i Rupture Mechanisms of Mucous Vesicles from the Slime of Pacific Hagfish (Eptatretus stoutii): Functional Properties of Mucin-like Glycoproteins By Shannon N. Ferraro A Thesis Presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Integrative Biology Guelph, Ontario, Canada © Shannon N. Ferraro, January 2016 ii ABSTRACT RUPTURE MECHANISMS OF MUCOUS VESICLES FROM THE SLIME OF PACIFIC HAGFISH (EPTATRETUS STOUTII): FUNCTIONAL PROPERTIES OF MUCIN-LIKE GLYCOPROTEINS Shannon N. Ferraro Advisor: Univeristy of Guelph, 2015 Dr. D.S. Fudge Pacific hagfish (Eptatretus stoutii) produce copious amount of viscous slime when physically threatened or agitated. The slime is composed of threads (produced by gland thread cells) and glycoproteins (produced by gland mucous cells) which interact to form an integrated gel network that acts as a defence against gill-breathing predators. This thesis investigates the mechanisms that drive vesicle swelling and slime formation. I tested two hypotheses, the Hofmeister hypothesis and the cationic gel hypothesis. The Hofmeister hypothesis predicts that swelling depends on the Hofmeister properties of the solutes in solution; “kosmotropes” stabilize proteins while “chaotropes” solubilize proteins and should cause swelling. My findings were not consistent with these predictions; my results show that swelling occurs even in the presence of strong kosmotropes. I also found that solutions containing multi-charged anions stabilized the glycoproteins and monovalent anions induced rapid swelling. The cationic gel hypothesis states that the glycoproteins are positively charged and are stabilized in vivo by multivalent anions. This hypothesis predicts that altering the ionization state of ions in solution should alter the swelling response. Indeed, this is what I found. Overall, these results are consistent with the idea that the glycoproteins are positively charged although this hypothesis needs to be tested more thoroughly before it can be accepted. iii ACKNOWLEDGEMENTS This project was made possible with the help and encouragement of several people. I would like to thank my advisor, Dr. Doug Fudge, for guiding me to become both a meticulous and creative researcher. I’d like to thank my advisory committee members Dr. Pat Wright and Dr. Janet Wood for their assistance and advice. I owe a big thank you to Dr. Andreas Heyland and André Hupé for their help and advice regarding statistical analyses. I also thank Mike Davies and Matt Cornish at the Hagen Aqualab for their care of the hagfish. I thank my collaborators, Jonathan Krieger (McMaster Children’s Hospital), Alex Clifford (University of Alberta), and Greg Goss (University of Alberta). I owe a thank you to Ryne Herkimer and Guylaine LaRochelle, who volunteered their time for video analysis and data collection. I would also like to thank the people who assisted me with experimental techniques, brainstormed ideas with me, and have given me moral support. These people include Sarah Schorno, André Hupé, Jean-Luc Stiles, Julia Herr, Tegan Williams, Gillian Priske, Elizabeth Johnston, Elizabeth Sears, Laura Dindia, Angela Safko, Tim Clark, Dr. Todd Gillis, Dr. Oualid Haddad, and Dr. Atsuko Negishi. Lastly, I would like to thank the hagfish for their time and their slime. iv TABLE OF CONTENTS List of Tables………………………………………………………………………….…...……. vi List of Figures…………………………………………………………………………..………. vii List of Abbreviations…………………………...……………………………………….…..… viii 1.0 Introduction……………………………………...………………..…………………..…...…. 1 1.1 Biology of hagfishes……………………………………………………………………. 2 1.2 Hagfish slime components and slime formation………………..……………………...... 3 1.2.1 Mucin vesicle composition and deployment……….……………………...... 4 1.2.2 Membrane pores and ion channels…………………………..………..….…. 6 1.3 The swelling of hydrogels………..……………………………………………..…….… 8 1.3.1 Electrostatic repulsion of polyelectrolyte gels……………………………… 8 1.3.2 The jack-in-the-box hypothesis for gel expansion…………...….………….. 9 1.3.3 Ionic influence in the swelling of gels ...………………….……….………. 10 1.4 The Hofmeister ion series…………………………………………………..………... 10 1.5 Thesis objectives…………………………………………………………….….…..... 12 2.0 Methods and Materials…………………………………………………………….……....... 14 2.1 Chemicals………………………………………….……………………………….… 15 2.2 Animals, anaesthesia, and slime collection………..……..………….………………..15 2.3 Mucin swelling assay………………….………………………………………..…… 16 2.3.1 Hofmeister ion series….…………..………….…………….……..……… 17 2.3.2 Ionization state adjustment ………………..……..……..………….….…. 18 2.4 Mucin titration………………………..…………………………………………...… 19 v 2.4.1 Gel fraction isolation………………………...…………………..……… 19 2.4.2 Glycoprotein gel dialysis…………….............……………………..…… 19 2.4.3 Glycoprotein gel solubilization…………...…….……….…….….…..…..19 2.4.4 Base titration…………………...…………...….………………...……… 20 2.5 Statistical analysis……………………….………………………………………..… 20 3.0 Results…………………………………………………………………..………………… 22 3.1 Hofmeister effects do not explain mucin granule swelling and stabilization ......……23 3.2 Effects of anionic charge on vesicle swelling……...……………………..….…….... 23 3.3 Analysis of titratable groups…………….……………..…...……………………….. 24 4.0 Discussion………….……………………………...……………………………………..… 26 Literature Cited…..…………………………………………………………………………….. 35 Tables ………………….……………………………………………………………………… 46 Figures ……………………...…..……………………………………..………………….…… 54 vi LIST OF TABLES Table 1.1: The Hofmeister series of ions and other solutes……………………………………..46 Table 2.1: Final ion concentrations of Hofmeister solutions……………………………………47 Table 2.2: Final ion concentrations of pH series solutions……………………………………...49 Table 3.1: The salts used to test the Hofmeister hypothesis, organized from most to least stabilizing, and their effects on hagfish mucus. …………………………………………….…..53 vii LIST OF FIGURES Figure 1.1: Slime gland pores on a hagfish……….……………………………………………..54 Figure 1.2: Main cell types in hagfish slime gland exudate ...…………..…………….….…….55 Figure 1.3: Holocrine secretion of exudate from the hagfish slime glad pore……………….....56 Figure 3.1: Effects of pH on granule swelling in sodium solutions of citrate, sulphate, phosphate, and carbonate……………………………………………………………………….……….......57 Figure 3.2: Minor swelling effects in sodium salt solutions of citrate, sulphate, phosphate, and carbonate above pH 10……………..……………………………………..………………….….58 Figure 3.3: Estimation curves based on a logistic curve modelling the granule swelling behaviour due to changes in pH of sodium solutions of citrate, sulphate, phosphate, and carbonate.......................................................................................................................................59 Figure 3.4: Estimated vs. theoretical transition pH values for each sodium solution of citrate, sulphate, phosphate, and carbonate ………………………………….……………………….....60 Figure 3.5: Titration of mucus glycoproteins……………………..……………………….….....61 Figure 4.1: Abundance of anionic species in solution of varying valencies plotted against granule swelling data……………………………………….…………………………………………….62 viii LIST OF ABBREVIATIONS ANOVA Analysis of variance ASW Artificial seawater ATP Adenosine triphosphate AQP Aquaporin DIC Differential interference contrast DTT Dithiolthreitol ECARS Environmentally controlled aquatic recirculation systems ESAQP3 Eptatretus stoutii aquaporin 3 ESAQP4 Eptatretus stoutii aquaporin 4 GMC Gland mucous cell GTC Gland thread cell IAA Iodoacetamide IPG Immobilized pH gradient IEF Isoelectric focusing MLD Mucin-like domain MLP Mucin-like protein MW Molecular weight PIPES Piperazine-N,N'-bis(ethanesulfonic acid) SB Stabilization buffer SDS Sodium dodecyl sulphate TCEP Tris(2-carboxyethyl)phosphine 1 INTRODUCTION 2 1.1 Biology of hagfishes Hagfishes (Chordata: Myxinidae) are primitive jawless chordates closely related to sea lampreys that possess a notochord and a cartilaginous skull (Fernholm, 1998; Forey and Janvier, 2000; Potter and Gill, 2003). Hagfishes and lampreys have been the subjects of many evolutionary studies due to their phylogenetic position as the modern archetypes of the agnathan (jawless) stage in vertebrate evolution (Potter and Gill, 2003; Zintzen et al., 2011). Often described as ‘eel-like’, the bodies of hagfishes are elongate and do not possess paired fins (Hardisty, 1979). Hagfishes are gill-breathers, taking water in at the pharynx and pushing it through internal gill pouches (Fernholm, 1998). They have primitive eyespots that can detect light, but are unable to form complex images as they possess neither a lens nor extrinsic musculature (Fernholm, 1998). Hagfishes are benthic creatures and are mainly scavengers, feeding on decomposing organic material which has fallen to the ocean floor or stealing prey captured by other predators (Martini et al., 1997; Fernholm, 1998; Auster and Barber, 200; Zintzen et al., 2011). Predatory behaviour has also been observed, however, by a slender hagfish (Neomyxine sp.) on a red bandfish (Cepola haastii) (Zintzen et al., 2011). Stomach content data also suggest that hagfishes prey on soft-bodied invertebrates (Zintzen et al., 2011). When physically agitated, hagfishes release a slime exudate from slime gland pores located laterally along the entire length of the animal (Fig. 1), which mixes with the surrounding seawater to produce copious amounts of slime (Koch et al., 1991; Janvier, 1996; Fernholm 1998; Winegard and Fudge, 2010). This behaviour is thought to be used as a defence mechanism to ward off gill-breathing predators, as the slime adheres to the gills