Allorecognition proteins in an invertebrate exhibit homophilic interactions

Abstract

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.

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.

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.

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.