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MMBL proteins: from lectin to bacteriocin
Maarten G. K. Ghequire1, Remy Loris2,3, and René De Mot1#
1
Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20, 3001
Heverlee, Belgium
2
Molecular Recognition Unit, Department of Structural Biology, Vlaams Instituut voor
Biotechnologie, Pleinlaan 2, 1050 Brussel, Belgium
3
Structural Biology Brussels, Department of Biotechnology (DBIT), Vrije Universiteit
Brussel, Pleinlaan 2, 1050 Brussel, Belgium
#
: author of correspondence, rene.demot@biw.kuleuven.be, Tel. +32 (0) 16 329681, Fax: +32
(0) 16 321963
Running title: MMBL lectins and bacteriocins
Abbreviations used: GNA, Galanthus nivalis agglutinin; MACPF, membrane attack complex
component/perforin ; MMBL, monocot mannose-binding lectin
Keywords: LlpA, antagonism, chimeric lectin, MMBL, bacteriocin, phylogeny
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Abstract
Arguably, bacteriocins deployed in warfare among related bacteria, are among the most
diverse proteinacous compounds with respect to structure and mode of action. Identification
of the first prokaryotic member of the so-called “monocot mannose-binding lectins”
(MMBLs or GNA lectin family) and discovery of its genus-specific killer activity in the
Gram-negative bacteria Pseudomonas and Xanthomonas has added yet another kind of toxin
to this group of allelopathic molecules. This novel feature is reminiscent of the protective
function, based on antifungal, insecticidal, nematicidal or antiviral activity, assigned to or
proposed for several of the eukaryotic MMBL proteins that are ubiquitously distributed
among monocot plants but also occur in some other plants, fishes, sponges, amoebas and
fungi. Direct bactericidal activity can also be effected by a C-type lectin but this is a
mammalian protein that limits mucosal colonization by Gram-positive bacteria. The presence
of two divergent MMBL domains in the novel bacteriocins raises questions about task
distribution between modules and the possible role of carbohydrate binding in specificity of
target strain recognition and killing. Notably, bacteriocin activity was also demonstrated for a
hybrid MMBL protein with an accessory protease-like domain. This association with one or
more additional modules, often with predicted peptide-hydrolyzing or –binding activity,
suggests that additional bacteriotoxic proteins may be found among the diverse chimeric
MMBL proteins encoded in prokaryotic genomes. A phylogenetic survey of the bacterial
MMBL modules reveals a mosaic pattern of strongly diverged sequences, mainly occurring
in soil-dwelling and rhizosphere bacteria, which may reflect a trans-kingdom acquisition of
the ancestral genes.
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MMBL-type lectins: what’s in a name?
Mannose-binding B-lectins have been purified from numerous monocot plants. The hallmark
of these so-called MMBLs (monocot mannose-binding lectins) is the presence of a domain
with three potential carbohydrate-binding pockets, each generated by a QxDxNxVxY motif.
Due to variable degeneracy of this signature sequence, some of these binding sites may not be
active [1-3]. Most plant MMBLs are built from two to four identical or homologous
protomers, though some are monomeric [4,5] or have a tandem domain structure [6]. Crystal
structures of several of them, some with complexed mannose/mannoside oligomers, have
been determined, revealing a common β-prism fold (Figure 1) [3,5-12]. Typically these plant
lectins bind mannose only weakly and display a somewhat higher affinity for
oligomannosides or high-mannose N-glycans [12,13].
It has been proposed that these lectins serve a defensive role, providing protection against
plant predators or phytopathogens [4]. Indeed, some members of this family possess
antifungal activity (e.g. gastrodianin from the orchid Gastrodia elata) and several others
display insect-killing capacity (e.g. ASAL from Allium sativum) or are nematicidal (e.g. RVL
from Remusatia vivipara). These protective potentials have been demonstrated in transgenic
crop plants [14,15]. Notably, by converting the homodimeric insecticidal ASAL into a
monomeric form, antifungal properties were acquired [16]. Additional interest in the plant
MMBLs stems from their therapeutic potential for suppressing enveloped viruses [17] and
triggering apoptosis in cancer cells [18]. Though lacking any detectable lectin activity,
neoculin, a heterodimeric MMBL protein from Molineria (Curculigo) latifolia fruit,
possesses sweet-tasting and taste-modifying properties by interacting with the human taste
receptor T1R2-T1R3 [19,20].
Eukaryotic MMBLs revisited
The ubiquitous occurrence in monocots, lending the original family name, contrasts with a
rare distribution across other plants, as inferred from isolated reports on MMBL-like proteins
found in the liverwort Marchantia polymorpha [21], the dicot Hernandia moerenhoutiana
[22], and the gymnosperm Taxus media [23]. Plant-like MMBLs were also identified in the
fresh-water sponge Lubomirskia baicalensis [24], in the ascomycetous fungus Fusarium
verticillioides and basidiomycete Marasmius oreades [25,26], and in the slime mold
Dictyostelium discoideum (comitin; [27]). A comitin-deficient amoeba mutant appeared more
susceptible to infections by the intracellular bacterial pathogen Legionella [28]. A protective
function has also been proposed for some of the MMBL lectins that were identified in fishes.
Pufflectin-s from skin mucus of Takifugu rubripes, a homodimeric lectin containing one
functional mannose-binding site [29,30], was found to bind the parasitic trematode
Heterobothrium okamotoi, suggesting that it contributes to the parasite-defense system in
fugu [29]. More recently, the homotetrameric lectin plumieribetin was isolated from skin
mucus and fin stings of Scorpaena plumieri. An integrin-inhibiting effect was demonstrated
and thought to contribute to some of the local and systemic effects of envenomation by
scorpionfish [31]. Another MMBL family member was also identified in skin mucus of
Atlantic cod (Gadus morhua) by proteomic analysis [32].
The MMBL domain is also found in several types of multi-domain proteins from both
monocot and dicot plants, in particular S-locus glycoproteins and S-locus receptor kinases,
involved in self-incompatibility [22]. To reflect the wider distribution beyond monocots, an
alternative family name, GNA, referring to the first described characterized member
(Galanthus nivalis agglutinin) has been proposed [22].
Prokaryotic MMBLs: in search of a function
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While not yet detected in Archaea, genes encoding MMBL-like (hypothetical) proteins have
been identified in several bacterial genomes. Different domain architectures can be
distinguished in these prokaryotic proteins: some are built from a single or tandem MMBL
domain only, whereas others carry carboxy- or aminoterminally fused polypeptides with one
or more additional domains. Figure 2 depicts the phylogenetic diversity of MMBL modules
extracted from bacterial proteins with representative architectures, in comparison with those
present in eukaryotic proteins from the major lineages. Separate clusters are apparent for the
plant, fish and fungal lectins. In contrast to these well-delineated eukaryotic clades, the
bacterial MMBL domains are found on several separate small or bigger branches indicating
increased sequence divergence. A highly patchy taxonomic distribution with extensive
sequence divergence, even among strains from the same species, is apparent. However, some
genera of Proteobacteria (Burkholderia, Pseudomonas) and actinomycetes, with larger-thanaverage genome sizes, seem to be relatively enriched in MMBL-containing proteins, which
may reflect acquisition of MMBL-encoding genes by horizontal gene transfer.
Bacterial killer MMBLs
LlpA (‘lectin-like putidacin A’) from banana rhizosphere isolate Pseudomonas putida
BW11M1 was the first bacterial MMBL protein to be characterized. Built from an MMBL
tandem, it was shown to function as a bacteriocin with a genus-specific target spectrum,
inhibiting growth of several phytopathogenic P. syringae strains. LlpA does not require a
cleavable signal sequence for secretion nor an immunity protein, the latter characteristic
being often observed for proteins exhibiting similar bacteriotoxic activities [33].
Subsequently, narrow-spectrum bacteriocin activity was also assigned to two proteins with a
similar tandem MMBL architecture in biocontrol strain P. fluorescens Pf-5, called LlpA1 and
LlpA2, with near identical amino acid sequences and indistinguishable target strain spectrum
[34]. More recently, antibacterial activity of two tandem MMBL bacteriocins from
phytopathogenic Pseudomonas syringae and Xanthomonas citri has been demonstrated. In
the latter case, activity against several xanthomonads was observed, but not against
Pseudomonas and vice versa, confirming lectin-like bacteriocins to represent genus-specific
killer proteins but acting across species borders [35]. We are currently investigating whether
this concept can be extended beyond -Proteobacteria, using the equivalent proteins encoded
by Burkholderia cenocepacia and Burkholderia ambifaria (β-Proteobacteria) as test cases.
The phylogenetic analysis of individual MMBL modules in these bacteriocins reveals a
clustering of N-terminal domains disparate from branches with C-terminal domains (Figure
2). Such independent evolution of MMBL domains within these tandems probably points
towards a yet unresolved dedicated function contributing to their antibacterial activity. A
notable exception to this within-tandem module divergence is found for a predicted protein
from the actinomycete Arthrobacter sp. FB24, currently the sole representative of this type
from a Gram-positive bacterium, for which biological activity also remains to be assessed.
In this context it will also be of interest to functionally characterize those ‘minimal’ bacterial
MMBL family members containing a single MMBL module without apparent additional
domain. Genes encoding such mono-MMBLs are common in fishes, fungi and some monocot
plants, but the bacterial representatives of bacilli (Firmicutes) and pseudomonads are
assigned to two well-resolved branches in the phylotree, separate from the eukaryotic
sequences (Figure 1). In the case of mono-MMBLs from Pseudomonas, they show obvious
sequence relationship with the N-terminal domains from the tandem MMBLs encoded by
other strains of the genus.
Bacterial chimeric MMBLs: toxic proteins as well?
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The MMBL module has been integrated, individually or as a tandem of closely related
domains, in several bacterial multi-domain proteins representing various domain topologies
(Figure 2). However, so far only one such hybrid MMBL protein has been functionally
characterized. Albusin B, secreted by the Firmicutes member Ruminococcus albus 7, consists
of a single aminoterminal MMBL module fused to a putative peptidase domain and inhibits
growth of another ruminal bacterium, Ruminococcus flavefaciens [36]. The contribution of
individual domains to this bacteriocin-like activity has not been further investigated.
From inspection of the various hybrid architectures, the association with domains potentially
conferring hydrolytic activities emerges as a recurrent theme (Figure 2). Sequence-based
clustering of several of the corresponding fused MMBL domains indicates evolutionary
relatedness, despite their occurrence in taxonomically unrelated prokaryotic genera:
associated domains include subtilase (subtilisin-like serine protease) in Burkholderia and
Stigmatella (β- and δ-Proteobacteria, respectively), unspecified hydrolase (GDSL-like
lipase/acylhydrolase) in Granulicella and Terriglobus (Acidobacteria), trypsin-like protease
and NLPC/P60-type cysteine peptidase in Streptomyces and Cellulomonas, respectively (both
Actinobacteria). In the Burkholderia ambifaria and Stigmatella aurantiaca polypeptides, the
presence of a propeptide domain preceding the actual protease domain suggests involvement
of proteolytic processing for biological activity [37,38]. The trypsin-related MMBL proteins
are equipped with an additional carboxyterminal module potentially involved in adhesion (βpropeller domain VCBS, [39]) or sugar-binding (ricin-type β-trefoil lectin).
In several nocardioform actinomycetes (Mycobacterium, Nocardia, Rhodococcus,
Segniliparus, Tsukamurella), a fusion with a carboxyterminal peptidoglycan-binding LysM
domain [40] is prominent. The MMBL module of the corresponding Mycobacterium
smegmatis protein, devoid of the C-terminal domain, has been crystallized, awaiting further
functional characterization [41]. It has been shown that the mammalian peptidoglycan
recognition proteins (PGRPs) upon binding to the bacterial cell wall can trigger lethal
activation of stress-responsive two-component systems [42].
In a Mucilaginibacter paludis strain (Bacteroidetes), two hybrid MMBL proteins are found:
one module is joined to a papain family cysteine protease domain, while a quite similar
module occurs in combination with a MACPF domain. Among prokaryotes, the MACPF
domain is particularly abundant among Chlamydiae and Bacteroidetes. In the latter group,
MACPF also occurs in combination with the carbohydrate-binding module BACON [43].
Originally, the MACPF designation refers to its occurrence in mammalian components of the
complement cascade (MAC), targeting Gram-negative bacteria, and in perforin (PF), killing
virus-infected cells [44]. These protective functions rely on the ability to form large
membrane pores [45]. However, no such lytic activity on eukaryotic cells could be
demonstrated for the MACPF protein of the insect pathogen Photorhabdus luminescens (Proteobacteria) that contains an additional β-prism domain [46]. No pathogenicity has been
attributed to members of the Mucilaginibacter genus that was described recently [47] and
now accommodates several new species isolated from soil and rhizosphere. Possibly, a
MACPF-MMBL hybrid may have evolved to serve as an antagonistic factor in competition
with other (micro)organisms residing in these environments.
Bacteriocins with a novel mode of action?
The identification of MMBL proteins with a role in warfare among closely related bacteria
has assigned another type of defense-related function in addition to the antifungal,
insecticidal, nematicidal and antiviral activities mainly associated with its plant members.
The narrow target spectrum, with allelopathic activity confined within genus borders, is
reminiscent of the killing range of a bacteriocin. Another lectin fold (C-type) has also been
recruited to serve a bactericidal function and enables mammalian RegIII proteins (such as
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mouse RegIIIγ and human HIP/HAP) to bind to the surface-exposed peptidoglycan layer of
Gram-positive bacteria [48] and restrict colonization of the small intestinal mucosal surface
[49].
Antibacterial activity has not yet been reported for a prokaryotic mono-domain MMBL
protein but has been described for tandem-MMBL proteins (demonstrated in the Gramnegative bacteria Pseudomonas and Xanthomonas), as well as for a hybrid architecture with
an adjacent protease domain (as found in the Gram-positive bacterium Ruminococcus). In the
uncharacterized prokaryotic hetero-domain MMBL proteins, different types of peptidehydrolytic modules are frequently found as an accessory domain. These are good candidates
for novel MMBL members with antibacterial function. Characterization of such new
bactericidal members will assist in elucidation of the contribution of individual domains to
the killing process. Conceivably, separate modules may be involved in target recognition and
binding, and in subsequent lethal action. How carbohydrate-binding to a mannose-containing
ligand or N-glycan would be involved in this process is currently unclear. The export route
followed by some the (candidate) bacteriocins is also not known. Some are equipped with a
cleavable N-terminal signal peptide for Type II secretion, whereas others lack an identifiable
export motif. These features seem not to be linked with phylogenetic affiliation.
The MMBL family is also intriguing from an evolutionary viewpoint. These proteins are
particularly abundant in monocot plants but display a mosaic distribution among other
organisms, including bacteria. As the prokaryotic family members are predominantly found
in soil-dwelling and plant-associated bacteria, it may be hypothesized that these proteins
evolved from genes originally acquired from plants. Compared to the eukaryotic proteins, the
bacterial MMBL domains have diverged more extensively, even within some tandemly
organized members, and they cannot be readily traced back to a specific origin.
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Funding
This work is financially supported by the FWO Vlaanderen [Grant G.0393.09N, to R.D.M.
and R.L.], the Onderzoeksraad VUB and VIB.
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Figure 1
Structures of representative plant MMBL proteins
Individual domains or protomers are shown in different colours and the monomer colored
green is always shown in the same orientation. (A) Monomeric single-MMBL domain
gastrodianin from Gastrodia elata (PDB entry 1XD5). (B) Heterodimeric curculin from
Curculigo latifolia lacking mannose-binding capacity (PDB entry 2DPF). The dimer is
formed through a swap of the C-terminal -strands (indicated by the black arrows). (C)
Fetuin-binding tandem-MMBL SCAfet from Scilla campanulata (PDB entry 1DLP).
Although the two domains are covalently attached, the swap of the C-terminal -strands is
retained. The N-terminal domain is in green, the C-terminal domain in cyan. (D) Tandem
MMBL from Allium sativum with bound mannose (PDB entry 1KJ1). The N-terminal domain
is in green, the C-terminal domain in cyan. Mannoses bound to the three QxDxNxVxY motifs
are shown in black. An additional mannose bound outside these motifs and of unclear
biological relevance is shown in red. This lectin is encoded as a tandem consisting of two
very similar MMBL domains, but after synthesis is cleaved to produce an apparent domainswapped heterodimer. (E) Homotetrameric GNA from Galanthus nivalis in complex with the
trimannose Man(1-3)[Man(-6)]Man (PDB entry 1JPC). In four out of the twelve binding
sites, only the Man(-6)Man moiety is observed, the third mannose being disordered.
Figure 2
Phylogenetic analysis of bacterial and eukaryotic MMBL modules
Unrooted maximum-likelihood phylotree constructed from an amino acid sequence alignment
of MMBL modules in representative proteins. The different domain topologies (single or
tandem MMBL, alone or combined with other domains) are indicated. A broken-line box
represents a domain, part of a tandem MMBL, that is found elsewhere in the tree. Pfam
domains indicated on the figure: subtilase peptidase S8 (PF00082; PeptS8), trypsin
(PF00089; Tryp), papain family cysteine protease (PF00112; PeptC1), ricin-type lectin
(PF00652; Ricin), peptidase NLPC/P60 (PF00877; NPLC), monocot mannose-binding lectin
(PF01453; MMBL), LysM (PF01476; LysM), MAC/perforin (PF01823; MACPF), Oglycosyl hydrolase family 30 (PF02055; Glyco Hydro), peptidase inhibitor I9 (PF05922; I9),
peptidase M15 (PF08291; PeptM15), pro-kumamolisin activation domain (PF09286; PK),
GDSL-like lipase/acylhydrolase (PF13472; Hydro), VCBS (PF13517; VCBS), unknown
function (PF14220; DUF4329). Color coding of tree branches is used to highlight the major
eukaryotic clusters with MMBL domains from plant lectins (green), fishes (light blue), fungi
(orange) and the prokaryotic clusters with chimeric MMBLs containing a LysM domain (teal)
or peptidase/hydrolase module (pink). For the LlpA-like proteins, the clusters with Ndomains (N; red) and C-domains (C; dark blue) are differentiated. The plant lectin protomers
are indicated with [A], [B], or [D]. Proteins with proven bacteriotoxic activity are labeled
with an asterisk. Organisms are specified by abbreviated names. Plants: Allium sativum
(Asat), Crocus vernus (Cver), Galanthus nivalis (Gniv), Gastrodia elata (Gela),
Hyancinthoides hispanica (Hhis) Molineria latifolia (Mlat), Polygonatum cyrtonema (Pcyr),
Remusatia vivipara (Rviv); fishes: Esox lucius (Eluc), Gadus morhua (Gmor), Lophiomus
setigerus (Lset), Oplegnathus fasciatus (Ofas), Salmo salar (Ssal), Scorpaena plumieri
(Splu), Takifugu rubripes (Trub); fungi: Aspergillus flavus (Afla), Aspergillus oryzae (Aory),
Coccidioides immitis (Cimm), Fusarium oxysporum (Foxy), Gibberella zeae (Gzea), Nectria
haematococca (Nhem); bacteria: Arthrobacter sp. FB24 (Arth), Brevibacillus laterosporus
(Blat), Burkholderia ambifaria (Bamb_AMMD; Bamb_MEX5), Burkholderia cenocepacia
(Bcen-1; Bcen-2), Cellulomonas flavigena (Cfla), Gordonia araii (Gara), Granulicella
mallensis (Gmal), Mucilaginibacter paludis (Mpal), Mycobacterium abscessus (Mabs),
Mycobacterium smegmatis (Msme), Mycobacterium xenopi (Mxen), Nocardia farcinica
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(Nfar), Paenibacillus larvae subsp. larvae (Plar), Paenibacillus sp. (Paen), Pseudomonas
fluorescens (Pflu_Pf5; Pflu_A506), Pseudomonas putida (Pput_BW; Pput_W619),
Pseudomonas syringae pv. aesculi (Psyraes), Pseudomonas syringae pv. aptata (Psyrapt),
Pseudomonas syringae pv. syringae (Psyrsyr), Rhodococcus erythropolis (Rery),
Ruminococcus albus (Ralb), Segniliparus rotundus (Srot), Stigmatella aurantiaca (Saur),
Streptomyces clavuligerus (Scla), Streptomyces lividans (Sliv), Streptomyces sp. (Stre_C;
Stre_SPB78), Tsukamurella paurometabola (Tpau), Terriglobus saanensis (Tsaa),
Xanthomonas axonopodis pv. citri (Xcit). The taxonomic affiliations and sequence accession
numbers are specified in Table S1. The multiple sequence alignment of the MMBL modules
used to build this phylogenetic tree is represented in Figure S1.
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Supplemental information
Table S1. Representative proteins containing a single MMBL domain or MMBL tandem,
either alone or combined with other domains.
Figure S1. Multiple-sequence alignment of MMBL modules extracted from representative
proteins containing a single MMBL domain or MMBL tandem, either alone or combined
with other domains. Differential shading reflects the extent of sequence conservation. The
location of the three QxDxNxVxY motifs is indicated. Abbreviated organism names are
explained in Figure 2. Sequences from bacteriocins are labeled with an asterisk.
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