Lessons in Immunity: From Single-cell Organisms to Mammals

Lessons in Immunity: From Single-cell Organisms to Mammals

Editors

Loriano Ballarin

University of Padova, Padova, Italy

Matteo Cammarata

University of Palermo, Palermo, Italy

Table of Contents

Cover image

Title page

Copyright

List of Contributors

Preface

Chapter 1. Ciliate Pheromones: Primordial Self-/Nonself-Recognition Signals

Introduction

Pheromone Identification

Pheromone Structures

Pheromone Activity

Pheromone Structure–Function Relationships with Other Signaling Proteins

Conclusions

Chapter 2. Cell Death Pathways in an Unconventional Invertebrate Model

Introduction

Signaling Pathways in Apoptotic Cell Death of the IPLB-LdFB Insect Cell Line

Autophagy-Mediated Cell Death in IPLB-LdFB

Concluding Remarks

Chapter 3. Immunotoxicology Approaches in Ecotoxicology: Lessons From Mollusks

Mollusk Hemocytes: Types and Functions

Interaction Between Hemocytes and Toxicants: The Immunomarker Approach

Mollusk Immunomarkers: The Role of Confounding Factors

Chapter 4. New Aspects of Earthworm Innate Immunity: Novel Molecules and Old Proteins With Unexpected Functions

Essentials of Earthworm Immunity: A Concise Introduction

Stars and Stripes or Pattern Recognition in Earthworms

Lysenin: A Multitasking Protein in Eisenia Earthworms

Lysenin Meets Nanoparticles: Unexpected Rise of a New Function

Conclusions and Perspectives

Chapter 5. Neuroprotection and Immunity in the Medicinal Leech Hirudo medicinalis: What About Microglia?

The Medicinal Leech Central Nervous System

Microglia as Brain Immune Cells

Immune Response Against Pathogens

Microglia Recruitment

Neuroinflammatory Markers

Toward Nerve Repair: Microglia/Neurons Crosstalk into the Spotlight

Conclusions

Chapter 6. Specificity of Innate Immunity in Bivalves: A Lesson From Bacteria

Innate Immunity in Bivalves: Diversity and Complexity

Bivalves and Marine Bacteria

Immune Recognition

Immune Signaling

Immune Effectors

Specificity of the Immune Response to Pathogenic Vibrios: Role of Surface Interactions and Serum-Soluble Components

Conclusions

Chapter 7. Immune-Related Signaling in Mussel and Bivalves

Premise

Multiple Layers of Biological Signaling

Immune Signaling and Related Cell Processes

How Small RNAs Can Influence the Immune Host Response

Perspectives

Chapter 8. Crustacean Immunity: The Modulation of Stress Responses

Sources of Stress

Indicators of Stress

Stress Responses Mediated by Neuropeptides

Upstream Modulators Triggering CHHs

The Crustacean Hyperglycemic Hormone Neuropeptides

Perspectives

Chapter 9. How Insects Combat Infections

Characteristics of Insect Immune Response

Interaction Between Insect Host and Pathogen

Chapter 10. Aedes aegypti Immune Responses to Dengue Virus

Introduction

Transmission to Human Hosts: Vectors

Dengue Virus in the Vector: Innate Immune Responses

Mitigating Dengue: Control Measures

Conclusion

Chapter 11. Protective Responses in Invertebrates

Background

Mediators of Immune Responses

Degree of Intercellular Reactive Oxygen Species Levels and Immune Responses

Prophenol Oxidase System Activation

Cellular Responses

Emerging Concepts About New Players Involved in Protective Responses

Chapter 12. Echinoderm Antimicrobial Peptides: The Ancient Arms of the Deuterostome Innate Immune System

Introduction

Echinoderm Immunity

Antimicrobial Peptides

Mode of Action

Antimicrobial Peptides in Echinoderms

Antimicrobial Biofilm Peptides in Echinoderms

Conclusion

Chapter 13. Inflammatory Response of the Ascidian Ciona intestinalis

Introduction

Self/Nonself Recognition

Inflammatory Responses

The Pharynx is Promptly Involved in the Inflammatory Reaction

Conclusions

Chapter 14. Cytotoxic Cells of Compound Ascidians

Introduction

Ascidian Circulation

Hemocytes of Compound Ascidians

Cytotoxic Cells

Cytotoxicity

Phenoloxidase

Allorecognition and Inflammation

Immunorecognition and Immunomodulation

Future Perspectives: New Roles for Old Cells

Chapter 15. Fish Transcriptomics

Introduction

Transcriptomics Analysis of Fish Leukocytes

Conclusion

Chapter 16. Developmental Biology of Teleost Lymphocytes

Introduction

T Cell Development

B Cell Development

Perspectives

Chapter 17. Cathelicidins: An Ancient Family of Fish Antimicrobial Peptides

The Presence of Cathelicidin in Bony Fish Genomes

Structure of Cathelicidin Gene and Transcripts

Conclusions

Chapter 18. Evolution and Immune Function of Fish Lectins

Introduction

Fish Lectins

The Lectin Repertoires in Fish: Genomic, Structural, and Functional Diversity

Rhamnose-Binding Lectins

Rhamnose-Binding Lectins in Fish: Biochemical and Molecular Features

Rhamnose-Binding Lectin—Molecular Structure, Phylogeny, and Evolution

Fucose-Binding Lectins

Fish FTLs: Biochemical and Molecular Features

F-Type Lectins—Phylogeny and Evolution

Galectin Structure and Evolution

C-Type Lectins

Mannose-Binding Lectins in Fish

Mannose-Binding Lectin Phylogeny and Evolution

Chapter 19. Teleost Immunoglobulins

Introduction

Teleost IgM

Teleost IgD

Teleost IgT

Teleost IgL Chain

Conclusions

Chapter 20. Immunity and Wound Healing: Regeneration or Repair?

Introduction

Injury-Induced Inflammation and Its Regulation During Tissue Repair in Diverse Model Organisms

Inflammation is Not All Bad for Repair

The Relationship Between Immune System Development and Regenerative Capacity

Role of Lymphocytes in Repair Mechanisms

Conclusion

Chapter 21. Marine Mammal Immunity Toward Environmental Challenges

Adaptation of Mammals to the Marine Environment

Marine Mammals, Sentinels for the Health of the Ecosystem

Immunotoxic Effects of Environmental Challenges

Molecular Effects of Environmental Changes on Marine Mammal Immunity

Index

Copyright

Academic Press is an imprint of Elsevier

125 London Wall, London EC2Y 5AS, UK

525 B Street, Suite 1800, San Diego, CA 92101-4495, USA

50 Hampshire Street, 5th Floor, Cambridge, MA 02139, USA

The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK

Copyright © 2016 Elsevier Inc. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions.

This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.

To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein.

The cover image for this book was inspired by the Italian Association of Developmental and Comparative Immunobiology (IADCI) logo.

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library

Library of Congress Cataloging-in-Publication Data

A catalog record for this book is available from the Library of Congress

ISBN: 978-0-12-803252-7

For information on all Academic Press publications visit our website at https://www.elsevier.com/

Typeset by TNQ Books and Journals

www.tnq.co.in

List of Contributors

Luigi Abelli,     University of Ferrara, Ferrara, Italy

Vizzini Aiti,     University of Palermo, Palermo, Italy

Claudio Alimenti,     University of Camerino, Camerino, Macerata, Italy

Vincenzo Arizza,     University of Palermo, Palermo, Italy

Loriano Ballarin,     University of Padova, Padova, Italy

Silvia Battistella,     University of Trieste, Trieste, Italy

Kornélia Bodó,     University of Pécs, Pécs, Hungary

Francesco Buonocore,     University of Tuscia, Viterbo, Italy

Paola Caicedo,     CIDEIM, Cali, Valle del Cauca, Colombia

Matteo Cammarata,     University of Palermo, Palermo, Italy

Laura Canesi,     University of Genova, Genova, Italy

Heather Coatsworth,     Simon Fraser University, Burnaby, BC, Canada

Maria R. Coscia,     Institute of Protein Biochemistry, National Research Council of Italy, Naples, Italy

Małgorzata Cytryńska,     Maria Curie-Sklodowska University, Lublin, Poland

Parrinello Daniela,     University of Palermo, Palermo, Italy

Magda de Eguileor,     University of Insubria, Varese, Italy

Stefania Domeneghetti,     University of Padova, Padova, Italy

Francesco Drago,     University of Lille – Science and Technology, Villeneuve D’Ascq, France

Péter Engelmann,     University of Pécs, Pécs, Hungary

Antonella Franchini,     Modena and Reggio Emilia University, Modena, Italy

Nicola Franchi,     University of Padova, Padova, Italy

Michela Furlan,     University of Trieste, Trieste, Italy

François Gagné,     Emerging Methods Section, Aquatic Contaminants Research Division, Environment Canada, Montréal, QC, Canada

Marco Gerdol,     University of Trieste, Trieste, Italy

Stefano Giacomelli,     Institute of Protein Biochemistry, National Research Council of Italy, Naples, Italy

Piero G. Giulianini,     University of Trieste, Trieste, Italy

Annalisa Grimaldi,     University of Insubria, Varese, Italy

Yuya Hayashi

Aarhus University, Aarhus, Denmark

Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany

Teresa Jakubowicz,     Maria Curie-Sklodowska University, Lublin, Poland

Christophe Lefebvre,     University of Lille – Science and Technology, Villeneuve D’Ascq, France

Simonetta Lorenzon,     OGS (National Institute of Oceanography and Experimental Geophysics), Sgonico (TS), Italy

Carl Lowenberger,     Simon Fraser University, Burnaby, BC, Canada

Pierangelo Luporini,     University of Camerino, Camerino, Macerata, Italy

Davide Malagoli,     University of Modena and Reggio Emilia, Modena, Italy

Annalaura Mancia,     University of Ferrara, Ferrara, Italy

Chiara Manfrin,     University of Trieste, Trieste, Italy

Valerio Matozzo,     University of Padova, Padova, Italy

Cammarata Matteo,     University of Palermo, Palermo, Italy

László Molnár,     University of Pécs, Pécs, Hungary

Parrinello Nicolò,     University of Palermo, Palermo, Italy

Clara Ocampo,     CIDEIM, Cali, Valle del Cauca, Colombia

Umberto Oreste,     Institute of Protein Biochemistry, National Research Council of Italy, Naples, Italy

Enzo Ottaviani,     University of Modena and Reggio Emilia, Modena, Italy

Alberto Pallavicini,     University of Trieste, Trieste, Italy

Maria G. Parisi,     University of Palermo, Palermo, Italy

Carla Pruzzo,     University of Genova, Genova, Italy

Giuseppe Scapigliati,     University of Tuscia, Viterbo, Italy

Domenico Schillaci,     University of Palermo, Palermo, Italy

Cole Schonhofer,     Simon Fraser University, Burnaby, BC, Canada

Marco Scocchi,     University of Trieste, Trieste, Italy

Nidhi Sharma,     University of Padova, Padova, Italy

Gianluca Tettamanti,     University of Insubria, Varese, Italy

Adriana Vallesi,     University of Camerino, Camerino, Macerata, Italy

Gerardo R. Vasta,     University of Maryland School of Medicine, Baltimore, MD, United States

Paola Venier,     University of Padova, Padova, Italy

Jacopo Vizioli,     University of Lille – Science and Technology, Villeneuve D’Ascq, France

Iwona Wojda,     Maria Curie-Sklodowska University, Lublin, Poland

Preface

Animals constitute the greatest part of eukaryotic biodiversity, with more than 2 million known species grouped in approximately 35 phyla.

The exploitation of only a limited part of this great variety of species was fundamental for the advancement of immunobiology, starting with the original experiments of Elie Metchnikoff with sea star larvae, which posed the basis for the phagocytosis theory, up to the recent studies on Toll receptors in Drosophila by Jules A. Hoffmann, which led to comprehension of the role of Toll-like receptors in innate immunity.

Animals have evolved a wide range of approaches to cope with foreign, potentially pathogenic organisms, and today, the increasing need to extend our knowledge of immune responses requires new, suitable, and simple model organisms for the study of the variety of defense strategies present in metazoans, besides the few species investigated so far. This is of general biological interest and might reveal new adaptive solutions and unknown recognition and effector mechanisms, useful for further progresses in immunology. It also has an applied interest, as a solid knowledge of invertebrate immunity is fundamental for setting up biological methods to control invertebrates, which are vectors of diseases or pests for crops. In addition, a better knowledge of the immune system of farmed species can be of great help in the optimization of the rearing strategies in cattle breeding and aquaculture. Furthermore, focusing on mammals only, as most immunologists do, does not allow for the study of the evolution of immune defense and host–parasites coevolution.

This book stems from the activity of the Italian Association of Developmental and Comparative Immunobiology, born in 1997, which coordinates the activity of many research teams on the common theme of immune responses in no mouse, no man models through periodic scientific meetings. We invited part of its members and other international collaborators with consolidated competence in their respective fields to write on specific aspects of their research. The outcome is a series of short chapters (lessons), in the form of overviews, in which a wealth of new and reviewed information, concerning many aspects of immunity in invertebrates and vertebrates, can be found. The aim is to provide scientists and teachers with an easy and updated tool reporting, in an evolutionary perspective, the state of the art in relevant fields of immunobiology.

We thank all the authors and reviewers for their important contributions and their patience with the process that, finally, brought this book to fruition.

Matteo,  and Loriano

Chapter 1

Ciliate Pheromones

Primordial Self-/Nonself-Recognition Signals

Adriana Vallesi, Claudio Alimenti,  and Pierangelo Luporini     University of Camerino, Camerino, Macerata, Italy

Abstract

As is common among multicellular life forms, single-cell organisms also use pheromones to communicate among members of the same species. In protist ciliates, pheromones have been identified in functional association with mating systems. Consistent with this association, they have been regarded only as nonself-signals committed to eliciting a mating response of cells to which they bind in paracrine-like (heterologous) fashion. However, their spectrum of activity has revealed wider borders. It also includes a self-activity, which promotes the vegetative growth of the same cells from which pheromones are secreted and to which they continuously bind in autocrine fashion. This double self- and nonself-activity is made possible by the pheromones’ ability to compete with one another in cell-binding reactions. In the Euplotes species, which synthesizes pheromone families under the control of multiple series of alleles at a single locus, this ability is ensured by the relationships of the structural homology that link these molecules into species-specific globular, disulfide-rich protein families having a common three-helix fold.

Keywords

Cell–cell communication; Ciliate mating and growth; Diffusible signaling proteins; Globular helical proteins; Protein–protein interactions

Introduction

Independently of their prokaryotic or eukaryotic nature, cells are evolutionarily driven to socialize to acquire the temporary or stable multicellular organization necessary to increase their size and engage in functional specialization and partition of labor.¹ This ancient impulse to socialize implies a very early evolution of the cell’s ability to discriminate between self and nonself, which is a sine qua non condition for pursuing effective cell–cell communication, cooperation, and structure–function integration. In its most basic aspects, this ability is primarily directed to the recognition of self-signaling molecules, as exemplified by the quorum-sensing phenomena in which bacteria perceive their environmental population densities and activities and, consistently, vary their behavior and metabolism.²,³ It then undergoes vast functional diversification among the unicellular eukaryotes in which it governs impressive phenomena of cell aggregation based on kin discrimination in social amoebas.⁴,⁵ Its maximal complexity is eventually shown by animals with the evolution of innate and adaptive immunity systems that are primarily directed to recognizing and processing nonself molecules for the defense of body integrity.⁶,⁷

This chapter focuses on the biology and structure of diffusible protein signals, nowadays known as pheromones and earlier as mating-type factors/substances or gamones, that control self/nonself recognition in protist ciliates. Together with Dinoflagellates and Apicomplexa, ciliates (Ciliophora) form the group of Alveolata, which clusters with those of Stramenopiles (diatoms) and Rhizaria (foraminifers and radiolarians) into the so-called SAR supergroup.⁸

Pheromone Identification

Ciliate pheromones are the chemical markers of genetically distinct vegetative cell classes—only two of the same sex in some species or multiple with indefinite numbers in others⁹,¹⁰—that have been described as mating types because their mixing determines a ciliate-specific mating phenomenon of conjugation. Ciliate conjugation is a sexual phenomenon as it involves a temporary union in mating pairs between cells, which mutually exchange gamete nuclei, undergo fertilization, and develop new micro- and macronuclei from the synkarya. However, it has nothing to do with the general phenomena of fertilization between gametes of opposite sexes. One major reason for opposing this parallelism is that many species of ciliates may form mating pairs between genetically different cells (heterotypic pairs), as well as between genetically alike (clonal) cells (homotypic mating pairs). Although destined to generate self-fertilization, these homotypic pairs are fully fertile (the Blepharisma case excepted) just like the heterotypic ones.

It is precisely by virtue of this unique capability of ciliates to form homotypic (intraclonal or selfing) mating pairs that ciliate pheromones were identified more than half a century ago.¹¹ By investigating mating interactions in Euplotes patella—the first Euplotes species used to study the Mendelian genetics of ciliate multiple mating systems controlled by a series of alleles codominantly expressed at a single genetic locus (annotated as mat locus)—Kimball¹¹ observed that an experimental condition sufficient to induce the formation of homotypic mating pairs was the simple suspension of cell cultures with cell-free filtrates from other cultures of different mating types. From this observation it became evident that in E. patella, and ciliates in general, the mating-type factors (pheromones) can be freely released into the extracellular environment and that the presence of these molecules in solution can be promptly detected by assaying the mating-induction activity of cell-free filtrates.

By applying this mating-induction assay to cell-free filtrates, pheromones have been identified in species of Blepharisma, Dileptus, Oxytricha, Ephelota, and Tokophrya in addition to other Euplotes species (Refs 12, 13, as reviews). However, studies of structure–function characterization have been carried out to varying degrees of complexity only on pheromones isolated from Blepharisma japonicum, Euplotes raikovi, Euplotes octocarinatus, Euplotes nobilii, and Euplotes crassus. These studies are reviewed in this chapter with particular attention to the cross-reactions that some Euplotes pheromones show with the signaling system components of more modern organisms.

Pheromone Structures

The first two pheromones to be purified and structurally characterized belong to the pink-colored freshwater ciliate, B. japonicum. Although they are of two mating types (I and II) defined as complementary, of which nothing is known about their genetic determination, their molecular structures are chemically unrelated. The pheromone isolated from the type-I cells (originally designated as gamone 1 or blepharmone) is an unstable glycoprotein of 272 amino acids and 6 N-linked sugars.¹⁴ It is particularly rich in polar and aromatic amino acids (Asn 10.3%, Ser 10.3%, Tyr 8.1%, Trp 5.1%), synthesized in the form of a cytoplasmic precursor, and active in inducing mating between type-II cells at picogram concentrations. In contrast, the second pheromone distinctive of type-II cells (also designated as gamone 2 or blepharismone) is a very stable tryptophan derivative, namely a calcium-3-(2′-formyl-amino-5′-hydroxybenzoyl) lactate.¹⁵ This has also been obtained by chemical synthesis and is active at nanomolar concentrations in attracting type-I cells besides those for inducing their mate.¹⁶

Quite different is the picture that emerges for the pheromone structures determined in four species of Euplotes, namely E. raikovi, E. nobilii, E. octocarinatus, and E. crassus. In all these species, pheromones have been shown to form species-specific families of structurally homologous proteins. Among these pheromone families, a more in-depth structural knowledge has been obtained for E. raikovi and E. nobilii, which secrete comparatively larger amounts of pheromones. Up to 200  μg of pure protein per liter can be recovered from E. raikovi cell-free filtrates.¹⁷ This has greatly facilitated chemical and genetic approaches to the determination of significant numbers of pheromone primary amino acid sequences. It subsequently opened up the way to NMR and crystallographic analyses of the pheromone three-dimensional structures based on the use of proteins at a natural isotope composition purified from cell-free filtrate preparations.

In E. raikovi, nine distinct amino acid pheromone sequences in their form of cytoplasmic precursors (pre/propheromones) and six three-dimensional structures of the secreted molecules are known¹⁸–²⁴ (Fig. 1.1). In contrast to the tight structural conservation of the strongly hydrophobic presegment (or signal peptide) of 19 amino acids and (to a slightly lesser extent) of the prosegment of 16–18 amino acids, the secreted pheromones of 37–40 amino acids (51 only in pheromone Er-23) show minimal sequence conservation. Only the positions of six cysteines are fully conserved, and the sequence dissimilarity between any two pheromones may be as high as 74%. In any case, despite this wide variability in the amino acid sequence, all the pheromones (pheromone Er-23 in part excepted) mimic one another in their three-dimensional conformation. This is based on a fold of three nearly parallel, right-handed α helices (increased to five with the addition of two single-turn helices in Er-23) that remain tightly associated with each other by the close proximity and strategic disposition of their disulfide bonds. As is usually required for waterborne signaling molecules that must maintain long-term activity in the environment, the stability of the globular conformation of E. raikovi pheromones is such that even heating these proteins up to 95°C does not cause unfolding and disruption of their secondary structures.²⁵ However, in spite of showing closely comparable molecular architectures and equivalent stabilities, each pheromone has its own unmistakable cell type structural earmarks that are likely deputed to confer specificity to its signaling activity. These earmarks are imposed by the evolution of variations that are most apparent for the shape, extension, and spatial arrangement of the carboxy-terminal domain, as well as for the geometry of the interhelix loops of the molecules.

Figure 1.1   Euplotes raikovi pheromone family. (A) Alignment of nine distinct sequences of pheromone precursors, as deduced from their DNA-coding sequences. Sequence alignment is based on the Clustal W algorithm and optimized manually by gap insertions. Residues conserved among all sequences, or all but one sequence, are shadowed. The positions of the cysteine residues are highlighted in bold. The numbers of residues of the pre, pro, and secreted regions are reported on the right of the sequences. (B) Ribbon presentations (as visualized by the program MOLMOL) of the six pheromone structures (side view) that have been determined by NMR analysis of native protein preparations. The Protein Data Bank codes are as follows: 1erc (E r -1), 1erd (E r -2), 1erp (E r -10), 1ery (E r -11), 1hd6 (E r -22), and 1ha8 (E r -23). The amino and carboxyl termini of each molecule are labeled N and C, respectively, and the sulfur atoms involved in the disulfide bonds are indicated as spheres. The three more regular α-helices common to all six molecules are labeled h1–h3 starting from the N -terminus.

Figure 1.2   Euplotes nobilii pheromone family. (A) Alignment of seven distinct sequences of pheromone precursors, as deduced from their DNA-coding sequences. (B) Ribbon presentations (as visualized by the program MOLMOL) of the four pheromone structures that have been determined by NMR analysis of native protein preparations. The Protein Data Bank codes are as follows: 2nsv (E n -1), 2nsw (E n -2), 2jms (En-6), and 2kk2 (En-A1). Indications and symbols as in Fig. 1.1 .

The overall structural organization of E. nobilii pheromones strictly matches the E. raikovi pheromone organization with the close phylogenetic kinship that links these two species.²⁶,²⁷ Nevertheless, E. nobilii pheromones, for which seven amino acid sequences of the precursor forms²⁸ and four NMR structures have been determined²⁹–³¹ (Fig. 1.2), have longer sequences (from 52 to 63 amino acids), include eight instead of six cysteines, and, more importantly, show structural specificities that appear closely correlated with the different ecology that distinguishes E. nobilii from E. raikovi. While E. raikovi thrives in temperate waters, E. nobilii has colonized the Antarctic and Arctic seas.³²,³³ Hence, E. nobilii pheromones are cold-adapted, psychrophilic proteins. With respect to E. raikovi pheromones, their overall contents of polar and hydrophobic amino acids are markedly different, most likely in functional correlation with their improved interactions with the solvent.³⁴ They are much richer in Thr (11.7% vs 5.7%), Asn (7.7% vs 4.2%), and Ser (8.6% vs 5.7%) residues and poorer in Leu (1.1% vs 7.3%), Pro (4.7% vs 8.9%), and Ile (1.6% vs 5.7%). Structurally more apparent are, however, the reduced extensions that E. nobilii pheromones show for the three helices of the molecular architecture with respect to E. raikovi pheromones. These reductions determine a spatial preponderance of regions devoid of a regular secondary organization, implying that the activity of E. nobilii pheromones in the thermodynamically adverse environment of the polar waters is facilitated by an improved flexibility of their molecular backbones. Indeed, E. nobilii pheromones have been measured to be much less thermostable than E. raikovi pheromones, unfolding upon heating to only 55–70°C.²⁵–³⁵

Much less information is available on the structures of E. octocarinatus and E. crassus pheromones for which only the amino acid sequences are known on the basis of their respective DNA-coding sequences. The determination of three-dimensional conformations of these pheromones has been made impractical by the very low concentrations in which they are secreted: 0.5  μg of pure protein per liter of cell-free filtrate are usually obtained in E. octocarinatus and 15–20  μg in E. crassus.³⁶,³⁷

Nine distinct pheromone amino acid sequences have been determined in E. octocarinatus (Fig. 1.3). They are twice as long as the E. raikovi and E. nobilii pheromone sequences, extending from 85 to 109 amino acids with 10 cysteines (8 in the pheromone Phr4).³⁸,³⁹ The positions of only 4 of the 10 cysteines have counterparts in E. raikovi and E. nobilii pheromones, implying that the E. octocarinatus pheromones have only two disulfide bonds and few structural motifs in common with those of E. raikovi and E. nobilii. They also appear to be unique among Euplotes pheromones in functioning as chemoattractants between cells of different mating types.⁴⁰

In E. crassus, pheromones have been thought to be represented by insoluble membrane-bound proteins³⁷,⁴¹ and as such are difficult to extract and characterize from cell membrane preparations. Evidence that E. crassus pheromones are, instead, constitutively secreted into the extracellular environment as in other Euplotes species was obtained from interspecific mating-induction assays,³⁷ suggested by the notion that cross-mating reactions are relatively frequent between Euplotes species in correlation with their high-multiple (virtually open) mating systems.⁹,⁴² The genetic control of these E. crassus pheromones appears not to conform with the general Euplotes pattern provided by multiple series of alleles at a single mat locus. Results from molecular approaches have been found to be more consistently explained by assuming that this control involves a phenomenon of mat gene duplication responsible for the production of two distinct E. crassus pheromone subfamilies. One includes the cell type-specific pheromones, and the other includes a new pheromone species that appears to be synthesized in common among cells of compatible mating types.³⁷,⁴¹ The functions of this shared pheromone, of which only a single 56 amino acid sequence with eight cysteines (designated Ec-α) has been determined, are still obscure. Preliminary evidence suggests that it may behave like an adaptor or scaffold protein that selectively interacts with the cell type-specific pheromones to mediate and/or reinforce their receptor-binding reactions.⁴¹ This possibility is supported by an unusually strong propensity of the Ec-α pheromone to oligomerize when it is purified by chromatographic separation and is reinforced by the comparison of its sequence with the three 45 amino acid sequences with 10 cysteines (designated Ec-1, Ec-2, and Ec-3) that have been determined for the subfamily of the cell type-specific pheromones (Fig. 1.4). This comparison makes it evident that a major distinctive structural trait of the Ec-α pheromone is the evolution of an unusually extended hydrophilic domain lying centrally in the molecule. This domain lacks Cys residues, abounds in Gly residues, and likely takes a random-coil conformation, which is presumptive for a specific capability of the Ec-α pheromone to interact with the other proteins.

Figure 1.3   Euplotes octocarinatus pheromone family. Alignment of nine distinct sequences of pheromone precursors, as deduced from the determination of their DNA-coding sequences. Indications as in Fig. 1.1 .

Figure 1.4   Euplotes crassus pheromones. Multiple alignments of four distinct sequences of pheromone precursors, as deduced from their DNA-coding sequences. Three sequences (E c -1, E c -2, and E c -4) are cell type specific and one (E c -α) is shared identically among different cell types. Indications as in Fig. 1.1 .

Pheromone Activity

As outlined earlier, it is essentially due to the application of mating-induction assays that ciliate pheromones have been identified. This application has thus decisively contributed to supporting the generalization that ciliate pheromones function exclusively as sexual factors committed to stimulating cell mating by binding in paracrine (or heterologous) fashion only to cells other than those from which they are synthesized and secreted.¹² However, observations on the physiology of pheromone secretion in E. raikovi turned out to counter this view, because cells were shown to secrete their pheromones throughout their life cycle independently of their inability to mate during the immaturity stage to vary their rates of pheromone secretion in relation to the environmental concentrations of their pheromone and to refrain from forming homotypic mating pairs in the presence of environmental excess of their pheromone.⁴³ A more coherent view with these observations thus appeared to be one predicting that ciliate pheromones, in addition to acting as nonself-mating signals, also (and most likely, primarily) act as self-signals committed to promoting the vegetative proliferation (mitotic growth) of their same source cells to which they bind in autocrine (or autologous) fashion.⁴³,⁴⁴

A decisive support for an autocrine, cell growth-promoting activity of ciliate pheromones was provided by the finding that E. raikovi cells bind their pheromones to membrane-bound isoforms that arise by a splicing mechanism of intron-like sequences from the same genes encoding the pheromones in the transcriptionally active cell macronucleus.⁴⁵,⁴⁶ These isoforms (functionally regarded as pheromone receptors) incorporate the full sequence of the cytoplasmic pheromone precursor and, using the signal (pre) peptide (normally expected to be proteolytically removed before the pheromone secretion) as transmembrane-anchoring domain, take a spatial orientation typical of membrane proteins of type-II, which have the C-terminal end directed to the cell outside and the N-terminal end directed to the cell inside. Given this structural equivalence between pheromones and extracellular binding domains of the twin receptor molecules, the way pheromones interact with their receptors on the cell surface has been thought to be fully mimicked by the protein–protein interactions that determine the molecular packing of the E. raikovi pheromone Er-1 into crystals.²² In the bidimensional plane of the crystal, the Er-1 molecules (that may be figured as pyramids with a triangular base) associate with one another by means of all three of their faces (a, b, and c delimited by helices 1 and 2, 2 and 3, and 1 and 3, respectively) through the cooperative utilization of initially weak interactions that arise from the formation of two distinct types of dimer. One of these involves face a of each monomer and is symmetrical with the two monomers related by a twofold axis; the other involves faces b and c and is asymmetrical with the two monomers related by a twofold screw axis (Fig. 1.5). This association is such that half of the molecules direct their carboxy-terminus to one side of the plane and may thus reflect the spatial orientation of the pheromone receptors on the cell surface. The second half of the molecules, instead, direct their amino-terminus to the opposite side of the plane and may thus be regarded as free pheromone molecules that bind to their receptors. In line with a cooperative model of protein–protein interactions, the homotypic pheromone/pheromone receptor complexes formed by cells, which grow suspended with their own (self) pheromone, have been observed to undergo clustering and internalization via endocytotic vesicles, whereas the heterotypic complexes formed by cells that have been suspended with a nonself-pheromone for being induced to mate are not internalized and are believed to be in some way involved in bridging cells into mating pairs.⁴⁷

Figure 1.5  Intermolecular helix–helix interactions in the crystal structure of the Euplotes raikovi pheromone E r -1. (A) Structure of the symmetrical dimer 1 in which two molecules are related by a twofold axis (→). Each monomer involves helices 1 and 2, delimiting face a ). Each monomer stacks helix 3, and its adjacent faces b and c , in an antiparallel fashion to form a linear structure without symmetrical contacts. The E r -1 molecule packing into the crystal lattice is shown in the boxed diagrammatic presentation. Shadowed molecules mimic ligand-binding (pheromone receptor) molecules oriented with their C-terminus toward the cell outside, while light molecules mimic soluble pheromone molecules oriented with their C-terminus toward the cell inside. After