Carbohydrate-binding modules (CBM) are protein domains that recognize and bind to oligo- and polysaccharide ligands. Within the Carbohydrate-Active Enzyme Database (CAZy; CAZyme database,
), CBMs are divided into 64 families, based on amino acid sequence similarity, and members of each family display common ligand specificity. Regarding reported specificities, characterized CBMs have been found to bind to crystalline or non-crystalline cellulose, chitin, β-1,3-glucans, β-1,3-1,4-mixed linkage glucans, xylan, mannan, galactan or starch, while others behave like lectins, binding to a variety of cell-surface glycans
As protein domains, one or more CBMs are generally associated with other protein domains, typically glycosyl hydrolase (GH) modules, and can be localized at either the N or C-termini of proteins, although proteins formed exclusively of single CBMs have been described
[3, 4]. Many CBMs have been biochemically characterized and structural data are now available providing insight into structure/function relations (for review see
). Since it is known that many CBMs are able to bind to polysaccharides, it is thought that when these are attached to catalytic domains their presence ensures intimate contact with the substrate and thus potentiated catalysis
[5–7]. Moreover, it has been postulated that some CBMs might possess the ability to locally destruct polysaccharide structure (e.g. lower crystallinity in cellulose), thus improving enzyme accessibility
Family 1 CBMs are small modules composed of 32 to 36 amino acids and are known as a “fungal CBM family”, because they were first detected in fungal cellulases and are exclusively produced by eukaryotes. The first characterized CBM1 was the cellobiohydrolase I from Trichoderma reesei. Since then, numerous CBM1 proteins from various fungi have been reported
[10–12]. The role of CBM1 in cellulases has been studied by separating the catalytic domain from its CBM, thus facilitating the study of the activity of the isolated catalytic domain on one hand and, on the other hand, the binding ability of the CBM. Data acquired in this way has indicated that CBM1 binds strongly to crystalline cellulose and that its presence is required for full activity of the enzyme against the insoluble polysaccharide
[6, 13, 14]. A structural study of CBM1 from T. reesei cellobiohydrolase I have shown that overall architecture forms a wedge shape that is formed by irregular antiparallel triple-stranded β-sheet, which is stabilized by 2 disulfide bridges
. A flat face of the wedge bears three aligned aromatic residues (Y5, Y31 and Y32) that, along with polar residues (Q7 and N29), appear to form the cellulose binding interface
[16, 17]. This is corroborated by the fact that the removal of any of these residues reduces the ability of the enzyme to degrade crystalline cellulose
. Nevertheless, the role of CBM1 at the molecular level is not fully characterized.
Interestingly, CBM1-containing proteins have also been identified in fungal-like organisms called oomycetes
. Like fungi, oomycetes are ubiquitous in marine, freshwater and terrestrial environments
. They have similar modes of nutrition and ecological roles to true fungi and form tip-growing branching hyphae. Oomycetes were initially classified within the kingdom of Fungi, but molecular phylogenetic studies have now firmly established the distinct taxonomic positions of true fungi and oomycetes. Oomycetes belong to the Kingdom Stramenipila, which includes diatoms, chromophyte algae, and other heterokont protists
[20–22]. Numerous oomycete species are plant pathogens, such as the causal agent of potato blight Phytophthora infestans or the sudden oak death pathogen Phytophthora ramorum. Features characterizing oomycetes are usually based on biochemical studies focused on Phytophthora sp. and particularly, the presence of cellulose rather than chitin in their cell wall. However the presence of either chitin or chitosaccharides was observed in the Saprolegniale oomycetes Saprolegnia monoica and Aphanomyces euteiches, where these compounds play an essential structural role
The first oomycetal CBM1- protein described is the cellulose-binding elicitor lectin (CBEL) from Phytophthora parasitica, the causal agent of tobacco black shank disease
. This non-enzymatic cell wall glycoprotein harbors two CBM1s associated to two PAN/Apple modules known to interact with polysaccharides or proteins
[26, 27]. Knockdown P. parasitica-CBEL transformants are affected in cell wall polysaccharide deposition and adhesion to cellulosic substrates, including plant cell walls
. In addition to structural and adhesive roles, CBEL also induces plant defense responses, such as the production of reactive oxygen species, cytosolic calcium variation, expression of PR-proteins, and necrosis in several plant species
[28, 29]. The mutation of a recombinant CBEL has revealed that functional CBM1 is required for the full elicitor activity of CBEL
. Moreover, it has been suggested that interaction of CBM1 module with the plant cellulose microfibrils is perceived by plant cells as a warning signal
[30, 31]. Similar results have been obtained with a fungal CBM1 from T. reesei suggesting that plants are able to perceive oomycetal as well as well fungal CBM1s
The discovery of CBM1-containing proteins in oomycetes has raised the question of their origin in a lineage distantly related to fungi. It has been recently suggested that some genes encoding proteins involved in the breakdown of plant cell wall components have been acquired by oomycetes from fungi through horizontal gene transfer
. However, CBM1-containing proteins were not detected in this analysis.
To better understand the origin, evolution and biological role of CBM1-containing proteins in oomycetes, we have performed data mining of fungal and oomycetal genomes and compared the protein organizations of different CBM1-containing proteins. In this way, we have revealed that oomycete-unique association of CBM1 with PAN/Apple domains.
Moreover, using CBEL from P. parasitica, which was shown to be a bona fide cellulose-binding protein, we propose a model structure of an oomycetal CBM1 and a role for the PAN/Apple domain in binding of the protein to additional carbohydrates. Accordingly, we present experimental evidence for a galactose or N-acetylgalactosamine-specific lectin activity associated with CBEL. Taken together, the results suggest that oomycetal CBM1-containing proteins have an ancient origin in the oomycete lineage and are involved in specific roles including adhesion to self and non-self components rather than in substrate degradation.