The CTLs are a family of related proteins that bind carbohydrates in a calcium dependent manner [1
]. In mammals, CTLs have been shown to affect a variety of important functions mediated by cell-cell interactions, including inflammation, thrombosis, development, and reproductive physiology [6
]. There are more than 120 C-type lectin domain containing proteins predicted from the genome sequence of the non-parasitic nematode C. elegans
, some of which have been characterized for their physiologic roles [4
]. In parasitic nematodes, it has been hypothesized that secreted C-type lectins function to subvert the host immune response by binding to specific cell-surface carbohydrate moieties [30
]. Compelling evidence for a role of C-type lectins in the pathogenesis of parasitic nematode infections includes the fact that they are abundant protein components of pooled excretory/secretory products collected from live parasites, are expressed at very high levels in host-dwelling stages, and localize to the cuticle where they might interact with host immune effector cells [26
]. Although it is likely that secreted lectins play an essential role in the biology of parasitic nematodes, thus far little experimental data exist to define the role(s) of specific members of this protein family in host-parasite interactions.
As part of an ongoing strategy aimed at identifying novel hookworm genes and proteins [17
], a number of partial cDNAs were amplified from adult A. ceylanicum
RNA using reverse transcription PCR (RT-PCR). Although an original 3’ oligonucleotide primer was designed in order to amplify nematode serine protease inhibitors [17
], the translated amino acid sequence of a partial cDNA was found to have homology to the CTL family of proteins, including members from both the free living nematode C. elegans
and other parasitic species [4
]. Using standard techniques, the full length cDNA corresponding to Ancylostoma ceylanicum
C-type lectin 1 (AceCTL-1) was successfully amplified from adult hookworm RNA. Analysis of the AceCTL-1
cDNA reveals an open reading frame of 550 nucleotides, which corresponds to a 167 amino acid mature protein with a 17-amino acid secretory signal sequence (). Based on sequence data, the mature AceCTL-1 protein is predicted to have a molecular mass of 18706 Da and a calculated pI of 8.14.
Figure 1 A. The translated amino acid sequence of the AceCTL-1 cDNA predicts a 17-amino acid secretory signal sequence (in bold), followed by a mature protein of 167 amino acids with an expected mass of 18706 Da. The predicted Carbohydrate Recognition Domain (CRD) (more ...)
CTLs are distinguished from other lectins by structural homology in the carbohydrate-recognition domain (CRD), a 115–130 amino acid segment with a conserved spacing and number of cysteine residues, which form disulfide bonds that stabilize a double loop structure [1
]. Using the Conserved Domain Search service (CD-Search) available through the NCBI [35
], we confirmed the similarity of AceCTL-1 to other C-type lectins. As shown in , the predicted AceCTL-1 protein contains a characteristic WIG motif (amino acids W69
), along with a predicted Carbohydrate Recognition Domain (spanning amino acid residues C102
). Within the predicted CRD, there are a number of amino acid residues that are also conserved within the CTL family, including A103
, and M124
. Additional residues predicted to mediate Ca2+
and carbohydrate binding include the tripeptide E94
Having confirmed that AceCTL-1 is most likely a C-type lectin, we then compared the translated amino acid sequence with those previously isolated by Loukas et al from the hookworm N. americanus
(NaCTL-1, NaCTL-2) and the canine roundworm Toxocara canis
(TES-70, TES-32) () [26
]). Using the Blast alignment tool available through the NCBI [22
], the CTL domain of AceCTL-1 was found to have 26–33% amino acid sequence identity (36–50% amino acid similarity) to NaCTL-1, TES-70, and TES-32. Multi-sequence alignment using the ClustalW algorithm [23
] shows that, despite the relatively modest sequence identity between AceCTL-1 and these three nematode CTLs, many of the residues that are essential for structural integrity and carbohydrate binding are conserved (). In contrast, the software analysis identified no significant amino acid sequence similarity with the N. americanus
lectin NaCTL-2. These data confirm the broad divergence of amino acid sequences within the CTL family, and are consistent with potentially wide spectra of biological activities and functions in parasitic nematodes.
A cDNA corresponding to the mature AceCTL-1 protein was expressed in Drosophila
S2 cells, and the recombinant protein purified using a combination of nickel resin affinity and rpHPLC. Following purification, we analyzed the binding of rAceCTL-1 to agarose beads crosslinked with individual carbohydrates [26
]. As shown in , rAceCTL-1 effectively bound to only one (GlcNAC) of 4 carbohydrates tested. By comparison, rAceCTL-1 expressed in a prokaryotic (E. coli
) system with a poly-histidine fusion tag failed to bind any of the carbohydrate containing beads (not shown), suggesting that post-translational modification is necessary for functional activity. It is also interesting to note that the carbohydrate binding signature sequence of AceCTL-1 (E94
) is distinct from that of the mammalian Mannose Binding Lectin (EPN), which binds both GlcNAc and mannose [5
]. Thus, it is possible that the substitution of Gly for Pro at residue 95 in AceCTL-1 accounts for the distinct binding pattern of the hookworm lectin.
Figure 2 A. Carbohydrate binding specificity of recombinant AceCTL-1. The rAceCTL-1 protein was incubated with sepharose beads crosslinked to individual carbohydrates, followed by washing and separation of bound protein by SDS-PAGE. After blotting to nitrocellulose (more ...)
Selective binding of rAceCTL-1 to GlcNAC is consistent with that described for C-type lectins that mediate sperm-egg recognition and fusion during fertilization, including a related sperm membrane protein from the sea urchin, Strongylocentrotus purpuratus
]. Although little is known about molecular mechanisms of fertilization in hookworms, the in vitro
binding specificity of rAceCTL-1 provided the first evidence of its potential role in reproductive physiology, as GlcNAC is a likely to be a major component of the nematode egg membrane [6
The rAceCTL-1 protein was used to immunize a rabbit with Freund’s adjuvant, and the purified α-rAceCTL-1 IgG was then used to characterize the life cycle stage and gender specificity of the lectin in A. ceylanicum. As shown in , AceCTL-1 protein was detected in sperm and soluble extracts of adult males, but not females, eggs/L1, or L3 stages of the parasite. We also did not detect the native AceCTL-1 protein in pooled adult worm ES products (not shown), suggesting the lectin is not secreted. Taken together, the data confirm that AceCTL-1 is primarily an adult male specific protein, and that sperm represent at least one source of its production in vivo.
Further evidence for a role of AceCTL-1 in hookworm reproductive function was obtained using immunohistochemistry. As shown in , sagittal sections of adult A. ceylanicum
probed with the polyclonal α-rAceCTL-1 IgG exhibited a distinct gender-specific staining pattern. In male worms, the α-rAceCTL-1 IgG localized exclusively to the testes. At higher magnification (100X), prominent staining of the rachis, a nematode structure that generates spermatocytes in the male, was clearly visualized [10
]. The rachis serves as a site of early sperm maturation, ultimately releasing spermatocytes that develop to mature sperm prior to insemination of the female via copulation. These data suggest that AceCTL-1 is incorporated at a very early stage into the developing spermatid. Of note, adult worm sections probed with a pre-immune rabbit IgG showed no staining of any structures. Preliminary data suggest that the antibody raised against the rAceCTL-1 protein also localizes to the male testes of the dog parasite A. caninum
(not shown), suggesting the presence of conserved epitopes in related lectins from at least two hookworm species. In fact, a review of the A. caninum
EST database [42
] identified multiple nucleotide sequences that predicted proteins with CTL domains.
Figure 3 Immunolocalization of AceCTL-1 in adult hookworms. Sagittal sections of adult males (left panels) or females (right panels) were probed with the α-AceCTL-1 IgG, followed by detection with a fluorescent labeled α-rabbit IgG. The native (more ...)
In contrast to the recognition pattern of male A. ceylanicum
, two very distinct structures were highlighted upon probing of adult female hookworm sections with α-AceCTL-1 (). One is the spermatheca, which lies between the nematode ovary and uterus [10
]. Both hermaphroditic (eg C. elegans
) and non-hermaphroditic nematodes utilize the spermatheca, a specialized structure located at the junction of the uterus and oviduct, to store sperm following copulation but prior to fertilization. Soon after deposition within the female genital tract, nematode sperm migrate up the uterus to the spermatheca, eventually anchoring themselves until binding a passing oocyte [10
]. Nematode eggs are sequentially fertilized by stored sperm as they pass through the spermatheca, a process in Ancylostoma
that likely occurs and requires a series of sperm-egg interactions based on recognition of specific cell surface molecules.
Once fertilization is initiated, the amoeboid sperm extend pseudopodia around the oocyte. In nematodes and other invertebrate species, successful fertilization results in fusion of the cell membranes, with the resulting zygote cell wall comprised of proteins derived from both sperm and egg [45
]. Consistent with this phenomenon, strong signal was also noted by immunohistochemistry in hookworm embryos visualized within adult females (). It is likely that these images represent recently fertilized oocytes that have passed through the spermatheca. These data suggest that Ancylostoma
eggs, similar to other nematode ova, incorporate sperm proteins into the egg membrane upon fertilization.
Our observation that AceCTL-1 protein can be detected in female worms and embryos by immunohistochemistry, but not by immunoblot, deserves comment. First, it is possible that there is significantly more AceCTL-1 contained in extracts of adult males, due to the fact that the testes are large structures literally filled with spermatocytes. In contrast, in adult females AceCTL-1 appears only in scattered embryos and within the spermatheca, a relatively small structure. Alternatively, it is possible that while native AceCTL-1 is present in the recently fertilized embryos, at the time of excretion by the adult female worm the protein has been degraded to the point that it is no longer present in quantities sufficient to be detected by immunoblot. Lastly, it is also possible that once incorporated into the egg membrane following fertilization, the native AceCTL-1 protein is no longer present in soluble protein extracts. Work is underway to determine the kinetics of AceCTL-1 expression, and potentially degradation, following fertilization of hookworm embryos.
In summary, the data presented here describe the molecular characterization of a C-type lectin with a putative role in the reproductive physiology of the hookworm A. ceylanicum
. While secreted lectins from parasitic nematodes, including hookworm, also function as secreted immunomodulatory proteins [30
], to our knowledge, this is the first member of the CTL family with a potential role in nematode sperm-egg recognition and fertilization. Work is currently underway to more clearly define the function of AceCTL-1 in the biology of Ancylostoma
, as well as to target hookworm reproductive mechanisms as a means of reducing the risk of infection. Strategies to reduce nematode fecundity have previously been suggested as a means to control human and veterinary nematode disease, including hookworm [47
]. Considering that female hookworms pass tens of thousands of fertilized eggs per day, strategies aimed at decreasing hookworm fecundity may ultimately lead to reduced transmission, as well as disease, within highly endemic communities.