Topic 6 - Evolution of Immune Systems & Protostome Immunity

1. Topic 6 - Evolution of Immune Systems & Protostome Immunity • Recurrent themes in comparative immunology: • Gene duplications lead to diversified functions • Evolutionarily related molecules can assume distinct functions - divergent evolution • Unrelated molecules can develop similar functions - convergent evolution • 4) Systems unrelated by function or evolution may have same effect - analogous systems to fight pathogens

2. The mammalian complement system is an example of a part of the immune system that got more complex with evolutionary time It also has several examples of gene duplications leading to discrete functions of the products of new gene copies ….

3. Duplication of genes provides 'spare' copies that can diversify Intrachromosomal gene duplication evolutionary divergence of duplicated genes with one copy often retaining original function

4. Evolution of duplicated genes Structural divergence & gene family lineages intra- and inter-chromosomal gene duplications evolutionary time Ancestry of Key Complement components (C3, C4 & C5)

5. Vertebrate complement systems - evolutionary elaboration of an ancient (immune) defense system mannose non-sialated membranes via C3, C4 & C5 MAC Janeway, Immunobiology

6. The 'core' of the modern C' system is derived from duplicated genes C3, C4 & C5 have a common ancestral gene which was C3-like C3 & C4 contain thioester groups that allow covalent bonding to target C5 has lost the thioester site but is structurally similar to C3 & C4 There are other thioester containing proteins (TEPs) These other TEPs share common ancestry with C3

7. C3 shares ancestry with another ancient TEP protein - a2M* a2M is a protease inhibitor - once the protease attacks the bait peptide the a2M thioester forms a covalent bond with the protease, trapping it. Protease may be from pathogen or part of host response a2M protease * Covalently bound, but active protease Bait peptide * Reactivethioester *a2-Macroglobulin

8. C3 has evolved some distinct (from a2M) biochemical features …. Kuby 13-6 C3(H2O) anaphylotoxin unreactive thioester reactive C3 convertase

9. … that lead to C3b forming ester bonds to pathogen membranes Kuby 13-6 C3(H2O)

10. Evolution of duplicated genes & structural divergence a2M- or C3-like? early metazoans C3 inverts verts a2M "TEP" a2M/"TEP" lineage C3 C4 C5 Complement lineage Phylogenetic trees look for lineage traits......

11. Phylogenetic tree of TEPs - close branch roots = closely related a2M & "TEPs" more closely related to each other Sponge C3-like (SeC3) more related to deuterostome C3 "TEPs" found in C. elegans & arthropods - so far

12. The "TEPs" are an offshoot of the a2M lineage but function like C3 Structural divergence & functional convergence !? Invertebrate C3 & "TEPs" both make thioester bonds to pathogens TEPs promote phagocytosis through opsonization, and may also be involved in a melanine encapsulation response. TEPs bind Gram +ve, -ve & macroparasites TEP1 in mosquito may operate in an analogous fashion to C3 in the alternative pathway of activation ….

13. reminder of the alternative C’ pathway Sialylated surfaces (such as eukaryotic membranes) attract Factor H which along with Factor I, cleaves up C3b so it can't complete C3 convertaseformation on self membranes kuby 7-10

14. In the vertebrate Alternative Pathway, Factor B stabilizes C3(H2O) until Factor D cleaves Factor B (joined to what is now C3b) into Bb which joins C3b in forming a covalently bound C3 convertase so far the only alternative pathway components identified in invertebrates are Factor B (Bf), Factor H and C3

15. Mosquito TEP1 also is spontaneously hydrolyzed in vivo This hydrolyzed form of TEP1 may be highly reactive like C3(H2O) factors analogous to Factor B and Factor H may stabilize the reactive TEP1 thioester, and prevent it from binding self. These mosquito Factor B/H analogues are called LRIM & APL1c

16. Some gene lineages are lost over time or adopt new functions (or - not yet found) Chordates & Vertebrates a2M & C3 Deuterostomes - echinoderms (urchins) • Protostomes • including • insects "TEPs" – new functions or lost (CD109?) C3 lost in insects a2M, "TEP" & C3 Coelomates animals with hemocoel (‘blood cavity’) "TEP" C. elegans nematodes C3-like Sponges Ancestral TEP duplications --> TEP, a2M & C3 lineages (?) Metazoans - multi-cellular critters

17. Many complement proteins evolved from common ancestral genes Bf/C2; C1s/C1r/MASP; MBL/C1q; C3/C4/C5 factor (Bf)

18. MAC - C6, C7, C8 & C9 share a common ancestor - also with Perforin The vertebrate complement systems are an elaboration of the C3 based opsonization system of invertebrates - that evolved gradually(?)

19. The complement system evolved gradually over time… C3 as an opsonin [as well as factor B (Bf)] in proto- & deuterostomes Putative recognition components but no MBL found yet Evidence of lectin pathway not yet found outside of chordates?? Early chordates (eg. ascidians) have MASP & a putative MBL But these systems may only be part of opsonization process MAC mediated lytic system only observed in sharks & higher vertebrates But a MAC/Perforin ancestor is found in ancestral vertebrates

20. A single report takes the Mannose response pathway back to among the earliest metazoans – but still no structural MBL homologue …. Multi-component complement system of Cnidaria: C3, Bf, and MASP genes expressed in the endodermal tissues of a sea anemone, Nematostella vectensis. Ayuko Kimura, Eri Sakaguchi, Masaru Nonaka; Immunobiology (2009) 214; 3: 165-178

21. Complement components found through phylogeny 'C1q' Bf 1st MAC/Perforin member "TEP" protostomes 2001, 13: 69-73 (modified) a2M, TEPs & C3-like in all eumetazoans (sponges, anemones, etc) & MASP etc?

22. Analogous approaches for dealing with pathogens - isolating or sealing • A wound or a pathogen can be isolated by these approaches • - Plants seal off infected leaves - hypersensitive response • - Mammals seal off capillaries adjacent to sites of inflammation • & granulomas form around parasites or infected macrophages • The response in coelomate invertebrates is akin to a combination of • coagulation and granuloma formation

23. Plant Hypersensitive response to pathogens • Detection via distinct plant PRRs – ‘R genes’ leading to ion influx • Oxidative burst that generates reactive oxygen species (ROS) • Cell death and deposition of additional cell wall

24. Wound healing in inverts has similarities to vertebrate blood clotting Protostomes & deuterostomes have open circulatory systems (unlike us). The heart pumps hemolymph thru veins into open spaces called the hemocoel. Hemocytes act as combination red and white blood cells hemolymph ≈ serum hemocytes ≈ leukocytes hemocoel ≈ nothing in us

25. Encapsulation & melanization of bacteria in wounded arthropods - A coagulation cascade coupled with the prophenoloxidase (PPO) system 1 min. after injection 30 minutes after of bacteria matrix ≈ clot + 30 minutes 24 hours after Fig. 5.33 from: The Insects, R.F. Chapman, Cambridge Press Arthropods are crabs, insects, etc

26. PRRs recognize pathogen & signal degranulation of hemocyte: this leads to release of anti-microbials, PPO and coagulation cascade Large Granules Proclotting Enz 2-Macroglobulin Clotting Factors Defensin TachylectinsCoagulogen Serpins Small Granules Defensin Tachystatin Tachyplesin Tachycitin Frontiers in Bioscience, 1998 3:D973-84

27. Our blood coagulation system is a serine protease cascade culminating in the conversion of fibrinogen to x-linked fibrin Route 1 – contact with extracellular matrix (collagen) Route 2 – contact with tissue fibroblasts & tissue leukocytes

28. Endotoxin (LPS) pro-factor C Factor C 1-3-b-D-Glucan pro-factor B Factor Factor G pro-factor G Pro-clotting enzyme clotting enzyme Coagulogen Coagulin (clotting agent) Protostome hemolymph coagulation also involves a serine protease cascade culminating in conversion of coagulogen to x-linked coagulin as worked out in Horseshoe crabs From chitin - own exoskeleton or fungal infection Route 1 Route 2 Frontiers in Bioscience, 1998 3:D973-84

29. More convergence & divergence in clot formation systems Coagulogen is functionally analogous to our Fibrinogen but coagulogen is a structural homologue of Nerve Growth Factor Fibrinogen is structurally related to Tachylectin in arthropods Tachylectin functions as a PRR & bacterial agglutinating compound The take-home: structural homologues do not necessarily share function and functional homologues do not necessarily share structure The 2.0-Å crystal structure of tachylectin 5A provides evidence for the common origin of the innate immunity and the blood coagulation systems. PNAS 2001 98:13519-24 Crystal structure of a coagulogen, the clotting protein from horseshoe crab: a structural homologue of nerve growth factor. EMBO J. 1996 15:6789-97

30. Another component of arthropod wound healing and innate immune responses is the melanin forming prophenoloxidase (PPO) pathway which is the culmination of another serine protease cascade irregular black marks on the shells of crabs and lobsters are often signs of past injuries & the prophenoloxidase melanization process

31. chitin Gram +ve Gram -ve PGRP The prophenoloxidase (PPO) system is also a serine protease cascade Toll & other PRR’s Serpins Serine protease inhibitors (PPO activating proteases: PAPs) Buffers against damage from RNIs & ROIs These and other intermediates have anti-microbial properties Current Opinion in Immunology 1998, 10:23-28.

32. Melanization (PPO system) occurs both at wound sites and around individual pathogens parasitic wasp larvae emerging from a host caterpillar leave black marks The PPO system is only (?) in invertebrates wasps

33. Arthropods also use the PPO system to make and harden their exoskeleton http://insected.arizona.edu/manduca/graphics/lifecycle.jpg. http://www.entomology.wisc.edu/manduca/images/pupa-stages.jpg

34. A melanin encapsulated malaria parasite (Plasmodium) in mosquito gut Encapsulation can occur independent of coagulation & vice versa Current Opinion in Immunology, 2001, 13:1:79-88 Encapsulation may also 'smother' the pathogen - in its own wastes

35. In wound healing PPO can be derived from other sources – at a cost Non-insect protostomes (crabs, clams, worms, etc.) use hemocyanin as an oxygen carrying molecule (like our hemoglobin). Hemocyanin is structurally related to phenoloxidase and can be diverted to the PPO pathway to amplify wound healing response. This represents a re-allocation of resources from oxygen transport to wound healing (J.Biol.Chem. 2001 276:27166-70)

36. In crabs the hemocyanin a-subunit is converted to phenoloxidase by an anti-microbial peptide - tachyplesin Nagai T et al. J. Biol. Chem. 2001;276:27166-27170

37. At least 6 types of crab AMP can convert Hc to PPO – to varying extents controls + - anti-microbial peptides Nagai T et al. J. Biol. Chem. 2001;276:27166-27170

38. The hemocyanin is directed to the exposed exoskeleton (chitin) by AMPs which bind to both the chitin and the hemocyanin chitin chitin + AMP chitin + lectin alone (neg, control) PPO helps to repair, seal & harden damaged exoskeleton Nagai T et al. J. Biol. Chem. 2001;276:27166-27170

39. Compound(s) released from hemocytes during clot formation also convert hemocyanin to phenoloxidase – particularly when chitin (1,3-b-glucan) is present + 1,3-b-glucan PPO activity  HLS – hemocyte lysate; Hc - hemocyanin The compounds responsible had features of serine proteases (not AMPs) Comp Biochem Physiol B Biochem Mol Biol. 2003 Jan;134(1):135-41

40. Summary of arthropod immune responses in the context of Mosquito responses to Plasmodium (malaria parasite) …. Malaria parasite stages - in blue ookinetes hemocoel gut hemocoel sporozooite Next slide Salivary gland oocysts

41. malaria ookinetes, but not oocysts, are susceptible to killing via TEP Gut lumen gut "TEP" "TEP" parasite ookinetes & oocysts are susceptible to melanin encapsulation

42. Similarities in serine protease cascades of several systems led to the proposal that they evolved following block duplications of gene clusters Toll dorsal-ventral polarity Protostome hemolymph coagulation Complement cascade vertebrate blood clotting system Krem & Cera (2002) Trends in Biological Sci. 27(2):67-74