Rupture Mechanisms of Mucous Vesicles from the Slime of Pacific Hagfish (Eptatretus Stoutii): Functional Properties of Mucin-Like Glycoproteins

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Rupture Mechanisms of Mucous Vesicles from the Slime of Pacific Hagfish (Eptatretus stoutii): Functional Properties of Mucin-like Glycoproteins

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

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.

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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.

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

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

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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

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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

INTRODUCTION

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 of the predators and reduces water-flow across them, potentially causing suffocation (Lim et al., 2006). As hagfishes are also gill-breathers, the secretion of slime poses a threat to them as well. To avoid suffocating on their 3 own slime, hagfishes are able to tie themselves into a knot, usually beginning at the anterior end of their body, and proceed to move the knot down the length of their body to remove the slime

(Zintzen et al., 2011). Hagfishes also secrete epidermal mucus, but its properties are quite different from the slime. Slime exudate is secreted via the holocrine mode, with entire cells being excreted. Epidermal mucus is released via merocrine secretions; the secretory cells release the mucus by exocytosis into epithelial ducts that lead to the surface of the body. The epidermal mucus is also thought to have antibacterial properties and is used by the hagfish for protection against microbes in its external environment (Ellis, 2001; Subramanian et al., 2008). My thesis research focused on the slime exudate of the Pacific hagfish. In particular, I investigated the swelling mechanisms of the slime vesicles found in the slime exudate secretion.

1.2 Hagfish slime components and slime formation

The slime gland exudate is composed of secretions from two main cell types: gland thread cells (GTCs) and gland mucous cells (GMCs) (Fig. 1.2) (Blackstad,1963; Spitzer and

Koch, 1998). Thread cells generate thread skeins, which are elaborately coiled protein threads, while GMCs produce large quantities of mucin vesicles, which are each bound by a membrane and filled with gel-forming mucin-like glycoproteins (Blackstad, 1963; Koch et al., 1991; Fudge et al., 2009). Slime exudate is released by holocrine secretion from the slime gland, which occurs when the hagfish contracts the muscles surrounding these glands (Downing et al., 1981b; Patzner et al., 1982; Spitzer and Koch, 1998.) The slime gland duct opening is approximately 100 µm in diameter, allowing the passage of only one GMC or GTC at a time (Fernholm, 1981; Downing et al, 1981a; Downing et al., 1981b). The size restriction of the duct causes the cells to be stripped of their membrane during the secretion process, releasing the mucin vesicles and thread skeins into the external environment (Fernholm, 1981). 4

Upon exudate secretion into seawater, the vesicle membranes lyse and release the mucin glycoproteins into the external environment, while the thread cells unravel up to lengths of 10-

17cm. The glycoproteins and thread skeins become entangled via hydrodynamic mixing forces, producing mature slime that traps large volumes of water via viscous entrainment (Fudge et al.,

2005; Koch et al., 1991). The mature slime is composed of 99.996% seawater, with the mucin vesicles and thread skeins each making up 0.002% of the slime (Fudge et al., 2005).

Slime volume is reduced if thread skeins do not unravel provided the mucin vesicles rupture, however no slime will form if the vesicles do not rupture, regardless of whether the thread skeins unravel (Koch et al., 1991; Fudge et al., 2005). Although the thread skeins are vital to the strength and integrity of the mature slime, the mucin vesicles must rupture in order for any slime to form, as they contain the glycoproteins that provide its gel-like properties (Koch et al.,

1991; Fudge et al., 2005). In Atlantic hagfish slime, the thread skeins unravel only in the presence of hydrated mucins, with unravelling being facilitated by the transferring of hydrodynamic forces between these two components (Winegard and Fudge, 2010).

1.2.1 Mucin vesicle composition and deployment

The GMCs are filled with disc-shaped vesicles that are approximately 7µm in diameter along the major axis and are encompassed in a single lipid- bilayer membrane (Luchtel et al.,

1991b). The interior of the vesicle is composed of glycoproteins similar to mucins, which are defined as high- molecular weight glycoproteins that contain O-glycan attachment sites, termed

‘mucin-like domains’ (MLDs) (Thornton and Sheehan, 2004). These glycoproteins typically have a very high carbohydrate content, upwards of 80% of the molecule’s weight in some cases.

Interestingly, this number is closer to 12% in hagfish mucins, making them different from other 5 known mucins (Salo et al., 1983; Spitzer and Koch, 1998). MLDs contain serine and threonine in their peptide core, which act as attachment sites for linkage sugars, usually N- acetylgalactosamine, but N-acetylglucosamine, galactose, fructose, sialic acid, and sulphate may also be present. When the oligosaccharide attaches to the polypeptide, the polypeptide stiffens, causing a volume expansion of the MLDs on the molecule, thus giving mucins their distinctive swelling and gel-forming properties when exposed to water (Thornton and Sheehan, 2004). The formation of mucus involves the interaction of mucins with water, as well as with each other through covalent disulphide bonds and entanglements with neighbouring polymers, forming a complex hydrophilic, viscoelastic polymer gel-network (Verdugo, 1990; Verdugo, 1991).

Although the exact mechanisms of hagfish mucin vesicle deployment remain largely unknown, it seems apparent that ions present in seawater as well as water influx provide vital interactions with the mucin vesicle to aid in its rupture (Luchtel et al., 1991b; Herr et al., 2010;

Herr et al., 2014). Mucous granules or vesicles in other species, such as in slugs, rupture due to certain ion sensitivities of the mucin granules and, in the case of the Pacific banana slug A. columbianus, interact with substances such as adenosine triphosphate (ATP), which induce rupture (Luchtel et al., 1991a). There is also evidence that this interaction with ATP is dependent on calcium-sensitive channels and proton gradients across the mucus granule membrane

(Deyrup-Olsen, 1996). Although the mucous granules in slugs and snails differ structurally from mucin vesicles in hagfish, their modes of deployment may be comparable regarding sensitivities to certain ions and molecules as Herr et al. (2014) found evidence that hagfish slime vesicles possess ion channels that require Ca2+ to be functional.

1.2.2 Membrane pores and ion channels

The permeability of the hagfish mucin vesicle membrane has not been fully characterized, although studies suggest the membrane possesses selective permeability to certain ions and almost certainly contains pores and ion-regulated channels (Salo et al., 1983; Luchtel et al., 1991b; Herr et al., 2010; 2014). Luchtel et al. (1991b) suggest that cations are able to permeate the membrane regardless of their valency, while only monovalent anions can do the same. In the same study, Luchtel et al. found that the vesicles did not rupture when exposed to 1 mol L-1 solutions containing polyvalent anions such as citrate and sulphate, and concluded that the membrane is impermeable to these types of ions. Evidence for calcium-regulated channels was found in this study as zinc, a known inhibitor of calcium channels, had a stabilizing effect on the vesicles (Luchtel et al., 1991; Büsselberg et al., 1994; Gore et al., 2004).

Nguyen et al. (1998) found that mucin vesicles isolated from rabbit goblet cells possess an inositol-1,4,5-trisphosphate sensitive calcium channel as well as an apamine-sensitive calcium-activated potassium channel (where certain ions are exchanged through the membrane).

Hagfish mucin vesicles may possess similar calcium-activated channels, as proposed by Herr et al. (2014) who found that the proportion and speed at which the vesicles ruptured significantly

-1 2+ increased with the addition of as little as 3 mmol·L CaCl2. The presumed Ca -regulated channels would require Ca2+ ions to bind to them to be functional (ie. open). In the absence of

Ca2+, the channels would remain closed and impermeable to the external environment. Once opened, however, these channels would act as non-specific pores, allowing the influx and efflux of any ion or molecule that complies with its size restrictions and charge selectivity (which are currently unknown). According to Herr et al. (2010; 2014), these “Ca2+-activated” channels are thought to be present in approximately 60% of mucin vesicles, which will rupture in 7 hyperosmotic solutions only if Ca2+ is present, while the other 40% of vesicles are “Ca2+- insensitive” and rupture in most solutions. Furthermore, Herr et al. (2014) showed that when the vesicle membrane was dissolved with detergent, this swelling dichotomy was no longer seen; the vesicles swelled at a constant pace. This provides further evidence that the membrane is the source of the differences between the two vesicle types.

Herr et al. (2014) also found that by treating the vesicles with HgCl2, a known aquaporin inhibitor, the swelling rate of the vesicles decreased by an order of magnitude. This suggests that aquaporins may be present in the vesicle membrane and assist in the rupture process. Aquaporins allow between 10 and 100 times more water flux through a membrane in a given amount of time than would be possible in a membrane without them (Agre et al., 2002). Recent work by Herr et al. (2014) provides molecular evidence for the presence of two types of aquaporins, EsAQP3 and

EsAQP4. Furthermore, this molecular evidence suggests close relatedness of EsAQP3 to AQP4 in the inshore hagfish (Eptatretus burger), and relatedness of EsAQP4 to AQP3 in Cope’s gray tree frog (Hyla chrysoscelis) and AQP10a in the spectacled lemur (Trachypithecus obscurus).

However, HgCl2 is also a non-specific thiol-reactive agent, and may inhibit membrane channel proteins other than aquaporins as well (Leach, 1960; Utey et al., 1967; Kimimura and Katoh,

1972; Maggio and Joly, 1995).

Although the swelling behaviour of mucous vesicles from hagfish slime is clearly governed in part by the properties of the vesicle membrane, the properties of the glycoprotein gel itself may be equally important in discovering the mechanisms responsible for vesicle stabilization in the gland and swelling in seawater.

1.3 The swelling of hydrogels

In a network of cross-linked polymers, the behaviour of the gel relies on the elasticity of the molecules and the electrostatic interactions of the system, which depend heavily on the degree of ionization of the polymer, the concentration of the salt solution it is exposed to, and the nature of the free ions in solution (Katachalsky and Michaeli, 1955). Tanaka (1978) observed and characterized the phase shift from a condensed state to a swollen state in polyacrylamide hydrogels; this occurrence is commonly referred to as volume transition, and has also been observed in polyelectrolyte gels in saline solutions (Dusek, 1993; Budtova and Navard 1998; Yin et al., 2009). At swelling equilibrium, the osmotic forces of the glycoproteins and of the free ions in solution are balanced by the network’s contractility (Katachalsky and Michaeli, 1955).

1.3.1 Electrostatic repulsion of polyelectrolyte gels

Early research of charged polymers demonstrates that the degree of ionization in macromolecules influences the viscometric behaviour of polymeric acids (Arnold and Overbeek,

1950; Markovitz and Kimball, 1950; Katchalsky and Eisenberg, 1951). Fuoss et al (1949) showed that when a weak polymeric acid was ionized to a high degree, the viscosity of the solution was over 1000 fold greater than when the polymeric acid only experienced a low degree of ionization. Later studies showed similar trends in polyelectrolyte gels, where changing the pH of the gel solution influenced the transition between swollen and deswollen states (Horbett et al.,

1983; Albin et al., 1990; Dong and Hoffman, 1991).

Polyelectrolytic gels are made up of crosslinked polymers with ionizable side groups attached to the core (Flory, 1953; Chu et al., 1995). In solution, the side groups dissociate, resulting in the network containing charged groups along the polymer chains. These charged 9 groups electrostatically repel each other, causing expansion of the gel network (Chu et al., 1995).

Not surprisingly, gel polymers with greater charge densities will exhibit a greater degree of electrostatic repulsion in solution (Chu et al., 1995; Rubinstein and Colby, 1996). The pH- dependant swelling process of acidic polyelectrolyte gels involves an increase in the electrostatic repulsion force of the network, driven by a decreased concentration of protons in the external solution; the protons dissociate from the acidic polymer side groups resulting in an increased negative charge density of each polymer, tipping the electrostatic balance of the network, resulting in swelling of the gel (Chu et al., 1995; Rubinstein and Colby, 1996).

1.3.2 The jack-in-the-box hypothesis for gel expansion

The interactions between hagfish slime glycoproteins and free ions are vital to the formation of mature slime in seawater (Herr et al., 2010; 2014). Verdugo et al. (1987) and

Verdugo (1991) suggest that exocytosed mucin granules rupture due to a ‘jack-in-the-box’ mechanism, in which mucin polymers swell rapidly due to electrostatic repulsion. This hypothesis proposes that calcium and other polyvalent cations act as charge-shielding and cross- linking ions between the negatively charged mucin molecules, allowing them to exist in a condensed state within the vesicle. When exposed to seawater or a solution containing monovalent cations, the Ca2+ ions are exchanged for cations (such as sodium) that are less effective at charge-shielding and unable to cross-link anionic functional groups, causing sudden increase in electrostatic repulsion among mucin molecules and rapid swelling (Verdugo et al.

1987). This hypothesis is supported by observations of the release of Ca2+ from the mucous vesicles of the Pacific banana slug Ariolimax columbianus before and during swelling, suggesting that the loss of Ca2+ from the vesicles prompts the expansion of the mucin network

(Verdugo et al. 1987). 10

1.3.3 Ionic influence in the swelling of gels

The degree to which a glycoprotein gel will swell is determined by the free energy of the gel network, which is dictated by the composition, temperature and osmotic pressure of the experimental solution (Vasheghani-Farahni et al., 1990). Horkay and Basser (2003) demonstrated a hierarchy of ionic interaction strength with polyelectrolyte gels according to specific ionic groupings. Alkaline earth metals such as Ca2+, Sr2+ and Ba2+ and transition metals such as Co2+ and Ni2+ were found to have stabilizing effects on the gel, presumably by forming bridges within the polymer network. However, these bridges were weak and easily broken.

Trivalent cations such as La3+ and Ce3+ also form stabilizing bridges within the gel, however these bridges are much stronger due to the greater binding affinity of these ions to the polymers.

Alkali metal ions (Li+, Na+ K+) did not have any stabilizing effect on the gel. The destabilizing ions observed by Horkay and Basser (2003) are also classified as protein destabilizers in the

Hofmeister series - a spectrum of solutes categorized by their ability to precipitate or destabilize proteins (Kunz et al., 2004).

1.4 The Hofmeister Ion Series

The Hofmeister series was proposed in 1888 by Franz Hofmeister who characterized the interactions between globulin proteins from hen eggs and several salts, providing the scientific community with an important development in electrolyte chemistry due to its broad application value (Kunz et al., 2004; Zhang and Cremer, 2006; 2010). Hofmeister categorized various ions according to their ability to precipitate the globulins (Table 1). As defined by Hofmeister,

“kosmotropic” species increase protein aggregation and stability, a process commonly referred to as “salting-out”. “Chaotropic” ions have the opposite effect and are thought to promote protein solubility by disrupting hydrogen bonds between water molecules, weakening the hydrophobic 11 effect of proteins in solution. This process is termed “salting in” (Baldwin, 1996; Collins, 2004;

Kunz et al., 2004; Zhang et al., 2005).

Although the Hofmeister series has received considerable attention due to its broad relevance, ranging from the behaviour of protein interactions, protein stability and crystallization, and enzyme activity, the operative mechanism remains largely unknown (Collins

2004; Perez-Jimenez et al., 2004; Broering and Bommarius, 2005; Pinna et al., 2005; Vrbka et al., 2006; Zhang and Cremer, 2008). The effects of anions seem to be more consistently arranged within the series, while cation effects fit much more loosely and do not appear to have a definitive ranking order (Weissenborn and Pugh, 1995; 1996). Hofmeister effects are additive and become stronger with increasing ion concentration (Broering and Bommarius, 2005).

Pegram and Record (2007) observed that a salt’s anion influences the effects of the cation. When part of a chloride salt, the precipitation strength of Na+ is stronger than K+ and Li+, both of which have near equal ranking; however, when these cations are part of a sulphate salt, the opposite is true. Furthermore, in iodide salts, the effects of K+ and Na+ are both stronger than Li+.

Additionally, the observations that Ca2+, Ba2+, and Li+ stabilize proteins whereas Na+ and K+ do not contradict the typical order of the Hofmeister series.

Along with organization and ranking order, the interactions of the ions on both bulk water and macromolecules are largely undefined. Multiple studies propose that Hofmeister ions do not interact with the structure of water molecules or bulk water characteristics such as surface tension or viscosity, but instead interact only with the molecules in solution to influence protein stability (Kropman and Bakker, 2003; 2004; Omta et al., 2003). However, several other studies suggest that the ionic interactions that disrupt hydrogen bonding between water molecules and 12 alter the surface tension of the solution contribute significantly to the mechanism (Collins 2004;

Marcus, 2009; Xie et al., 2013; Xie and Gao, 2013).

1.5 Thesis Objectives

In this study, I asked the following questions: what mechanism drives the rupture of hagfish slime vesicles in seawater? What allows the vesicles to remain stable in the slime glands of the animal? What are the characteristics of the mucin glycoproteins, and how do these characteristics influence vesicle swelling?

I investigated the mechanisms that regulate mucin vesicle rupture in hagfish slime. I examined how characteristics of the mucin-like glycoproteins (MLPs) affect swelling mechanics of the vesicles. My goal was to characterize the properties of the glycoprotein gel in the absence of the membrane, and investigate what causes the gel to expand in seawater and how it remains stabilized in the gland. I explored two main hypotheses: the Hofmeister ion series hypothesis and the cationic gel hypothesis.

The first hypothesis states that the MLPs obey Hofmeister-like behaviour. The

Hofmeister ion series arranges ionic species in a spectrum, ranging from “kosmotropes”, known for their ability to stabilize proteins, to “chaotropes”, which promote solubility of proteins

(Baldwin, 1996; Collins, 2004; Zhang et al., 2005). This hypothesis predicts that the glycoproteins should swell in the presence of chaotropic ions, and remain condensed when exposed to kosmotropic ions. I tested this hypothesis by exposing MLP granules to a wide range of salts containing kosmotropic and chaotropic ions that are well documented in the Hofmeister series (tested separately, using a Hofmeister-neutral ion as the ‘control-ion’ in the salt). 13

The second hypothesis states that the gel obeys the jack-in-the-box mechanism for swelling and collapse, but with the charges reversed, with the glycoproteins being positively charged instead of negatively charged as previously believed. This hypothesis states that the glycoproteins make up a cationic gel that is stabilized by multivalent anions and is swollen by the exchange of the multivalent anions for monovalent ones. This hypothesis predicts that regardless of composition, any multi-charged anion should condense the MLPs, and swelling should occur if they are exchanged for a mono-charged anion. I tested this by exposing MLP granules to multi-charged anion-containing solutions (sodium citrate, -phosphate, -sulphate, and

-carbonate) over a series of pH values designed to expose the glycoproteins to anions at all possible ionization states. Decreasing the pH results in the anion becoming progressively more protonated, decreasing its charge. To identify the acid and base properties of ionisable groups on the vesicle glycoproteins, I produced a titration curve of solubilized glycoproteins to find the pKa values of the titratable groups they might possess.

The data presented in this thesis are consistent with the hypothesis that hagfish mucin vesicles contain a cationic gel, rather than an anionic gel as was previously assumed. I demonstrate that multi-charged anions are effective at keeping the mucin gel condensed, while monovalent anions, mono- and divalent cations, and neutral solutes cause mucin swelling.

These results are consistent with the hypothesis that polyvalent anions are involved in the stabilization of a cationic mucin-like gel in the slime gland and that swelling in seawater is governed by an exchange of these polyvalent anions for monovalent anions such as Cl-.

MATERIALS AND METHODS 15

2.1 Chemicals

Sodium citrate, Tris base, NaCl, NaBr, NaI, NaF, KCl, NH4Cl, NH4Ac, NH4F, NaNO2, sodium acetate, citric acid, guanididium chloride, Na2SO4, (NH4)2SO4, MgSO4, Na3PO4, Tris-

HCl, glycerol, and sucrose were obtained from Fisher Scientific (Ottawa, ON). Dowex ion exchange resin was also obtained from Fisher Scientific. Clove oil, PIPES [piperazine-N,N'- bis(ethanesulfonic acid)], urea, IAA, and SDS were obtained from Sigma-Aldrich (St. Louis,

MO). DTT and TCEP [tris(2-carboxyethyl)phosphine] were produced by Acros Organics and obtained through Sigma-Aldrich (Oakville, ON).

2.2 Animals, anaesthesia, and slime collection

Pacific hagfish (E. stoutii) were obtained from the Bamfield Marine Science Center

(Bamfield, British Columbia, Canada) and maintained in the Hagen Aqualab at the University of

Guelph (Guelph, Ontario, Canada). The hagfish were kept in Environmentally Controlled

Aquatic Recirculation System (ECARS) tanks filled with 2000 liters of recirculating artificial seawater (34ppt) at 10°C.

To anesthetise the hagfish prior to sample collection, a 1:9 dilution of clove oil in ethanol was added to artificial seawater for a final concentration of 50 µL clove oil per liter of seawater

(Herr et al., 2010). The hagfish were placed in the diluted clove-oil and seawater and left to absorb the anaesthetic for 20-30 minutes prior to experimentation. The hagfish were deemed ready for experimentation when they ceased to respond to touch. The hagfish were rinsed with deionized water and dried before the slime glands were stimulated to expel slime exudate. The glands were stimulated by mild electrostimulation from a GRASS Instruments SD9 stimulator

(6V, 80 Hz; Quincy, MA). Once expelled through the slime pore, the exudate was collected into a stabilization buffer (SB) solution composed of 0.9M sodium citrate and 0.1M PIPES, pH 6.7 16

(modified from Downing et al., 1981, Fudge et al., 2003). After exudate collection, hagfish were transferred to seawater free of clove oil to allow them to recover from the anaesthetic, and then returned to the ECARS. All experimental protocols were approved by the University of Guelph

Animal Care Committee (Protocol #2519).

The stabilized exudate was filtered through a nylon mesh with pores 53 µm in size, in order to separate mucin vesicles (7 µm diameter) from the thread skeins (150 µm diameter)

(Downing et al., 1984; Luchtel et al., 1991b). The thread skeins were then discarded and the mucin vesicles were quantified using spectrometry. Sample absorbance levels were measured at

350 nm with an Ultrospec 3100 pro spectrophotometer (Biochrom Ltd., Cambridge, England).

These values were used to estimate the number of mucin vesicles present in the sample, using the linear relationship between absorbance at 350 nm and number of mucin vesicles found by Herr et al. (2014). All samples were then diluted to a concentration of 100 vesicles per µL and stored at

4°C in SB. Prior to experimental use, isolated vesicles were treated with 1% Triton X-100 detergent to solubilize their membranes. Vesicles were referred to as ‘granules’ post-membrane solubilization. The sample was allowed to sit for 90 minutes to ensure full effect of the detergent.

2.3 Mucin swelling assay

Open-ended flow-through chambers were constructed in order to view the swelling behaviour of the glycoprotein granules. The chambers were constructed by drawing two parallel lines approximately 6 mm apart of petroleum jelly containing 0.1 mm diameter glass beads onto a glass slide, onto which a coverslip was placed (Herr et al., 2010; 2014). This created a chamber with a volume of approximately 20 µL. A 30 µL aliquot of stabilized granules were added to each chamber and allowed 2 minutes to settle and adhere to the glass slide. The chamber was then washed with 60 µL of SB in order to rid the sample of any loose granules. The remaining 17 granules adhering to the glass slide were then exposed to 30 µL of experimental solution by adding the solution to one end of the chamber using a micropipette, and pulling it through the chamber via capillary action with a strip of filter paper on the opposite end of the chamber (Herr et al., 2020; 2014).

Swelling behaviour was observed from time lapse videos captured by a monochrome digital camera (Q-imaging Retiga Exi Fast1394) on a Nikon Eclipse 90i microscope (Nikon

Instruments Inc., Melville, NY) with a 20X DIC objective. The time lapse videos were set to capture 5 frames per second (fps) for 30 seconds, followed by 1 fps for 160 seconds (Herr et al.,

2010; 2014). This was done using NIS-Elements A.R. 3.0 software (Nikon Instruments Inc.).

Videos were analyzed using the same software. The area of each granule was calculated using a circumference measurement, collected by manually tracing the circumference of each granule.

2.3.1 Hofmeister Ion Series

To test the hypothesis that vesicle rupture is dependent on interactions involving chaotropic ions, mucin granules were exposed to varying solutions containing ions varying in

- 2- 2 - - Hofmeister effect. Kosmotropic (F , acetate, SO4 , citrate, PO4 ) and chaotropic (Br , I ) anions were tested by keeping the corresponding cation constant; sodium was used as it is listed as having neutral effects in the Hofmeister series. Ammonium salts of -fluoride, -chloride, and - acetate were used to compare these anionic effects with a kosmotropic cation.

All solutions were buffered with 5 mmol·L-1 Tris base and adjusted to a final pH of 8.0 using Dowex ion exchange resin to lower pH in sodium salts or 1.0 molˑL-1 HCl to lower pH of ammonium salts, or 1.0 molˑL-1 NaOH to raise pH. All solutions were adjusted to have a final osmolarity between the range of 2795-2805 mOsm using a Vapro Vapor Pressure Osmometer 18

(model 5520, Wescor Inc., Logan, UT). Final ion concentrations in each solution are listed in

Table 2.1a-k.

2.3.2 Ionization state adjustment

The cationic gel hypothesis was tested by creating a pH series for four known stabilizing polyatomic anions: citrate, phosphate, sulphate, and carbonate. Dowex ion exchange resin was added to each solution to lower the pH; pH was monitored using an Accumet Basic pH meter

(model 5520, Wescor Inc., Logan, UT). The pH of each solution was lowered to the pH values

+ + corresponding to each anion’s pKa values. The Dowex resin exchanges H ions for Na at an equal ratio, removing Na+ from the solution and adding H+, thus decreasing the pH. The Dowex resin absorbs a near negligible amount of Na+; lowering the pH of sodium citrate from 5 to 4 requires the exchange of only 9.0X10-5 mol·L-1 of Na+. This method was chosen to lower the pH of each solution so that no additional anions were added.

A pH above an anion’s highest pKa corresponds to a pH where the ion possesses its maximum charge. As the pH is lowered, the proportion of the anions with the greater charge decreases until most have a lesser charge, and is neutral below its lowest pKa. Aliquots of each solution were taken at each pKa point. Mucins were then exposed to each treatment via the mucin swelling assay. All solutions were buffered with 5 mmol·L-1 Tris base and adjusted to have a final osmolarity in the range of 2795-2805 mOsm·L-1 using a Vapro Vapor Pressure Osmometer

(model 5520, Wescor Inc., Logan, UT). Final ion concentrations in each solution are listed in

Table 2.2.

2.4 Mucin titration

2.4.1 Gel fraction isolation

To separate the vesicle membrane from the mucin gel, isolated mucin vesicles were washed with SB before being treated with 1% Triton X-100 detergent. The sample was allowed to sit for 90 minutes to ensure full effect of the detergent. The sample was then centrifuged at

5000 g for 5 minutes and the gel-pellet was collected. The pellets weighed 0.178±0.003g, with a glycoprotein concentration of 0.0947±0.01g·mL-1.

2.4.2 Mucin gel dialysis

The mucin gel fraction was dialyzed in deionized water to remove SB salts. The gel pellet was placed into regenerated cellulose dialysis tubing with pore sizes of 12,000 – 14,000 Daltons

(Fisher Scientific, Fair Lawn, NJ). The gel was then exposed to a serial dilution; the gel and tubing were placed in 2 L of deionized water (dH2O) which was refreshed every 24 hours for 3 days. The volume of gel collected post-dialysis was 5 mL, with a final glycoprotein concentration of 0.0172 g·mL-1. The final concentrations of sodium citrate and PIPES, assuming full equilibrium was reached for each dialysis step, was 0.9x10-9 mol·L-1 and 0.1x10-9 mol·L-1, respectively. The dialyzed gel was then collected and solubilized.

2.4.3 Mucin gel solubilization

A 5.0 mL aliquot of the dialyzed gel was collected into a small beaker, to which 25 µL of

20 mmol·L-1 TCEP, a disulphide bond reducing agent, was added. The gel was then sonicated to ensure complete solubilization.

2.4.4 Base titration

A 3.0 mL sample of solubilized mucin was titrated with 10 µL aliquots of 1 mol·L-1

NaOH. The pH of solution was measured after the addition of each base aliquot using an

Accumet Basic pH meter (model AB15, Fisher Scientific). Plateaus were distinguished by visual interpretation of the titration curves (Roxby and Tanford, 1971; Righetti et al., 1978; Besnier et al., 2002).

2.5 Statistical analysis

To extract a quantitative value for the transition pH for the anions in each solution, I

퐿 fitted the swelling data to a logistic curve of the form 푦 = + ℓ where L represents the 1+푒−푘(푥−푧) maximum asymptote height of the curve (13.51, 5.40, 5.05, and 9.91 for the sodium salts of carbonate, phosphate, sulphate, and citrate, respectively). The parameter 푘 represents the vertical slope at the transition pH, 푧 represents the horizontal shift of the curve, and ℓ represents the curve’s minimum asymptote (1.0 for all solutions. A swelling ratio of 1.0 indicates no change in area, thus is the smallest ratio possible). The 푘 parameter was assigned a constant value of 4.2 after a series of least squares non-linear regressions were performed for each replicate titration data set to estimate which 푘 -value resulted in the smallest total fit error (sum of squares) for all the salts. 푘 – values were tested over a range of 1.0-10.0 in 0.1 increments. The value 푘 =4.2 gave the smallest fit error value for all four solutions (0.31, 0.36, 0.25, and 0.76 for the curves of sodium -carbonate, -phosphate, -sulphate, and -citrate, respectively). A non-linear least squares regression was then also done to estimate the most accurate transition pH value (parameter z) for each individual salt from this model equation. All regressions were done using scripts written in 21

Python. Mean estimated transition pH values were then compared using a one-way ANOVA followed by a TukeyHSD post-Hoc test.

RESULTS 23

3.1 Hofmeister effects do not explain mucin granule swelling and stabilization

To test the idea that the swelling of hagfish slime mucins is governed by Hofmeister effects, isolated hagfish slime glycoproteins were exposed to eleven salt solutions containing ions representing a range of well documented Hofmeister effects (Table 2.1). Analysis of granule swelling behaviour after exposure to each experimental solution revealed that anionic effects did not always follow the pattern predicted by the Hofmeister series hypothesis (Table 3.1).

Ammonium salts of fluoride, acetate, and chloride, as well as sodium salts of fluoride and acetate were predicted by the Hofmeister hypothesis to have kosmotropic effects, however these salts induced swelling of the hagfish slime granules. Sodium salts of iodide and bromide were predicted to swell the glycoproteins, which was indeed observed. Sodium salts of citrate, phosphate, and sulphate were predicted to promote stabilization of the proteins, and this was observed in the granules as they remained in a condensed state. Solutions that induced swelling resulted in 100% of the granules swelling, while solutions that induced stabilization resulted in

100% of the granules remaining condensed

3.2 Effects of anionic charge on vesicle swelling

Results from the Hofmeister trials revealed that stabilization occurred only when polyvalent anions were present, which led me to consider the possibility that the glycoproteins are positively charged and therefore can be condensed by the presence of anions with multiple charges via a cross-linking effect. To test this idea, hagfish slime glycoproteins were stripped of their vesicle membranes and exposed to sodium salt solutions of citrate, sulphate, phosphate, and carbonate, each titrated to pH values ranging from approximately 2-13 (with the exception of carbonate, with a pH range of 5-13). Granules showed a high degree of swelling at low pH and 24 little to no swelling at higher pH values (Figure 3.1). At pH values above 10, minor swelling

(1.27-1.49 fold area increase) was observed (Figure 3.2). The pH values where the low-pH transition occurred differed widely among the four solutions. Fitting the swelling data to a logistic model curve allowed me to quantitatively estimate the transition pH for each salt (Figure

3.3). Estimates of the transition pH for sodium citrate, -sulphate, -phosphate, and –carbonate were 4.03±0.02, 1.77±0.005, 5.41±0.07, and 10.08±0.04, respectively, all of which were significantly different from each other (TukeyHSD, p<0.05). A linear regression of the transition pH values against the theoretical pKa values for each respective anion showed a strong correlation (R2=0.9543, p = 0.0231, linear regression) (Figure 3.4). I also observed a small amount of swelling in all four salt solutions at pH values greater than 10, showing swelling of

1.27-1.49 fold increase in granular area.

3.3 Analysis of titratable groups

Titration of the isolated glycoprotein gel with NaOH revealed three plateau areas in the pH ranges of 7.0-8.8, 10.0-10.6, and 11.2-11.6, which were not observed in the water-control curve or the TCEP control curve (Figure 3.5). To neutralize the first group, 6.0X10-5 moles of

NaOH was needed. Assuming the OH- from the NaOH solution would exchange with H+ from the functional group on a 1:1 molar ratio, the first functional group was calculated to be present at a concentration of 0.02 mol·L-1. The group that buffered between pH 10-10.6 required

4.0X10-5 moles of NaOH; assuming a 1:1 molar exchange ratio between OH- and H+, this substance was present at 0.0133 mol·L-1. The final group was neutralized with 2.0X10-5 moles of

NaOH and calculated to be present at a concentration of 6.67X10-3 mol·L-1. Based on these calculations, each kDa of glycoprotein theoretically contains 2.23 titratable groups. This was 25 calculated from the ratio of the concentration of each functional group to the concentration of glycoprotein.

The only plateau seen in the TCEP control curve and the water control curve is the plateau caused by the electrode’s limit (beginning at pH 11.6).

DISCUSSION 27

4.0 Discussion

Hagfish slime has thus far been assumed to be negatively charged, and mucins found in other animals are negatively charged (Allen et al., 1984; Aspden et al., 1995; Periera-Chioccola et al., 2000; Adikwu 2005; Herr et al., 2010; Herr et al., 2014 ). The studies in this thesis present novel evidence that slime of the Pacific hagfish is comprised of a cationic mucous gel. The data presented here suggest that a multi-charged anion is present in the vesicles and is responsible for keeping the vesicles stable in vivo. In seawater, these stabilizing anions are likely displaced by

Cl-, a monovalent anion, resulting in the loss of cross-bridging and charge-shielding among glycoproteins, which leads to electrostatic repulsion of the glycoproteins from each other and the swelling of the vesicle. The results presented in this thesis significantly add to our understanding of how hagfish slime is formed in seawater and gives further insight into its mechanisms of deployment.

The Hofmeister hypothesis states that the swelling or stabilization of the vesicle glycoproteins is dictated by their interactions with ions in their environment, and where those ions occur in the Hofmeister series. “Kosmotropic” ions were predicted to have a stabilizing effect on the granules, and “chaotropic” ions were predicted to induce swelling. This hypothesis was not supported by my findings. I observed that treating isolated glycoproteins from hagfish slime with strongly kosmotropic solutions (NH4F, NH4Ac, NH4Cl, NaF, and NaAc) induced swelling, a chaotropic effect. These observations allowed me to reject the Hofmeister mechanism as a driver of swelling and stabilization in the case of glycoproteins from hagfish slime vesicles.

The only solutions tested in this experiment that kept the granules in a condensed state were those that contained multi-charged anions, raising the possibility that it is the magnitude of the charge on the anions that determines whether that ion will be stabilizing or not. 28

Prior studies investigating the Hofmeister series have not included solubility of glycoprotein gels such as hagfish slime mucus, but primarily focus on aqueous solutions of globular proteins such as albumin, digestive enzymes, and hormones (Baldwin 1996; Broering and Bommarius, 2005; Collins 2004). Mucins are heavily glycosylated proteins more similar to scleroproteins which frequently contain cysteine and disulfide bonds that form cross-links between molecules (Sikorski 2001). The structural differences between these two types of proteins may explain why Hofmeister effects were not always observed in the hagfish slime glycoproteins.

I proposed the cationic-gel hypothesis, which states that swelling is primarily driven by the displacement of stabilizing multi-charged anions by monovalent anions, which leads to electrostatic repulsion among the supposed positively charged glycoproteins (Figure 4.1).

According to this hypothesis, multivalent anions stabilize positively charged glycoproteins by shielding the positive charges and also induce electrostatic cross-linking. This hypothesis predicts that swelling and stabilization of the gel can be affected by changing the concentration of polyvalent anions in solution. Indeed, I found that reducing the negative charge of an anionic species in a normally stabilizing solution resulted in swelling. By lowering the pH of each solution, the anionic species were protonated, decreasing their negative charge. Exposure of solutions under these conditions resulted in swelling and expansion of the glycoproteins. At high pH, the multivalent anions were deprotonated, making their net charge more negative; this had a stabilizing effect on the glycoproteins, resulting in the proteins remaining in a condensed state.

The transition pH values correspond quite closely with the pKa values for the equilibrium between the anion being in a multi-charged state to one in which it is mono-charged (4.76, 1.90,

7.23, and 10.31 for citrate, sulphate, phosphate, and carbonate, respectively) (Martell and Smith, 29

1982; 1989). These observations are potentially explained by the notion that the vesicle glycoproteins are positively charged, and therefore condense due to charge-shielding and cross- linking effects by multivalent anions, and swell when these effects are lost. In seawater, swelling is likely driven by the exchange of a multi-charged stabilizing anion in the vesicle (possibly sulphate, as it is abundant in seawater) for an abundant mono-charged anion from the environment, such as chloride. Such an exchange would lead to electrostatic repulsion and swelling among the positively charged groups on the glycoproteins.

Katachalsky et al. (1951) showed that swelling and contracting of charged polymers can be reversible, theoretically by transforming ionization energy into mechanical energy. This occurs when the ionization state of the polymer is altered. In the case of positively charged glycoproteins, high pH causes deprotonation, neutralizing their charge. Under these conditions, the negatively charged charge-shielding ions will not be able to form electrostatic cross-links, allowing the glycoproteins to swell due to diffusion. This is also supported by my observations of slight swelling (1.27-1.49 fold increase in granular area) in solutions of pH 10 or higher.

An alternative explanation for these observations is that the glycoproteins are actually negatively charged as previously assumed, and the increased amount of positively charged Na+ ions that accompanies multivalent anions is what stabilizes the mucins. While these solutions contain a considerably greater concentration of Na+ than solutions containing monovalent anions, it appears unlikely that the Na+ concentration is the stabilizing agent. Preliminary experiments showed that monovalent anion-containing solutions such as NaCl and NH4Cl induced rapid swelling even at very high concentrations (6 mol·L-1) (data not shown). For these reasons, it is unlikely that the positive counter-ions are responsible for the condensing effects that I observed. 30

It is also possible that the multivalent anions are essentially repelled by the negatively- charged glycoproteins due to their large charge density and are unable to enter the matrix. For example, a deprotonated citrate ion contains three negative charges per molecule and sulphate has two charges per ion. In contrast, chloride contains only one charge per ion. Anions with similar rupture-inducing effects such as bromide and iodide each have one charge per ion. The increased charge density of the multi-charged anions could prevent them from penetrating the gel network, thus resulting in a lack of ion displacement in the glycoprotein matrix. Multi-charged ions have large hydration shells, a shell of water molecules that act as a solvent. This shell typically needs to be shed in order for the ion to move through membrane channels; if this is energetically unfavourable, the ion would not enter the channel and would be excluded to the external solution (Aqvist and Warshel, 1989; Lynden-Bell and Rasaiah, 1996). If the anions are unable to enter the network, the concentration gradient for the surrounding aqueous solution would lead the water out of the vesicle. Thus, water would leave the vesicle by osmosis instead of entering it, allowing the gel to remain in a condensed state. However, due to the low charge of monovalent anions, these species are able to enter the matrix, bringing with them water by osmosis and other solutes down their diffusion gradients, thus disrupting the glycoproteins and resulting in swelling.

Titrating the glycoprotein gel also provided evidence that supports the cationic gel hypothesis. Three buffer zones were visually observed from the base titration at pH 7.0-8.8,

10.0-10.6, and 11.2-11.6. It is likely that these buffer zones correspond to the pKa values of three different chemical groups on the glycoproteins (at pH 8.2, 10.3, and 11.4, respectively). A possibility is that these ionizable groups are the carboxyl and amino termini on the mucin-like glycoproteins, however Salo et al. (1983) demonstrated that amino sugars and neutral sugars 31 make up 8.7 % and 2.0 % of hagfish mucus respectively; amino sugars are therefore a good candidate for the positively charged groups on the glycoproteins. A performic acid oxidation done by Salo et al. (1983) revealed that hagfish slime contains cysteine and histidine (3.4% and

5.4% of residues, respectively), which are both contenders for the side group responsible for the plateau at pH 7.0-8. Cysteine’s thiol group has a pKa of 8.37 and histidine’s imizadole group has a pKa of 6.04 (Martell and Smith, 1982; 1989). Analysis of the first titration plateau reveals that

-1 this substance was present at a concentration of 0.02 mol·L . Both cysteine and histidine’s pKa values correspond to the pH of the first buffer zone in the titration. However, cysteine is not positively charged at high pH; the thiol group becomes deprotonated from its neutral form to become negatively charged. Based on these characteristics, it is more likely that histidine is the chemical group side chain of the glycoprotein corresponding to this first titration plateau.

Candidates for the chemical group responsible for the second buffering zone at pH 10-

10.6 include lysine (pKa 10.54) and tyrosine (pKa 10.07), both of which are relatively abundant in hagfish mucus (2.6% and 0.92% of residues, respectively) (Salo et al., 1983; Martell and

Smith, 1982; 1989). However, tyrosine is deprotonated to a net negative charge at high pH and therefore is not a viable candidate for a source of positive charges. N-acetylgalactoseamine is an

O-linked glycan commonly found in glycoproteins, and has a pKa of 10.8 on its amine surface group (Medina et al., 2011). Calculations from the titration data reveal that 0.0133 mol·L-1 of this chemical was present. Thus, both N-acetylgalactosamine and lysine may be contenders for this side group.

The titration plateau at pH 11.2-11.6 may be due to an arginine side group on the mucus glycoprotein. Arginine has a pKa of 12.5 (above which it is cationic), and makes up 1.4% of oxidized hagfish slime mucus residues (Salo et al., 1983; Martell and Smith, 1989). Another 32

possibility is N-acetylglucosamine, an O-linked glycan with a pKa of 11.56. Both arginine and

N-acetylglucosamine’s characteristics align with the pH values of the titration plateau. Analysis of the third plateau shows that the group responsible is present at a concentration of 6.67X10-3 mol·L-1.

Mucous glycoproteins typically occur in the size range of 100-500 kDa (Bansil and

Turner, 2006). If the average molecular weight of the glycoproteins is assumed to be 100 kDa, their concentration in the titrated sample would be 1.78X10-4 mol·L-1 and each molecule would have 223 titratable groups. If the MW of each glycoprotein was twice as large (ie. 200 kDa), their sample concentration would be 8.9X10-5 mol·L-1, and each molecule would have 446 titratable groups. At 500 kDa, the glycoproteins would be present at 3.6X10-5 mol·L-1 with 1115 titratable groups. This gives a ratio of 2.23 titratable groups per kDa of glycoprotein. This is typical of more well-studied mucins such as gastric mucins, and therefore shows validity in the estimates of the side group abundances calculated from the titration (Bansil and Turner, 2006).

The above estimates of hagfish mucous composition are putative and require further investigation, as discrepancies exist in their validity. For example, Salo et al. (1983) demonstrate that histidine is in lesser abundance than arginine, however if histidine corresponds to the first titration plateau and arginine corresponds to the second plateau, my estimates for the glycoprotein side groups suggest that the mucus contains more histidine (0.02 mol·L-1) than arginine (6.67X10-3 mol·L-1). Furthermore, a sulphitolysis experiment of hagfish vesicular mucus performed by Salo et al. (1983) revealed different abundances of the amines tested than was revealed by a performic acid oxidation in the same study, and describes the mucus as being free of histidine altogether. Further studies examining the composition of hagfish slime vesicular 33 mucus using techniques such as mass spectrometry will be needed to more rigorously test the cationic gel hypothesis.

The evidence produced in this study suggests that hagfish slime is comprised of a cationic glycoprotein gel, but confirmation will require further investigation. If the mucus is indeed cationic, it is likely condensed with a multivalent anion in vivo. Knowledge of this counter-ion’s identity would allow for increased understanding of the biochemical mechanism behind vesicle rupture and slime formation in seawater. Similar investigations regarding the composition of the gel would also give further insight into the identities of the hypothesized positively-charged groups. Measurements of charge potential would be greatly beneficial, and can be done using isoelectric focusing and mass spectrometry of the mucous glycans; This would indicate whether the mucus has a positive or a negative charge. This analysis can also be done using a zeta potential analyzer, which can give a more specific indication of charge.

Because hagfish are such primitive organisms, it may be possible that ancestral gels began as cationic substances, and evolved to become negatively charged, as is now conventional to our knowledge. Conversely, the cationic gel of hagfish slime may have secondarily evolved from a negatively charged mucus due to environmental circumstances; the abundance of the monovalent anion Cl- in seawater may be imperative for gel expansion, by displacing the intravesicular stabilizing anion in order for rupture to occur. The Ca2+ in seawater is also important to this process, as the vesicles contain putative ion channels that require binding of

Ca2+ in order to become functional and allow the influx of ions such as Cl- (Herr et a. 2014). This raises the question of whether there are other unrecognized cationic gels in biology, and if a mucus-producing organism’s environment has an influence on the gel’s properties. While I have presented a novel theory of hagfish slime properties, the full characterization of slime 34 deployment and formation is far from complete and requires further investigations to answer the several questions raised by this theory.

In conclusion, my data in this thesis are consistent with the hypothesis that the defensive slime of the Pacific hagfish consists of a cationic gel. The glycoprotein gel was condensed in the presence of multivalent anions, and expanded or swelled when monovalent anions were present.

In seawater, this process could take place when stabilizing, multi-charged anions in vivo are replaced by Cl- during an influx of these ions through Ca2+-activated ion channels in the vesicle membrane (Herr et al., 2014). The identities of the positively charged chemical groups attached to the gel’s glycoproteins are thus far unknown, however histidine, lysine, and arginine are notable candidates. The findings in this thesis raise questions about the evolution of mucus and slime, possible advantages to producing a positively-charged mucus rather than the more common negatively-charged variety, and raises the question of whether there are other cationic gels in biology that have yet to be recognized.

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Tables

Table 1.1: Ions and other solutes organized according to their stabilizing and destabilizing effects as denoted by the Hofmeister series. From Cacace et al. (1997).

A F + -8 [H+] 1.00X10-8 [H ] 1.00X10 + [Na+] 1.87 [NH4 ] 1.40 - 2- [F ] 1.40 [SO4 ] 0.933

[Na+] 9.99X10-7

G B [H+] 1.00X10-8 [H+] 1.00X10-8 [Na+] 1.4 + [NH4 ] 1.40 [F-] 1.4 [Ac-] 1.40

[Na+] 9.00X10-7 H

[H+] 1.00X10-8 C [Na+] 1.4 [H+] 1.00X10-8 [Acetate-] 1.4 + [NH4 ] 1.40

[Cl-] 1.40 I [Na+] 9.99X10-7 [H+] 1.00X10-8 [Na+] 1.4 D [Cl-] 1.4 [H+] 1.00X10-8

[Na+] 2.1 J [Citrate3-] 0.7 [H+] 1.00X10-8 [Na+] 1.4 E [I-] 1.4 [H+] 1.00X10-8

[Na+] 1.87 K [HPO 2-] 0.805 4 [H+] 1.00X10-8 - [H PO ] 0.128 + 2 4 [Na ] 1.4 [Br-] 1.4

Table 2.1: Final calcuated concentrations assuming complete dissociation of ions in ammonium fluoride (A), ammonium acetate (B), ammonium chloride (C), sodium citrate (D), sodium phosphate (E), sodium sulphate (F), sodium fluoride (G), sodium acetate (H), sodium chloride (I), sodium iodide (J), and sodium bromide (K). All values are mol·L-1. Osmolarity of each solution was 2800±5 mOsm. Tris was included in all solutions at a range of 4.72X10-3 – 5.00X10-3 mol·L-1. Tris concentration was altered while adjusting osmolarity. All solutions had a pH of 8.0.

A pH 1.2 2.2 4.2 5.2 6.2 8.2 10.2 11.2 12.2

[H+] 0.0631 0.00631 6.31X10-5 6.31X10-6 6.31X10-7 6.31X10-9 6.31X10-11 6.31X10-12 6.31X10-13

[Citrate 3-] 0 0 1.39X10-2 0.299 0.636 0.700 0.700 0.700 0.700

[HCitrate 2-] 0 0 0.136 0.292 6.23X10-2 0 0 0 0

1- -3 -2 [H2Citrate ] 7.70X10 7.00X10 0.505 0.109 0 0 0 0 0

-2 [H3Citrate] 0.692 0.630 4.48X10 0 0 0 0 0 0

[Na+] 1.97 2.08 2.10 2.10 2.10 2.10 2.10 2.10 2.11

B pH 5.0 6.7 8.0 9.0 10.0 11.0 12.0

[H+] 1.00X10-5 2.00X10-7 1.00X10-8 1.00X10-9 1.00X10-10 1.00X10-11 1.00X10-12

2- -3 -2 [CO3 ] 0 0 4.67X10 4.16X10 0.298 0.769 0.913

1- -2 -2 [HCO3 ] 4.01X10 0.645 0.909 0.889 0.635 0.164 1.96X10

-2 -3 [H2CO3] 0.893 0.288 2.05X10 1.87X10 0 0 0

[Na+] 1.87 1.87 1.87 1.87 1.87 1.87 1.88

C pH 1.5 3.5 5.5 8.0 9.0 10.0 11.0 12.0

[H+] 0.316 3.16X10-3 3.16X10-5 1.00X10-8 1.00X10-9 1.00X10-10 1.00X10-11 1.00X10-12

3- -3 -2 [PO4 ] 0 0 0 0 0 2.80X10 3.01X10 0.252

2- [HPO4 ] 0 0 0.137 0.604 0.689 0.696 0.670 0.484

1-] -2 -2 -3 [H2PO4 0.128 0.670 0.686 9.59X10 1.09X10 1.12X10 0 0

-2 [H3PO4] 0.572 3.01X10 0 0 0 0 0 0

[Na+] 1.24 1.86 1.87 1.87 1.87 1.87 1.87 1.87

D pH 1.0 2.5 3.5 5.5 7.5 9.5 10.5 11.5 12.5

[H+] 1.00 0.0316 3.16X10-3 3.16X10-5 3.16X10-7 3.16X10-9 3.16X10-10 3.16X10-11 3.16X10-12

2- -2 [SO4 ] 9.33X10 0.739 0.909 0.933 0.933 0.933 0.933 0.933 0.933

1- -2 [HSO4 ] 0.833 0.194 1.87X10 0 0 0 0 0 0

[H2SO4] 0 0 0 0 0 0 0 0 0

[Na+] 0.902 1.84 1.87 1.87 1.87 1.87 1.87 1.87 1.90

Table 2.2: Final concentrations of ions after pH adjustments with Dowex ion exchange resin for sodium citrate (A), sodium carbonate (B), sodium phosphate (C), and sodium sulphate (D). All values are in mol·L-1. Osmolarity of each solution was 2800±5 mOsm. Tris was included in all solutions at a range of 4.72X10-3 – 5.00X10-3 mol·L-1. Tris concentration was altered while adjusting for Osmolarity.

Table 3.1: Effects of salt solutions on proteins as predicted by the Hofmeister series and as experimentally observed with hagfish slime vesicle glycoproteins.

Figures

Figure 1.1: Illustration of a hagfish. The slime gland pores are located ventro-laterally along both sides of the animal. From Herr et al. (2014). 55

Figure 1.2: Hagfish slime exudate contains two main cell types. Gland mucous cells (GMCs) are denoted by arrowheads, and gland thread cells (GTCs) are indicated by arrows. GMCs and GTCs produce glycoprotein-containing vesicles and intermediate filament-based fibers, respectively. From Winegard (2012). 56

Figure 1.3: Holocrine secretion of exudate from a hagfish slime gland. When physicallyagitated, the muscle surrounding the gland pore contracts and the exudate is ejected from the gland (a). During this process, the GMCs and GTCs are forced through a narrow pore duct opening, which strips them of their plasma membranes, and releases their contents (vesicles and thread skeins, respectively) (b). Upon contact with seawater, the vesicles swell and lyse, releasing the glycoproteins they contain, and the thread skeins unravel (c). The threads and glycoproteins entangle in each other to form a sieve that entraps water, forming mature hagfish slime. Modified from Herr (2012).

Figure 3.1: Normalized granule area vs. pH of 2800 mOsm sodium salt solutions. Normalized granule area is defined here as the ratio of vesicle area pre:post exposure to experimental solution. Degree of swelling is higher across salts at lower pH, but drastically decreases at increasingly higher pH values for each salt. Above pH 10, granule area increases slightly in all four salts. Ratio of 1 indicates no change in granular area. Error bars represent S.E.M. N=10 hagfish, 5 granules from each, total= 50 granules.

Figure 3.2: Normalized granule area vs. pH of sodium salt solutions, focused on pH values above 10. Normalized granule area is defined here as the ratio of granule area pre:post exposure to experimental solution. Minor swelling (1.27-1.49 fold area increase) is seen in all four salt solutions. Carbonate does not allow for complete stabilization; its highest pKa occurs at such a lower pH than is required for the glycoproteins to deprotonate. The deprotonation of cationic chemical groups on the glycoproteins to their neutral state causing a loss of electrostatic cross-linking and results in a slight spacial expansion of the glycoproteins due to diffusion. 59

Figure 3.3: Normalized granule area vs. pH of each salt solution (markers) plotted alongside fitted logistic model curves (lines) that were generated to estimate the transition pH of the four salts tested. A least squares regression gave a significant fit accuracy of sodium salts of -sulphate, -citrate, -phosphate, and -carbonate data, respectively (p<0.05). Error bars represent S.E.M. N=10 hagfish, 5 granules measured for each, total= 50 granules.

Figure 3.4: Transition pH values for sodium-salt-anions as estimated from the model logistic curve. Transition pH estimates for each salt are significantly different from each other as indicated by different letters (TukeyHSD, p<0.05). Experimental transition pH estimates are significantly correlated with their respective theoretical transition pH (Linear regression, R2=0.95, p=0.02). Error bars represent S.E.M. N=10 hagfish, 5 granules measured from each, total=50 granules. 61

Figure 3.5: Titration curves of deionized water (negative control), 3 mM TCEP (blank control), and dialyzed mucin + 3 mM TCEP. The three plateaus of the mucin + 3 mM TCEP (at 7.0-8.8, 10.0-10.6, and 11.2-11.6) are not seen in either of the control samples, and likely correspond to basic chemical groups, although the identity of these groups is not yet known. Data points are pH of solution±SEM. N=3.

Figure 4.1: Collected and modelled data plotted against the abundance of anions in neutral, mono-, di-, and tri-valent ionization states. The transition between swelling and stabilization occurs at the pH values where the anion in solution transitions from a multi- to a mono- charge. The primary y-axis depicts the swelling ratio of the granule area post:pre exposure to the experimental solution. A ratio of 1.0 indicated no change in granule area. The secondary y-axis depicts the proportion of the respective anion’s valency abundance. A value of 1.0 indicates that the species with that valency makes up 100% of the species in solution. N=10 hagfish, 5 granules measured from each, total=50 granules.