The biophysical and structural basis of allorecognition specificity
How do Alr1 and Alr2, by themselves, distinguish self from non-self? We hypothesize the answer is isoform-specific homophilic binding. This hypothesis is based on in vitro observations demonstrating that allelic isoforms of the Alr1 and Alr2 proteins can bind to themselves across opposing cell membranes. Each isoform is capable of binding to isoforms with highly similar amino acid sequences, but does not bind to isoforms with divergent sequences.
Discovery of isoform-specific binding is remarkable. Natural populations of Hydractinia maintain hundreds of unique Alr1 and Alr2 alleles, resulting in natural fusion rates of 0-2% between randomly selected colonies. How natural selection could create hundreds of unique binding specificities within a single locus is a puzzle for population geneticists, molecular evolutionists, and structural biologists alike. We are attempting to answer this question by unraveling the structural basis of this isoform-specific homophilic binding using a combination of crystallographic, biochemical, and computational approaches.
Discovery of isoform-specific binding is remarkable. Natural populations of Hydractinia maintain hundreds of unique Alr1 and Alr2 alleles, resulting in natural fusion rates of 0-2% between randomly selected colonies. How natural selection could create hundreds of unique binding specificities within a single locus is a puzzle for population geneticists, molecular evolutionists, and structural biologists alike. We are attempting to answer this question by unraveling the structural basis of this isoform-specific homophilic binding using a combination of crystallographic, biochemical, and computational approaches.
The allorecognition signaling pathway
In rejection, tissues contacting another colony become swollen with nematocysts (the stinging organelles characteristic of cnidarians) and differentiate into specialized fighting stolons that grow over their opponent. In fusion, these same tissues adhere and reorganize themselves to create a continuous epidermis and gastrovascular system, all within minutes. How are these disparate responses regulated? To answer this question, one must first define the allorecognition signal transduction pathway. We hypothesize that the cytoplasmic tails of Alr1 and Alr2 initiate allorecognition responses because they are relatively large and bear a number of potential phosphorylation sites. Our current work focuses on identifying the intracellular binding partners of Alr1 and Alr2 as a first step toward unraveling this pathway.
Characterizing the Hydractinia allorecognition gene complex
Alr1 and Alr2 predict allorecognition phenotypes in inbred strains of Hydractinia, but wild-type animals sometimes display unexpected phenotypes. This raises the question: Are there additional allorecognition loci in Hydractinia? We think so. Recently, we have succeeded in generating sequences for three haplotypes of the ARC. These sequences reveal at least 10 conserved Alr-like genes, of which one, Alr4, appears to be as polymorphic as Alr1 and Alr2. In addition, these sequences reveal ample evidence of copy number variation, recombination, and gene conversion across the ARC. We are currently working to dissect this interval via classical genetic approaches combined with high throughput sequencing and CRISPR/Cas9-mediated genome editing.