Strongylocentrotus purpuratus Unexpected Diversity in the Innate Immune System of the Purple Sea Urchin L. Courtney Smith Department of Biological Sciences George Washington University Washington DC 202-994-9211 Strongylocentrotus purpuratus

Deuterostomes Animal Phylogeny Protostomes and the Evolution of Immunity Chordates Vertebrates Mammals Birds Reptiles Amphibians Fish Sharks & Rays Agnathans Lampreys Hagfish Protostomes Arthropods Molluscs Annelids Protochordates Ascidians Echinoderms Sea Urchins

Sea Urchin Genome Annotation Incomplete Complement System Missing Classical and Terminal pathways Duplication of Alternative pathway Large gene families Toll-like receptors, 222 gene models (humans have ~11) expressed in coelomocytes (immune cells) NOD-like receptors, 203 gene models (humans have ~20) expressed in gut Scavenger receptors, 218 gene models expressed in coelomocytes C-type lectins, 408 gene models (humans have ~100) encoding 108 small lectins, 300 mosaic proteins (Cohen & Smith, unpublished) Highly Diverse Innate Immune System Hibino et al., Dev Biol 300:349, 2006 TSUGSC, Science 314:941, 2006 Rast et al., Science 314:952, 2006

Gene Expression in Sea Urchin Immune Cells Responding to LPS Of 1025 randomly chosen ESTs, 72% matched to two unknown sequences. EST333 (Smith et al., 1996) DD185 (Rast, Pancer & Davidson, 1999) 185/333 All others Nair et al., Physiol Genomics 2005.

Terwilliger et al., Physiol Genomics 2006 Alignment of 185/333 cDNAs Leader and elements Groups based on element 15 Sets of multiple cDNAs with the same element patterns Variable position of stop codon one with a frame shift and early stop codon Variation in elements Protein gly rich and his rich regions histidine patches (purple) repeats RGD and glycosylation no Cys and little discernable secondary structure 300 nt Gaps define blocks of sequence called elements. Alternative splicing? Terwilliger et al., Physiol Genomics 2006

185/333 Gene Structure < 2 kb 407bp Leader Elements 1 - 25 E2 < 2 kb Quantitative PCR estimates 80 - 120 alleles per genome “However, BLAST searching the partially assembled genome at the Baylor College of Medicine’s website, there was near perfect matches for clones matching different contigs. The line represents the contig from the website while the box represents the cDNA from our studies. The % identity is within each box. This suggest that the each transcript is encoded by a single exon and a leader and no alt splicing occurs.” BLAST results gave near perfect matches = one exon  suggesting one gene per pattern Terwilliger et al., Physiol Genomics 2006

Gene Element Patterns Exon 1 Exon 2 elements Intron elements Group Pattern Source 9 12 16 21 # L Int. 1 2 3 4 5 6 7 8 10 11 13 14 15 17 18 19 20 22 23 24 25 1 3 B A b A 6* 1 S A b 5* 1 S B b 2 1 S C b 3d 5 S B b B 3b 5 B B b 6* 2 S D b 7* 1 S C b C 3 2 S C b 3* 1 S C b 4* 1 S D b D 1 28 B C b 5* 3 C C c 6 1 C C a 3f 6 C E a 3c 1 C C a 2 14 B E a E 7* 1 C E b 8* 1 C E b 9* 1 C E c 1* 1 C B b F 2* 4 C B b 2* 1 C E a 1f 12 B E b 1c 1 S C b 1e 1 S D b 300 bp Intron elements A B C D E 1 2 3 4 5 6 7 8 9 100 bp Buckley & Smith, in prep

185/333 Sequence Diversity Element diversity is a result of SNPs and indels Sequences of identical genes are rarely identified within an individual sea urchin Identical genes are never shared among sea urchins. Some element sequences are shared among dissimilar genes

Element Scrambling L 38 unique genes from animal 10 Exon 2 Elements 57 L 46 12 67 3 11 11 2 2 4 4 37 9 11 3 3 8 5 2 7 3 2 2 19 13 1 1 1 1 Intron Elements 20 3 3 Exon 2 Elements 61 21 1 1 1 2 14 1 2 1 19 2 2 2 20 9 2 2 2 1 1 19 1 10 11 2 1 1 5 17 5 10 4 10 16 1 1 1 1 16 17 1 1 1 1 1 1 11 6 3 17 6 19 3 6 2 7 6 2 3 2 3 12 6 34 3 4 3 2 2 3 1 2 2 4 4 6 4 17 6 7 3 3 30 13 6 18 39 8 35 9 13 4 5 5 3 1 2 1 5 3 Numbers in the element bubbles mean Numbers of element bubbles indicate number of different sequences 9 6 3 13 9 5 6 7 9 5 9 23 2 1 4 6 4 2 1 14 11 10 17 29 18 10 7 8 22 25 4 7 3 5 13 8 6 7 7 8 4 41 19 14 18 21 10 25 1 1 1 5 5 6 8 5 10 2 12 14 12 12 3 1 7 15 1 9 9 5 20 28 7 Buckley & Smith, in prep

Conclusions Future Problems Complex immune system Large gene families Unknown mechanisms for diversification Do all metazoa have mechanisms for immune diversification? Future Problems 185/333 locus structure 185/333 protein function

Sea urchin immunology is supported by the National Science Foundation Acknowledgments George Washington University David Terwilliger Katherine Buckley Virginia Brockton Rebecca Easley Young-Ok Kim Heather Del Valle Lindsay Edwards Macquarie University David Raftos Sham Nair Nolwen Dheilly Sea urchin immunology is supported by the National Science Foundation Duke University Tom Kepler Supriya Munshaw Dickinson College John Henson Medical Univ. of S. Carolina Paul Gross