4.3.1 Role of galectins in immune responses
Lectins sharing both their preference for binding β-galactosides and significant sequence similarity in the carbohydrate recognition domain (CRD) are commonly termed galectins (Barondes, 1994). Each galectin contains one or two highly conserved carbohydrate recognition domains (CRDs) made up of about 135 amino acid residues (Figure 4.3.1-1). The prototype galectins contain a single CRD which can form two-fold symmetric homodimers through inter-actions between the hydrophobic β-strands of each subunit. Thus, under normal conditions these lectins are bivalent molecules. The tandem galectins contain two homologous CRD domains separated by a proline-glycine-enriched linker peptide of about 20-30 amino acids. The chimera type galectins contain one CRD and a domain of about 110-130 residues, depending on species, that includes multiple repeat sequences rich in proline, tyrosine, glycine and glutamine (Hughes, 1999). In humans, 15 galectins have been described within the galectin family. In C. elegans there are 11 galectins, CeLec1 – CeLec11.
Figure 4.3.1-1 Galectin structures. The homologous carbohydrate recognition domain (CRD) present in all galec-tins, the unique glycine-thyrosine-glutamine-proline-rich repeat sequence of the chimera type galectins and the link regions of tandem CRD galectins are shown. (Hughes, 1999)
Various biological roles of human galectins have been proposed, for example in regulation of immunity and inflammation, progression of cancer and in specific developmental processes (Leffler, 2004). In the past galectins and helminth infections have rarely been associated with each other but new data from S. ratti as well as from other parasitic nematodes showed high abundance of different galectins in the parasitic stages. The possible involvement of galectins in parasite infections becomes obvious when considering two further aspects. Firstly, galectins are involved in parasite infection and allergic inflammation, two very different but immunologically
CRD Repeats CRD CRD Link CRD
Prototype Chimera Tandem repeat
linked phenomena. Secondly, the hygiene hypothesis which suggests that allergic responses rep-resent a misdirected activation of the arm of the immune system responsible for parasite attrition and that parasite infection may prevent the development of some allergic conditions (Yazdan-bakhsh, 2004).
Host galectins are implicated in both establishing and combating parasite infections. The first line of recognition in an immune response is the parasites surface which is in general highly glycosylated and thus offers potential galectin binding sites. In this respect, there are examples of host galectins binding directly to glycoconjugates on the surface of parasites, leading to both positive and negative regulation of host immunity. For the protozoal parasite Leishmania major for example it has been shown that host galectin-9 binds specifically to the parasites surface lipophosphoglycans and thus is assisting in parasite binding to macrophages, promoting the cell invasion and finally facilitating infection (Pelletier, 2003). Human galectin-10 is present in eosi-nophils and basophils. The only other vertebrate galectin showing such restricted expression is the ovine galectin-14 that has recently been shown to be present only in ovine eosinophils (Young, 2008). Mucus collected from both lung and stomach of sheep with induced tissue eosi-nophilia from either allergen exposure with house dust mite or parasite infection with H. contor-tus, a parasitic nematode of ruminants, contains large quantities of galectin-14. This indicates that host galectins play a role during the immune response to parasitic infection.
On the other hand, the specific functions of the parasites galectins` is not known (Gree-halgh, 2000), regardless of the variety that have been identified in helminth parasites so far.
However, it has been shown that parasite galectins are involved in host-parasite interactions. For example a galectin from O. volvulus was recognised by sera from the majority of filaria-infected patients and was able to bind IgE (Klion, 1994). Furthermore two tandem-repeat type galectins have recently been discovered in a proteomic analysis of secretions from adult B. malayi stages (Hewitson, 2008). Johnston et al. (2009) stated that those galectins bind galactose containing glycoconjugates and may protect adult stages from the host’s eosinophil and neutrophil mediated damage. A vaccination of goats with recombinant galectin antigen induced partial protection against H. contortus infection (Yanming, 2007). In another study it was shown that H. contortus produces a not further identified galectin, or possibly a mixture of galectins, which have potent chemokinetic activity for ovine eosinophils in vitro (Turner, 2008). This is interesting because experimental helminth infections have shown that eosinophils accumulate in the gastrointestinal tract, where it is thought that they help eliminate the parasite and for S. stercoralis infection
eosinophil chemotaxis may have a central role in immunity (Mir, 2006). For the parasitic nema-tode Dirofilaria immitis it has been shown that in atopic individuals, resident in an area of canine endemia, the specific IgE response is stimulated mainly by two molecules, one of them being a member of the galectin family (Pou-Barreto, 2008).
4.3.2 Galectins identified in S. ratti E/S products and extracts
As reported in the result section 3.4.1 the proteomic analysis and the subsequent screening of S. ratti and S. stercoralis EST databases led to the identification of seven different galectins.
The assigned names and the corresponding Strongyloides EST clusters are shown in table 4.3-1.
The resulting sequences were blasted and named according to their closest relationship with C. elegans galectins. Four of these sequences, Sr-Gal-1, -2, -3 and -5, were found in the super-natants and three of the sequences, Sr-Gal-11, -21 and -22, remain hypothetical since the proteins were not found. However, PCR experiments with primers for all seven galectins showed the presence in cDNA of iL3. Using the nematode SL-1 primer and RACE-PCR it was possible to obtain the full length sequences of four galectins, Sr-Gal-1, -2, -3 and -22, showing that unlike the other galectins Sr-Gal-22 is composed of only one CRD. The attempts to obtain the full length sequences of Sr-Gal-5, -11 and -21 were not successful. To capture the missing fragments experiments should either be repeated using different primer sets or as soon as the S. ratti ge-nome becomes available it will be possible to compose the sequences by BLAST search and multiple sequence alignment.
Table 4.3-1 Seven S. ratti galectin sequences and the corresponding cluster numbers
Assigned Name S. ratti EST Cluster
S. stercoralis EST
Cluster Homology
Sr-Gal-1 SR00627 SS00732 96
Sr-Gal-2 SRX0840
(SR05051) SS00840 96
(93)
Sr-Gal-3 SR00900 SS00593 90
Sr-Gal-5 SR00857 SS02629 83
Sr-Gal-11 SR00257 SS01127 94
Sr-Gal-21 SR00838 / /
Sr-Gal-22 SRX1190 SS01190 90
An explanation for the identification of only four of the galectins might be that Sr-Gal-11, -21 and -22 are not being secreted. The secretion mechanism for galectins is ectocytosis in which cytosolic proteins concentrate in the cytoplasm underlying plasma membrane domains. The membrane then forms protrusions (‚blebs‘) including the previously formed protein aggregates.
The blebs finally detach from the plasma membrane and are released as extracellular vesicles from which soluble proteins are released (also see section 1.7). Then, however, it should be pos-sible to identify them in the extract samples but also the extracts revealed only sequences of Sr-Gal-1, -2, -3 and -5. Supposed that all mRNAs of the galectins are actually being transcribed the only remaining reason would be the underrepresentation of Sr-Gal-11, -21 and -22 among the vast number of different proteins in the samples. This opinion led to the attempts to enrich exclu-sively the galectins using lactose affinity separation, a method in which galectins are bound to lactose molecules. The lactose molecules are themselves covalently bound to agarose beads. Af-ter the separation process the galectins can be eluted by treating the beads with a lactose solution.
This method was previously applied successfully to extracts of H. contortus iL3 (Turner, 2008).
Since the preparation of sufficient amounts of E/S products is too time consuming for perform-ing preliminary tests with this newly established method in the laboratory it was decided to use iL3 extracts instead. The test presented in section 3.4.1.4 showed a successful enrichment of Sr-Gal-1, -2 and -3. However it was not possible to remove some other proteins like the immu-nodiagnostic antigen L3NieAg, a metalloprotease, a petidyl-prolyl cis/trans isomerase and a tro-ponin fragment. Among the mentioned proteins Sr-Gal-1 and -2 showed a high abundance ac-cording to their unused protein scores of 26.31 and 37.77 respectively. The results show that the galectins do bind β-galactoside structures but it was not possible to capture the previously uni-dentified galectins Sr-Gal-11, -21 and -22. In case the reason lies in the expression of these galectins at a very low level it should be possible to quantify the relative expression levels by using a comparative real time PCR method in future studies. Even though it seems attractive to isolate native galectins or galectin mixtures for immunological studies and assays it is still neces-sary to increase the amounts of worm material in order to obtain sufficient amounts of protein at the end.
A method that shows both the carbohydrate binding properties and the immunogenicity of S. ratti secreted galectins is the carbohydrate microarray method (Horlacher, 2008) presented in section 3.4.1.7. The test showed that galectins present in iL3 extracts are capable of binding to different carbohydrate structures. To some extend these results could apply for the E/S products due to the fact that Galectins were found in E/S products as well as in extracts. Since rat sera
from infected rats were used to visualise the bound galectins it was shown that the host develops galectin specific antibodies during the course of the infection. In order to further study the subtle differences in carbohydrate-binding patterns it is necessary to further fractionate the native galectins or to express them singularly and test them as pure protein. This will lead to the identi-fication of different endogenous ligands recognised by the various galectin types and, ultimately, their distinct biological roles. By doing so it has for example been shown that laminin is the most likely endogenous ligand for human galectin-1, a lectin proposed to mediate muscle development and induce apoptosis of activated T-cells by binding with CD45 glycoprotein (Perillo, 1995).
Since it was one aim of this work to recombinantly express an identified protein contained in the culture supernatants a member of the galectin family was chosen for expression in E. coli.
First attempts to express galectin sequences Sr-Gal-1 or -2 were not successful due to unsatisfac-tory cloning results. Sr-Gal-3 was the first sequence that was successfully cloned and expressed as shown in section 3.4.1.5. The recombinant Sr-Gal-3 was tested against rat sera using ELISA.
The results showed an immune recognition of the recombinant protein. Interestingly also sera from humans previously infected with S. stercoralis were capable of recognising the S. ratti galectin. This is probably related to the fact that the Sr-Gal-3 shows a 90% homology to the cor-responding S. stercoralis EST cluster. Since the remaining galectins all show high homologies between 83–96% it is suitable to recombinantly express the remaining galectins in the future in order to study their immunogenic potential in both S. ratti and S. stercoralis infection.
The results show that S. ratti galectins are interesting candidates for further studies. Com-bining these findings with data on the different immunogenic capacities of nematode galectins that can be found in the literature they seem interesting for two reasons. Firstly they seem to be good candidates for vaccination strategies. For example Yanming et al. (2007) have shown that the application of recombinant H. contortus galectin let to a partial protection to homologous infection in goats. Secondly they might be good candidates to study inflammatory reactions since it has been shown that different members of the human galectin families provide inhibitory or stimulatory signals to control intestinal immune response under intestinal inflammatory condi-tions. Due to the fact that parasitic nematodes are also capable of masking or tuning the host immune response it is likely that galectins are involved in these processes.