Allorecognition proteins in an invertebrate exhibit homophilic interactions

Summary

Sessile colonial invertebrates—animals such as sponges, corals, bryozoans, and ascidians—can distinguish between their own tissues and those of conspecifics upon contact [1]. This ability, called allorecognition, mediates spatial competition and can prevent stem cell parasitism by ensuring that colonies only fuse with self or close kin. In every taxon studied to date, allorecognition is controlled by one or more highly polymorphic genes [2–8]. However, in no case is it understood how the proteins encoded by these genes discriminate self from non-self. In the cnidarian Hydractinia symbiolongicarpus, allorecognition is controlled by at least two highly polymorphic allorecognition genes, Alr1 and Alr2 [3, 5, 9–12]. Sequence variation at each gene predicts allorecognition in laboratory strains such that colonies reject if they do not share a common allele at either locus, fuse temporarily if they share an allele at only one locus, or fuse permanently if they share an allele at both genes [5, 9]. Here, we show that the gene products of Alr1 and Alr2 (Alr1 and Alr2) are self-ligands with extraordinary specificity. Using an in vitro cell aggregation assay, we found that Alr1 and Alr2 bind to themselves homophilically across opposing cell membranes. For both proteins, each isoform bound only to itself or to an isoform of nearly identical sequence. These results provide a mechanistic explanation for the exquisite specificity of Hydractinia allorecognition. Our results also indicate that hydroids have evolved a molecular strategy of self-recognition that is unique among characterized allorecognition systems within and outside invertebrates.

Results and Discussion

Invertebrate allorecognition systems have evolved in response to two selective pressures. First, most colonial invertebrates begin their lives as motile larvae that settle on densely populated surfaces, metamorphose into sessile adults, and spend the rest of their lives competing with each other for space [13, 14]. At the same time, colonies routinely encounter themselves when they grow around objects, recover from disease, or regrow following fragmentation. Rather than compete, these isogeneic colonies peacefully coexist and, in some taxa, will even fuse to form a larger colony [15, 16]. Allorecognition genes allow colonies to accurately distinguish self from non-self and mount appropriate competitive responses. These genes are highly polymorphic. Tens to hundreds of alleles can exist in populations, with natural selection maintaining rare ones because they are reliable markers of self. In several species, correctly identifying “self” also serves a second, critical function; it prevents germline parasitism [17–19]. These species possess pluripotent stem cells capable of differentiating into germ cells throughout their lifespan. Fusion permits these stem cells to migrate between colonies and compete for access to the germline, with potentially catastrophic fitness costs for the loser [20, 21]. Allorecognition loci mitigate this risk by restricting fusion to self or close kin.

The molecular basis of allorecognition has been intensely studied in two model systems, the protochordate, Botryllus schlosseri, and the hydroid, Hydractinia symbiolongicarpus. In these species, strikingly similar programs of inbreeding, genetic mapping, and positional cloning resulted in the discovery of strikingly dissimilar gene complexes [3, 5, 22–24]. In Botryllus, this gene complex is called the fuhc (for fusion/histocompatibility) [2]. Colonies fuse if they share one or both fuhc haplotypes and reject if they share none. Five genes within the fuhc have thus far been shown to play a role in allorecognition: cfuhc^tm, which encodes a single-pass transmembrane protein with three tandem Ig-like domains in its extracellular portion [6, 25]; cfuhc^sec, which encodes a predicted secreted protein with two EGF domains [6, 25]; fester and uncle fester, each encoding transmembrane proteins with single extracellular sushi domains [7, 26]; and BHF, which encodes a small intracellular protein with no obvious homologs [8]. Three of these, cfuhc^tm, cfuhc^sec, and BHF, bear polymorphisms that co-segregate with allorecognition responses, suggesting they could function as allodeterminants [6, 8, 25]. The fourth gene, fester, is polymorphic, but this variation does not co-segregate with allorecognition responses [7, 27]. The fifth gene, uncle fester, is not polymorphic, making it unlikely to be involved in self-discrimination [26]. Although the mechanism of self/non-self discrimination in Botryllus is not known, it has been hypothesized that fester could undergo extensive alternative splicing to acquire specificity for the particular cfuhc^tm alleles present in a colony, thus creating a colony-specific receptor-ligand pair [7]. Further details on Botryllus allorecognition can be found in a recent review [28].

In Hydractinia, allorecognition is controlled by a gene complex containing at least two allorecognition genes, Alr1 and Alr2 [5, 9, 10]. Colonies fuse if they share at least one allele at both genes, fuse temporarily if they share an allele at only one gene, and reject if they do not share any alleles at either gene. Alr1 and Alr2 both encode single-pass transmembrane proteins with extracellular regions containing a membrane-proximal “spacer” and either two or three N-terminal immunoglobulin superfamily (IgSF)-like domains, respectively [14, 15]. The extracellular regions of both proteins are highly polymorphic. In laboratory strains and several pairs of wild-type colonies, polymorphisms in Alr1 and Alr2 co-segregate with allorecognition responses in a manner consistent with their role as allodeterminants [11, 12]. As in Botryllus, the Hydractinia allorecognition molecules do not bear strong homologies to other proteins, except for the presence of IgSF-like folds. The mechanism by which Alr1 and Alr2 discriminate self from non-self has also remained elusive. Additional details of the Hydractinia allorecognition system have been reviewed elsewhere [1, 29].

In Alr1 and Alr2, the conspicuous concentration of amino acid variation in the extracellular region suggests that the interactions responsible for self-discrimination occur at the cell surface. Moreover, the domain architectures of both proteins resemble that of several types of receptors and cell-adhesion molecules known to act as self-ligands by binding to themselves across opposing cell membranes (homophilic trans interactions) [30, 31]. We therefore hypothesized that Alr1 and Alr2 might function via homophilic trans interactions and, further, that the protein isoforms encoded by different alleles would only bind to very similar or identical partners, providing a molecular mechanism for self-discrimination.

To test whether Alr1 and Alr2 are capable of homophilic trans interactions, we used a mammalian cell-aggregation assay, which can detect whether cells that normally do not adhere to each other do so upon expression of an ectopic surface protein [32]. Full-length alleles were isolated from two laboratory strains that fuse to themselves but reject each other and are homozygous for alternative alleles at Alr1 and Alr2 (genotypes: Alr1^f/fAlr2^f/f or Alr1^r/rAlr2^r/r). We then created mammalian expression constructs containing FLAG-epitope tagged fusion proteins for Alr1^f, Alr1^r, Alr2^f, or Alr2^r (Figure 1A). Transient transfection of these constructs into CHO cells demonstrated that each isoform was successfully expressed on the cell surface (Figure 1B–C). When transiently co-transfected into CHO cells along with a GFP reporter construct, GFP⁺FLAG⁺cells were detectable within 24 hrs, (Figure 1D). These cultures were subsequently dissociated into single cell suspensions, then incubated with agitation and examined for the presence of multicellular aggregates. Within 4 hrs, GFP⁺ cells formed multicellular aggregates, while GFP⁻ cells remained solitary (Figure 1E–H). Control cells transfected with GFP alone did not form aggregates (Figure 1I). These data indicate that Alr1 and Alr2 are capable of mediating homophilic trans interactions.

Figure 1. Ectopic expression of single Alr1 and Alr2 proteins causes CHO cells to aggregate.

(A) Domain architecture and cloning strategy for Alr1 and Alr2. Coding sequences of Alr1 or Alr2 were cloned into the mammalian expression vector pFLAG-CMV3 such that the expressed protein bore a mammalian N-terminal pre-protrypsin leader sequence and a FLAG octopeptide followed by the sequence of Alr1/2 starting immediately after the Hydractinia signal peptide. See also Table S1. (B–C) Surface expression of FLAG-Alr1/2 on CHO cells. CHO cells were transiently co-transfected with pmCherry-C1 (red) and either pFLAG-Alr1^f (B) or pFLAG-Alr2^f (C), then subsequently fixed and stained with a mouse anti-FLAG primary antibody followed by an Alexa488 conjugated goat anti-mouse secondary antibody (green) and DAPI (blue), then imaged with confocal microscopy. Scale bars = 10 μm. (D) CHO cells co-express GFP and FLAG-Alr1^f. Contour plot validating co-expression of GFP and FLAG-Alr1^f in a representative flow cytometry experiment. CHO cells were transiently co-transfected with pFLAG-Alr1^f and pmaxGFP, incubated for 24 hrs, stained with mouse anti-FLAG primary antibody and an Alexa647 conjugated goat anti-mouse secondary antibody, and visualized via flow cytometry. (E–H) CHO cells co-expressing FLAG-Alr1^f (E), FLAG-Alr1^r (F), FLAG-Alr2^f (G), or FLAG-Alr2^r (H) and GFP form multicellular aggregates. (I) CHO cells expressing only the GFP reporter construct do not aggregate. In panels E–I, left image is bright-field image, right image is blue-light fluorescence, and scale bars = 100 μm.

To test whether homophilic binding is isoform-specific, we repeated the cell aggregation assays by mixing cell lines expressing different Alr1 or Alr2 isoforms that were color-coded by co-transfection with either a GFP or RFP reporter. As expected, cells expressing the same isoform but different fluorescent proteins formed bicolored aggregates (Figure 2A–B, D–E). However, cells expressing different isoforms and reporters segregated to form single-color aggregates, with no evidence of co-localization of the two fluorescent markers (Figure 2C,F). This provided further evidence that the Alr proteins can mediate homophilic trans interactions and demonstrated that these homophilic interactions only occur between the same isoform. Thus, the Alr alleles that determine allorecognition responses in our laboratory strains encode cell surface proteins that discriminate self from non-self via isoform-specific homophilic binding.

Figure 2. Homophilic binding of Alr1 and Alr2 is isoform-specific.

(A) Bi-colored cell aggregate formed when CHO cells transiently transfected with FLAG-Alr1^f and GFP were co-incubated with CHO cells transiently transfected with FLAG-Alr1^f and RFP. (B) Same as (A) but with Alr1^r. (C) Single-color aggregates formed when CHO cells transiently transfected with Alr1^f and GFP and were subsequently co-incubated with cells transiently transfected with Alr1^r and RFP. (D) Bi-colored cell aggregate formed when CHO cells transiently transfected with FLAG-Alr2^f and GFP were co-incubated with CHO cells transiently transfected with FLAG-Alr2^f and RFP. (E) Same as (D) but with Alr2^r. (F) Single-color aggregates formed when CHO cells were transiently transfected with Alr2^f and GFP and subsequently co-incubated with cells transiently transfected with Alr2^r and RFP.

Natural populations of Hydractinia maintain hundreds of distinct Alr2 alleles [33, 34], and Alr1 is expected to be similarly polymorphic [12]. To test whether isoform-specific homophilic binding is a general property of Alr proteins, we cloned four additional Alr1 alleles and two additional Alr2 alleles with sequences that differed from the alleles above. Each allele was then co-transfected into CHO cells with a GFP or RFP reporter and tested in an aggregation assay. In all cases, cells expressing an Alr gene product formed multicellular aggregates, indicating that homophilic binding is a general property of ALR proteins (Figure 3A). The specificity of this binding was investigated in further assays in which the six Alr1 and four Alr2 alleles were tested in all pairwise combinations (Figure 3A). The results confirmed that each protein isoform was capable of mediating homophilic but not heterophilic trans interactions. The lone instance of heterophilic binding was between Alr1^f and Alr1^LH07-82a, which differ by only four amino acids in their extracellular regions (Figure 3B). This indicates that self-recognition does not require a perfect sequence match, but instead that binding specificities are likely determined by amino acids at key positions in the extracellular region.

Figure 3. Multiple Alr1 and Alr2 alleles exhibit isoform-specific homophilic binding.

(A) Summary of aggregation assays performed with additional Alr1 and Alr2 isoforms. (B) Number and location of amino acid differences between isoforms tested in (A). Each row represents a pairwise combination of Alr1 (top) or Alr2 (bottom) isoforms tested. For each pair, the number and location of mismatched amino acids is shown with respect to the IgSF-like domains and spacer region, as illustrated in the cartoon. Outcome of each binding assay is shown in right hand column. The average length of each extracellular domain is shown in parentheses. Size of domain 1 excludes FLAG tag and linker. Intensity of red coloration indicates relative amount of variation.

Taken together, these studies indicate that isoform-specific homophilic binding is the mechanism by which the Hydractinia allorecognition proteins discriminate self from non-self. This mechanism appears to be unique among the genetic systems that have evolved for intraspecific recognition. In social amoebae [35, 36], basidiomycete fungi [37, 38], and three classes of angiosperms [39], the protein-protein interactions responsible for self-discrimination have been elucidated. Although the identity of these recognition molecules varies widely, their genomic organizations and molecular logic are similar. Each involves two or more linked genes encoding protein products that directly bind to each other. Each gene is polymorphic, and the proteins encoded by these genes can only mediate self-recognition in certain allelic combinations. Moreover, tight linkage ensures these allelic combinations are inherited as haplotypes. In contrast, Hydractinia has evolved a completely different molecular strategy: a system of highly polymorphic genes, each encoding a cell surface protein that binds only to itself or very similar isoforms.

It may seem implausible for such a large number of Alr1 and Alr2 alleles to evolve if each new gene product must simultaneously preserve the ability to bind to itself but not others. However, insects have evolved precisely this strategy as the basis of neural self-avoidance [40]. Drosophila neurons express a cell adhesion molecule called Dscam. Alternative splicing of Dscam can generate 19,008 different ectodomains, each of which binds only to itself [41, 42]. Neurons stochastically express different isoforms, and homophilic binding between identical isoforms on sister neurites causes them to repel and avoid forming self-synapses [43]. This isoform-specific binding is the result of variation at three extracellular Ig-like domains. Each is encoded by an exon that is independently spliced into the full-length gene from a block of 12, 48, or 33 alternative sequences. Each Ig-like domain binds only to the identical domain in an opposing molecule [44, 45], and just one mismatched domain is sufficient to prevent full-length Dscam isoforms from binding [41, 42]. Thus, the Dscam locus has evolved 93 Ig-like domains, each with a unique binding specificity. We suspect further study of the Hydractinia system will demonstrate that it has similarly evolved hundreds of binding specificities via the IgSF-like domains of Alr1 and Alr2. In fact, the only salient difference between these two systems appears to be that all variation in Dscam is encoded in the genome, while in Hydractinia this polymorphism is maintained in populations as a pool of alleles, each with a remarkable ability to distinguish self from non-self.

Experimental Procedures

Alr1 and Alr2 sequences

To clone Alr1 and Alr2 alleles from inbred strains, RNA was extracted from whole colonies using the RNAqueous Total RNA Isolation Kit (Life Technologies) and cDNA synthesized using Superscript III Reverse Transcriptase (Life Technologies) with either random hexamers or gene-specific primers according to the manufacturer’s instructions. Full-length alleles were amplified by PCR with Phusion DNA Polymerase (New England Biolabs) and cloned into pCR-BLUNT-II-TOPO (Life Technologies). For additional Alr1 alleles, coding sequences were amplified from cDNA libraries for four field-collected colonies, as reported in ref [33] using Phusion DNA polymerase with degenerate primers. For Alr1^LH06-49a and Alr1^LH06-82a, primers used were 5′-ATGTATGCTGCCTCAACCTTGACTT-3′ + 5′-TGCGATCGATATTGCACTAATTCAC-3′ in the initial PCR and 5′-ATGCTGCCTCAACCTTGACTTAGAC -3′ + 5′-TGCACTAATTCACGTGACAGCTTCT-3′ in a subsequent nested reaction. For Alr1^LH07-43a and Alr1^LH07-46a, primers used were 5′-TGTATGCTGCYTCAACCTTGA-3′ + 5′-TCATCTGGTGGTGRTGAACGT-3′ in the initial PCR, and 5′-TGCTGCYTCAACCTTGASTT-3′ + 5′-TGGTGGTGRTGAACGTCCTC-3′ in a subsequent nested reaction. Transcripts of Alr1 exhibit alternative splicing of exon 4, resulting in a long isoform and a short isoform that lacks 14 amino acids in the extracellular region immediately proximal to the transmembrane helix. We isolated and tested the long isoforms of each allele. Additional Alr2 alleles were a gift from Dr. Leo Buss (Yale University) and were from the collection of full-length Alr2 alleles reported in ref. [34]. Table S1 lists the Genbank accession of each full-length allele used in this study.

Mammalian expression

Allorecognition alleles were cloned into the mammalian expression vector pFLAG-CMV3 (Sigma), which includes an N-terminal FLAG tag. For each allele, a coding sequence was amplified that included the extracellular region beginning immediately after the predicted signal peptide cleavage site, the transmembrane domain, and a portion of the cytoplasmic tail. The Alr1^f, Alr1^r, Alr2^f, and Alr2^r alleles were cloned into the multiple cloning site of pFLAG-CMV3 (Sigma) via restriction-enzyme based cloning. Wildtype alleles were cloned into a version of pFLAG-CMV3 modified to allow cloning to be performed via ligation independent cloning [46]. Table S1 provides annotated amino acid sequences of the Alr1/2 ectodomains that were created. Plasmids were transiently transfected into CHO-K1 cells using TransIT-CHO (Mirus Bio) according to the manufacturer’s instructions. To fluorescently label the cells that took up the ALR expression construct, prior to transfection the ALR expression constructs were mixed with plasmids encoding either GFP (pmaxGFP, Lonzo BioSciences) or RFP (pmCherry-C1, Clontech Laboratories) at molar ratios of 10:1 ALR construct:fluorescent reporter construct. CHO cells were cultured in Ham’s F12, Kaighn’s Modification Media (Hyclone) supplemented with 10% Fetal Bovine Serum, 100 units/ml penicillin and 100 μg/ml streptomycin (Sigma).

Confocal Microscopy

CHO-K1 cells were grown on 22 mm² sterilized glass coverslips (Fisher) coated with poly-L-lysine solution (Sigma catalog no. P4707). Cells were then co-transfected with pmCherry-C1 (Clontech) and either pFLAG-Alr1^f or pFLAG-Alr2^f. Transfected cells were washed with PBS and fixed in 2% paraformaldehyde (VWR, 100503-916) for 15 minutes at room temperature. Fixed cells were blocked in 2% BSA (Sigma, A9418) for 45 minutes at room temperature and stained with a 1:1000 dilution of mouse anti-FLAG antibody (Sigma F1804) for 60 minutes at room temperature, washed with PBB (PBS + 0.5% BSA), stained with a 1:1000 dilution of Alexa 488 conjugated goat anti-mouse antibody (Life Technologies A21235), and finally washed with PBB. Nuclei were counterstained with 1mg/ml DAPI for 1 minute at room temperature. Coverslips with stained cells were washed with PBS, briefly air dried and mounted on glass microscope slides using a homemade gelvatol (PVA + glycerol + sodium azide) mounting medium. Cells were imaged using a Nikon A1 s216.6 confocal microscope. Figures were constructed using a single 0.5um scan slice.

Flow Cytometry

CHO cells transfected as described above were incubated for 24 hrs, then resuspended by incubating for 5 minutes with 0.25% Trypsin/0.02%EDTA (Sigma), followed by the addition of cell culture media. Cells were then washed and resuspended in staining buffer (PBS + 2% FBS + 2 mM EDTA), and stained for 20 minutes on ice with a mouse anti-FLAG primary antibody (clone M2, Sigma cat. number F1804) at 1:500 dilution, washed, then stained for 20 minutes on ice with an Alexa647 conjugated goat anti-mouse secondary antibody (Life Technologies Cat. #A21235) at a 1:500 dilution. Cells were then washed in staining buffer and analyzed on a Becton Dickson LSR II flow cytometer.

Aggregation Assays

Previously transfected cells were dissociated via 5 minute incubation with 0.25% Trypsin/0.02% EDTA solution (Sigma), and washed in complete CHO culture medium (Ham’s F/12 Kaign’s Modification + 10% fetal bovine serum + 100 U/ml penicillin + 100μg/ml streptomycin) and counted. For each aggregation assay, a total of 5 x 10⁴ cells were added to one well of a 24-well low-adhesion plate (Corning) in a total volume of 500μl complete medium + 70U/ml DNAse I (Sigma) + 2 mM EGTA (Sigma). For tests of two alleles, 2.5 x 10⁴ cells from each transfection were mixed together. The plate was then incubated for 4 hrs at 37°C in 5% CO₂ on an orbital rotator at 80–90 RPM. Assays were then visualized with an inverted fluorescence microscope. Assays of each allele were repeated a minimum of three times.

Highlights.

• alr1 and alr2 allow Hydractinia colonies to distinguish self from conspecifics.

• ALR1 and ALR2 proteins bind homophilically across opposing cell membranes.

• Allelic isoforms of these proteins only bind to themselves or similar isoforms.

• Isoform-specific binding could be the mechanism of allorecognition specificity.