1. 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
Strongylocentrotus purpuratus

2. 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

3. 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(Cohen & Smith, unpublished)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

4. 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.

5. 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

6. 185/333 Gene Structure < 2 kb
407bp
Leader
Elements E2
< 2 kb
Quantitative PCR estimates 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

7. 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

8. 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

9. Element Scrambling L 38 unique genes from animal 10 Exon 2 Elements57 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

10. 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

11. 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