1, homologous to the family 5 glycoside hydrolases, was previously isolated from the plant parasitic root-knot nematode Meloidogyne incognita. We describe here the detection of the enzyme in the nematode homogenate and secretion and its complete biochemical characterization. This study is the first comparison of the enzymatic properties of an animal glycoside hydrolase with plant and microbial enzymes. MI-ENG1 shares many enzymatic properties with known endoglucanases from plants, free-living or rumen-associated microorganisms and phytopathogens. In spite of the presence of a cellulose-binding domain at the C-terminus, the ability of MI-ENG1 to bind cellulose could not be demonstrated, whatever the experimental conditions used. The biochemical characterization of the enzyme is a first step towards the understanding of the molecular events taking place during the plant–nematode interaction.
Natural plant cellulose is a polysaccharide composed of β-d-glucopyranosyl units joined by 1,4-glycosidic bonds. It is a carbon and energy source for a large number of cellulolytic free-living and rumen symbiotic microorganisms, which have developed a large panel of plant cell-wall degrading enzymes [1–3]. Moreover, the plant cell wall is a major mechanical barrier to the propagation and development of plant pathogens, many of which also produce cellulolytic and pectinolytic enzymes able to disrupt cell-wall polymers. The root-knot nematode, genus Meloidogyne, is an endoparasite that has evolved very tight interactions with its plant host. The success of parasitism depends for a large part on the capacity of the infective larva to penetrate the root tip and to migrate intercellularly towards the vascular cylinder.
Cellulolytic enzymes from microorganisms have been studied extensively in terms of structure and enzymatic properties. Cellulases catalyse the hydrolysis of β-1,4-glycosidic bonds. They are divided into endoglucanases that cleave the glucan chains at interior sites and cellobiohydrolases that release cellobiose from the chain ends. Microorganisms have developed two main strategies to degrade cellulose. Aerobic fungi and bacteria secrete batteries of individual but synergistically acting enzymes, while anaerobic microorganisms associated with the rumen of chewing animals utilize highly structured multienzyme complexes (cellulosomes) operating at the cell surface. Most cellulases secreted by aerobic organisms have a characteristic modular structure composed of a catalytic domain linked to a functionally independant cellulose-binding domain (CBD) by a flexible linker peptide . In plants, most cellulases lack the ancillary CBD domain. Cellulase catalytic domains have been classified according to sequence-based homologies into 12 of the 77 glycoside hydrolase families  (http://afmb.cnrs-mrs.fr/~pedro/CAZY/ghf.html). Similarly, CBDs have been classified into 13 families according to similarities in primary structure .
Only a few cellulases have been purified from animals. Cellulase activities have been detected in molluscs [7,8] and snails , but whether cellulase is produced by the animal itself or an associated microorganism is difficult to establish until isolation of the corresponding gene. Endogenous production of cellulase by animals has only recently been demonstrated for plant parasitic nematodes [10,11] and termites . In termites, two family 9 β-1,4-endoglucanase cDNAs have been cloned consisting of a single catalytic domain . Concerning cyst nematodes, two categories of family 5 β-1,4-endoglucanase genes have been cloned in each of the two genera analysed, one with and one without CBD . The first glycoside hydrolase gene, named MI-ENG1, isolated from the root-knot nematode Meloidogyne spp. encodes a β-1,4-endoglucanase composed of a catalytic domain and a CBD joined by a linker rich in proline and hydroxyamino-acid residues . The MI-ENG1 catalytic domain belongs to the family 5 of glycoside hydrolases, which is the largest cellulase family. The MI-ENG1 CBD belongs to family II, which so far has been composed of bacterial hydrolase CBDs.
Here we present the biochemical characterization of MI-ENG1. The enzymatic properties of MI-ENG1 are described in terms of physico-chemical properties, mode of action, substrate specificity and cellulose binding ability. This work presents the first complete biochemical characterization of an animal family 5 glycoside hydrolase, and allows the comparison of its enzymatic properties with those of enzymes from microbe and plant origin. The role of this enzyme in nematode parasitism is discussed.
- Top of page
- Materials and methods
Cellulases have been extensively studied in the case of cellulolytic microorganisms from the soil and rumen of animals. However, the enzymatic properties of plant or phytopathogen cell-wall degrading enzymes have been poorly analysed. This study is the first extended biochemical characterization of a family 5 endoglucanase produced by a phytoparasitic animal. The biochemical characteristics of MI-ENG1 were analysed in order to have a better understanding of its role in parasitism. Moreover, this study allows the comparison of an animal cellulase with microbial cellulases from the same glycoside hydrolase family.
MI-ENG1 shares many enzymatic properties with known endoglucanases from plants, free-living microorganisms and phytopathogens. By analysing the products of enzymatic reaction and by measuring the liberated reducing sugars, we have demonstrated that MI-ENG1 is an endoglucanase devoid of any exoglucanase activity. Furthermore, substrate specificity analysis showed that MI-ENG1 cleaves β-1,4 linkages but is unable to cleave β-1,3 linkages.
The predicted molecular mass of MI-ENG1 is 53.4 kDa. However, zymogram and Western-blot analysis together suggested that MI-ENG1 is produced as an active endoglucanase with an approximate molecular mass of 62 kDa in total nematode homogenate and stylet secretions. This result suggests that MI-ENG1 undergoes post-translational modifications such as glycosylation when synthesized in the nematode. This hypothesis is supported by the presence of several potential N- and O-glycosylation sites in the deduced protein sequence of MI-ENG1. Glycosylation of family 5 glycoside hydrolases has been demonstrated in several cases [34,35]. In plant pathogens, it is assumed that such a glycosylation would protect hydrolases from proteolysis . During infestation, plant pathogens secrete cellulases that are subjected to degradation by plant proteases. Protection of cellulases from proteolysis could then play a major role in the success of pathogen development in planta.
MI-ENG1 shares a lot of physico-chemical properties with family 5 endoglucanases characterized. Optimal MI-ENG1 activity was observed at pH 5.0, 50 °C. Several cellulases from plants , fungi [38,39] or bacteria [40,41] show optimal hydrolytic activities between pH 4.0 and pH 6.0. Similarly, extracellular endoglucanases secreted by the phytopathogenic enterobacteria Erwinia sp. have an optimal activity at pH 5.0 , or retain 80% of activity at pH 5.0 . These pH conditions for enzymatic activity are in accordance with the pH 5.5 of the plant cell apoplasm. Furthermore, although no thermostabilizing domain could be shown in MI-ENG1, the enzyme is particularly active at high temperatures (up to 60 °C). This is also the case for several bacterial [40,44], fungal  or phytopathogen hydrolases such as the endoglucanase CELB from E. carotovora whose optimal temperature is 50 °C . Optimal temperature and pH conditions revealed that MI-ENG1 is active over a large spectrum of conditions, as it retains 80% of its activity between pH 4.5 and 6.5 and 50% of its activity between 30 and 60 °C. This could be related to the fact that root-knot nematodes are wide-spread in tropical and temperate regions. Therefore, they are subjected to various edaphic and climatic conditions and are able to infect more than two hundred plant species. The ability of MI-ENG1 to be active at diverse pH and temperature conditions could reflect an ability of the enzyme to degrade host plant cellulosic materials in a large variety of environmental conditions.
The inhibitory effect of Zn2+ and Cu2+ metallic cations on MI-ENG1 activity is a common feature of cellulases and xylanases [46–48], including plant cellulases [24,49]. Furthermore, several family 5 glycoside hydrolases, especially from ruminal cellulolytic bacteria, are also inhibited by these cations [47,50]. Enzyme inhibition by metallic cations usually suggests the presence of at least one sulfhydryl group in the active site, usually a cysteine amino acid, whose oxidation by the cations destabilizes the conformational folding of the enzyme , or leads to the formation of disulfide bonds at an irregular position of the protein . Five cysteine residues in the catalytic domain of MI-ENG1 could provide potential metallic cation reactive sites causing enzyme inhibition.
The endoglucanase activity of MI-ENG1 is not subjected to feed-back inhibition by soluble sugars such as glucose or cellobiose, suggesting that enzyme regulation could be provided by other polysaccharides or/and that regulation of the enzyme synthesis may exist. Such a regulation has been observed in most cellulolytic organisms  and in phytopathogenic organisms [27,52], where cellulase gene expression is regulated by soluble carbon sources which are easily metabolized.
The MI-ENG1 endoglucanase is not significantly active against crystalline cellulose, like all the family 5 glycoside hydrolases yet characterized. Up to now, cellulases able to hydrolyse microcrystalline cellulose have been found in families 9 and 48 of glycoside hydrolases from ruminal cellulolytic microorganisms [53–56] and termites . No plant or biotrophic phytopathogen cellulase is yet known to degrade crystalline cellulose. A large number of cellulases are inactive or poorly active on this substrate when acting separately, but an efficient degradation can be observed when glycoside hydrolases act synergistically or when they are associated in multienzymatic complexes . Whereas cellulolytic ruminal microorganisms hydrolyse efficiently the plant biomass to produce energy and a carbon source, it is assumed that root-knot nematodes only need limited hydrolysis of the cell wall in order to separate adjacent cells when migrating intercellularly in the root tissue. Furthermore, one could hypothesize that plant parasitic nematodes do not produce a large hydrolytic multienzyme complex, but rather a more simple and qualitative panel of hydrolytic enzymes [10,59] (M. N. Rosso, L. Arthaud, T. N. Ledger and P. Abad, unpublished results).
One peculiarity of MI-ENG1 as compared to characterized microbial or plant cellulases is its inability to bind to cellulose under the experimental conditions tested, in spite of the presence of a CBD at the C-terminus. Whether the native MI-ENG1 binding features are different from the enzyme expressed in bacteria still has to be determined. Few data are available concerning the binding ability of CBDs isolated and binding in some cases is weak and depends on very specific physico-chemical conditions (J. T. Pembroke, personal communication). However, inability to bind to cellulose has been reported in the case of a family IIIc CBD . The MI-ENG1 CBD belongs to family II CBDs. Comparing the MI-ENG1 CBD protein sequence to family II CBD sequences gave no explanation for the absence of binding ability on crystalline cellulose. Tryptophan residues have been shown to be involved in cellulose binding . A peculiarity of CBDs isolated so far from nematodes is the presence of only two conserved tryptophan residues instead of three or four residues in subfamilies IIb and IIa, respectively ; the ability of such CBDs to bind to cellulose has been demonstrated in the case of the M. incognita cellulose binding protein (MI-CBP1) , and the cyst nematode endoglucanase GR-ENG1 (G. Smant, personal communication). CBDs of nematode origin carrying two tryptophan residues could then constitute a new subfamily inside the family II. Some CBDs have been shown to play an important role in cellulase activity and thermostability [60,61]. More studies such as CBD deletion or directed mutagenesis will be necessary in order to analyse whether the MI-ENG1 CBD has any effect on enzyme activity and whether residue substitutions restore cellulose binding ability.
To conclude, the enzymatic properties of M. incognita MI-ENG1 are in accordance with the environment the nematode faces during plant invasion. MI-ENG1 shares biochemical characteristics with the family 5 glycoside hydrolases characterized so far and with cellulases isolated from pathogens invading plant tissues intercellularly, such as bacteria or biotrophic fungi. The only peculiarity of the enzyme is its inability to bind efficiently to cellulose under the experimental conditions tested, which may reflect a weak binding or an absence of binding in planta. The lack of observed binding to cellulose together with the little effect of MI-ENG1 on crystalline cellulose or natural cell walls, suggest that MI-ENG1 plays a supporting role to other secreted cellulases. The ongoing isolation of new β-1,4-endoglucanase genes from M. incognita indeed suggests that the nematode secretes several cellulases during parasitism, some of them sharing identical molecular masses (M. N. Rosso, L. Arthaud, T. N. Ledger and P. Abad, unpublished results). Similarly, several cellulase genes have recently been cloned from plant-parasitic cyst nematodes . The biochemical and molecular characterization of these new cell-wall degrading enzymes should lead to the understanding of their respective role in parasitism and the molecular events leading to nematode-specific plant tissue alterations.