The Pennsylvania State University the Graduate School College Of

The Pennsylvania State University

The Graduate School

College of Medicine

MEPRIN METALLOPROTEASES CLEAVE AND

INACTIVATE INTERLEUKIN-6

A Dissertation in

Integrative Biosciences

Timothy R Keiffer

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

May 2012 The dissertation of Timothy R. Keiffer was reviewed and approved* by the following:

Judith S. Bond Evan Pugh Professor and Chair of Biochemistry and Molecular Biology Dissertation Advisor Chair of Committee

Channe Gowda Professor of Biochemistry and Molecular Biology

Chris Norbury Professor of Microbiology and Immunology

Ira Ropson Associate Professor of Biochemistry and Molecular Biology

Gavin P Robertson Professor of Pharmacology, Pathology, Dermatology, and Surgery

Cara-Lynne Schengrund Emeritus Professor of Biochemistry and Molecular Biology

Peter Hudson Director, Huck Institutes of the Life Sciences Willaman Professor of Biology

*Signatures are on file in the Graduate School.

ABSTRACT

The inflammatory response is influenced in part by a class of small molecular-weight proteins called cytokines. There is a complex and changing mixture of cytokines at the sites of inflammation during every stage of the inflammatory process, from initiation to resolution.

Proteases can influence the inflammatory response by altering cytokine bio-activity, either through inactivating the cytokine via degradation or activating the cytokine by converting the cytokine from a “pro” form to an active form.

Meprins are zinc metalloproteinases of the metzincin superfamily that are comprised of α and/or β subunits that interact with each other to form unique membrane-bound and secreted isoforms. Meprins are expressed in the brush-border membranes of kidney and intestinal epithelial cells along with the epidermal layers of the skin and certain populations of leukocytes.

The meprin metalloproteinases have been implicated in the pathogenesis of several inflammatory diseases. It is hypothesized that one mechanism by which meprins modulate and regulate inflammation is by direct proteolytic action on pericellular inflammatory mediators such as cytokines. Interleukin-6 (IL-6) is an important pro-inflammatory mediator and several lines of in vivo data indicate that IL-6 is an in vivo substrate for meprins. The current work focused mainly on characterizing the interaction between meprins and interleukin-6, with the hypothesis that meprins cleave and bio-inactivate IL-6.

This work demonstrates that the homomeric isoforms of meprin A and B cleave IL-6 at the C-terminus and remove a five amino acid segment, thereby inactivating the cytokine. Both the homomeric human meprin isoforms and the rodent meprin isoforms cleave human and mouse isoforms of IL-6, thereby decreasing the bio-activity of IL-6. Furthermore, IL-6 inactivated by meprin A or B does not act in an antagonistic manner towards intact IL-6. The homomeric rodent

iii meprin A and B isoforms of meprins cleave human IL-6 with high affinity (apparent) (Km) and

-1 -1 -1 -1 efficiency (kcat/Km) values; 4.9 µM and 0.20 µM s , and 12.0 µM and 2.5 µM s for meprin A and B, respectively. Madin-Darby Canine Kidney Cells transiently transfected with meprin β and transfected with either meprin β or meprin α constructs cleave human IL-6. The kinetics of meprin cleavage of IL-6 and the finding that meprins cleave IL-6 in a cell-based system indicate that IL-6 is a good substrate for both meprin A and B and provide a kinetics-based rationale for proposing that meprins are capable of cleaving IL-6 in vivo.

While evaluating the interaction between meprins and IL-6 was the main aim of this thesis work, another aim was to study the interaction of endogenous inhibitors with meprins.

Endogenous proteinase inhibitors, such as alpha-2-macroglobulin and Tissue Inhibitors of

Metalloproteinases (TIMPs), are important for the regulation of some protease activity in vivo, however, they do not inhibit meprin activity. It has been proposed that mannose binding lectin

(MBL), which is also located at sites of inflammation along with meprins, inhibits meprin activity. Therefore, the interaction between MBL and meprins were studied further. It was found that while MBL did not significantly inhibit meprin peptidase or proteinase activity, the interaction between the glycans of meprin and MBL was confirmed. This interaction between meprins and MBL may have significance in vivo, as MBL recognition of meprins anchored to epithelial cells during inflammation may contribute to overall tissue damage via complement activiation.

Wound healing is a complex inflammatory process that involves several factors including

IL-6 and MBL. Furthermore, diabetic patients have compromised and impaired wound healing.

It is known that diabetic mice have down-regulated meprin expression. Pilot studies with wound healing exudates from humans were initialed in order to determine a potential role of

iv meprins in the process of wound healing. While no correlation was discovered between meprin presence and the diabetic status of the patient from which the wound samples were taken, meprin alpha and beta subunits were located in human wound healing exudates from both diabetic and non-diabetic patients.

This thesis expands the knowledge of the interaction between meprins and IL-6 and proposes a mechanism of meprin-mediated modulation of inflammation via direct proteolysis of inflammatory mediators. This body of work also further describes the interaction of meprins and

MBL and provides an impetus for studying meprins in the context of other inflammatory processes such as in complement-mediated cell damage and wound healing.

Table of Contents List of Figures ...... viii List of Tables ...... x List of Abbreviations ...... xi Acknowledgements ...... xiv

Chapter 1 INTRODUCTION 1. Proteases ...... 1 1. MMPs, ADAMs, and Astacin Metalloproteinases ...... 3 2. Control of Protease Activity in the Immune Response...... 8 2. Proteases in Inflammation and Disease ...... 10 3. The Meprin Metalloproteinases...... 13 1. Structural Characteristics ...... 13 2. Cleavage Site Specificies and Known Meprin Substrates ...... 18 3. The Role of Meprins in Inflammatory Disease ...... 20 4. Interleukin-6 ...... 22 5. Mannose Binding Lectin ...... 28 6. Rationale for Experimental Aims ...... 32 Chapter 2 MATERALS AND METHODS 1. Meprin Purification from Cell Culture Medium and Kidney Tissue ...... 38 2. Gel Electrophoresis, Gel Staining, and Western Blotting ...... 41 3. Meprin Activity Assays, Peptide and Protein...... 43 4. Cytokine Degradation ...... 44 5. Kinetic Parameter Determination of Interleukin-6 Cleavage by Meprins ...... 44 6. Determination of the Cleavage Site on Interleukin-6 by Meprin A ...... 45 7. B9 Cell Proliferation Assay ...... 45 8. Transient Transfections of Madin-Darby Kidney Cells and Activation of a Cell-Expressed Meprins ...... 47 9. Evaluating Cleavage of Interleukin-6 by Cell-Expressed Meprins ...... 48 10. Inhibition of Meprins by Lectins ...... 48 11. Mouse Urine Meprin Concentration Determination ...... 48 12. Handling of Would Healing Samples ...... 49 Chapter 3 RESULTS 1. 1. Interleukin-6 is a Meprin Substrate ...... 50 1. Human Meprin A and B Cleave Human Interleukin-6 ...... 50 2. Mouse Interleukin-6 is Cleaved by Rodent Meprin A and B...... 55 2. Meprin Cleaves Interleukin-6 at the C-terminus with High Affinity, Decreasing the Activity of Interleukin-6 ...... ……………………………57 1. Meprin A and B Cleave Human Interleukin-6

With High Affinity .. …………………………………………………….57 2. Meprin A Cleaves Human Interleukin-6 at the C-terminus ...... 61 3. Human Homomeric Meprin A and B Decreases the Activity of H Human Interleukin-6 ...... 63 4. Both Homomeric Mouse Meprin A and Rat Meprin B 3 M Decreases the Activity of Mouse Interleukin-6 ...... 66 5. Human Meprin-Cleaved Interleukin-6 Does Not Act as either an 5 d Agonist or an Antagonist ...... 69 3. Modeling Meprin Processing of Interleukin-6 in a Cell-Based System ...... 71 1. Meprins Expressed on the Surface of Madin-Darby 1 K Kidney Cells Cleave Exogenous Human Interleukin-6 ...... 71 4. Concanavalin A, but not Mannose Binding Lectin Inhibits the Proteolytic Activity of Meprin A ...... 74 1. Purification of Lectins from Human Blood Samples ...... 74 2. Native Human Mannose Binding Lectin Recognizes Active and 2 Latent Homomeric Meprin A ...... 76 3. Mannose Binding Lectin Does Not Inhibit the Proteinase or s P Peptidase Activity of Homomeric or Heteromeric Meprins ...... 78 4. Concanavalin A Partially Inhibits Meprin A Proteinase Activity .. 79 5. Characterization of Wound Healing Samples from Diabetic and Non-Diabetic Patients ...... 82 1. Wound Healing Samples from Diabetic and Non-Diabetic Patients 1 H Have Similar Total Protein Concentrations ...... 82 2. Human Meprin α was Detected in both Diabetic and Non-Diabetic 1 W Wound Healing Samples ...... 85 3. Human Meprin β was Detected in both Diabetic and Non-Diabetic 3 W Wound Healing Samples ...... 85 Chapter 4 DISCUSSION 1. Validation of Cleavage of Interleukin-6 by Meprins ...... 91 22. Meprin Cleavage Site on Interleukin-6……………………………………….. 96 33. Meprin Decreases Activity of Interleukin-6 ...... 100 44. Interleukin-6 is Cleaved by Cell-Associated Meprin ...... 104 a5. Effects of Lectin Interaction with Meprins ...... 115 q6. Meprin Involvement in Wound Healing ...... 118

REFERENCES ...... 121

vii

List of Figures:

Figure 1. Proteolytic mechanisms for the serine, cysteine, aspartic,

and metallo-proteinases ...... 2

Figure 2. Relationship of the metalloproteinases ...... 4

Figure 3. The domain structure of the meprin α and β subunits and their associated

meprin A and B isoforms ...... 15

Figure 4. Crystal structure of human interleukin-6 ...... 25

Figure 5. The classical signaling, trans-signaling, and inhibition of interleukin-6

trans-signaling pathways ...... 27

Figure 6. Subunits and structure of mannose binding lectin ...... 29

Figure 7. Mannose binding lectin-mediated complement activation ...... 31

Figure 8. Interleukin-6 is increased in the serum and colon of meprin KO-mice in an experimental model of inflammatory bowel disease...... 33

Figure 9. Interleukin-6 is increased in the colon of meprin double KO-mice in an

experimental model of inflammatory bowel disease...... 35

Figure 10. Human interleukin-6 is cleaved by both human and rodent homomeric

meprin A and B...... 51

Figure 11. Eukaryotic-cell derived human interleukin-6 is cleaved by human homomeric

meprin A and B...... 53

Figure 12. Eukaryotic-cell derived human interleukin-6 is cleaved by recombinant

homomeric mouse meprin A and rat meprin B ...... 54

Figure 13. Mouse interleukin-6 is cleaved by rodent homomeric meprin A and B...... 56

Figure 14. Kinetics of homomeric, rodent meprin processing of

human interleukin-6 (NCI) ...... 59

Figure 15. Meprin A cleaved human interleukin-6 lacks 5 C-terminal amino acids ...... 62

viii

Figure 16. Human meprin A cleaved human IL-6 has decreased activity...... 65

Figure 17. Mouse meprin A and rat meprin B decrease the activity of mouse IL-6 ...... 67

Figure 18. Meprin A and B cleaved mouse IL-6 has decreased activity, as seen

in a B9 cell bio-activity assay with CellTiter96...... 68

Figure 19. Human meprin A and meprin B treated IL-6 acts neither

as an antagonist or agonist...... 70

Figure 20. Meprins expressed by MDCK cells cleave exogenous human IL-6...... 72

Figure 21. Isolation and purification of mannose binding lectin from human blood ...... 75 Figure 22. Mannose binding lectin recognizes homomeric meprin A ...... 77 Figure 23. Mannose binding lectin does not inhibit peptidase or proteinase activity of either meprin A or B...... 80 Figure 24. Concanavalin A partially inhibits meprin A proteinase activity...... 81 Figure 25. Probe of human wound fluid samples for meprin α by Western blot...... 86 Figure 26. Probe for human meprin β in wound-healing samples...... 87 Figure 27. Location of glycosylation motifs on both human and mouse interleukin-6 ...... 93 Figure 28. Colon concentrations of IL-6 in meprin knock-out mice after DSS challenge are similar...... 95 Figure 29. Protease cleavage locations on human interleukin-6...... 98 Figure 30. Proteases from polymorphonuclear leukocytes efficiently decrease the activity of interleukin-6 ...... 102

List of Tables:

Table 1. Kinetic constants for meprin A and B processing of human IL-6 ...... 60 Table 2. Protein concentrations of wound healing samples...... 84 Table 3. Summary of the human meprin α and β subunits detected in wound healing samples...... 88 Table 4. Kinetic constants of meprin A and B cleavage of interleukin-6 compared with the cleavage of meprins against other protein and peptide substrates ...... 106 Table 5. Matrix metalloproteinase cleavage of various chemotactic cytokines alters their activity...... 109 Table 6. Matrix metalloproteinases cleave cytokines and change their biological activities...... 111

List of Abbreviations

Å Angstrom

α2m alpha-2-macroglobulin

ADAM A Disintegrin and Metalloprotease

APP Amyloid Precursor Protein

ARF Acute Renal Failure

BMP Bone Morphogenic Protein

CatG Cathepsin G

CSF Cerebrospinal Fluid

Con A Concanavalin A

COPD Chronic Obstructive Pulmonary Disease

CRD Carbohydrate Recognition Domain

CUB Complement C1r/C1s, Uegf, Bmp1

DMEM Dulbecco‟s modified Eagle‟s Medium

DSS Dextran Sulfate Sodium Salt

ECM Extra-cellular matrix

EDTA Ethylenediaminetetraacetic acid

EGF Epidermal Growth Factor

EGFR Epidermal growth factor receptor

ELISA Enzyme-linked Immuno-sorbent Assay

FBS Fetal Bovine Serum

FGF-19 Fibroblast Growth Factor-19 gp130 Glycoprotein 130

H2O2 Hydrogen peroxide

HEK Human Embryonic Kidney

HOCl Hypochlorous acid

I Domain Inserted Domain

IBD Inflammatory Bowel Disease

ICE IL-1β Convertase

IL Interleukin

IL-6 Interleukin-6

IL-6R Interleukin-6 Receptor

IFN-γ Interferon-gamma

LPS Lipopolysaccharide

KO Knock-out

2ME β-mercaptoethanol

MAM Meprin A5 protein tyrosine phosphatase μ

MBL Mannose Binding Lectin

MCP-1 Mouse Chemoattractant Protein

MIP-1α/β Macrophage inflammatory protein 1 α/β

MMP Matrix Metalloproteinase

MT-MMP Membrane-bound MMP

NCI National Cancer Institute

NE Neutrophil elastase

PAGE Polyacrylamide Gel Electrophoresis

PICS Proteomic Identification of Protease Cleavage Site Specificity

PMN Polymorphonuclear leukocytes

PR3 Proteinase 3

RANTES Regulated on activation normal T cell expressed and secreted

RPMI Roswell Park Memorial Institute

xii

SDS Sodium Dodecyl Sulfate

Serpin Serine proteinase inhibitor sgp130 Soluble Glycoprotein 130 sIL-6R Soluble IL-6 Receptor

TAILS Terminal Amine Isotopic Labeling of Substrates

TBS tris (tris(hydroxymethyl)aminomethane) buffered saline

TBS-T tris buffered saline with 0.1% Tween-20

TLR Toll-like receptor

TNF Tumor Necrosis Factor

TIMP Tissue Inhibitor of Metalloproteases

TM Transmembrane

TRAF Tumor Necrosis Factor Receptor Associated Factor

UTR Untranslated Region

WT Wild-Type

WHS Wound Healing Sample

xiii

Acknowledgements

I approached writing this thesis with more than a bit of trepidation. From rushing to meet deadlines, scheduling last minute meetings, enduring those long periods of time when one cannot conjure up the correct words on the page (if any words can be summoned at all), it is amazing I have retained even a bit of sanity after I completed this stage of my schooling. As with most difficult things in life however, I have emerged from this endurance trial more complete, with a feeling of real accomplishment.

However, one does not survive grad school alone without a good support network. First and foremost, I would like to thank my thesis advisor, Dr. Judith Bond, for the opportunity to complete my graduate studies. Dr. Bond is a consummate and very accomplished scientist, an excellent mentor, and a superb advocate of the basic sciences. During my stay in her lab, she allowed me to direct my own studies (mostly) free from interference; however, she was always trying to impress on me the importance of looking at the big picture and knew when and how to keep me on “the straight and narrow” path, particularly with all the projects I started!

Upon reflecting on my work, trying to fit seemingly divergent projects into one coherent narrative, this gave me a chance to remember all the people who had assisted me ever since my arrival in Dr. Bond‟s lab. The first person that came to mind was Dr. Susan Ishmael. During my rotation in the Bond Lab, Susan added to my knowledge about basic bench-work and also showed me how to maintain a proper lab-book. Another person who made a major contribution to my life in the Bond lab was Dr. Sanjita Banerjee. Sanjita always had a good perspective on science and on life in general. I consider her a good friend and an amazing scientist. And of course, my labmate Jialing Bao is also a very good friend. She and I have shared many

xiv experiences and much knowledge together, particularly from attending several conferences over the past two years.

While I highlighted only a few people from the Bond Lab, virtually everybody that I have ever worked with assisted me in many ways and contributed to my overall growth as both a scientist and as a person. The lab techs, Christine and Ge, were always good for some great advice. Drs Rene Yura, Elimelda Moige Okindo-Ongeri, Gail Matters, and John Bylander are all also great scientists and great people to know. They all tolerated my bothersome questions and requests and were always glad to help me out and tell me their perspective on things

My committee has also been a big help, providing me with the necessary guidance and encouragement throughout my stay here. They have also been very kind and very understanding, especially about last-minute meeting scheduling changes!

While all these people contributed significantly to both my scientific and personal growth, I definitely could not have gone through graduate school without the support of my family. My mother Nancy and my stepfather Bryan provided me with a home and supported me wholeheartedly throughout my entire stay here at the Penn State College of Medicine. I am eternally grateful for their words of encouragement and support, along with all the tangible things (home-cooked meals, washed laundry and the like) they have provided to me. I promise that I will not use the excuse of getting another degree to stay at home longer!

My experience in the Bond Lab and at the College of Medicine has been a blessing and a great opportunity for me, and so I thank everybody overall for helping me out along the way.

There is not enough space to list everyone who has helped me in all kinds of myriad ways through these years. May the effort all of you spent on me be rewarded several hundred-fold.

Chapter 1: INTRODUCTION

1.1 Proteases

Proteases, also known as peptidase and endo-or exoproteinases, are specialized proteins that facilitate the hydrolysis of peptide bonds within proteins and peptides. Currently, one of the largest known classes of genes in the genome of both mice and humans are the protease genes: over 600 genes encoding proteases have been identified in both genomes, comprising approximately 2% of total genomic content in humans [1]. There are six known classes of proteases, organized by the catalytic mechanism utilized by the protease to achieve proteolysis of their target proteins. These protease classes are: aspartic, cysteine, glutamic, metallo-, serine, and threonine proteases. Figure 1 illustrates the catalytic mechanisms utilized by four out of the six protease classes for which the most information is available [2]. The other two protease classes, the threonine and glutamic proteases, were discovered in 1995 and 2004 respectively.

Few threonine and glutamic proteases, relative to the other protease classes, have been identified and characterized to date. The main catalytic difference between the protease classes is the moiety that acts as the nucleophile, which attacks the carbonyl of the peptide bond, and thereby breaks the peptide bond. For serine and cysteine proteases, the nucleophile is an “activated” serine and cysteine residue, respectively, in their active sites while the metallo- and aspartic proteases use an “activated” water molecule as a nucleophile. Furthermore, the serine and cysteine proteases form a covalent bond with the protein in the active site prior to peptide bond cleavage while the aspartic and metalloproteases “activate” a water molecule via non-covalent interactions - it is this activated water molecule which facilitates splitting of the peptide bond for these protease classes [2].

Figure 1. Proteolytic mechanisms for the serine, cysteine, aspartic, and metalloproteinases. The catalytic mechanisms of the serine proteases (a); cysteine proteases (b); aspartic proteases (c); and metalloproteases (d) are shown, with the nucleophile of these four protease classes highlighted by a red circle. The electron shuffling between the nucleophile, the catalytic residues and molecules (Zn2+, which is a zinc molecule) of the active site cleft

(represented by the large semi-circles) and the peptide bond cleaved are illustrated by red arrows

[2].

1.1.1 MMPs, ADAMs, and Astacin Metalloproteinases

One of the larger classes of identified proteases are the metalloproteinases. Over 200 genes out of the ~ 600 protease genes code for metalloproteinases; these particular proteases are classified into 50 evolutionary families. The majority of the metalloproteinases are grouped into a superfamily called the “metzincins”, which themselves are further separated into discrete evolutionary families: Astacins, ADAMs (A Disintegrin and Metalloprotease), MMPs (Matrix

Metalloproteinases), Leishmanolysins, Pappalysins, and Serralysins [1, 3]. Figure 2 shows the linkage trees of the metalloproteinase family, with a highlight on the “metzincin superfamily”.

The protease domains of the different evolutionary families within the „metzincin superfamily‟ (e.g., astacins and MMPs) share minimal amino acid identity, but they have similar three-dimensional structures. For example, members of the astacin family have less than 10% amino acid similarly to the MMP family. However, all the metzincin protease domains have similar 3-dimensional structures. One of the similar 3-dimensional folds shared by the members of the “metzincin superfamily” is the Met-turn, which is a 1,4-β-turn containing a conserved methionine. This conserved methionine is essential for the maintenance of protease active site integrity [3-6]. Zinc is bound in the active site of these proteases by a conserved

HExxHxxG/NxxH/D sequence [5]. The Met-turn and the conserved zinc-binding sequence are connected together by an intervening segment. It is these two characteristics of proteases such as the ADAMS, astacins, and MMPs, that classify them as part of the bigger “metzincin superfamily”.

Figure 2. Proteases of the Metzincin Superfamily. More than half of the genes coding for metalloproteinases in both human and mouse genomes (as indicated by the numbers in subscript) are for proteases of the “zincin” superfamily. These proteases are classified as “zincins” due to the HEXXH motif in their active site. Furthermore, the vast majority of “zincins” themselves are classified into another protease superfamily called the “metzincins”, which are defined by a more precise active site motif – HEXXHXXG/NXXH/D. The “metzincin” superfamily is comprised of six family members; examples of proteases in these families are mentioned underneath their respective family name [1].

The MMP family, made up of over 25 secreted and membrane-bound proteases, has been the subject of keen interest due to their proposed roles in cancer metastasis via remodeling and breakdown of extra-cellular matrix (ECM) and basement membranes [7-10]. The protease domains of members of the MMP family are approximately 30 to 50% identical on the primary sequence level and MMPs share several domains and structural characteristics. Most MMPs contain a pro-domain, a protease domain, and one or more C-terminal domains [11, 12]. The C- terminal end of the majority of the MMPs, save for the matrilysins (MMP-7 and MMP-26), contains a hemopexin domain. The hemopexin domain is a domain comprised of four short repeats in tandem [13]. It is thought that this hemopexin domain gives the MMPs their differential substrate specificity and provides a platform for the protein-protein interactions for both substrate and inhibitor interactions [14-16]. The membrane-bound MMPs (MT-MMPs) have a C-terminal transmembrane domain which also contributes to the substrate specificity of the MT-MMPs. Several of the MMPs also possess a furin domain, which provides a site where other proteases can cleave MMPs in order to activate their proteolytic activity [11, 17, 18].

ADAMs are also members of the „metizincin superfamily‟. Approximately 23 ADAMs have been identified in humans [19]. In terms of domain structure, ADAMs are similar to the

MT-MMPs, except that the ADAMs lack the hemopexin-like domain and instead have several additional domains [20]. One of these domains is the disintegrin domain, the domain from which the ADAMs in part derive their unique name. The disintegrin domain is responsible for

ADAM binding to integrins, although the breadth of the importance of the integrin/ADAM interaction for ADAM in vivo action is still poorly understood [21, 22]. ADAMs also have another domain, the cysteine-rich domain, that binds to integrins independently of the disintegrin domain [23]. The cysteine-rich domain also binds to other components of the extra-cellular

5 matrix, such as heparin sulfate and fibronectin [23, 24]. A definite in vivo role for the EGF

(Epidermal Growth Factor)-domain, the last of the three major domain differences between the membrane-bound MMPs and the ADAMs, has not yet been determined, but it has been hypothesized that the EGF-like domain also assists ADAMs in binding to their substrates [25,

26].

Astacin proteases, mostly secreted proteases of the „metzincin superfamily‟, have been identified in animals, but have not been identified in either fungi or plants. Proteases of the astacin family include BMP1, hatching enzymes and meprin metalloproteinases [27]. Several astacin proteases, such as the one from crayfish, consist only of a pro-domain and a protease domain. However, many of the eukaryotic astacins have additional domains, C-terminal to the protease domain, such as one or multiple CUB (complement subcomponent Clr/Cls, embryonic sea urchin protein Uegf, BMP-I) and/or EGF domains [28]. Of the astacins, the meprin metalloproteinases have unique structural and functional characteristics that differentiate them from the other astacin proteases – these differences will be elucidated and expanded upon later.

The importance of proteases in a variety of biological processes cannot be overstated.

Proteases play a vital role in a vast number of physiological processes such as: cell homeostasis, tissue remodeling, cell migration, alteration of chemical gradients, signal transduction, the shedding of cell surface proteins, protein maturation, protein turnover, and cell death [18, 29-31].

Modification of proteins by proteolytic action can lead to either limited or extensive degradation of target protein or either activation or inactivation of the activity of different proteins, such as growth factors and cytokines [32, 33]. This type of differential processing of proteins involved in

6 biologically important process by different proteases is important for modulating cellular processes such as inflammation – this concept will be expanded upon in a later section.

Protease activity is tightly controlled by several mechanisms, starting with transcriptional regulation. Some proteases, such as several proteases of the MMP family, are found at very low levels under normal (i.e. unstressed) conditions but upon injury, disease, or other type of insult, the levels of these proteases are quickly increased [34, 35]. Another way to regulate protease activity is to keep the protease physically inactive until conditions make it appropriate for the protease to become active and start proteolysis of its targets. The MMPs and other proteases accomplish this type of regulated inhibition through the use of an N-terminal sequence called the

“pro-domain”, which interacts with the nucleophile (in the case of MMPs, the zinc) and also physically excludes potential protein substrates from the active site until the pro-domain is itself removed by another protease [36, 37]. However, this type of inhibition is not absolute - it has been determined that MMPs still possessing their pro-domain are capable of proteolysis under certain redox conditions, particularly in the presences of oxidants such as HOCl and H2O2 [38].

The third type of protease control involves endogenous inhibitors. One major endogenous inhibitor of protease activity, predominately metalloproteinase activity, is alpha-2-macroglobulin

(α2m). This particular inhibitor is synthesized as an ~800kDa protein in the liver and is a major endogenous protease inhibitor in the plasma, inhibiting the activity of MMPs and ADAMs, but not astacins [39]. Two other classes of endogenous inhibitors are the TIMPs (Tissue Inhibitors of Metalloproteinases) and the serpins (serine proteinase inhibitors). The TIMPs are comprised of 4 small (~ 21 kDa) proteins named TIMP-1, TIMP-2, TIMP-3, and TIMP-4. Collectively, the

TIMPs inhibit several ADAMs and all the known MMPs (albeit with different affinities), but the

TIMPs do not inhibit astacin metalloproteinases [39, 40]. The serpins are a superfamily of

7 proteins, with ~500 members, ranging in size from 40 to 50 kDa. While the serpins have multiple physiological functions, one of their primary roles is to act as endogenous proteinase inhibitors. The serpins mostly inhibit the activity of serine proteinases, but they also inhibit cysteine proteases such as cathespins L, K, and S [41-43]. All these aforementioned factors, transcriptional control, zymogen-forms of proteases, and endogenous inhibitors, work in concert and together play essential roles in keeping proteolytic activity under control.

1.1.2 Control of Protease Activity in the Immune Response

Immune cells express a variety of proteases including polymorphic nuclear leukocyte

(PMN)-dervied serine proteases such as neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CatG) along with several MMPs [44, 45]. Epithelial cells also express several proteases such as MMPs, ADAMs, and meprins; therefore, proteases are present normally and during the immune response, resulting in changing profiles of proteases during the various phases of the immune response [40]. One direct way proteases aid immunity is by producing/activating molecules that kill bacteria. The defensins and the complement-related proteins are examples of this type of protease. Some proteases such as NE directly kill bacteria, but there not many proteases that have this type of direct antimicrobial activity [46, 47].

The best studied members of the IL (interleukin)-1 cytokine family are IL-1α, IL-1β, and

IL-18. These cytokines are synthesized as a “pro-form” (proIL-1α, proIL-1β, proIL-18). While this “pro form” of proIL-1α has activity both when bound into the membrane and intracellularly

(as proIL-1α can act as a transcription factor), proIL-1β and proIL-18 have no activity in their

“pro forms” [48]. Caspase-1, which is also called IL-1β convertase (ICE), converts the majority of the synthesized proIL-1β and proIL-18 (intracellularly) to their active forms by removing a

8 portion of the N-terminus from proIL-1β and proIL-18 [49, 50]. The active IL-1β and IL-18 cytokines are then secreted outside the cell, where the cytokines can be cleaved by extracellular proteases [51].

However, a significant portion of proIL-1β and proIL-18 escape the cell during injury.

Furthermore, some cells types lack caspase-1 expression and thus express the pro-forms of the

IL-1 family of cytokines, thereby providing an opportunity for other proteases, both in the azophillic granules and also extracellular proteases, to convert these pro-cytokines into their active forms, thus increasing inflammation [52]. It has been shown that several proteases are able to convert proIL-18 to IL-18, such as proteinase-3, meprin A and B, and MMP-3 [52, 53].

Furthermore, MMP-2, MMP-3, MMP-9, and meprin A and meprin B can convert proIL-1β to

IL-1β [54-56]. However, if mature IL-1β is incubated with MMP-1, MMP-2, MMP-3, or MMP-

3 over a long period of time, these proteases degrade and inactive IL-1β [57].

Another way aside from modulating the amount of protease level/activity via cytokine control and spacial/time separation is by the aforementioned endogenous inhibitors.

Macrophages, monocytes, and neutrophils express serpin B1 to protect themselves from self- injury by their own expression of CatG, NE, and PR3 [58]. Furthermore, a major component in blood is the serpin Al-PI, a 52kDa protein which inhibits the protease activity of CatG, NE, PR3, along with other proteinases expressed by immune cells, which prevents these immunologically important proteases from acting outside inflammatory sites [42]. Immune cells such as eosinophils, macrophages, and neutrophils, also express TIMPs [35]. The concentrations of

TIMPs can also be modulated by cytokines, as TNF (Tumor Necrosis Factor)-α increases TIMP-

1 production [40].

1.2 Proteases in Inflammation and Disease

There are several main roles proteinases can play within the immune response. For example, proteases may aid immune cell migration to sites of infection and injury via degradation of ECM and basement membranes. MMPs have been implicated in this function because several MMPs are expressed by immune cells and these proteases are well known to degrade components of the ECM. There have been several models utilized to study cell migration. Studies with serine, cysteine, and aspartic acid proteases on migration of tumor cells through basement membranes ex vivo provide evidence showing that these proteases were not directly responsible because tumor cell migration was not arrested with specific protease inhibitors [59]. Several similar ex vivo studies showed that MMP-inhibitors arrested tumor cell migration, therefore it was concluded that MMPs, particularly the MT-MMPs were directly involved with tumor cell transmigration through the basement membrane. These experiments with the MMPs showed the presence of novel type IV collagen fragments in the disrupted basement membrane, furthermore confirming the potential role of MMPs in cell migration [59,

60]. Supplementing this cancer cell data, it has been shown that TIMP-1 inhibits neutrophil migration through basement membranes, indicating that one or more proteinases expressed by neutrophils are involved with its migration [35]. However, it is thought that proteases contribute to cell migration primarily through indirect means, such as through the generation of chemical attractant gradients, rather than by large-scale proteolytic cleavage of the ECM [61].

Another proposed way that proteases affect cell migration particularly that of immune cells is by creating and altering chemokine gradients. More and more chemokines are being discovered as in vitro substrates of proteases. MMPs and other proteases, such as the cathepsins, have been shown to modulate the activity of many chemokines. These proteases can either

10 activate or inactive these various chemokines by removing their N or C-termini, while extensive degradation of these chemokines predictability leads to the loss of chemokine activity [62]. Also, degradation products generated by protease-mediated ECM cleavage have been shown to act as chemoattractants. Given that leukocytes pass through barriers such as the basement membrane many times, it is thought that this migration does not involve large-scale remodeling of the ECM and basement membrane, but that these chemoattractants are essential for this immune cell migration [61].

Chemokines are not the only type of protein involved in immunity that are altered by proteolysis. The role of the MMPs in differentially cleaving cytokines such as the “pro- cytokine” members (proIL-1β and proIL-18) has already been discussed, but other cytokines have also been discovered as an expanding class of substrates for direct cleavage by several proteases. PMN-derived serine proteases have been shown to act upon several cytokines, including: IL-2, IL-6, IL-8, and TNFα [44, 63]. Cleavage of these cytokines decreases their activity, thus PMN-derived serine proteases act as a brake on inflammation.

When proteases accumulate at sites of infection or become active at inappropriate times, they can cause and exaggerate injury rather than preventing or attenuating it, showing the need to keep protease levels tightly controlled by temporal separation and anti-protease inhibitors. For example when high amounts of neutophil elastase are present in a disorder such as cystic fibrosis, this enzyme extensively degrades normal immune sentinels such as surfactant protein D.

The extensive loss of this important first-responder anti-microbial agent causes a niche in the lung that resident bacteria excessively fill, thereby causing chronic infections [64, 65].

Uncontrolled activity of neutrophil elastase has also been associated with another lung disorder,

COPD (Chronic Obstructive Pulmonary Disease) [66]. In general, for the case of lung disorders,

11 a prolonged inflammatory response leads to lung damage by uncontrolled protease activity damaging connective lung tissue and causing fibrosis via bioactivation of transforming growth factor-β [67, 68]. The neutrophil derived serine proteases are also known to degrade TIMPs, thereby allowing the MMPs to have more activity when neutrophils are chronically present in inflamed tissue [69].

Neutrophil elastase is not the only serine protease expressed by leukocytes that is linked with disease. Cat-G and PR3 have been found in cancer cells, breast cancer and myeloid leukemia cells respectively. It was originally thought that these proteases aid in cancer cell migration via direct remodeling of the ECM [44]. However, it is also possible that these proteases help cancer cell migration via cytokine function alteration. Another way proteases could help cancer cell survival is by altering and destroying existing chemokine gradients, preventing immune cells from homing in on the location of the tumor cells. This mechanism of immune system evasion by tumor cells has already been proposed for tumor-expressed MMPs

[70].

A major observation in many inflammatory diseases is that there is an imbalance of proteinases and antiproteases (inhibitors). Under normal conditions, levels of MMPs are very low to non-existent and any residual activity needed by the MMPs for normal homeostatis is kept under strict control by the TIMPs and other inhibitors. During the inflammatory response, the levels of MMPs are significantly increased and thus the MMP activity overcomes TIMP inhibition. For example, analysis of colon samples from IBD patients show that the ratios of

MMP-1/TIMP-1 and MMP-3/TIMP-1 are significantly skewed and increased towards the proteinase levels (MMP-1 and MMP-3) compared to normal inflammatory disease [35].

Typically, many MMPs are only upregulated in circumstances of injury or disease, in part because the expression of MMPs is cytokine-dependent in many cases [71, 72]. This can lead to cycles where upregulation of one cytokine leads to an expression of a protease such as MMP and the levels (and thus the activity) of the protease is controlled by a feedback mechanism where the protease concentration is decreased when the cytokine levels are decreased via clearance and degradation. Furthermore, this increase of MMP levels alters the concentration of other inhibitors. It has been shown that MMP-9 cleaves an inhibitor of NE, the serpin α-1 antiproteinase [73].

A wide variety of data has been collected on the contribution of proteases to disease by using knock-out animals to evaluate the role of proteases in disease pathogenesis. Interestingly, many of the MMP KO animals do not show a phenotype under normal conditions – the animals need to be stressed first before an observable phenotype is detected. The data accumulated from the single MMP KO mice thus far indicate that many of the MMPs have redundant proteolytic functions and thus the loss of function of one MMP is compensated by another MMP.

Furthermore, the various phenotypes seen in the challenged MMP KO mice show that while no single MMP is essential for normal homeostasis, the MMPs have specific roles in many biological functions such as cell migration and inflammation [18, 45, 74].

1.3 The Meprin Metalloproteinases

1.3.1 Structural Characteristics

The meprin metalloproteinases are unique mammalian proteinases of the astacin metalloproteinase family that are comprised of evolutionarily related α and/or β subunits. The primary amino acid sequences of these subunits are approximately 50% identical in humans and

13 rodents [28]. The domain structures of the meprin α and β subunits are nearly identical, save for the addition of an “I” (Inserted) domain after the TRAF (Tumor Necrosis Factor Receptor-

Associated Factor) domain in the α subunit (Figure 3). The consequence of this I domain is that the meprin α subunit is cleaved at this domain intracellularly in the endoplasmic reticulum during biosynthesis, thus the mature (secreted) form of meprin α lacks a segment of the C- terminus that the β subunit retains in its mature form: the EGF-like domain, the transmembrane domain, and the cytoplasmic tail [75, 76].

Due to the differential processing of meprin α and β subunits, the meprin metalloproteinases exist as multiple isoforms, both secreted and membrane-bound. These isoforms are labeled as meprin A and meprin B, depending on their subunit composition as shown in Figure 3 Homomeric (secreted) meprin A (containing α-α submits), homomeric

(membrane-bound) meprin B (containing β-β subunits), and heteromeric meprin A (membrane bound, containing α-β subunits). Meprins are the only known astacins that form homo- and hetero-oligomers. Furthermore, meprin B is the only astacin that has a transmembrane C- terminal domain [27, 75, 77].

Homomeric meprin A is a “multimer of meprin α dimers” by which covalently bound meprin α dimers associate noncovalently with other meprin α dimers. Studies with recombinant meprins have show that secreted meprin A is the largest known extracellular protease, but the molecular size of this multimer is dependent on protein and salt concentration and the activation status of the meprin proteinase. Latent homomeric meprin A can reach sizes of approximately

Figure 3. The domain structure of the meprin α and β subunits and their associated meprin A and B isoforms. The meprin subunits α and β are identical in domain structure, except for the I (“inserted”) domain present in the α subunit. This I domain is necessary and sufficient for meprin α to lose its C-terminal end during maturation, resulting in the three isoforms of meprin: membrane-bound meprin B (dimer of meprin β subunits), membrane-bound meprin A (tetramer of α and β subunits, found in α2β2 or α3β1 ratios), or secreted meprin A

(multimer of α-α dimers).

80 to 100 subunits (40 to 50 dimers associated together) under high meprin concentrations in low salt conditions. When the meprin α subunit is activated and lacks its pro-piece, the highest molecular size found is approximately 20 subunits (10 dimers associated with each together) under lower salt conditions regardless of protein concentration [77]. It is thought that this self- oligomerization property of meprin α serves to concentrate the meprin proteinase in the extracellular space.

The homomeric meprin B and heteromeric meprin A isoforms are membrane bound, thereby sequestering and concentrating the activity of these meprins at the cell surface.

Recombinant homomeric meprin B is a dimer of meprin β subunits regardless of activation status of the subunits and salt and protein concentrations. Heteromeric meprin A is comprised of a tetramer of meprin α and meprin β subunits. It is thought that the main form of this tetramer, as shown in Figure 3, is a dimer of the meprin β subunit covalently bound to the meprin α attached noncovalently to another covalently bound meprin β/α dimer though the meprin α subunits of the heteromeric dimers. The main form of this heteromeric meprin A tetramer appears to be α2β2 form, however meprin tetramer forms with α3β1 have been detected [27, 75, 77].

The MAM (meprin, A-5 protein, and receptor protein-tyrosine phosphatase mu) and

TRAF domains of the meprins contribute to these unique oligomeric structures by facilitating protein-protein interactions between the subunits. The MAM domain of meprin α and β contributes to inter and intra subunit disulphide bond formation. Meprin β dimers are comprised of 3 intersubunit disulphide bonds, 2 between the MAM domains and 1 between the TRAF domains [78]. Meprin α dimers possess only 2 intersubunit bonds, both in the MAM domain, because the cysteine forming the disulphide bond for the meprin β subunits is not conserved in

16 the meprin α subunit [27]. Certain charged residues within the MAM domain also contribute to both the stability and oligomerization properties of the meprin A homooligomer [79].

Meprins are also extensively glycosylated, with approximately 15 to 20 percent of the mass of meprin being comprised of carbohydrates [80, 81]. Mouse meprin α has 9 out of its 10 possible N-linked glycosylation sites occupied by sugars [82]. No individual glycan is essential for meprin A oligomerization, however, these glycans collectively are important for proteolytic activity and secretion of meprins. Furthermore, mutational analysis of the glycans of mouse meprin α show that the combined glycans located at two glycosylation sites in the meprin α protease domain (asparagine 152 and 270) are essential for both covalent and non-covalent interactions between the meprin α subunits comprising the meprin α dimers [82-84]. These sugars are important for meprin interaction with biologically important lectins such as mannose binding lectin (MBL) [85, 86]. Meprin glycosylation is also useful for meprin purification by lectin-laden resins, which led to the discovery of the MBL/meprin interaction [86].

Meprins were first discovered and purified from the brush-border of mouse kidneys, where they comprise roughly 5% of the total protein content of the brush border membranes [87,

88]. Since then, meprin expression has been detected in intestine, skin, and certain populations of leukocytes [89-92]. Meprins have also been detected in rodent and human urine [77, 93, 94].

Since meprins have been discovered at sites of inflammation and they have been determined to be involved in the pathogenesis of several inflammatory diseases, it is important to discover what type of substrates meprins hydrolyze and what type of substrates meprins act upon in vivo.

1.3.2 Cleavage Site Specificies and Known Meprin Substrates

Individual proteases were originally suspected to have thousands of substrates, but it is now thought that specific proteases only have a small subset of substrates (i.e. they are more specialized) [95]. However, meprins stand out among other proteases in that they can cleave a wide variety of both peptide and protein substrates. Amongst the better-studied meprin substrates in vitro are ECM proteins (collagen IV, fibronectin, gelatin, laminins, and nidogen) and active peptides (gastrin and bradykinin), [96, 97].

Meprins have approximately 40% sequence homology between the meprin α and β subunits, but there is ~55% homology between the protease domains of the meprin α and β subunits [28, 98]. Despite this homology, the meprin subunits have markedly different peptide bond cleavage specificites. Meprin α has a preference for small and aromatic residues at the P1 and P1‟ residues of substrates, with an additional preference for proline at the P2‟ position; meprin β prefers acidic residues at the P1 and P1‟ positions of its substrates. This residue discrimination is thought to be due to several key amino acids differences in the active sites of the meprin α and β subunits. The acidic residues of meprin β substrates are thought to interact with three key basic residues within the active site of meprin β, forming salt bridges [98]. In the meprin α subunit, these corresponding basic residues are aromatic residues, which are thought to explain differences in the cleavage preferences of meprin α and meprin β [99, 100].

Data published by Becker-Pauly et al in 2011 yielded new information about the active site preferences for astacin proteases in general. The astacin proteases, human meprin A, human meprin B, and LAST_MAM proteases, were incubated with chemically-protected oligopeptide libraries generated from HEK (human embryonic kidney) cells. This technique is called PICS

(Proteomic Identification of Protease Cleavage Site Specificity). The astacin proteases tested have a conserved preference for negatively charged residues (glutamic and aspartic acid) in the

P1‟ position of peptide bonds. This preference for negatively charged residues by meprin A was not previously seen in a peptide library screen of mouse meprin A for active site specificities, however there are known differences between human and mouse meprin A cleavage of substrates [100, 101]. Furthermore, astacin proteases exhibit negative cooperativity - proline in the P2‟ and P3‟ positions affects the amino actid cleavage preference of the astacins at the P1‟ position. It was also found that meprin α has a peptide bond cleavage-preference similar to that of meprin β and that the presence of aspartate and glutamate in the P1‟ position of a cleaved peptide bond decreases the frequency that proline will be in the P2‟ position and vice versa. This is the first documented observation of negative site cooperatively for meprin cleavage sites

[102].

One point of interest is finding new physiologically relevent substrates for meprins. A proteomic approach with cell culture (transfecting meprin in HEK293 cells) led to the identification of 17 potential meprin substrates such as clusterin and vinculin – further implicating involvement of meprins in processes such as immunity and tissue remodeling [103].

Studies with meprins utilizing other proteomic approaches such as the aforementioned PICS and

TAILS (Terminal Amine Isotopic Labeling of Substrates) have recently identified a novel meprin substrate, FGF-19. Meprin B processed FGF-19 (Fibroblast Growth Factor-19) showed less activity than full-length FGF-19; this form of FGF-19 decreased the ability of keratinocytes to proliferate and migrate. These proteomic techniques have also confirmed the cleavage site of several other meprin substrates, including IL-1β and bradykinin [102].

Cytokines are an expanding class of meprin substrates, further implicating meprins in the immune response. One initial cytokine finding was that meprin B (but not meprin A under the conditions tested) cleaves osteopontin. Osteopotein is a cytokine involved in bone growth which also acts as a transitional extracellular matrix protein [97, 104]. More recent data has shown that meprins cleave cytokines of the IL-1β family, such as proIL-1β and proIL-18. Furthermore, it was shown that homomeric meprin A and B and heteromeric meprin A process proIL-1β into a biologically active form. The cytokine proIL-18 is also processed from a latent to an active form by heteromeric A and meprin B [53, 55, 56]. It has been reported that meprin A cleaves and inactivates several chemokines, such as MCP-1 (Mouse Chemoattractant Protein), RANTES

(Regulated upon Activation, Normal T-cell Expressed, and Secreted), and MIP-1 (Macrophage

Inflammatory Protein), [104].

There are some limited data linking in vitro meprin substrates to in vivo models. For example, meprin β KO mice had less active IL-18 in their serum upon DSS (dextran sulfate sodium salt) administration than their WT counterparts. Meprin α KO mice had more active IL-

18 in their serum compared to WT controls, indicating that meprins are involved in processing proIL-18 in vivo [53]. A new meprin substrate has been identified in brain, APP (Amyloid

Precursor Protein). APP fragments approximately 11 and 20 kDa in size have been identified in meprin WT mice, but not meprin β KO mice, indicating that meprin also may process this substrate in vivo [105].

1.3.3 The Role of Meprins in Inflammatory Disease

Given that meprins cleave extracellular matrix proteins, it is hypothesized that meprins are involved in the mobility of cells. In this vein, meprins have been implicated in the

20 pathogenesis (metastases) of cancer. Meprins are expressed by several cancer cell lines, including breast cancer cell lines and prostate cancer cell lines. Several cancer cell lines have a alternatively spliced meprin β transcript, β‟ [106-108]. The physiologic role of this β‟ transcript is not yet understood.

Meprins have been implicated in the pathogenesis of several inflammatory diseases, such as acute renal failure. Mice lacking the meprin β gene had less inflammation when their kidneys were subjected to ischemia/reperfusion injury [109]. Also, mice with “low” meprin activity (e.g.

C3H/He mice lack meprin α expression in adult kidney) are less susceptible to this type of renal injury, either induced by sepsis or ischemia/reperfusion [110, 111] Meprin A was also shown to increase tissue damage in both bladder and kidney upon bacterial lipopolysaccharide (LPS) challenge [112].

Currently, attention is focused on meprin involvement in human disease and disorders.

Meprins were first implicated in diabetic nephropathy using mice studies. Expression of both meprin α and β subunits is decreased in diabetic mice [113]. This is in constrast to MMPs, as the meprin metalloproteases are downregulated in several instances of injury; both meprin α and

β expression is decreased in epithelial cells of the intestine during inflammation [94]. Unlike most of the MMPs, meprins are found constitutively expressed in tissues such as the kidney, intestine, and the skin [90, 114, 115].

There are several confirmed links between meprin and human disease. A polymorphism in the meprin B gene has been linked to diabetic nephropathy [116]. Meprins have also been implicated in the pathogenesis of inflammatory bowel disease (IBD) in both humans and mice.

In an in vivo mouse model of IBD, meprin α KO mice had more severe inflammation than WT

21 controls. The DSS-treated WT and meprin α KO mice displayed marked differences in both their colon and serum cytokine concentrations. However it was only a smaller subset of the all the 17 cytokines tested that were significantly different in the WT and meprin α KO mice subjected to the IBD model – this will be elaborated on in another section [53, 117]. A polymorphism in the human meprin α gene within the 3‟ UTR (untranslated region) has been linked to increased IBD susceptibility in certain human populations. Also, IBD patients had lower levels of meprin α mRNA expression in their colons than in the colons of healthy controls

[118]. The 3‟UTR of genes is responsible for stability of the transcript and it is hypothesized that this 3‟UTR polymorphism may be involved with the stability of the meprin α transcript

[119, 120].

1.4 Interleukin-6

Interleukin-6 (IL-6) is a multifunctional cytokine that is important for a number of various physiological processes including hematopoiesis and the inflammatory response. The gene encoding human IL-6 is located on chromosome 7p21 and is approximately 5 kb in length, and the mouse IL-6 gene is located on chromosome 5 [121]. IL-6 is produced by a number of immune cells including B and T-cells, monocytes, macrophages, eosinophils, and dendritic cells

[121-123]. IL-6 expression has also been reported in a variety of non-immune cells such as osteoblasts, skeletal and smooth muscle cells, fibroblasts, keratinocytes, and even tumor cells

[121, 124, 125]. IL-6 expression from these cell types is induced by other cytokines such as IL-

1, IL-17, and TNF-α [126-128]. Other major physiological stimuli for IL-6 expression are bacterial endotoxins and viral infections [129, 130].

The levels of IL-6 are low in healthy (and younger) individuals until there is infection or injury, after which the levels of IL-6 increase significantly in a short amount of time. Since IL-6 expression increases dramatically almost immediately after infection and injury, this cytokine is a major initiation factor of the acute phase response, a variety of biochemical changes that occur immediately after the initiation of inflammation in order to arrest pathogen growth and limit the damage caused by inflammation [121, 131]. As such, IL-6 has been found to be essential for the clearance of several pathogens; IL-6 KO mice cannot effectively clear infections from pathogens such as vaccinia virus, Listeria monocytogenes, and Pneumococcal pneumonia [132, 133].

Studies looking into the role of IL-6 in wound healing have also revealed that IL-6 is essential for proper wound resolution and closure [134, 135]. Other than the aforementioned inflammatory defects and abnormalities with acute phase induction, IL-6 KO mice exhibit no developmental or reproductive defects [121, 133].

There are several discernable differences between human and mouse IL-6. The human and mouse IL-6 genes share 65% homology. The human and mouse IL-6 gene products (both the precursor and the mature forms) have approximately the same amino acid length. However, human and mouse IL-6 proteins share only 42% homology [121]. Furthermore, human and mouse IL-6 can undergo a variety of post-translational modifications such as phosphorylation and glycosylation. IL-6 isoforms ranging from 21 to 28 kDa in size have been detected in vivo for both mice and humans. Human IL-6 has two sites for N-linked glycosylation and one site is continually occupied, while mouse IL-6 has no N-linked glycosylation sequons but has several potential sites for O-linked glycosylation [121, 136].

While human and mouse IL-6 greatly differ in their primary sequences and some of their post-translational modifications, these proteins have a common structure. The structure of IL-6

23 has been solved and is shown in Figure 4. The core structure of both mouse and human IL-6 is comprised of a 4-α helical bundle protein arranged by two pairs of antiparallel α-helices arranged in an up-up-down-down configuration. Both human and mouse IL-6 have 2 disulphide bonds within the core helical structure and the locations of these disulphide bonds are conserved among the two IL-6 isoforms [121, 136, 137].

Aside from the core α-helical structure and a small helix outside the main core bundle,

IL-6 possesses a short C-terminal region and a longer N-terminal region. Structure-function and mutagenesis studies with IL-6 show that while the N-terminus is not essential for the activity of this cytokine, the C-terminus of the cytokine is extremely important [138]. Both human and mouse Il-6 constructs lacking five amino acid from their C-terminal ends have significantly less activity (~1000x less) than full-length human and mouse constructs [139-141]. It is thought that these C-terminal residues are important for IL-6 binding with the IL-6 receptor Modifications to the C-terminus of IL-6 aside from truncation also decreased the activity of IL-6. While substitution of the C-terminal residues of mouse IL-6 with the equivalent C-terminal residues of human IL-6 did not adversely affect mouse IL-6 activity, semi-random mutagenesis of the C- terminal mouse IL-6 residues Leu181-Arg182-Qln183-Met184 significantly decreased the activity of this cytokine several-fold [142].

Interleukin-6 requires two other proteins for cell uptake and subsequent signal transduction. The first protein required is the membrane-bound IL-6 receptor (IL-6R), which is also referred to as IL- 6Rα, gp80, or CD126. IL-6R is important for IL-6 binding, but not for signal transduction. Furthermore, IL-6R also exists as a soluble form (sIL-6R) which is generated by proteolysis or by alternative mRNA splicing [121]. The second protein required for the IL-6 signaling complex is glycoprotein 130 (gp130), which is required for transduction of the

Figure 4. Crystal structure of human interleukin-6. Interleukin-6 is comprised of a small, core 4 α-helical structure, with the major helices labeled A-D. These helices are in an up-up

(helices A and B), down-down (helices C and D) orientation. The helix E is a smaller helical structure in a long loop between helices C and D. In this model of IL-6, the N and C-termini are in close proximity to each other. The first few N-terminal residues of IL-6 are unstructured, thus the N-terminus of IL-6 in this model starts at Leucine19, as indicated in the picture. The C- terminal end of IL-6 is also highlighted by “C-ter” in this model. This interleukin-6 presentation was adapted from [143].

25 signaling cascades needed for the biological function of IL-6. While gp130 is a ubiquitously expressed membrane-bound protein, this protein also exists as a soluble form (sgp130) generated by alternative mRNA splicing [121, 136, 144, 145]. There are several consequences with having these various soluble and membrane-bound forms of the IL-6 receptor/signaling complex, as summarized in Figure 5. “Trans-signaling” allows IL-6 to induce its biological effects at virtually every location in the body, even though the expression of the membrane-bound IL-6R is limited to T and B-cells and monocytes/macrophages, due to sIL-6R and the ubiquitous presence of gp130 [121]

IL-6 activity is controlled primarily at the expression level and through expression of the soluble and membrane-bound components of the IL-6 receptor. However, there is increasing evidence that IL-6 activity can be controlled directly via proteolytic cleavage. Several lysine and arginine-specific proteases, known as RGP-A, RGP-B, and KGP, expressed by Prophyromonas gingivalis, the bacteria that cause gingivitis, degrade both IL-6 and sIL-6R, thereby hindering the inflammatory response and preserving the life of the bacteria [146, 147]. The only mammalian proteases known to act directly on IL-6 are the PMN-derived serine proteases: CatG, PR3, and

NE. These proteases degrade IL-6 by cleaving either at the N-terminus or at the various loops between the 4 α-helices of IL-6. PMN-derived proteases degrade and thus decrease IL-6 activity

[63, 148].

Figure 5. The classical signaling, trans-signaling, and inhibition of interleukin-6 trans- signaling pathways. A) (Classical signaling) Under classical conditions, IL-6 (in red) binds in a membrane-bound complex with gp130 (in green) and the IL-6 receptor (IL-6 in blue), causing a downstream signaling pathway in the cell. Immune cells such as macrophages and neutrophils are the only cells that express the membrane-bound IL-6 receptor. (Trans-signaling) However, epithelial and endothelial cells can respond to IL-6 if they intake IL-6 pre-complexed with the solublized form of the IL-6R (sIL-6R), which binds to the membrane-bound gp130 on the cell surface. B) Trans-signaling of IL-6 is arrested by a molar excess of the solubilized gp130 protein, which is generated by alternative mRNA splicing. While the sgp130 does not interfere with the classical IL-6 signaling, the sIL-6/sIL-6R complex preferentially binds to the spg130

[145].

While IL-6 is an important cytokine for the inflammatory response, dysregulated production of IL-6 causes an over-abundant inflammatory response and thus contributes to the severity of disease. Over-production of IL-6 has been implicated in the pathogenesis of several inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease (IBD) [121].

IL-6 KO mice develop significantly less colitis upon experimental induction of IBD vs wild-type controls [149]. Furthermore, peripheral blood mononuclear cells and lamina propria mononuclear cells (isolated from human colon biopsies) have significantly increased IL-6 production as compared with the same type of cells isolated from control patients [121, 150,

151]. For these reasons, IL-6 is considered a therapeutic target for the treatment of IBD and several other inflammatory diseases [121].

1.5 Mannose Binding Lectin

Mannose binding lectin has been implicated as a potent, specific endogenous inhibitor for the meprins [86]. The structure of MBL is shown in Figure 6. MBL is comprised of a multimer of trimers that are synthesized in the liver, which then circulate in the blood as larger-order forms. This self-oligomerization property of MBL serves to help facilitate MBL binding to its targets. The CRDs (carbohydrate recognition domain) on one subunit, the 3 polypeptide chains comprising a complete subunit as seen on the bottom of Figure 6a, are approximately 45 Å

(Angstroms) apart from each other. The distances between the CRDs present on different trimers of the larger-order MBL forms are still unknown [152-154].

The main function of MBL is to act as an innate immune system sentinel. MBL recognizes certain terminal carbohydrate moieties, namely mannose, fucose, and N- acetylglucosamine on pathogens and mediates their destruction by opsinization or complement

Figure 6. Subunits and structure of mannose binding lectin. Mannose binding lectin (MBL) is comprised of a multimer of trimers and each trimer is comprised of three identical protein subunits, as shown in Figure 6a. A small N-terminal region (shown in blue) assists with protein- protein interactions to form the larger multimer of MBL. A collagen-like helix (shown in pink) connects the N-terminal portion with the C-terminal portion of the molecule, which is made up of an α-helical coiled coil region (also called the “neck” region, shown in green) and a CRD

(carbohydrate-recognition domain, shown in yellow) on its C-terminal end. Each trimer oligomerizes with other trimers, as shown in Figure 6b, to make larger-order multimers ranging from dimers to hexamers [155].

29 activation [152]. Many glycoproteins on mammailian cells terminate with sialic-acid and/or are not glycosylated in a spacial manner that facilitates efficient binding of MBL. These two mechanisms are the main ways by which MBL differentiates “self” structures from bacterial structures [154]. MBL was originally thought only to bind to non-self glycoproteins, but accumulating data indicates that MBL also recognizes self-proteins particularly in cases of injury. These self-proteins are called “altered self-ligands” [154]. Meprin was identified as an

MBL ligand during a search for MBL ligands in the kidney. During this study instituted by

Hirano, Ma et al. 2005, it was found that MBL inhibited meprin proteolysis of casein, gelatin, collagen IV, and parathyroid hormone via calcium-dependant recognition of the meprin glycans

[121]. While actinonin and a number of hydroxmate derivatives inhibit meprins, they also inhibit other proteases [101, 156]. Thus, there is a need to search for a specific meprin inhibitor.

Figure 7. Mannose binding lectin-mediated complement activation. One of the primary functions of MBL is to act as a “first responder” to invading microbes by recognizing and attaching to carbohydrate motifs on bacterial cell walls that are not present on “self” cells

(Figure 7a). Once MBL attaches to its microbial target, MASPs 1-3 (MBL-Associated Serine

Proteases – the role of MASP3 is unclear at this time) attach to the N-terminal portion of MBL and cleave complement proteins C2, C3, and C4 (Figure 7b). These cleaved proteins form complexes (such as C2aC4b) which congregate on the bacterial surface, causing a cascade of downstream reactions which ultimately lead to the death of the bacterial cell via lysis [154, 155].

1.6 Rationale for Experimental Aims

There is a well-established body of data implicating meprin in the pathogenesis of inflammatory diseases such as IBD and acute renal failure [109, 110, 117, 118]. In the course of evaluating and comparing the inflammatory response between the wild-type, α, and αβ meprin knock-out (KO) mice, it was observed that the concentration of several cytokines and chemokines were increased in both the serum and colon of the meprin α KO mice in the IBD model (Figure 8a and Figure 8b). The cytokines IL-6 and the chemokines MCP-1 and

RANTES were significantly higher in both the serum and colons of DSS (Dextran Sodium

Sulfate)-treated meprin α KO mice compared to DSS-treated meprin wild-type mice; the cytokines IL-1β and IL-18 and the cytokines IL-5, IL-6, IL-12, and interferon-gamma (IFN-γ), were also significantly increased in the serum and colon, respectfully, of DSS-treated meprin α

KO mice. Furthermore, interleukin-6 (IL-6) was the sole cytokine increased in the colon of double meprin KO mice (Figure 9). Interestingly, the 4 out of the 5 cytokines whose concentrations were increased in the DSS-challenged meprin α KO mice (IL-1β, IL-18, MCP-1, and RANTES) and the 2 cytokine levels that were increased in the colon of the DSS-challenged meprin α knock-out mice (MCP-1 and RANTES) are all already known meprin substrates in vitro [53, 55, 56, 104].

Given that meprins have been found to be implicated in the pathogenesis of several inflammatory diseases, discovering meprin substrates linked to inflammation is a high priority.

The differential cytokine profiles between the meprin null and meprin wild-type mice upon DSS treatment, as seen in Figures 8 and 9 show several things.

Figure 8. Interleukin-6 is increased in the serum and colon of meprin KO-mice in an experimental model of inflammatory bowel disease. WT (blue bars) and meprin α KO mice

(red bars) were challenged with DSS and at day 5, both serum and colon tissue were taken from the mice. From the panel of 17 cytokines evaluated in the biological samples, only the concentrations of cytokines in the blood serum (A) and colon tissue (B) that were statistically significant in the DSS-treated groups are shown (*, p<0.05 and **, p<0.001) [53, 117]. Five cytokines were significantly different in the blood serum and seven cytokines were different in the colon tissue between the WT and αKO mice treated with DSS; levels of IL-6, MCP-1, and

RANTES were consistently increased in both colon and serum of DSS-treated α-KO mice [53,

117]. Note: levels of IL-1α, IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12, TNF-α, GM-CSF, IFN-γ, and MIP-1α were not significantly different in serum samples quantified from DSS-treated wild-

33 type and meprin αKO mice. Also, levels of IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-9, IL-18 TNF-α,

GM-CSF, and MIP-1α were not significantly different in colon samples taken from DSS-treated wild-type and meprin αKO mice.

3500.0 * 3000.0 2500.0 WT 2000.0 αβ KO 1500.0 1000.0 500.0

Cytokine concentration (pg/mL) concentration Cytokine 0.0 IL-6

Figure 9. Interleukin-6 is increased in the colon of meprin double KO-mice in an experimental model of inflammatory bowel disease. WT and meprin αβ KO mice were treated with DSS and at 5-days after DSS challenge, colon tissue was taken from the mice and the levels of 17 cytokines in the tissue were quantified by ELISA. The figure shows the differential levels of the only cytokine that was elevated in the KO mice vs the WT – interleukin-

6 (*, p<0.05). The levels of IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-8, IL-9, IL-10, IL-12, TNF-

α, GM-CSF, IFN-γ, MCP-1, MIP-1α, and RANTES were not significantly different in colon samples tested from DSS-treated wild-type and meprin αKO mice. Furthermore, no IL-6 was detected in the colon samples from either the wild-type or meprin double-null control animals, which were given untreated water [53, 117].

The cytokine profile results from the WT and meprin-null mice in the IBD experiments show that meprins do not completely alter cytokine profiles and that only a few discrete cytokines are affected. The differential serum cytokine profiles between wild-type and meprin α null mice listed potential new cytokine substrates (such as IL-5, IL-6, IL-10, and IL-10) to test to determine if these cytokines could also be direct meprin substrates. Since interleukin-6 levels are elevated in both the meprin α null and the meprin double-null mice subjected to the IBD damage model, it was decided that IL-6 is a prime candidate for examining interactions with meprins in vitro.

The meprin null mice had increased IL-6 levels in inflammation compared to meprin wild-type mice in an experimental model of inflammatory bowel disease. It is proposed that a biological role of meprins is to keep IL-6 activity down by cleaving and degrading this cytokine.

From the cytokine profiles shown in Figure 8 and Figure 9, it is clear that the lack of meprin expression does not alter the levels of cytokines globally, only a select subset of cytokines/chemokines are altered in these cases. However, in both the meprin α and the meprin

αβ KO mice, looking at both systemic response (serum cytokine/chemokines levels) and the local inflammatory response (colon cytokine/chemokine levels), only the levels of IL-6 were consistently increased in the KO animals verses the WT controls in the IBD. These data lead to the hypothesis that meprins directly interact with IL-6 and keep the activity of this cytokine under control by direct proteolysis. The aim of this project was to characterize the interaction between the meprin isoforms and IL-6 kinetically, determine the meprin cleavage site on IL-6, and evaluate the consequences of meprin cleavage of IL-6 on the activity of the cytokine.

To investigate this hypothesis, IL-6 was incubated with recombinant homomeric meprins and the resulting fragmentation pattern of IL-6 was analyzed via SDS-PAGE and protein staining

36 by either Coomassie staining or Western blotting. The site of cleavage on IL-6 by meprins was determined by comparing the peptide maps between meprin-cleaved and uncleaved IL-6 using mass spectrometry. Finally, the biological consequence of meprin cleavage on IL-6 was investigated using a cell line that is only able to proliferate in medium supplemented by either mouse or human IL-6 [157]. Whenever possible, both human and mouse IL-6 were used during the course of these experiments in order to gain a fuller picture of how meprins might affect the course of inflammatory disease in both mice and humans by directly acting upon IL-6.

Chapter 2: MATERIALS AND METHODS

2.1 Meprin Purification from Cell Culture Medium and Kidney Tissue

Homomeric meprin A and homomeric meprin B were expressed in human embryonic kidney 293 (HEK 293) cells as previously described [98, 100]. The mouse meprin α construct

(coding for amino acids 1-615; possesses a C-terminal 6x His tag) and the truncated rat meprin β construct (coding for amino acids 1-648; possesses a C-terminal 6x His tag) were stably transfected into HEK 293 cells with Lipofectamine™ 2000 (Invitrogen) reagent using the transfection protocol provided by Invitrogen. These particular truncated meprin α and β constructs were used for the production of recombinant, homomeric mouse meprin A and rat meprin B, respectively, for two main reasons. First, the C-terminal 6x His tags aided in the purification of these meprin constructs. The C-terminal over the N-terminal of meprins was chosen to position these 6x His tags due to the loss of the signal sequence during maturation of the meprin protein in the ER. Furthermore, if the 6x His tag was placed on the C-terminal of the full-length mouse meprin α construct, the tag would also be lost during maturation as the meprin

α I domain is cleaved during maturation, leading to the lack of all meprin α domains C-terminal of the cleaved I domain site in the mature meprin α protein [76]. Second, these truncated constructs were used to ensure that the expressed meprin A and meprin B proteins would be secreted in the medium of the HEK 293 cells. If the full-length meprin β protein construct was used to transfect the HEK 293 cells, the expressed meprin B isoform would be retained on the cell surface of the HEK 293 cells due to its transmembrane (TM) domain, instead of being secreted into the cell culture medium. The truncated rat meprin β construct used for the HEK

293 cell transfection lacked the C-terminal domains past the TRAF domain, allowing HEK 293 cells to produce 6x His tagged meprin B, which is then secreted into the cell culture medium for ease of purification.

To obtain prodigious amounts of secreted homomeric mouse meprin A and rat meprin B for the purification step, the meprin-expressing HEK 293 cells were grown to confluency on 75 mm plates (Gibco) in Dulbecco‟s modified Eagle‟s Medium (DMEM; Gibco) supplemented with

10% FBS and 100x antibiotics/antimyotics (Gibco) - 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. Upon achieving full confluency, the stably- transfected meprin-expressing HEK293 cells were switched to serum-free DMEM medium and this serum-free DMEM medium was collected after 48 h to 72 h of incubation with the meprin- expressing cells. Serum-free DMEM medium was used in order to aid the meprin purification step, as it was observed that excessive FBS impeded metal-affinity chromatography. After the serum-free medium collection, FBS-containing DMEM medium was added back to the meprin- expressing HEK cells for 48 h to 72 h so that the HEK cells could recover from lack of FBS.

Then the meprin-expressing cells were again incubated with serum-free DMEM medium for 48 h to 72 h with the medium collected and stored at the end of the incubation time. This serum/serum-free collection cycle was repeated for the meprin A and meprin B expressing HEK

293 cells until there was less than 50% cell confluency on the 75 mm plates. For the meprin purification step, no less than 1 L of medium was put through the metal affinity columns.

Since the homomeric mouse meprin A and rat meprin B have the C-terminal 6x His tag, these proteins could be purified from the collected serum-free DMEM medium by metal-affinity chromatography. First, the meprin-containing medium was calibrated to 20 mM Tris, 150 mM

NaCl, and 10 mM imidazole and titrated to a pH 7.5 by HCl. This medium was then subjected to nickel-affinity chromatography using a HisTrap™ chromatography column (GE Biosciences).

The recombinant meprins in the medium were eluted from the affinity column by step-wise imidazole titration, with 10, 50, 150, and 500 mM imiadzole in 20 mM Tris, 150 mM NaCl, pH

7.5. The elution fractions were scanned by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); the presence of meprins in these fractions was determined by

Coomassie Blue staining (SimplyBlueTM SafeStain; Invitrogen) and Western Blotting with meprin polyclonal antibodies (HMC-14 for meprin A and PSU-57 for meprin B). Meprin- containing fractions were then pooled together and buffer-exchanged into 20 mM Tris, 50 mM

NaCl, pH 7.5 via Econo-Pac® 10 DG desalting columns (Bio-Rad). The activity of the purified meprins was confirmed using the BK+ and OCK+ peptidase activities while the purity of the meprins was ascertained by separating the meprins on 8% SDS-PAGE and staining with

Coomassie Blue. The protein concentration of the purified meprins was calculated using a micro

BSA kit (Bio-Rad). The yields of the meprin A protein were approximately 1 to 4 milligrams per liter of collected medium, while the meprin B protein yield was approximately 5 to 15 milligrams per liter of medium. The purified meprins were then flash-frozen by liquid nitrogen and stored at -80°C until use.

Recombinant, homomeric human meprin A and meprin B were kindly provided by Dr.

Christoph Becker-Pauly (University of Mainz). Human meprin A and meprin B were expressed, purified, and activated as previously described. These meprins were fully active as seen by their activity against the fluorogenic substrates BK+ and OCK+.

For use in the meprin/MBL studies, native heteromeric meprin A, consisting of the heterotetramer meprin A isoform with active α, but latent ß subunits, was purified from the brush borders of male C57BL/6 mice as previously described [97]. Briefly, 25g of whole C57BL/6 kidney tissue was homogenized in 20 mM Tris, pH 7.5 and subjected to ultracentrifugation

(100,000 g). The heteromeric meprin A located in the membrane fraction (pellet) was released by treatment and incubation with 0.1 mg/ml papain (Sigma-Aldrich) for 2 h at 37°C and 1 mM

40 iodoacetamide for 15 min. The now-solubilized meprin A was separated from insoluble proteins by centrifugation (26,000 g). Heteromeric meprin A was then purified further by precipitating out meprin using ammonium sulfate (Sigma-Aldrich) at 40% and 80% saturation. Meprin A was isolated from the kidney homogenate by another round of centrifugation (26,000 g), dissolved in

20 mM Tris, pH 8.5, and then subjected to ultracentifugation (100,000 g). The heteromeric meprin A was then subjected to two-step purification. The first purification step was done using ion-exchange chromatography with a Mono Q® (Pharmacia Biotech) chromatography column using FPLC, utilizing a salt gradient from 0 M to 1 M NaCl in 20mM Tris buffer, pH 7.5. The fractions were scanned for meprin using SDS-PAGE in the same manner as for the meprins purified from HEK 293 cells. Meprin-containing Mono Q fractions were pooled together and subjected to size-exclusion chromatography with a Superose® 12 (Pharmacia Biotech) chromatography column using FPLC. Since 20 mM Tris, 50 mM NaCl, pH 7.5 buffer was used for the size-exclusion purification, the heteromeric mouse meprin A did not require the desalting step as with the recombinant meprin A and B; meprin A-containing Superose 12 fractions were pooled together, concentrated with a YM-100 concentrator (MilliPore), flash frozen in liquid nitrogen, and stored at -80°C until use.

2.2 Gel Electrophoresis, Gel Staining, and Western Blotting

Proteins were separated by denaturing SDS-PAGE using 8 and 15% polyacrylamide gels at 30-35 mA in 25 mM Tris, 192 mM glycine, and 0.1% SDS, pH 7.5. To visualize proteins directly, SDS-PAGE gels were stained with SimplyBlueTM SafeStain Coomassie Blue stain

(Invitrogen) or Krypton™ (Thermo Scientific) fluorescent stain. For the Coomassie Blue staining method, SDS-PAGE gels were submerged in 100 ml deionized water and subjected to 3 cycles of 1.5 min microwave heating (on High) and 2 min of shaking on an orbital shaker, with

41 the deionized water deposed of and replaced with fresh deionized water prior to the start of the next heating/shaking cycle. At the end of the third circle, 30 mL of SimplyBlueTM SafeStain

Coomassie Blue stain (Invitrogen) was added to the SDS-PAGE gel instead of the 100 mL deionized water and then microwaved-heated for 1.5 min and agitated on an orbital shaker for 30 min. After this initial staining step, the SDS-PAGE gel was destained by addition of 100 mL deionized water.

The other main method for direct protein staining was with Krypton fluorescent stain.

SDS-PAGE gels were fixed twice for 10 min in 40% ethanol, 10% acetic acid solution. The gels were then washed in ultrapure water and stained with 1x Krypton™ fluorescent stain (Pierce) for

16 h at room temperature with agitation. The SDS-PAGE gels were then destained by two cycles of washing (for 30 min) with 5% acetic acid and then ultrapure water. Proteins on the

SDS-PAGE gel were visualized with a Typhoon 9400 Variable Mode Images – Amersham

Bioscience with the 680 nm laser light source and a preferred emission filter of 720 nm.

Another method utilized to visualize proteins was by using Western blotting. In brief, proteins separated on SDS-PAGE were transferred to nitrocellulose membranes by a Semi-Dry transfer apparatus (Bio-Rad) in buffer containing 25 mM Tris, 192 mM glycine, 20% methanol, and 0.1% SDS, pH 7.5. The proteins were transferred to. After protein transfer, the membranes were blocked with 10% dry milk in 20 mM Tris, 140 mM NaCl, and 0.1% Tween-20. Primary polyclonal antibodies were added at 1:5,000 dilution (polyclonal mouse IL-6 antibody; Abcam, ab9730 and polyclonal human IL-6 antibody; Thermo Scientific, P620 ) or at 1:3330 dilution

(polyclonal HMC-14 antibody for meprin A; polyclonal PSU 57 for meprin B) to the blots in 5% dry milk in 20 mM Tris, 140 mM NaCl, and 0.1% Tween-20 and incubated overnight at 4ºC.

Horseradish peroxidase-coupled anti-rabbit secondary antibodies (GE Biosciences) were added

42 to the blots, after washing with TBS-Tween, and the protein bands were visualized with West

Pico or West Dura chemiluminescent substrate (Pierce). Quanitation of Coomassie-stained gel images and Western blotters was accomplished with the Java-based image processing program

Image J [158].

2.3 Meprin Activity Assays, Peptide and Protein

Peptidase activity of meprin A and B was evaluated with the fluorogenic peptide analogues bradykinin (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Lys(Dnp)-Gly-OH; BK+) and orcokinin (Abz-Met-Gly-Trp-Met/Asp-Glu-Ile-Asp-Lys(Dnp)-Ser-Gly-OH; OCK+) as described previously [100, 159]. In short, meprins were incubated with 10 µM of fluorogenic peptide substrates in 20 mM Tris, 50 mM NaCl, pH 7.5. Meprin A and B process the BK+ and

OCK+ substrates respectively, releasing the fluorescent moiety (Abz) from quenching; the activity of the meprins was determined by monitoring the rate of abz release (by fluorescence increase) over time (60 sec). The degradation of BK+ and OCK+ were monitored by a Hitachi

F-2000 Fluorescent Spectrophotometer at excitation and emission wavelengths of 320 nm/417 nm and 326 nm/418 nm, respectively. The peptidase activity (specific activity) of meprins is listed as change in fluorescence (U) over meprin amount (µg).

The proteinase activity of meprin A and B was evaluated using the substrate azocasein, as described previously [98]. Briefly, meprins were incubated with 11 mg/ml azocasein in 20 mM ethanolamine, 150 mM NaCl buffer at pH 9.5, with the meprins and azocasein pre-incubated at

37°C for 15 min prior to azocasein proteinase assay. Meprin cleavage of azocasein was quenched by addition of 5% trichloroacetic acid and the reaction mixture was then centrifuged for 4 min at 13,000 rpms. The absorbance of this supernatant fluid was measured at 340 nm with

43 a DU® 800 Spectrophotometer (Beckman Coulter). Meprin azocasein proteinase activity is listed as units of activity (U) over meprin amount (mg). For calculation of units of azocasein activity, one unit of activity is equal to a change of 0.001 fluorescent intensity per min at 340 nm

[98].

2.4 Cytokine Degradation

Human and mouse interleukin-6 (Peprotech) or human IL-6 (NCI repository) were incubated with meprins in 20 mM Tris, 50 mM NaCl, pH 7.5 at 37°C. IL-6 (1 µM) and homomeric meprin A and meprin B (0.1 µM) were incubated together in Tris buffer (50 µL total volume), pH 7.5 for up to 20 h at 37°C. Enzyme and substrates were equilibrated to 37°C for 5 min prior to incubation. Meprin proteolysis was stopped with 100 µM actinonin (in 50%

DMSO/deionized water) prior to cytokine sample boiling in denaturing sample buffer. The cytokine degradation fragmentation pattern was visualized by direct protein staining or Western blotting as described in Section 2.2.

2.5 Kinetic Parameter Determination of Interleukin-6 Cleavage by Meprins

Samples of human IL-6 (NCI repository) incubated with meprin A and B were separated by 15% SDS-PAGE and stained with fluorescent Krypton™ stain as described in the previous section. The disappearance of the IL-6 substrate band was monitored by laser densitometry

(Typhoon 9400 Variable Mode Images – Amersham Biosciences) and the velocity was calculated with Image Quant 5.2 by dividing the change in OD over time. Five time-points in duplicate were used for each velocity calculation at IL-6 concentrations of 2 to 10 µM for mouse meprin A and 2 to 22 µM hIL-6 for rat meprin B. The meprin concentrations used to determine

44 the kinetics of meprin cleavage of IL-6 were 3 nM for homomeric mouse meprin A and 0.2 nM for homomeric rat meprin B.

The velocities vs. substrate (IL-6) curves were plotted using GraphPad Prism 5.

GraphPad Prism 5 was also used to calculate several kinetic values, such as the Vmax and Km by fitting the velocity data to the Michaelis-Menton equation using nonlinear regression analysis.

The kinetic values kcat and kcat/Km are given as “apparent” values, assuming that all of the meprins used in the kinetic studies are fully activated and that each meprin subunit has one active site. With this assumption, the enzyme turnover value kcat was calculated futher by dividing the derived Vmax value by the meprin concentration. The efficiency constant, kcat/Km, was calculated by dividing the kcat and the Km values.

2.6 Determination of the Cleavage Site on Interleukin-6 by Meprin A

Meprin A and recombinant human IL-6 (NCI) were incubated together for 1 h and separated on SDS-PAGE as described above. The gel silica containing the product bands were excised, dried, and treated with trypsin (Sigma). Uncleaved and homomeric mouse meprin A- cleaved rhIL-6 protein bands were processed for mass spectrometry analysis as previously described [160]. The peptides present in the uncleaved and meprin A-cleaved rhIL-6 samples were identified by ProteinPilot software.

2.7 B9 Cell Proliferation Assay

The biological activities of treated and untreated IL-6 were measured as a function of B9 cell proliferation. B9 cells are a mouse hybridoma cell line that absolutely requires interleukin-6, either mouse or human, for proliferation. Therefore, this cell line is commonly used in the literature to determine the activity of IL-6 in biological samples and other preparations [157].

The B9 mouse cell line was graciously provided by Dr. Tom Scott (Clemson University) [161].

These B9 cells were maintained in 1640 RPMI medium (Gibco) containing 5% FBS, 1/100 antibiotic/antimyotic (Gibco), 50 µM β-mercaptoethanol, and 2 ng/mL mouse IL-6. B9 cells were sub-cultured 1:10 every 3 or 4 days. Prior to treatment with IL-6 or meprin-cleaved IL-6,

B9 cells were washed twice with serum-free and IL-6 free RPMI media to remove any residual

IL-6 remaining from normal culturing/passaging.

The activity of uncleaved and meprin-cleaved IL-6 was measured in two ways. For the first method, the B9 cells were cultured in 96-well plates at a concentration of 20,000 cells per well in a final concentration of 100 µl (200 cells per µl) and incubated with uncleaved or meprin

A or meprin B cleaved human and mouse IL-6 (Peprotech; concentration of 0.5 ng/ml IL-6) for

72 h. B9 cells were treated with IL-6 that was incubated with meprin A or meprin B for several time points and then further meprin digestion was inhibited with 50 µM actinonin prior to addition to the B9 cells. Cell growth was measured 72 h after cytokine treatment by adding 20

µL of CellTiter 96® reagent (Promega) to each well and then incubating the plates for an additional 1-2 h at 37°C. The absorbance at 490 nm for each well was read with a UV/VIS Plate

Reader Spectra Max 190 – Molecular Devices. The proliferation of the B9 cells was expressed as the difference between the wavelength 490 nm value of B9 cells not treated with IL-6 and the wavelength 490 nm of cells treated with uncleaved and meprin-cleaved IL-6. The activity of the meprin-cleaved IL-6 was plotted as a ratio of the change in 490 nm of meprin A/B cleaved IL-6 over the change of 490 nm seen with uncleaved IL-6. Each IL-6 percentage value plotted is the average of 3 independent experiments, with each experiment consisting of an average reading derived from 6 wells.

In the other method of determing IL-6 activity, B9 cells were seeded into 10 ml tissue culture flasks (Corning) to a concentration of 200,000 cells/ml. Recombinant, homomeric mouse meprin A and rat meprin B were incubated with mouse IL-6 for 1 h as previously stated and then incubated with the B9 cells in the flask at a concentration of 0.5 ng/ml for 72 h. After 72 h, the cell concentration in the flasks was determined by direct cell counting via hemocytomer. The cell count values listed of every IL-6/meprin treatment of B9 cells in this manner are average values of 3 independent experiments with each individual experiment cell count itself the average of the cell count of 3 flasks.

2.8 Transient Transfections of Madin - Darby Kidney Cells and Activation of Cell-

Expressed Meprins

Constructs for meprin β and meprin α were transfected into Madin-Dary Kidney Cells

(MDCK) cells using Lipofectamine 2000™. MDCK cells were maintained in RMPI (Roswell

Park Memorial Institute) medium as described in section 2.1. DNA Constructs for the full-length meprin β or the meprin β plus the full-length meprin α construct were pre-incubated in Opti-

MEM® for 5 min at 25°C and then combined with pre-incubated Lipofectamine 2000™. This new mixture was incubated at room temperature for 30 min and then added to MDCK cells at approximately 30% confluency. The Lipofectamine 2000™ and DNA complexes were incubated with the MDCK cells at 37°C in a 5% CO2 incubator for 18 h. At 18 h post- transfection, the meprin expressed by the cells was activated by limited trypsin digestion as previously described [53]. Briefly, the cells were incubated with trypsin for 30 min and then washed twice with serum-free RPMI medium. To ensure there was no residual active trypsin with the cells, soybean trypsin inhibitor (Sigma) was added to the transfected cells and incubated for 1 h. The cells were then washed again twice with serum-free DMEM medium [53, 162].

2.9 Evaluating Cleavage of Interleukin-6 by Cell-Expressed Meprins

Recombinant human IL-6 (1 µg) from NCI was added to the plate containing MDCK cells transiently expressing membrane-bound, activated meprins as described in the above section. The MDCK cells were approximately 70-80% confluent prior to addition of exogenous

IL-6. After 24 h, the medium was removed from the well and the proteins were separated on

15% SDS-PAGE gels. The appearance of IL-6 was visualized using Western blotting (human

IL-6 polyclonal antibody, Thermo-Fisher).

2.10 Inhibition of Meprins by Lectins

Two lectins, mannose binding lectin (MBL) and concanavalin A (Con A - Sigma), were pre-incubated with meprins for 1 h 37°C in 20 mM Tris, 50 mM NaCl, and 10 mM CaCl2 buffer as described previously [86]. Recombinant human MBL was kindly provided by Dr Steffen

Thiel (Aarhus University). The peptidase and proteinase activity of the lectin-treated meprins was ascertained as described above in section 2.3 and compared to the activity of the meprins not treated with lectins.

2.11 Mouse Urine Meprin Concentration Determination

Congenic C57BL/J6 wild-type male mouse urine was collected from mice at 12 weeks of age, separated by 8% SDS-PAGE, and then transferred to nitrocellulose via Western blotting as previously described. The concentration of the meprin α subunit in the urine was determined using quantitative densitometry, with recombinant, homomeric mouse meprin A used as the standard. The meprin A concentration was calculated as nanograms per volume (ul) of urine and converted to molar concentration. Eight mice were used to determine the average concentration of meprin A in mouse urine.

2.12 Handling of Wound Healing Samples

Samples from the wounds of diabetic and non-diabetic patients were kindly provided by

Dr. Robert Grunfeld (Milton S. Hershey Medical Center). A Vacuum Assisted Wound Closure

(VAC®) canister was used to isolate wound fluids from 10 diabetic and 8 non-daibetic patients

(mean age of 54 years). These wound healing samples (WHS) were then cleared by centrifugation at 10,000 rpm for 10 min at 4°C. To prohibit any potential proteolysis in the

WHS as well as deactivate any clotting factors, a protease inhibitor cocktail with EDTA (Roche) was added to the WHS at a final concentration of 1x. These WHS were then concentrated 2-fold by YM-30 concentrators (Millipore) in order to both concentrate the samples and attempt to clear out some of the smaller molecular-weight proteins. The protein concentration of the wound healing supernatant fractions was then determined by the Micro BCA™ Protein Assay Kit.

These supernatant fractions were also subjected to electrophoresis via 8% SDS-PAGE, under both reducing (with SDS) and non-reducing (no SDS) conditions, and probed via Western blotting with polyclonal antibodies for both human meprin α and meprin β subunits (HMC 14 and PSU 57, respectively) as described above.

Chapter 3: RESULTS

3.1 Interleukin-6 is a Meprin Substrate

3.1.1 Human Meprin A and B Cleave Human Interleukin-6

In previous studies evaluating cytokines of the IL-1β family (proIL-1β, proIL-18) as potential meprin substrates, a variety of conditions were utilized for the fragmentation experiments [53, 55, 56]. For the meprin cleavage experiments herein with interleukin-6, conditions similar to those used for meprin cleavage of proIL-18 were used, except that the enzyme:substrate ratio used was 1:10 (mol:mol) rather than 1:20 for the meprin cleavage experiments with proIL-18 [53].

To determine whether IL-6 is a meprin substrate, recombinant human IL-6 (1 µM) from

Peprotech was incubated with 100 nM recombinant human and rodent homomeric meprin A and

B (the rodent meprins were mouse meprin A and rat meprin B). Meprin activity was stopped with 50 µM actinonin at the speicificed time point. Samples were then boiled in SDS sample buffer, containing β-mercaptoethanol (2ME), and separated on 15% SDS-PAGE gels. The fragmentation pattern of IL-6 produced by meprin cleavage was visualized by Coomassie staining.

Figure 10 shows a side-by-side comparison of fragmentation patterns generated by human and mouse meprin A (Figure 10a) and human and rat meprin B (Figure 10b) on unglycosylated human IL-6. While it is impossible to determine the site(s) of meprin processing on IL-6 from the fragmentation patterns, it appears that human/rodent A and B process meprin similarly under identical incubation conditions. The meprins process human IL-6 into a smaller product(s) over time; the smaller meprin-generated products of IL-6 vanish over time with extended incubation

Figure 10. Human Interleukin-6 is cleaved by both human and rodent homomeric meprin

A and B. Homomeric recombinant meprin A (A) and B (B), both human and rodent (100 nM), and human interleukin-6 (1 µM, Peprotech) were incubated together for the time points indicated under the gels, separated on 15% SDS-PAGE, and visualized by Coomassie Blue staining. All the meprin isoforms processed IL-6 into smaller product(s) over time. Both recombinant homomeric human and rat meprin B cleaved all human IL-6 to a small product after 0.25 h. One note - mouse meprin B has not yet been produced recombinantly, thus recombinant rat meprin B was used in its place. Act is an abbreviation for actinonin, an inhibitor used to inhibit meprin proteolysis of IL-6 at the noted times. Meprin reactions were inhibited with 50 µM actinonin.

51 with meprins. There also appears to be slight, but noticeable SDS-PAGE apparent size differences between the IL-6 products generated by meprin A and meprin B.

The form of IL-6 used for these experiments is derived from E. coli and thus is not glycosylated, but IL-6 found in humans is glycosylated. While the different glycosylation patterns along with other post-translational modifications found in different recombinant sources of IL-6 does not significantly affect the activity of IL-6 between these species, it is possible that glycosylation of human IL-6 could hinder and possibly prevent this form of IL-6 from being cleaved by meprins [136].

Figure 11 and Figure 12 show the fragmentation of human IL-6 (rhIL6) isolated and purified from a eukaryotic cell line. This form of recombinant IL-6 was obtained from NCI and is glycoslated. This form of rhIL-6 was cleaved by both recombinant, homomeric human meprin

A and B (Figure 11) and recombinant, homomeric mouse meprin A and homoeric rat meprin B

(Figure 12). Similar to the actions seen on the unglycosylated IL-6 form, human and rat meprin

B formed products more rapidly than meprin A, but in this case, the meprin B-generated product was more stable than that of the meprin A-generated product. Human meprin A fragmented the rhIL6 substrate band into smaller forms not easily discernable on 15% SDS-PAGE from 5 min of incubation onwards. Mouse meprin A did not fragment rhIL-6 as rapidly as human meprin A under the conditions tested, but Image J analysis of the meprin-A treated rhIL-6 bands in Figure

12 show that approximately 20% of the initial rhIL-6 substrate band was cleaved after 30 min incubation with mouse meprin A. Human meprin B fragmented rhIL6 into a form smaller than the initial rhIL6 band within 5 min of incubation, similar to how both human and rat recombinant, homomeric meprin B fragmented the unglycosylated recombinant form of human

IL6.

Figure 11. Eukaryotic-cell derived human interleukin-6 is cleaved by human homomeric meprin A and B. Homomeric recombinant human meprin A and B (100 nM) and recombinant human interleukin-6, rhIL6, (1 µM, NCI) were incubated together for the time points (to model more limited degradation of rhIL6 by meprins) indicated under the gels, separated on 15% SDS-

PAGE, and visualized by Coomassie Blue staining. “Act” is the abbreviation for actinonin – these lanes show IL-6 that was incubated with meprins which were pre-treated with 50 µM actinonin prior to incubation with IL-6.

Figure 12. Eukaryotic-cell derived human interleukin-6 is cleaved by recombinant homomeric mouse meprin A and rat meprin B. Homomeric recombinant mouse meprin A and rat meprin B (100 nM) and recombinant human interleukin-6, rhIL6, (1 µM, NCI) were incubated together for the time points indicated under the gels, separated on 15% SDS-PAGE, and visualized by Coomassie Blue staining. “Act” is the abbreviation for actinonin – these lanes show IL-6 that was incubated with meprins which were pre-treated with 50 µM actinonin prior to incubation with IL-6.

3.1.2 Mouse Interleukin-6 is Cleaved by Rodent Meprin A and B

While both human and mouse IL-6 share the same tertiary structure (IL-6 is a 4-helix bundle protein), they only share 40% sequence homology [163]. Human meprin A and mouse meprin A also have discernible differences from each other cleaving both protein and peptide substrates

[101]. In that the original rationale for testing IL-6 as a potential meprin substrate was from mouse in vivo data, it was essential to confirm that rodent meprins (mouse meprin A and rat meprin B) are also capable of cleaving mouse IL-6. Thus, mouse IL-6 was incubated with rodent meprins in the same manner as with both human and rodent meprin incubation with human IL-6.

To this end, recombinant, homomeric mouse meprin A and rat meprin B (100 nM) were incubated with mouse IL-6 (1 µM) from Peptotech. This isoform of mouse IL-6 (mIL-6) is expressed in and purified from E.coli and thus this mouse isoform of IL-6 is not glycosylated. mIL-6 was incubated with homomeric mouse meprin A and rat meprin B in the same manner as described for the human IL-6 incubations with meprins. Rodent meprin A and B incubation with mouse IL-6 (mIL6) was also inhibited with 50 µM actinonin at several different time-points (as indicated in Figure 13); the fragmentation of mIL6 was visualized by separating the meprin and mIL6 incubations on 15% SDS-PAGE and protein staining the gel with Commassie Blue stain.

Both homomeric mouse meprin A and rat meprin B cleaved mIL6 to smaller products discernable on SDS-PAGE over time, as shown in Figure 13. Over a 4 h incubation period, meprin A cleaved mouse IL-6 to a smaller product (Figure 13a). However a visible amount of the initial substrate mIL6 band persisted throughout all of the time-points (0.5 h to 4 h) meprin A was incubated with mouse IL-6. In a similar manner observed with recombinant human and rat

Figure 13. Mouse Interleukin-6 is cleaved by rodent homomeric meprin A and B.

Homomeric mouse recombinant meprin A (A) and rat meprin B (B), at a concentration of 100 nM, and mouse interleukin-6 (1 µM, Peprotech) were incubated together for various times. The meprin reaction with mIL-6 was stopped with actinonin and boiling in SDS with 2ME. The meprin/IL-6 incubations were then loaded onto gels and separated by 15% SDS-PAGE; the fragmentation pattern of mouse IL-6 by meprin A and B was visualized by Coomassie staining.

The lanes labeled with “+ Act” indicate the lanes on the 15% SDS-PAGE gel for which the incubation mixtures containing meprin were inhibited with actinonin prior to addition of IL-6 substrate. Both homomeric meprin A and B cleaved mouse IL-6 to a smaller form, demonstrating that mouse IL-6 is a meprin substrate.

56 meprin B incubations with rhIL6, rat meprin B had cleaved most if not all of the initial mouse

IL-6 substrate into a smaller form by the first time point (30 min, Figure 12b). Meprin A and meprin B were also pre-treated with 50 µM actinonin, as seen by the “+ Act” lanes (50 µM) actinonin labeled in red in both Figure 12a and 12b. The fragmentation pattern seen in Figure

12b shows that meprin B processes IL-6 to its subsequent product faster than meprin A if both are incubated with mIL6 under similar conditions. As was also seen in the meprin fragmentation patterns of both hIL6 and rhIL6, the mIL6 products generated by meprin A and B appear to differ from each other.

Overall, both meprin A and B cleave both human and mouse IL-6, confirming that IL-6 is a meprin substrate in vitro. The homomeric isoforms of the meprin metalloproteinses truncate the cytokine and also eventually degrade IL-6 products over an extended time of incubation.

This is the first evidence showing that IL-6 is cleaved by meprin metalloproteinases

3.2 Meprin Cleaves Interleukin-6 at the C-terminus with High Affinity, Decreasing 4 the Activity of Interleukin-6

3.2.1 Meprin A and B Cleave Human Interleukin-6 With High Affinity

Once it was determined that IL-6 is a meprin substrate, the next question posed was the potential fitness of interleukin-6 as an in vivo meprin substrate. Most of the known peptide substrates of meprins have micromolar affinities (in the range of 100-400 µM) towards meprin A and meprin B [97]. The cytokine proIL-18 is processed by meprin B and heteromeric meprin A to its active form with low micromolar affinity (1 µM), therefore proIL-18 is one of the better substrates for meprin B from a kinetic standpoint

One way to ascertain indirectly if IL-6 is a good meprin substrate in vivo is to characterize meprin cleavage of IL-6 kinetically. Due to the amount of cytokine needed to perform the kinetic studies, the recombinant form of human IL-6 derived from the eukaryotic cell line, obtained from the National Cancer Institute (NCI) repository, was used to perform the kinetic characterization of meprin cleavage of IL-6. This recombinant form of human IL-6 is glycosylated, thereby more accurately modeling the kinetics of meprin cleavage of IL-6 in human with post-translational modifications such as glycosylation present. The initial velocity of meprin cleavage of IL-6 with both meprin A and B was determined using laser densitometry

(the IL-6 bands were visualized by staining the SDS-PAGE gels by Krypton staining); the percentage loss of the IL-6 substrate band was measured over time. Figure 14 shows the velocity vs. substrate curves plotted for IL-6 degradation by recombinant, homomeric mouse meprin A and rat meprin B. Determination of the affinity (Km) values of meprin A and B cleavage of IL-6 was calculated using nonlinear regression analysis via Graphpad 5. The velocity values as a function of substrate concentration were fitted to the Michaelis-Menten equation and several kinetic parameters were derived, including the Km (affinity) and Vmax values. The Km values of mouse meprin A and rat meprin B cleavage of rhIL-6 are listed in

Table 1, along with other kinetically-relvant parameters such as kcat and the efficiency constant kcat/Km.

Figure 14. Kinetics of homomeric, rodent meprin processing of human interleukin-6

(NCI). These curves were derived by measuring the disappearance of hIL-6 on 15% SDS-PAGE using laser densitometry and then plotting the velocity (µM/min) vs the IL-6 concentration.

(N=2). Each velocity value listed is an average of two independent experiments and the error bars are the standard error of the velocity measurements. The kinetics of meprin cleavage were measured using rhIL-6 concentrations of 2 to 10 µM for homomeric mouse meprin A and 2 to

22 µM rhIL-6 for homomeric rat meprin B. The concentration of meprin enzymes used was 3 nM and 0.2 nM for meprin A and meprin B, respectively.

Meprin Isoform Km kcat (apparent) kcat/Km (apparent) -6 -1 -1 -1 6 (M x 10 ) s (M s ) x 10 Mouse meprin A 4.9 ± 1.1 1.0 0.20 ± 0.03 Rat meprin B 12.0 ± 1.2 30.4 2.5 ± 0.04

Table 1. Kinetic constants for meprin A and B processing of human IL-6. The rate of substrate (IL-6) degradation by meprins was determined by measuring the disappearance of the hIL-6 band on 15% SDS-PAGE gels with time using laser densitometry. The kinetic constants of IL-6 cleavage by meprins were determined by fitting the velocity values to the Michaelis-

Menton equation. IL-6 (2 µM to 10 µM) was incubated with mouse meprin A (3 nM) and meprin B (0.2 nM) in 20 mM Tris, 50 mM NaCl, pH 7.5 for various times (0 to 10 min). The error values listed for the Km values were calculated by the Graphpad 5 program, which are the standard error of the Km values. The error listed for the efficiency constants (kcat/Km) are the standard error of the individual efficiency values for each individual Km calculated. Both meprin

A and B have similar efficiency constants, while mouse meprin A has a lower affinity value and turnover rate (kcat) than rat meprin B. The kcat and kcat/Km values are listed as (apparent) values due to the assumption that all of the meprin used for the kinetic studies is fully active and that one meprin subunit has one active site.

The affinity (Km) of homomeric meprin A and B cleavage of IL-6 substrate was in the lower micromolar range, with Km values of 4.9 and 12.0 µM, for meprin A and B respectively

-1 (Table 1). kcat values (1.0 and 30.4 s for meprin A and B, respectively) for the meprin cleavages of human IL-6 were calculated by dividing the Vmax value ascertained using the non- regression analysis by the enzyme concentration used for the degradation. The efficiency

- constant, kcat/Km, was calculated by dividing the kcat value by the Km, which are 0.20 and 2.5 µM

1s-1, respectively for meprin A and meprin B cleavage of rhIL-6 (Table 1).

3.2.2 Meprin A Cleaves Human Interleukin-6 at the C-terminus

The fragmentation gels of meprin-treated hIL6, rhIL6, and mIL6, shown in Figures 10 through 13, indicate that the meprins initially cleave a small portion off of IL-6. This implies that the initial cleavage site of IL-6 on meprins is in proximity to either the N or the C-termini of the IL-6, for both the human and mouse isoforms of IL-6. This leads to the suggestion that the residues in the vicinity of either the N or C-termini of the IL-6 cytokines are the target of meprin cleavage.

The N and C-termini are important for the activity of IL-6. To determine the precise locations where meprins cleave IL-6 and also to gain insight into the effect meprin cleavage has on the activity of IL-6, the meprin cleavage sites on IL-6 were determined. The eukaryotic-cell derived recombinant form of human IL-6 (rhIL6, obtained from NCI – 1 µM) was incubated with recombinant, homomeric mouse meprin A (100 nM) for 10 min and the meprin-fragmented rhIL6 was separated on 15% SDS-PAGE. The meprin-treated and untreated IL-6 bands were excised from the gel and trypsinized. The trypsinized peptides were extracted from the gel plugs with acetonitrile/trifluoacetic acid washes. These peptides were then suspended in pure water and subjected to electrospray ionizing mass spectrometry analysis.

Figure 15. Meprin A cleaved human interleukin-6 lacks 5 C-terminal amino acids. The primary sequence of human interleukin-6 is shown, with the residues in green that were identified (80% confidence) in the samples (uncleaved and mouse meprin A – cleaved rhIL6 samples) sent for mass spectrometry analysis. The residues labeled in red are residues that were not identified in the cleaved sample; these are the residues that the meprin A removed from the

C-terminus of human interleukin-6. From what is known about the peptide bond cleavage specificity of meprin A, the data implicating that meprin A cleaves an arginine (R)/alanine bond outside the main helical bundle of rhIL-6 makes logical sense [97, 143].

The derived peptide maps of untreated and mouse meprin A-treated rhIL6 are displayed in Figure 15. These peptide maps were used to ascertain the site of meprin A cleavage on rhIL6.

The peptide mapping of the meprin A treated rhIL6 shows that the meprin treated IL-6 is missing

5 amino acids from the C-terminus (ALRQM – alanine, leucine, arginine, glutamine, and methoinine). Therefore, the putative meprin A site on rhIL6 is the arginine-alanine (R-A) bond proximal to the C-terminal of the cytokine, right after the 4th α-helix of IL-6 [143].

3.2.3 Human Homomeric Meprin A and B Decreases the Activity of 3

Human Interleukin-6

The fragmentation gels of meprin A and B-treated mouse and human IL-6, along with the peptide map analysis of mouse meprin A cleavage of rhIL6, showed that limited meprin cleavage of IL-6 removes a small portion of the C-terminus from IL-6 and leaves the remainder of the cytokine intact. There are published data indicating that the C-terminus of IL-6 makes a considerable contribution to both the stability of the cytokine and the ability of IL-6 to bind to its receptor [136, 139]. It is thought that truncation or mutation of the C-terminus may lead to significant changes in other parts of the IL-6 structure, which may explain why removing only 5 amino acids causes such a large apparent molecular weight change on SDS-PAGE [139]. In order to ascertain if meprin cleavage of IL-6 decreases its activity, meprin A and meprin B- treated human and mouse IL-6 (Peprotech) were incubated with B9 cells in order to measure the amount of activity the meprin-cleaved IL-6 retained compared to the uncleaved IL-6 [157, 161].

To determine what effect meprin cleavage has on IL-6 activity, human IL-6 (1 µM) was treated with recombinant, homomeric human A and B (100 nM) for 3 time points – 30 min, 60 min, and 120 min. After incubation, meprin activity was inhibited with 50 µM actinonin. The

63 meprin/IL-6 incubation mixture was added to RPMI 1640 medium, supplemented with 50 µM β- mercaptoethanol and 5% fetal bovine serum (FBS). The final cytokine concentration was 2 ng/ml; this mixture was added to 96-well plate (50 µl) and diluted serially 1:1 with RPMI medium lacking cytokine, yielding final cytokine concentrations of 1, 0.5, 0.25, and 0.125 ng/ml.

The mouse hybridoma cell line B9 was added to each well (50 µl) to yield a final cell concentration of 20,000 cells per well (400,000 cells per ml). The cells and treated medium were incubated at 37ºC in 5% CO2. After 72 h, 20 ul of CellTiter96 (Promega) reagent was added to each well and incubated in the same conditions for 2 h. The color of the formazan product produced by the B9 cells is purple. The absorbance at 490 nm was monitored for each well with a plate reader; the greater the absorbance at 490 nm, the greater the proliferation of the B9 cells.

The proliferation of B9 cells incubated with meprin A and B-treated IL-6 was compared

(normalized) to the proliferation of B9 cells incubated with untreated IL-6.

Human IL-6 cleaved by either human homomeric meprin A or B significantly decreases the activity of IL-6. As seen in Figure 16, human IL-6 treated with human meprin A for 30 min has approximately 30% of the activity of uncleaved human IL-6. With extended incubation, meprin A decreases the activity of IL-6 the longer the cytokine is incubated with meprin A, with the activity of human IL-6 incubated with human meprin A for 120 min possessing approximately 10% of the activity of uncleaved human IL-6. Human meprin B cleavage also decreases the activity of IL-6, but to a lesser extent than human meprin A over the 2 h time frame. After 120 min of incubation with human meprin B, the human IL-6 has approximately

30% of the activity of uncleaved human IL-6. This is the first evidence showing that meprin A or meprin B cleavage of human IL-6 decreases the activity of this cytokine, and that IL-6 activity decreases with length of time of incubation with meprins.

Figure 16. Human Meprin A cleaved human IL-6 has decreased activity. Human IL-6 (1

µM) was incubated with meprin A (100 nM) for 30, 60, or 120 min and then added to B9 cells

(20,000 cells per 100 µl, in a 96 well plate). The IL-6 cytokine, untreated and meprin B treated, was incubated with B9 cells for 72 h. The proliferation difference between the treated cells was determined by adding 96 Cell-Titer reagent to the wells, incubating for 1 to 2 h at 37°C, and monitoring the wavelength at 490 nm. The meprin A treated IL-6 showed significantly less activity (at all incubation times; meprin A activity is shown by the blue bars) than the untreated human IL-6 and the meprin B-treated human IL-6 (activity is shown by the red bars), **- p<0.001 (N=3). From the kinetic data, it is assumed that all of the meprin B-treated hIL-6 is in its smaller product form and that

3.2.4 Both Homomeric Mouse Meprin A and Rat Meprin B Decreases the Activity of

4 Mouse Interleukin-6

To relate in vivo and in vitro studies on IL-6, it is necessary to show a similar activity experiment with rodent meprins and mouse IL-6 on B9 cells. The B9 cells were seeded into 10 mL flasks (Millipore) at a concentration of 100,000 cell/ml (5 ml total) with meprin A or meprin

B-treated mIL-6 at a final concentration of 0.025 ng/ml mIL-6. After 72 h, the number of cells in the flasks were counted by hemocytometer and the cell proliferation was plotted against the proliferation of B9 cells incubated with untreated mIL-6. Treating mIL-6 with homomeric mouse meprin A and rat B significantly decreases the activity of this cytokine (Figure 17).

Interestingly, meprin B treatment of mouse IL-6 exhibited significantly less activity than meprin

A treatment, the exact opposite of what was observed with human meprin A and B treatment of human IL-6.

In Figure 17, the proliferation of B9 cells was measured by cell counting via hemocytometer. However, the bio-activity of meprin-treated human IL-6 in Figure 16 was measured with CellTiter96 (Promega) reagent, so a similar B9 cell proliferation experiment with rodent meprin-treated mouse IL-6 was performed in order to better compare the loss of bio- activity between meprin-treated human and mouse IL-6. As seen in Figure 18, both recombinant homomeric mouse meprin A and recombinant homomeric rat meprin B decreased the activity of mouse IL-6, although the bio-activity decrease of IL-6 after 60 min by both meprin A and meprin B was only approximately 25% of baseline activity. Furthermore, there was a significant increase of mouse IL-6 activity after 5 minutes of incubation with mouse meprin A, but this bio-activity increase from the baseline of untreated mouse IL-6 bio-activity disappeared by the 15 min incubation time point. In contrast to the results seen in Figure 17,

Figure 17. Mouse meprin A and rat meprin B decrease the activity of mouse IL-6. The meprin A and B treated mouse IL-6 (1 hour of incubation of meprins with IL-6 displayed less activity as seen via decreased B9 proliferation than the untreated mouse IL-6 (n=3).

Furthermore, the B9 cells treated with the meprin B-cleaved IL-6 proliferated significantly less than the B9 cells treated with the meprin A-cleaved IL-6. This further confirmed that incubations of IL-6 with meprins causes a decrease of IL-6 activity. The cell counts of B9 cells incubated with uncleaved meprin were significantly different than the B9 cell counts with both meprin A and meprin B treated mIL-6. The cell proliferation of the B9 cells treated with meprin

B-cleaved mouse IL-6 was significantly lower than the proliferation of the B9 cells treated with meprin-A cleaved mouse IL-6. *, p<0.05 (N=3), of 3 independent experiments.

Figure 18. Meprin A and B cleaved mouse IL-6 has decreased activity, as seen in a B9 cell bio-activity assay with CellTiter96. Mouse IL-6 (1 µM) was incubated with meprin A (100 nM) for several time-points (5 min, 15 min, 30 min, and 60 min), and added to B9 cells (20000 cells per 100 µl, in a 96 well plate) at several concentrations. The IL-6 cytokine, untreated and meprin B treated, was incubated with B9 cells for 72 h. The proliferation difference between the treated cells was determined by adding 96 Cell-Titer reagent to the wells, incubating for 1 to 2 h at 37°C, and monitoring the wavelength at 490 nm (N=3).

Figure 18 shows that meprin A and meprin B both decreased the bio-activity of mouse IL-6 to the same extent after 60 min of incubation with meprins.

3.2.5 Human Meprin-Cleaved Interleukin-6 Does Not Act as either an 5

Agonist or an Antagonist

From the studies of MMPs with chemokines, it was found that several fragments from

MMP-mediated cleavage of cytokines can act as antagonists by binding to the chemokine receptor and preventing the receptor from propagating cellular signals further. Conversely,

MMP processing of chemokines may also increase their activity [18].

To test the possibility that meprin-truncated IL-6 acts as either an antagonist or an agnonist, a 1:1 mixture of uncleaved hIL-6 and human meprin A and meprin B-cleaved hIL-6 were used to stimulate B9 cells in a manner similar to the B9 cell assay set up used to ascertain the activity of meprin-cleaved hIL-6. If the human meprin-cleaved IL-6 acts an antagonist, the activity of the 1:1 mixture would be expected to be approximately that of the negative control; conversely, if the meprin-cleaved hIL-6 acts as an agonist, the activity of the 1:1 mixture would be expected to approach that of the untreated hIL-6. Accordingly, if the activity of the 1:1 mixture is approximately half of the untreated hIL-6, than the meprin-truncated hIL6 would be said to act neither as an agonist or an antagonist.

As seen in Figure 19, both the meprin A and meprin B treated human IL-6, mixed 1:1 with the untreated IL-6 showed, approximately 62% and 55% activity, respectively, to untreated

IL-6. There was no significant difference between the percentage activity between the 50:50 mixes of the human-meprin A and human meprin-B IL-6 with uncleaved IL-6. This indicates that meprin-cleaved IL-6 acts neither as an antagonist or agonist.

100 90 80 Human meprin A 70 treated IL-6, 50:50 60 % Bioactivity compared to 50 Human meprin B untreated IL-6 treated IL-6, 50:50 40 30 20 10 0

Figure 19. Human meprin A and meprin B treated IL-6 acts neither as an antagonist or agonist. Both treatment groups had approximately half the activity of the uncleaved IL-6, showing that the meprin-cleaved IL-6 does not either stop IL-6 from binding to the IL-6 receptor on the B9 cells or increase the activity of the cytokine by increasing binding to the receptor.

(N=3).

3.3 Modeling Meprin Processing of Interleukin-6 in a Cell-Based System

3.3.1 Meprins Expressed on the Surface of Madin-Darby Kidney Cells 1

Cleave Exogenous Human Interleukin-6

To model more effectively the interaction between meprins and IL-6, meprins were expressed on the surface of MDCK cells. The ability of these cell-based meprins to cleave exogenous human IL-6 was determined by Western blotting of culture medium containing IL-6 that had been exposed to active, MDCK-cell based meprins. A similar cell-based experiment was done with proIL-18 to show that cell-based meprins were capable of cleaving exogenous substrates in a system that is better suited to mimic the context in vivo by which meprins and meprin substrates such as proIL-18 may interact with each other [53].

In this particular cell system, MDCK (Madin-Darby Canine Kidney) cells were grown to

80-90% confluency on 24-well plates. Full-length DNA constructs for rat meprin α and β were transfected transiently into the MDCK cells using Lipofectamine 2000® and Opti-Mem reagents.

To activate the cell-surface bound meprins, MDCK cells were trypsinized in a limited fashion and then washed to remove any residual trypsin. Exogenous rhIL6 (1 µg in 1 ml of MEM medium) was added to the MDCK cells. Twenty-four hrs later, the medium was removed and the fragmentation pattern of the rhIL6 observed using PAGE to separate the proteins in the medium. Proteins were then transfered to a nitrocellulose membrane and rhIL6 was identified by

Western blotting.

Figure 19 shows a Western blot of samples from three similar experiments. MDCK cells that were transfected with both the rat meprin α and rat meprin β constructs express the rat heteromeric meprin A isoform on the surface, while the MDCK cells transfected with only the

Figure 20. Meprins expressed by MDCK cells process exogenous human IL-6. Full-length

DNA constructs of rat meprin β or α were transiently transfected into MDCK cells. The expressed meprins were activated by limited tryspin digestion; exogenous recombinant human

IL-6 (NCI) was added to these meprin-expressing MDCK cells. This is a Western blot of 3 similar experiments showing the fragmentation pattern of exogenous rhIL6 after incubation with cell-expressed meprins. The chart below the representative Western blot shows if trypsin was added to the cells post-transfection ( - or +) to activate the meprins and also tells which meprin

DNA construct(s) were introduced into the MDCK cells; rat meprin β for the homomeric meprin

B isoform, both rat meprin β and α for the heteromeric meprin A isoform, or only rat α isoform.

Some MDCK cells were transfected with a 2:1 molar ratio of rat α (as indicated by the ++) to rat meprin β DNA construct in order to induce the MDCK cells to express more secreted rat meprin

A vs the membrane-bound heteromeric form of rat meprin A.

72 rat β construct expressed the homomeric meprin B isoform on their surface. Lanes 2, 6, and 10 of the Western blot shown in Figure 19 indicate that trypsin-actived meprin B expressed from

MDCK cells is capable of cleaving rhIL6 to a smaller form in a similar manner as meprin B did with rhIL-6, hIL-6, and mIL-6 in the in vitro fragmentation assays. When the MDCK cells express activated heteromeric meprin A (meprin comprised of the heterotetramer of rat meprin α and β subunits), this cell-based meprin is also capable of fragmenting exogenous rhIL6 to a smaller form identical in their apparent weight (on SDS-PAGE) to the meprin B-cleaved IL-6, as seen in lanes 3 and 4, lanes 7 and 8, and lane 10.

The next question asked was whether the fragmentation of IL-6 is due to the activated meprins or from some unknown factor in the medium that cleaves IL-6 when active meprins are expressed by the MDCK cells. To answer this question, exogenous IL-6 was added to MDCK cells expressing both rat meprin B and heteromeric rat meprin A not activated by limited trypsin proteolysis. The rhIL-6 recovered from the medium from MDCK cells expressing latent meprin

A and B on their surface (lanes 13 and 11, respectively) migrated slower than the exogenous rhIL-6 incubated with MDCK cells expressing the active forms of rat meprin A and B (lanes 12 and 10, respectively), indicating that meprins must be activated on the MDCK cells before they can cleave exogenous rhIL-6.

Overall, this experiment with MDCK cells showed several things. First, meprins expressed from and anchored to the surface of cells are capable of cleaving exogenous proteins such as rhIL-6, but these meprins must be activated, via limited trypsin-digestion, before they can cleave the exogenous protein. This is also the first evidence that an active protease expressed on the surface of a brush-border cell line is capable of cleaving IL-6.

Hirano, Ma et al. 2005 have reported that mannan-binding protein (also known as mannose binding lectin, MBL), a serum lectin, binds to both subunits (α and β) of heteromeric meprin A isolated from mouse kidney. Furthermore, it was reported that MBL binding to heteromeric meprin A significantly decreased the proteolytic activity of meprins, thus leading to the proposition that MBL is an important endogenous meprin inhibitor [86]. Finding specific and potent inhibitors for the meprins is of great interest because they could be important regulators of protease activity in vivo. Pilot studies were conducted to further assess the possible role of MBL as an endogenous (and potent) inhibitor of the meprin metalloproteinases.

3.4 Concanavalin A, but not Mannose Binding Lectin Inhibits the Proteolytic Activity of

Meprin A

3.4.1 Purification of Lectins from Human Blood Samples

To assess MBL as an inhibitor of the meprin metalloproteinases, native human MBL was isolated and purified from serum samples of healthy blood donors (kindly provided by Nancy

Hinkle, Penn State Hershey Medical Center) in a similar fashion as described by Hirano et al.

Briefly, the blood serum was dialyzed against calcium-containing Tris buffer in order for the

MBL to bind to column-bound mannose in a calcium-dependant manner. The dialyzed serum was then applied to a Sepharose 4B-mannose column and the bound MBL was eluted with

EDTA (Ethylenediaminetetraacetic acid), which chelates the calcium and releases the MBL [86].

As seen in Figure 21, the elutant from the Sepharose 4B-mannose column contained two major bands. To ascertain the identity of these proteins, these bands were excised from the SDS-

PAGE gel and sent to the Penn State College of Medicine Proteomics CORE facility for

Figure 21: Isolation and purification of mannose binding lectin from human blood.

The elutants from the Sepharose 4B-mannose and Superose 12 FPLC column were analyzed via

15% SDS-PAGE and Coomassie Blue staining and Western blotting, respectively. As seen in the protein-stained SDS-PAGE gel on the left, the column elutant contained two main distinct protein species; the upper band shown on the gel was approximately 33 kDa in apparent size while the lower band on the gel was approximately 30 kDa in apparent size on SDS-PAGE. The

33 kDa and 30 kDa bands were identified as MBL and SAP respectively by mass spectrometry analysis. To purify these lectins further, the Sepharose 4B-mannose elutant was applied to a

Superose 12 FPLC column and 1 ml fractions were collected. As seen on the Western blot on the right, MBL signal was detected in the 8th through 10th 1 ml fraction, indicating that MBL eluted from the column as approximately a 300 kDa protein (a trimer form of the MBL complex). The * lane on the Western blot is a positive control of MBL. Since SAP forms complexes of only 125 kDa, the Superose 12 column was able to separate the MBL from the

SAP in the elutant from the Sepharose 4B-mannose column [164, 165].

75 identification via mass spectrometry analysis. The upper band in Figure 21 was identified as

MBL while the lower band in Figure 21 was identified as serum amyloid protein (SAP). These two lectins were further purified by size-exclusion chromatography; both this newly purified native human MBL and SAP were used in experiments to characterize the MBL/meprin interaction.

3.4.2 Native Human Mannose Binding Lectin Recognizes Active and 2

Latent Homomeric Meprin A

According to the data published on the MBL/meprin interaction by Hirano, Ma et al.

2005, native human MBL binds to the glycans of both the meprin α and meprin β subunit of heteromeric meprin A purified from mouse kidneys. However, the interactions between MBL and homomeric meprin A and B were not examined in detail by Hirano, Ma et al. 2005. To examine further the interaction between homomeric meprin A and B with MBL, recombinant mouse mouse meprin A and homomeric rat meprin B were separated on 15% SDS-PAGE, transferred to nitrocellulose membrane, and the proteins were probed with native human MBL in buffer containing 10 mM CaCl2. As seen in Figure 22a, native human MBL reacted with homomeric mouse meprin A, with little to no evidence detected showing an interaction between native human MBL and homomeric, recombinant rat meprin A and rat meprin B. Given that native human MBL (nhMBL) reacted most strongly to homomeric mouse meprin A, as visualized via lectin blotting, the native human MBL/homomeric meprin A interaction was characterized further.

Figure 22. Mannose binding lectin recognizes homomeric meprin A. Homomeric, recombinant mouse and rat meprin A, and rat meprin B (0.2 µg and 0.3 µg mouse meprin A, 0.5

µg and 1 µg) were probed with native human MBL (nMBL) as previously described via lectin blotting in 20 mM Tris, 50 mM NaCl, and 10 mM CaCl2, pH 7.5. The native human MBL did not recognize rat meprin B, reacted weakly with rat meprin A, but definitely reacted with mouse meprin A (as shown by the red arrow) (Figure 22a). To further characterize the interaction between homomeric mouse meprin A and nMBL, active and latent homomeric mouse meprin A were probed by nMBL lectin blotting (Figure 22b) as previously described. The nMBL recognized both latent (- trypsin) and activated (+ tryspin) mouse meprin A (Figure 22b, lanes

1-4), but the interaction between homomeric mouse meprin A and nMBL was ablated when 20 mM mannose (+ mannose) was added to the lectin blot prior to addition of nMBL (Figure 22b, lanes 5-8). The red arrows also indicate the positive interaction between mouse meprin A and nMBL via lectin blotting in Figure 22b.

As seen in Figure 21b, nMBL recognized increasing amounts (0.2 and 0.5 µg) of both latent and active meprin A (Figure 21b, lanes 1-4). However, the native human MBL interaction with both active and latent homomeric mouse meprin was ablated when the meprins on the membrane were pre-incubated with 20 mM mannose (Figure 21b, lanes 5-8), showing that nMBL interacts with active and latent homomeric meprin A in a glycan-dependent way.

3.4.3 Mannose Binding Lectin Does Not Inhibit the Proteinase or Peptidase Activity of

Homomeric or Heteromeric Meprins

While the interaction between meprin and MBL was confirmed and further clarified with the nMBL lectin blotting, the ability of MBL to inhibit the proteolytic activity of the different isoforms of meprins still needs to be clarified, confirmed, and expanded. The published data on the nMBL/meprin interaction, as published by Hirano, Ma et al 2005 indicates that nMBL inhibits mouse kidney heteromeric mouse meprin A from cleaving both larger ECM protein substrates such as collagen IV and gelatin along with smaller protein substrates such as casein and PTH (approximately 30 kDa and 10 kDa, respectively). However, the ability of MBL to inhibit the peptidase activity of meprins was not evaluated.

Because of the low yield of native human MBL from human blood serum, there was inadequate material availible for the scope of the inhibition studies required to evaluate MBL as a potential meprin inhibitor. Recombinant human MBL (rhMBL) was kindly provided by Dr.

Steffen Thiel (University of Aarhus, Denmark) for use in evaluating MBL inhibition of both proteinase and peptidase activity of meprins.

The MBL isoforms, both the native and recombinant forms of human MBL, were pre- incubated with the recombinant, homomeric forms of mouse meprin A and rat meprin B, along

78 with heteromeric kidney meprin A isolated and purified from C57BL/6 mice, in calcium- containing buffer. Both the peptidase and proteinase activity of the MBL pre-treated meprin isoforms were evaluated with the BK+ and OCK+ assays (for both meprin A and meprin B isoforms, respectively) and the azocasein assay as previously described.

As was seen in Figure 23, no significant decrease in either peptidase (Figure 23a) or proteinase activity (Figure 23b) was seen with any isoform of meprin tested upon pre-incubation with increasing amounts of rhMBL. While these studies did not confirm the finding by Hirano,

Ma et al. 2005 that MBL is a potent inhibitor of meprins, several other lectins have been shown to bind to meprins. It is possible that one or more of these other lectins might be capable of inhibiting meprin in the manner first proposed for MBL-mediated inhibition of meprins, that lectin-binding to the glycans of meprin physically occludes substrates from the active site of meprins.

3.4.4 Concanavalin A Partially Inhibits Meprin A Proteinase Activity

The lectin concanavalin A (Con A) has been previously shown to interact with meprins

(both meprin α and β in mouse kidney) in a glycan-dependent manner [166]. To test if lectins could inhibit meprin proteolytic activity, Con A and heteromeric kidney meprin A as well as recombinant meprin B were pre-incubated together in the same manner as was

Figure 23. Mannose binding lectin does not inhibit peptidase or proteinase activity of either meprin A or B. Recombinant human mannose binding lectin (rhMBL), at increasing concentrations (0 to 5 µM and 10 µM), and the meprin A (115 nM) and meprin B (110 nM) isoforms were pre-incubated together for 30 min at room temperature in 20 mM Tris, 50 mM

NaCl, and 10 mM calcium chloride buffer at pH 7.5. The peptidase (BK+ assay and OCK+ assays for meprin A and B, respectively) (A) and proteinase (via azocasein assasy) (B) activity of the meprins were then determined as described in the Materials and Methods section. No significant decrease of either the proteinase or peptidase activity of the MBL-incubated meprins was seen.

Figure 24. Concanavalin A partially inhibits meprin A proteinase activity. Concanavalin A and heteromeric mouse kidney meprin A (NmA) and homomeric rat meprin B (RmB) were pre- incubated as previously described with rhMBL; the meprin peptidase (BK+ and OCK+) and proteinase activity (azocasein) of the treated meprins were assayed as described in the Materials and Methods section. As determined with rhMBL, Con A did not inhibit the peptidase activity of any meprin isoform tested (A). However, Con A decreased the proteinase activity of NmA at each concentration tested, with a significant difference in proteolytic from untreated NmA seen with 1.0 and 5.0 µM Con A treatment (**, p<0.01, in comparison to 0 µM Con A). At the final

Con A concentration tested, 5.0 µM, meprin protein proteinase activity was decreased to approximately 50% of untreated heteromeric kidney meprin A (B).

previously described above. The peptidase activities of the pre-incubated heteromeric meprin A and recombinant meprin B were evaluated with the BK+ and OCK+ assays, respectively; the proteinase activity of both meprins was tested with the azocasein assay. Con A did not significantly inhibit the peptidase activity of either recombinant meprin B or heteromeric meprin

A (Figure 23a). However, while Con A did not inhibit the proteinase activity of recombinant meprin B, this lectin significantly decreased the proteinase activity of the heteromeric meprin A.

At 5 µM Con A, the activity of heteromeric meprin A was decreased approximately 50% compared to untreated heteromeric kidney meprin A (Figure 23b).

3.5 Characterization of Wound Healing Samples from Diabetic and Non-Diabetic 4

Patients

3.5.1 Wound Healing Samples from Diabetic and Non-Diabetic Patients 1

Have Similar Total Protein Concentrations

Given that meprins have been shown to interact with both IL-6 and MBL, also that proteases in general have been implicated in cell egress, along with meprins being implicated in several inflammatory diseases, wound healing samples from both diabetic and non-diabetic patients were isolated. Wound healing samples (WHS) used in these studies, collected from both diabetic and non-diabetic patients, were kindly provided by Dr. Robert Grunfeld (Penn State).

Wound healing is an inflammatory process, with proteases contributing to each stage of wound healing. Diabetic mice and humans have severe deficiencies in their wound healing processes, leading to improperly healed wounds. Limb amputation is common for diabetic patients due to chronic wounds becoming chronically infected and gangrenous. In a collaborative project with a member of the Surgery Dept, PSUCOM, we asked whether meprin

82 are present in human wound healing fluids and and whether there is a difference in meprin expression in diabetic vs non-diabetic wounds

In order to find differences between the diabetic and non-diabetic WHS, the protein content of the WHS was quantitated first. For this purpose, the WHS were centrifuged at 10,000 rpm and the resulting supernatant fluid was isolated in order to remove any insoluble particulates in the sample; the total amount of protein in this fraction was measured by the Micro-BSA assay

(Thermo Scientific). As seen in Table 2, the WHS from the patients possessed a wide range of protein concentrations, with one sample having a total protein concentration as low as 10.0 µg/µl

(WHS 128-1) and the highest protein concentration detected was 57.4 µg/µl (WHS 110-2). The average protein concentration of the WHS isolated from the diabetic patients was 29.7 ± 3.9

µg/µl, while the average protein concentration of the WHS isolated from the non-diabetic patients was 28.0 ± 4.5 µg/µl, showing that there was no significant difference between the WHS by this metric (total protein concentration).

Wound Healing Sample Protein Concentration (μg/μl) Diabetic? 45-1 12.2 ± 0.5 Yes 45-2 24.7 ± 2.3 Yes 45-3 26.6 ± 1.4 Yes 46-1 36.3 ± 3.4 Yes 46-2 46.0 ± 3.7 Yes 46-3 29.8 ± 1.5 Yes 49-1 55.0 ± 5.7 No 56-1 17.2 ± 1.2 No 56-2 27.2 ± 3.4 No 56-3 24.1 ± 2.1 No 61-1 56.6 ± 3.6 Yes 61-2 15.7 ± 1.0 Yes 61-3 30.1 ± 1.0 Yes 98-1 22.7 ± 0.8 No 99-1 47.4 ± 6.6 Yes 101-1 11.6 ± 0.4 No 108-1 11.6 ± 0.8 No 108-3 22.7 ± 1.1 No 110-2 57.4 ± 0.4 No 110-3 29.9 ± 3.9 No 112-1 40.8 ± 3.2 No 113-1 20.3 ± 2.3 Yes 120-3 15.2 ± 1.8 No 126-2 30.6 ± 0.4 Yes 128-1 10.0 ± 5.8 Yes

Table 2. Protein concentrations of wound healing samples. The supernatants of both the diabetic (yes) and non-diabetic (no) wound healing samples (WHS) were isolated by centrifuging the WHS at 4°C for 10 min at 10,000 rpm in order to keep insoluble particulates from interfering with the protein quantitation. The protein concentration of these supernatants was determined by Micro BSA kit (Thermo Scientific). The protein concentrations of each supernatant were determined in triplicate (N=3).

3.5.2 Human Meprin α was Detected in both Diabetic and Non-Diabetic 1

Wound Healing Samples

The next question that needed to be answered was whether or not the meprin α and/or meprin β subunit could be detected in WHS. To answer this question, WHS supernatants were concentrated 2-fold by YM-30 Millipore concentrators, separated on 8% SDS-PAGE gels, and probed for the meprin α subunit by Western blotting.

As seen in Figure 25, at least 8 of the WHS samples evaluated (lanes 1-4, 6-7, and 10-

11, corresponding to WHS 97-3, 98-1, 113-1, 115-1, 118-3, 120-3, 128-1, 118-3) showed a band on the Western blot having approximately the same apparent size on SDS-PAGE as the recombinant human meprin α subunit (lane 13, apparent size indicated by the red arrow on

Figure 24). This is indirect evidence showing that these WHS contain the human meprin α subunit. The human α subunit was found in both diabetic and non-diabetic WHS, as indicated in

Table 3. While the polyclonal antibody used for this Western blotting probe for meprin α is also capable of detecting the human meprin β subunit, a separate Western blot, utilizing a more specific antibody for the human meprin β was used to determine if the same WHS had human meprin β.

3.5.3 Human Meprin β was Detected in both Diabetic and Non-Diabetic 3

Wound Healing Samples

In the same manner as for the human meprin α subunit, the WHS samples listed in Table

3 were probed for the human meprin β subunit. As seen in Figure 25, 8 out of the 12 WHS lanes showed a band (lanes 1-4, 7-8, and 11-12, corresponding to WHS 97-3, 98-1, 113-1, 115-1,

120-3, 123-2, 118-3, 120-3) approximately the same apparent size on 8% SDS-PAGE as the

Lane: 1 2 3 4 5 6 7 8 9 10 11 12 13

Figure 25. Probe of human wound fluid samples for meprin α by Western blot. Eight of the lanes (1-4, 6-7, 10-11) had visible bands with the same approximate size as the human α control band (lane 13). The antibody used for the human meprin α probe, HMC-14, is a polyclonal antibody and thus multiple bands were seen. It is thought that the lower bands and the higher molecular weight band (above 131 kDa) recognized in this Western are serum proteins. The identity of this protein band seen at 86 kDa as the human meprin α subunit needs to be confirmed via mass spectrometry.

Lane: 1 2 3 4 5 6 7 8 9 10 11 12 13

Figure 26. Probe for human meprin β in wound-healing samples. The signal with an apparent size of the human meprin β subunit (lane 13, as highlighted by the red arrow), was detected in 8 of the WHS tested. It is thought that the lower bands and the higher molecular weight band (above 131 kDa) recognized in this Western are serum proteins. The identity of this protein band seen at approximately 90 kDa as the human meprin β subunit needs to be confirmed via mass spectrometry.

WHS Diabetic Status Meprin α Meprin β 97-3 Unknown Yes Yes 98-1 Yes Yes Yes 113-1 Yes Yes Yes 115-1 Unknown Yes Yes 116-9 Unknown n/d n/d 118-3 Unknown Yes Yes 120-3 No Yes Yes 123-2 No n/d Yes 126-2 No n/d n/d 128-1 No Yes n/d

Table 3: Summary of the human meprin α and β subunits detected in wound healing samples. Out of the 10 wound healing samples (WHS) tested, both human meprin α and β were detected in 7 of them. In the 7 WHS samples tested, 6 of them contained both subunits, while for

WHS 128-1 and 123-2, only the human meprin α and human meprin β were detected, respectively. The human meprin subunits were detected in both the diabetic and non-diabetic

WHS, showing that there is no direct correlation between the presence of meprin and the diabetic status of human patients. n/d = meprin band not detected.

In summary, as seen in Table 3, both the human meprin α and β subunits were detected in the WHS regardless of the diabetic status of the patient. Furthermore, in 6 out of the 7 WHS tested (97-3, 98-1, 113-1, 115-1, 118-3, 120-3), both the meprin α and β subunits were detected together, while in WHS 128-1 and 123-2, only the meprin α or the meprin β subunit was detected, respectively. Based on the signal on these Western blots given by the putative meprin

α and meprin β bands and the detection limit of the meprin proteins by the meprin antibodies, it is estimated (considering that these WHS were concentrated 2-fold) that the amount of meprin α and meprin β ranges approximately from 5 ng to 50 ng per 3µL. This would indicate that the concentration range of meprin A and B in the WHS is from 10 nM to 100 nM.

Chapter 4: DISCUSSION

This work characterizes a direct interaction between meprin metalloproteinases and the pro-inflammatory cytokine interleukin-6 (IL-6). Homomeric meprin A and meprin B both cleave IL-6, not only in vitro but also in a cell-based system; active homomeric meprin B and heteromeric meprin A expressed and localized on an epithelial cell line are capable of cleaving exogenous IL-6. Furthermore, meprin cleavage of IL-6 kinetics indicates that IL-6 is probably a meprin substrate in vivo as the concentration of meprins in tissues is sufficient to cleave this cytokine. Meprin A removes the C-terminus from IL-6 and since the C-terminal portion of IL-6 is important for the activity of this cytokine, it was predicted that meprin cleavage of IL-6 would decrease its activity. This prediction was confirmed via a B9-cell based activity assay that meprin cleavage of IL-6 decreased its activity. Finally, it was found that both human and mouse meprins cleave IL-6 and decrease the activity of both human and mouse IL-6. This implicates meprin in the pathogenesis of inflammatory disease via modulation of IL-6 not only in mouse inflammatory diseases, but also for inflammatory disease of humans. While all the in vitro data contained within this body of work strongly implicates a role for meprin modulation of IL-6 in vivo, this assertion is still only based on correlation until rigorious in vivo studies are done to confirm the meprin/IL-6 interaction in vivo.

Meprins are highly regulated metalloproteinases that participate in a wide range of processes in healthy, injured, and diseased animals, including humans. The isoforms of meprin have overlapping capabilities but also activities that serve in counterpoising roles [117]. Of particular note, meprins are able to digest extracellular matix proteins and disrupt epithelial tight junctions. However, meprins are also capable of activating some cytokines, notably the cytokines of the IL-1β family while inactivating others, such as RANTES, MCP-1, MIP-1α and

MIP-1β (these are all inactivated by meprin A) [53, 55, 56]. The cytokine IL-6 can now be added to the list of cytokines cleaved and deactivated by meprins.

This work demonstrates for the first time that meprin metalloproteinases cleave a very important pro-inflammatory mediatator, interleukin-6. Uncontrolled levels of this cytokine has a determinative role in the exacerbation of maladies such as inflammatory bowel disease [135,

151]. Rodent, homomeric meprin A and B cleave both mouse and human interleukin-6; human homomeric meprin A and B also cleave human interleukin-6. The meprins cleave the proteolytically vulnerable C-terminus of interleukin-6 and then extensively degrade the molecule. The overall biological effect of meprin cleavage of IL-6 is to cause the cytokine to lose activity. The meprins do not act alone but in a milieu of many other metalloproteinases, especially the MMPs and the PMN-derived proteases, which have different temporal and tissue profiles.

4.1 Validation of Cleavage of Interleukin-6 by Meprins

In vitro studies using biologic materials of diverse biological origins may not predict in vivo outcomes. The current studies were designed to relate processes in mice with clinical relevance in humans using in vitro assays. Accordingly, it was necessary to ascertain whether or not the sources of the biological reagents influenced outcomes. Critical variables included the source of IL-6 and meprins.

While published data indicates that the bio-activity of recombinant human and mouse IL-

6 are not different from native IL-6 forms because of differential glycosylation (or lack thereof in the case of E.coli-generated IL-6 isoforms) patterns on the recombinately-expressed IL-6, these differential glycosylation patterns might protect certain peptide bonds from protease cleavage

[136]. Therefore, the rhIL-6 from NCI (which is mono and di-N-glycosylated) was used for the kinetic and meprin-cleavage site analysis, as to better model meprin cleavage of IL-6 in humans and mice in vivo. Native mouse IL-6 is not N-glycosylated, but is O-linked glycosylated [137].

Figure 27 shows the location of the glycosylation motifs of both mouse and human IL-6.

Confirming that meprin cleavage of IL-6 was not limited to only human IL-6 was important to link the in vivo finding that meprin KO mice have both increased inflammation in the DSS- challenge model of IBD and the finding that a polymorphism of the human meprin α gene, suspected to cause decreased levels of meprin expression, is a susceptibility gene for IBD together [94, 117, 118].

The other variable that had to be considered throughout these in vitro studies were the meprins themselves. Homomeric human and mouse meprin A (recombinant) have different affinities for several key substrates, as it was observed that homomeric mouse meprin A has a

12-fold and a 9-fold higher specific activity to the fluorogenic peptide substrate BK+ and to gelatin, respectfully, than homomeric human meprin A [101]. This is another reason it was important to determine if both human and rodent homomeric meprins catalyzed cleavage of both human and mouse IL-6. The differences seen in tissue distribution of meprins in human vs rodents, the differences seen in human and mouse meprin inhibition by various inhibitors, and the differences seen in the affinity of cleavage of different meprin substrates by both human and mouse meprins all imply that human and rodent meprins may play different roles from each other in their associated species [101].

That rodent meprins, both homomeric meprin A and B, cleave mouse IL-6 was especially important to ascertain, as the in vivo cytokine data implicating IL-6 as a substrate for meprins originated from a mouse IBD model. Furthermore, the finding that human meprins

Figure 27. Location of glycosylation motifs on both human and mouse interleukin-6. The

N and C-termini of IL-6 are depicted by black arrows and text. Human has two potential N- linked glycosylation sites, one at Asn 45 (located at the C-terminal end of the 1st helix, shown by the bottom red arrow and text) and the other site at Asn 144 (located in the middle of the small helix, noted by the other red arrow and text) [163]. Mouse IL-6 has no N-linked glycosylation site, but is O-linked glycosylated at Thr 140, which is also located in the small helix located outside the main 4-helical bundle (noted by the blue arrow and text) [167].

93 cleave both E.coli-derived and mammalian-cell derived IL-6 is an important way to link meprin- involvement in human inflammatory disease via IL-6 control in vitro data. The pathogenesis of

IBD is complex and IL-6 plays a central role for both human and mouse colitis. IL-6 has been directly linked to the level of disease severity in IBD, both in mouse models and human patients.

IL-6 KO mice are resistant to IBD induced with DSS [144, 149, 151].

There is in vivo data implicating meprins in the pathogenesis of human IBD. A polymorphism in the 3‟-UTR of the human meprin α gene has been significantly linked to IBD in human patients [118]. The 3‟ UTR is important for stability of the gene transcript and so the current thought is that this particular polymorphism in the meprin α gene causes a decrease in levels of meprin A protein [119, 120]. In support of this hypothesis, human intestine samples from IBD patents have decreased mRNA levels of the meprin α transcript vs control samples.

Also, it has been shown in a mouse model of IBD that meprin α KO mice have greater inflammation, both locally (in the colon) and systemically, than their wild-type counterparts when given DSS to induce IBD [117, 118].

Lack of meprin A may be responsible for the increased inflammation in both mice and humans suffering from IBD by allowing more IL-6 activity in the inflamed intestine. However, there most likely are other factors explaining the increased inflammation in the meprin null mice and the human patients with the meprin α polymorphism. The colon levels of IL-6 in the meprin

α and the meprin αβ KO mice were similarly elevated from WT meprin colon concentrations and not statically different from each other, as seen in Figure 28. This finding is consistent with the knowledge that meprin β is not expressed in mouse colon in mouse colon. However, not all the levels of known cytokines were evaluated and the cytokine levels between the WT and meprin

94 consistent with the knowledge that meprin β is not expressed in mouse colon in mouse colon

3500.0 3000.0 2500.0 2000.0 1500.0

1000.0 6 Concentration (pg/mL) Concentration 6

- 500.0 IL 0.0 WT α KO αβ KO Mouse Meprin Genotype

Figure 28: Colon concentrations of IL-6 in meprin knock-out mice after DSS challenge are similar. Wild-type (WT) and meprin α and αβ KO mice were subjected to DSS treatment to induce intestinal inflammation (n=5 to 10 mice per genotype group). On day 5 after DSS administration, the mice were killed and the colonic concentration of fourteen cytokines was calculated via ELISA. This figure compares the colonic IL-6 concentrations between the three meprin genotypes. While the concentrations of IL-6 in the colons of meprin αKO and meprin

αβKO mice were not significantly different 5 days post-DSS administration, the levels of IL-6 in

WT colon were significantly different from meprin αKO and meprin αβKO mice, p< 0.005 and p<0.05, respectively. Furthermore, IL-6 was the only cytokine whose levels were different in the colon tissue of DSS-challenged WT, meprin α and meprin α/β KO mice [162].

KO mice were only determined at one timepoint during the experiment. Still, these combined data strengthen the hypothesis that meprin A is responsible for keeping IL-6 levels under control during inflammatory insults such as DSS-induced IBD, especially since IL-6 was the only cytokine tested which was significantly increased in the meprin αβ KO animal [118, 168]. This does not mean that IL-6 is the only mediator by which meprins (or lack of them) mediate pathogenesis. For example, in the urinary tract infection model, IL-6 levels between infected

WT and meprin KO mice were statistically insignificant [112]. There are other methods by which meprins modulate the immune response other than direct cytokine cleavage, such as through affecting cell migration and cleavage of other non-cytokine substrates [27, 94].

4.2 Meprin Cleavage Site on Interleukin-6

The gel fragmentation experiments with meprins and IL-6 show that both meprin A and

B initially cleave the cytokine, resulting in the removal of a small portion of the the molecule.

Subsequently, meprins degrade the cytokine further with no additional product observed on gels.

Several other proteases important for the immune response have been shown to cleave IL-6, such as the PMN-derived proteases neutrophil elastase (NE), proteinase-3 (PR3), and cathepsin-G

(CatG), but not in the same manner as the meprins. Also, several bacterial proteases called gingipains, which are trypsin-like cysteine proteases produced by the bacteria Porphyromonas gingivalis (a bacterial strain which is the major cause of adult periodontitis), also cleave IL-6 at several of the same positions as the PMN-derived proteases do. Gingipains cleave and inactivate

IL-6 in order to impede the immune response in the gum tissue [146, 147].

The only apparent sites of vulnerability are the N and C-termini of the cytokine and the loops between the helices [143]. As expected, these PMN-derived proteases NE, CatG, and PR3,

96 as well as KGP, RGP-A and RGP-B, do not cleave IL-6 at the helices themselves, but cleave IL-

6 at positions more vulnerable to proteolytic attack. Neutrophil elastase first cleaves IL-6 at the

N-terminus (first at residue 11 and then at residue 19). Further cleavage products of NE- treatment of IL-6 were detected after the initial cleavage at the N-terminus, although the sites of cleavage for these further products were not determined. PR3 cleaves IL-6 between helices C and D, while CatG cleaves IL-6 between helices A and B [63, 148]. RGP-A removes a portion from the N-terminus (residues 1-18); both RGPs and KGP cleave the C-terminus of IL-6 and then further degrade the cytokine [146]. Figure 29 shows pictorially the locations of these cleavage sites on the interleukin-6 molecule. The purpose of illustrating all of these sites of cleavage by these various proteases upon IL-6 is that there are multiple ways the activity of IL-6 can be decreased, but due to timing of when certain proteases appear in the inflammatory milleu and due to local inflammatory environment conditions, meprins are likely to be the major proteolytic contributor in keeping IL-6 activity under control directly.

From the peptide maps of the meprin-generated IL-6 products, it was determined that meprin A removes a small portion, 5 amino acids, from the C-terminus portion of IL-6.

Therefore, meprins are the first mammalian proteases identified that cleave IL-6 at the C- terminus. Past experiments with IL-6 show that the cytokine lacking its 5 C-terminal residues displays decreased stability [139]. Human IL-6 constructs lacking only 3 amino acids from the

C-terminus had dramatically less activity than wild-type, full length human IL-6. However, these IL-6 bio-activity experiments were done with truncated IL-6 constructs, not with IL-6 lacking the C-terminal residues post-translation via specific cleavage [140, 141]. Given that these C-terminal residues are important for stability and activity of IL-6 it is possible that the loss

Figure 29. Protease cleavage locations on human interleukin-6. The locations of cleavage by the PMN-derived proteases (CatG, NE, PR3 – noted with green text and arrows), meprin A and

B (noted with blue text), and the proteases produced by Porphyromonas gingivalis, KGP, RGP-

A and RGP-B (noted with purple text and arrows), are shown superimposed on a ribbon model of human IL-6. The numbers listed below the various proteases are the residues where the proteases cleave IL-6. The helices of the human IL-6 are (A through E) labeled with red text and arrows. The only protease known to cleave anywhere with the main 4-α helical bundle (helices

A through D) is KGP, which cleaves the Lys173-Glu174 bond at the C-terminal end of helix D.

The protease PR3 cleaves the Ala145-Ser146 bond located within a smaller helical structure

(helix E) which is outside the main 4 α-helical bundle. The N-terminal and C-terminal ends, designated on this model with black text and arrows, are the most susceptible sites to proteolytic attack. Both cleavage sites of NE on IL-6 (Val11-Ala12 and Leu19-Thr20) are on the N- terminus just before the N-terminal end of helix A; RGP-A and B cleave IL-6 at both the N and

C-termini of IL-6, at Arg16-Gln17 and Arg181-Ala182. Meprin A and B cleave IL-6 at the same location on the C-terminus of IL-6 as RGP-A and RGP-B, at Arg181-Ala182 proximal to the C- terminal end of helix D. CatG cleaves IL-6 at the Phe78-Asn79 peptide bond, which is located near the N-terminal portion of helix B. The hIL-6 model is a PBD (Protein Databank) rendering of human interleukin-6 (using model 1ALU) taken from: http://en.wikipedia.org/wiki/Interleukin-6.

99 of these residues leads to loss of activity – which could confirm the original hypothesis that meprin cleavage of IL-6 causes the cytokine to lose its activity.

4.3 Meprin Decreases Activity of Interleukin-6

Cleavage by both meprin A and B decreases the activity of IL-6, as confirmed by the B9 hybridomia proliferation assay. Fortunately, both human and mouse IL-6 act on mouse cells, thus the mouse-derived B9 cells could be used to analyze the effects of meprin cleavage of both

IL-6 species [169]. Human IL-6 treated with human recombinant homomeric meprin A and B lost its bio-activity over time of incubation with meprins, with less than 10% of hIL-6 bio- activity remaining after 1.5 h incubation with human meprin A. Recombinant human homomeric meprin B did not decrease the bio-activity of hIL-6 as dramatically as human meprin

A, but only 30% of hIL-6 activity (as compared to untreated hIL-6) remained after incubation with human meprin B. Decrease in activity of mouse IL-6 after incubation with meprins was also seen with the B9 cell proliferation assay, but after 1 hr of incubation with both meprin A and

B, mouse IL-6 retained approximately 65% of activity compared with uncleaved mouse IL-6.

After 1 hr of incubation with human meprin A and B, human IL-6 only had 10% and 40% activity, respectively. However, a different activity pattern was seen in the experiment with the

B9 cells incubated in the 10 ml flasks, treated with uncleaved and rodent meprin A and B cleaved mouse IL-6, and counted by hemocytomer after 72 hours of incubation. In this experiment, meprin A-treated mouse IL-6 had approximately 70% activity after 1 hr of incubation while meprin B-treated mouse IL-6 had approximately 40% activity after 1 hr of incubation. Between these two experiments with mouse IL-6, both methods of measures showed approximately the same decrease of mouse IL-6 activity by meprin A after 1 hr of incubation

(about 65-70% activity) while a greater activity decrease of mouse IL-6 by meprin B incubation

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(65% vs 40%) was seen when the B9 cells were counted directly via hemocytomer. All of these activity experiments show that IL-6 activity is decreased with increasing time of incubation with meprins and that IL-6 does not completely lose its activity immediately after meprin incubation.

The next question that needs to be asked is how well meprin decreases the activity of IL-6 when compared to other proteases that also decrease the activity of IL-6.

The PMN-derived proteases NE, CatG, and PR3 also dramatically decreased the activity of human IL-6, as seen in Figure 30. Comparing the data on deactivation of IL-6 bio-activity by NE, CatG, and PR3 proteases with that of the meprins might imply that incubation with the

PMN-derived proteases deactivates IL-6 more rapidly than meprins. However, the B9 experiments with the PMN-derived proteases used 0.2 µg/µl IL-6 and 0.01 µg/µl protease, corresponding to ~8.0 µM substrate (assuming a molecular weight of 26 kDa for IL-6) and ~ 330 nM for each enzyme (assuming a molecular weight of 30 kDa for NE, CatG, and PR3). The B9 bio-activity assays with meprins were performed with 8-fold less substrate (1.0 µM IL-6) and

3.3-fold less enzyme concentration (100 nM meprin A and meprin B).

When comparing the relative contribution of IL-6 activity control by meprins and the

PMN-derived proteases, the pH of the inflammatory loci must be considered. At sites of inflammation, the pH is considerably decreased from normal. At some inflammatory loci, the extracellular pH can decrease to as low as 5.5 [170, 171]. PMN-derived proteases have pH optima of 7.5, 8.0, and 8.0-8.5, for NE, PR3, and CatG respectively [172]. Meprin A isoform also has a neutral pH optimum (7.5), but meprin B has a more acidic pH optimum (pH 6.2)

[100]. Thus, while there will be an abundance of PMN-derived proteases at sites of inflammation due to the influx of neutrophils, the inflammatory environment may favor meprin

B cleavage of IL6 over PMN- derived cleavage of IL-6 (even over meprin A cleavage of IL-6).

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Figure 30. Proteases from polymorphonuclear leukocytes efficiently decrease the activity of interleukin-6. This data is from Bank, Kupper et al. 1999. The bio-activity of unglycosylated (A) and glycosylated (B) human IL-6 after treatment with CatG (circles), NE

(squares), and PR3 (triangles) was evaluated using the B9 hybridoma cell assay as previously described [148]. The bio-activity of protease-treated IL-6 is displayed as a percentage of untreated IL-6 (on the Y-axis), with the treatment time of IL-6 with the protease listed in minutes on the X-axis. All three proteases decreased the activity of human IL-6 to less than 10% of uncleaved hIL-6 within 60 min, regardless of the glycosylation status of IL-6 [148].

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There also could be an additive effect on IL-6 at sites of inflammation from both the PMN- derived proteases and meprins. For instance, since meprin would already be present in the intestine, expressed from the intestinal epithelial cells as both membrane-bound and secreted meprin, meprins have an opportunity to cleave IL-6 before the PMN-derived proteases arrive to the site of inflammation. Meprins may make the first proteolytic cut on IL-6 while the PMN- derived proteases further degrade IL-6, which would limit the inflammation induced by the increased concentration of IL-6. Since meprin α expression, which decreases during IBD in humans, meprin cleavage as a form of control for IL-6 may be more important during the initial phase of inflammation induction than during the continual phase of inflammation [118].

No protease-derived fragments of IL-6 have been successfully isolated from any inflammatory sites to date. However, the bulk IL-6 activity at several inflammatory exudates

(ascites, synovial fluids, pleural effusions, drained CSF) is lower than anticipated, based on the concentration of IL-6 detected in the exudates and the ability of said exudates to stimulate B9 cells [63, 148]. Approximately 76% of the samples (87 samples out of 115) tested had lower bio-activity than expected. There are several possible explanations for this contradiction. First, there may be other proteins at the inflammatory site that block IL-6 bio-activity. For example, solubilized gp130 (sgp130) acts as a mild antagonist of IL-6 activity [173]. Alternatively, the cytokine could be cleaved by proteases in places that leaves the bulk of a cytokine intact (and be detected via ELISA), but dramatically decreases their activity. In fact, this was the rationale used to determine whether IL-6 is a substrate for the PMN-derived proteases.

Several pro-inflammatory cytokines were increased in both the serum and in the colon of the meprin α KO mice vs the meprin WT mice that were subjected to the IBD model. As the meprin α KO mice had increased inflammation compared to WT mice, the increased IL-6 levels

103 seen in the serum and colon of the meprin α and meprin αβ KO animals are most likely fully active [118]. To confirm the increased IL-6 activity in the meprin KO animals, IL-6 from several inflammatory sites, such as in serum and colon homogenate, could be analyzed via B9 cell assays and compared with the IL-6 activity from meprin WT mice. It would be expected that the IL-6 from the meprin knock-out mice would have significantly higher activity than the

IL-6 from wild-type mice when the activity of the cytokine is normalized to the IL-6 concentration.

The data from the fragmentation, meprin cleavage-site determination, and activity assays give in vitro support for the hypothesis that meprins are an important factor for maintaining control of activity of IL-6. This lack of IL-6 control may explain in part why the meprin knock- out animals have increased inflammation in the IBD model in particular. Without either meprin

A or B to help cleave IL-6, the levels of this cytokine persist even in the presence of other proteases such as the PMN-derived proteases and MMPs (and no MMPs have been identified to date that cleave IL-6 directly), thus leading to increased inflammation. However, it still needed to be determined if cell-based meprins are capable of cleaving IL-6. While meprin β is not expressed in the human colon, there are sections of the upper intestine where the intestinal epithelium cells express both the meprin α and meprin β subunits, and it is also thought that the infiltrating monocytes also express both meprin subunits, thus the meprins in both these cellular locations would be the heteromeric meprin A isoform.

4.4 Interleukin-6 is Cleaved by Cell-Associated Meprin

Meprins and IL-6 are both found at sites of inflammation, particularly in the intestine for

IBD and in the kidney for acute renal failure (ARF) induced by isochemia/reperfusion injury

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(ARF is considered an inflammatory disorder) [94, 174]. Co-localization of meprin and cytokine does not show or guarantee that these two proteins interact with each other. However, the data gleamed from the kinetic studies are promising in demonstrating that should these two proteins encounter each other in vivo, meprins would be able to cleave IL-6.

From the kinetic studies of IL-6 with meprin A and B, it has been shown that IL-6 is one of the “better” substrates for meprin A and B, with affinities, Km, in the lower micromolar range and efficiency constants (kcat/Km) better than most known meprin substrates. Table 4 shows the kinetic constants of other protein (villin, actin, proIL-18) and peptide (GRP, glucagon, and gastrin 17) meprin substrates compared with the calculated kinetic constants of meprin cleavage of IL-6. As seen in Table 4, meprin A cleaves proIL-18 and IL-6 with similar affinities, but since the turnover number of proIL-18 to IL-18 by meprin A is approximately 16 times higher than that for IL-6, making the specificity constant (kcat/Km) of meprin A for proIL-18 15 times greater than that for IL-6. While the affinity of IL-6 for meprin B is approximately 3-fold lower than the affinity of IL-6 for meprin A, meprin B has a 30-fold higher turnover number than meprin A for IL-6, and so the specificity constant of meprin B cleavage of IL-6 is 25 times higher than that of meprin A. Still, meprin B has a 2-fold higher specificity constant for proIL-

18 than for IL-6, but overall, IL-6 is still one of the more kinetically competent substrates for meprin amongst all the known meprin substrates.

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Substrate Meprin A Meprin B Reference

Km kcat kcat/Km Km kcat kcat/Km

(M x 106) s-1 (M-1s-1) x106 (M x 106) s-1 (M-1s-1) x106

GRP-(14-27) 116 88.3 0.76 48.1 12.6 0.26 [97]

Glucagon 223 17.4 0.078 220 26.8 0.13 [97]

proIL-18 5.49 16.4 3.0 1.31 6.79 5.19 [53]

Villin 0.96 13.3 13.9 0.73 150 128.2 [160]

Non-muscle 0.6 0.75 1.25 1.17 0.63 0.86 [160] actin

Gastrin 17 ND ND ND 1.04 11.0 10.6 [77]

Human IL-6 4.9 1.0 0.20 12.0 30.4 2.5

Table 4. Kinetic constants of meprin A and B cleavage of interleukin-6 compared with the cleavage by meprins of other protein and peptide substrates. The kinetics of meprin A and B cleavage of recombinant human IL-6 are in bold. Meprins have a higher affinity for rhIL-6 than for the peptide substrates, but meprin A has comparable affinities towards IL-6 and proIL-18.

Meprin B has a 10-fold higher affinity toward proIL-18 than IL-6. Comparisons of the specificity constant show that rhIL-6 is a better substrate for meprin B than meprin A, although proIL-18, gastrin 17, and villin are also better substrates for meprin B than IL-6. ND – Not detected.

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While IL-6 is one of the “better” substrates for meprin, this kinetic information by itself cannot prove whether or not meprin is capable of cleaving IL-6 in vivo at sites of inflammation.

It is proposed that the concentration of meprin is sufficient to cleave IL-6, since the kidney and intestine are the tissue locations where the meprin concentration is the highest, especially during inflammation when there are two sources of meprins, one source being the meprins expressed from the epithelial cells and the other source the meprins expressed on the infiltrating macrophages [92, 94]. The concentration of secreted meprin in rat urine is 20 nM and it is proposed that the amount of meprin localized at the mouse kidney brush border is in the higher micromolar range [77]. The concentration of IL-6 in the colon of the WT and KO meprin mice increases rapidly from an undetectable level under normal conditions to 735.8 pg/ml, 3057 pg/ml, and 2649 pg/ml in the WT, meprin α KO, and the meprin αβ KO animals, respectively, upon DSS administration [117]. Assuming a molecular size of approximately 30 kDa for mouse

IL-6, the concentration of IL-6 is only in the pM range, from 25 pM in the inflammed colon of the meprin WT mice to 90-100 pM IL-6 in the inflammed colon of both the meprin α and meprin

αβ KO mice [136]. However, while the bulk concentration of IL-6 in the colon might not reach the micromolar concentrations needed for the most efficient cleavage by meprins, it is proposed that in the micro-enviroment of the colon during inflammation, the concentration of IL-6 might be further increased into the micromolar range. In the case of proIL-18, it was seen that during inflammation, the serum concentration of IL-18 was approximately 50-60 pM [53]. While the concentration of proIL-18 ostensibly did not reach micromolar range in the bulk serum, allowing for the most efficient conversion of proIL-18 to IL-18, it was still observed that the meprin β KO mice had significantly lower IL-18 serum concentration than WT meprin mice and conversely,

107 meprin α KO mice had significantly higher IL-18 serum concentrations than WT meprin mice subjected to the IBD model [53].

Finding physiological substrates for meprin has always been of interest, given that meprin has been linked to inflammatory disease both in mouse models and for humans (IBD and acute renal failure as well) [53, 109]. Extending the panel of cytokines known to be meprin substrates will aid in future investigations into ascertaining the complete role meprin plays in modulating the pathogenesis of inflammatory disease. Thus far, meprins have been shown to cleave several chemokines and cytokines, mostly cytokines of the IL-1β family (including IL-18) and the chemokines RANTES, MCP-1 and now IL-6 [53, 55, 56, 104].

Cytokines and chemokines are an expanding class of substrates not only for the meprins, but also for the MMPs. Table 5 and Table 6 show the effects MMP cleavage has on both chemokines and cytokines, respectively. To date, there is much more data on MMP cleavage of chemokines than cytokines. MMPs can either activate or deactivate chemokines. Furthermore,

MMP-derived products of chemokine cleavage can act as antagonists or agonists. It was not the case that meprin-cleaved human IL-6 acted as either an agonist or antagonist against uncleaved human IL-6, but it is possible that meprin-derived products of chemokine cleavage might act in such a manner.

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LIX CXCL1 CXCL 4 CXCL5 CXCL CXCL7 CXCL8 CXCL9 CXCL 6 12 A

MMP-1 +++ ---

MMP-2 +++ ---

MMP-3 ---

MMP-8 +++ +++ No +++ --- Effect

MMP-9 +++ ------No --- +++ ------Effect

MMP- +++ +++ --- 13

MMP- +++ --- 14

B CX3CL1 CCL2 CCL7 CCL8 CCL13

MMP-1 antagonist antagonist antagonist

MMP-2 antagonist antagonist

MMP-3 antagonist antagonist antagonist antagonist

MMP-8 antagonist

MMP-9

MMP- antagonist 13

MMP- antagonist 14

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Table 5. Matrix metalloproteinase cleavage of various chemotactic cytokines alters their activity. This table summarizes the information published about MMP interactions with chemokines. Depending on where the MMP truncates the chemokine, a) the activity of the chemokine can be increased (+++) or decreased (---) to the point of inactivity. In some cases, the

MMP fragments a chemokine, yet the activity of the chemokine is unaffected (no effect). Some

MMP-inactivated chemokines (and the subsequent fragments) may still be able to bind to their receptor but are unable to facilitate chemotaxis, thus they act as antagonists, as seen in Table 5b

(antagonist). Blank spots indicate that no proteolysis of the chemokine was detected upon incubation with the indicated MMP [33].

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MMP Cytokine Substrate Biological Effect Reference

3, 9, 14 TGF-β1 Cytokine activation [18]

2, 3, 9 proIL-1β Cytokine activation [18]

1, 2, 3, 9 IL-1β Cytokine deactivation [18, 175]

1, 2, 3, 9, 17 proTNF Cytokine activation [18, 175]

Table 6. Matrix metalloproteinases cleave cytokines and change their biological activities.

The MMPs also cleave cytokines, although the list of known cytokine substrates for MMPs is far less than the known chemokine substrates of MMPs. Interestingly, MMPs 2, 3, and 9 convert the cytokine proIL-1β to its active form IL-1β, however, further incubation of IL-1β with MMP 1, 2,

3, and 9 degrade and thus deactivate the IL-1β cytokine. This demonstrates the dual nature of proteases acting on a single substrate.

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MMP cleavage of cytokines is one way to regulate the expression of MMPs, as one method of transcriptional regulation of MMPs is through cytokines [176, 177]. For example, IL-

1 potently induces the expression and activity of several MMPs [178, 179]. As seen in Table 6,

MMPs 1, 2, 3, and 9 degrade and thus deactivated IL-1β after an extended incubation. MMP cleavage of cytokines acts a control mechanism during inflammation, as cytokines upregulate

MMP expression and when the concentration and activity of these MMPs are sufficient, the cytokine is then degraded, completing this “feedback loop” [175]. No MMPs have been identified to date that cleave IL-6. While the PMN-derived proteases are capable of cleaving and inactivating IL-6 in vitro, the inflammatory environment may not be suitable for efficient in vivo cleavage due to the aforementioned pH optimum differences between these proteases and meprins [100, 172]. While the concentration of neutrophil-derived proteases is estimated to be quite high at sites of inflammation, about 40 µg/ml (approximately 1.3 µM for the smaller 30 kDa proteases), these proteases might have lower kinetic constants for IL-6 than meprins [63,

180].

MMP and other proteases are also capable of modulating cytokine and chemokine concentrations by means other than direct cleavage. MMPs are capable of liberating active cytokines from the surface of macrophages, such as TNF-α, IL-1 cytokines, and IL-6 [181].

Furthermore, MMPs can release chemokines sequestered in the ECM and form chemokine gradients for immune cells. An example of this is seen with MMP-7 cleavage of syndecan-1, which is a cell-bound heparin sulfate proteoglycan, and the subsequent release of the syndecan-1 ectodomain into the extracellular space [182]. When epithelial cells are damaged during mucosal injury and inflamamtion, they synthesize and deposit chemokines such as CXCL1 (the human analogue to this mouse chemokine is CXCL8) onto this syndecan-1 molecule. During

112 injury and inflammation, the level and activity of MMP-7 is increased, which causes the cleavage of syndecan-1 and the subsequent release of the CXCL1 chemokine into the extracellular space, which causes a gradient for neutrophils to migrate to the site of injury [18].

This example with MMP-7 and syndecan-1 shows how proteases affect inflammation indirectly, aside from the effects proteases may have on inflammation by direct cleavage of cytokines and chemokines.

From the animal studies done with the meprin KO animals, it is clear that meprins play a role in the modulation of inflammation at mucosal surfaces, as seen with the meprin contribution towards IBD (inflammation of the mucosa of the gastrointestinal tract) and urinary tract infections (inflammation of the mucosa of the urinary tract) [53, 112, 117, 118]. There is also some data implicating meprins in the progression of lung inflammation (inflammation of the mucosa of the pulmonary system). Recent data shows that NE converts meprin A to an active form, allowing meprin A to cleave and release membrane-bound proTGF-α to its soluble, active form. The proposed effect of this NE-mediated meprin A is to stimulate IL-8 producation and to trans-activate the EGFR (Epidermal Growth Factor Receptor) and TLR-4 [183]. Since meprins are highly expressed at and from these particular mucosal surfaces and they have been implicated in several inflammatory disorders at these locations, this gives some support to the idea that the mucosal-lined epithelium is not a “passive bystander” during instances of injury and disease. The epithelium itself may make a significant contribution to the inflammatory response by deploying its own assortment of molecules upon challenge or injury [94, 184].

While these studies with IL-6 further elaborate on how meprins could modulate inflammatory disease by direct interaction with IL-6, the potential meprin interactions with other molecules involved with IL-6 trans-signaling, such as the souble IL-6R (sIL-6R) and the soluble

113 gp130 (sgp130), were not evaluated. Figure 5 showed how IL-6 produces a signaling response in cells that lack expression of the membrane-bound IL-6R and how this form of IL-6 trans- signaling is blocked. Meprins could potentially modulate IL-6 related responses further by cleaving and rendering either (or both) sgp130 and sIL-6R unable to bind to IL-6 or bind with less affinity. During IBD in humans, concentrations of both sIL-6R and sgp130 are elevated, although the peak concentration of both these soluble receptors occurs during different phases of

IBD. The concentration of sIL-6R peaks and declines prior to disease remission and sgp130 peaks after disease resolution [144].

Proteases have been shown to act on IL-6R, releasing the alpha chain of the IL-6R subunit from the cell membrane. ADAM-10 and ADAM-17 (TACE) both cleave IL-6R at the membrane [185]. The PMN-derived proteases CatG, NE, and PR3 all completely degrade sIL-

6R upon extended incubation, although sIL-6R was protected from degradation by the NE and

PR3 proteases if the receptor was pre-complexed with IL-6. It is possible that these PMN- derived proteases are also capable of degrading sgp130, but that has not been confirmed in vitro

[186]. Thus, it may be possible that the meprin metalloprotenases are also capable of cleaving and altering the activity of these protein components involved in IL-6 trans-signalling.

There is another significance of the finding that cell-anchored meprins are able to cleave

IL-6. Monocytes and macrophages express both IL-6 and the α and β subunits of meprin. This means that not only are the epithelial meprins immediately available to keep the activity of IL-6 in check upon initiation of inflammation, but also macrophages themselves have meprins available to keep IL-6 activity curtailed. The next step would be to evalauate the relative contributions that immune-cell based meprins and epithelial based meprins have on keeping the activity of IL-6 under control.

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4.5 Effects of Lectin Interaction with Meprins

Meprins are unique amongst proteases with their ability to form multiple membrane- bound and secreted isomers. This also means that determining the roles of meprins in both the progression of inflammation and the pathogenesis of inflammatory disease requires more discernment than for other proteases, as knocking out the expression of one meprin subunit may decrease the concentration of one inflammatory mediator while knocking out the expression of the other meprin subunit may cause the opposite effect [53]. The data gleaned from the meprin

KO in the IBD model has led to the supposition that the balance of meprin expression is important for the proper maintance of inflammation and that diseases such as IBD and urinary tract infections are exasperated when this balance of meprin expression is disrupted [112, 117].

While the contention of this thesis work is that the significant increase of IL-6 concentration seen in the DSS-administered meprin KO mice is due to meprins playing a role in controlling the inflammatory response by cleaving and inactivating IL-6, there is another way meprins could possibly contribute indirectly towards this increase in IL-6. Meprins are also known to cleave extracellular proIL-1β and proIL-18, converting them to their active forms [53, 55, 56]. It is possible that in the meprin α KO mice, the imbalance of meprin B production might lead to increased levels of active IL-1β, and it is known that IL-1β stimulates production of IL-6 [187].

Since the colonic concentrations of IL-6 in the meprin α and the meprin αβ KO mice were similar, there is a low likelihood that increased IL-1β is responsible for the IL-6 increase seen in the DSS-treated meprin α KO mice. This interplay between meprins and cytokines illustrates the potential need for specific meprin inhibitors for homomeric meprin A and meprin B as well as the heteromeric meprin A isoform.

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The known methods of controlling meprin activity are: down-regulation during inflammation, the presence of a “pro-piece” that needs to be removed by trypsin-like proteases before meprins can become active, and spacial separation [94]. However, there are no known endogenous inhibitors for meprin. Several endogenous proteins have recently been proposed as potential meprin inhibitors, such as cystatin C (an endogenous inhibitor of cysteine proteinases) and fetunin–A (a plasma binding protein) [188]. The most potent inhibitor of meprins identified to date is the hydroxamate compound actinonin. Actinonin is a potent antibacterial chemical secreted by Streptomyces, but actinonin has also been found to inhibit virtually all MMPs and several of the serine proteases [27, 156, 189]. Since meprins have been implicated in cancer progression, there is an interest in finding potent and specific inhibitors of meprins [94].

Hirano, Ma et al. 2005 published data implicating MBL as a meprin inhibitor. To evaluate further the potential of MBL as an in vivo inhibitor of meprins, the interaction of MBL with homomeric isoforms of meprin A and B and the subsequent effect on meprin activity were evaluated. While these studies confirmed that MBL interacts with recombinant homomeric meprin A, no inhibition of any meprin isoform tested was seen at any concentration of MBL incubation. However, another lectin, Con A partially inhibited the proteolytic activity of heteromeric meprin.

There are several potential reasons for this discrepancy between these studies and those of Hirano, Ma et al 2005. The mouse kidney meprin used for the Hirano, Ma et al 2005. inhibition studies was the same meprin that was isolated from an MBL-affinity column. It was never determined what percentage of the total meprin in the kidneys bound to the MBL-affinity column and how much passed through the column without binding. Given that the meprins can be extensively glycosylated (15% of the mass of meprin is made up of carbohydrate) and that

116 there are probably multiple sub-populations of differentially glycosylated meprins, it is possible that the meprin used for the Hirano, Ma et al. 2005 inhibition studies was a sub-population of meprin optimally N-glycosylated for MBL-mediated recognition and binding. The glycans on meprin are primarly N-linked, lack a terminal salic acid, and are made up primarly of N- acetylglucosamine, mannose, galactose [27, 79, 94]. In contrast, the meprins used for these

MBL-interaction studies were recombinantly expressed and purified from HEK293 cells, not isolated from mouse kidney.

The findings that confirmed that meprins are recognized by MBL in a glycan–dependent manner may have greater implications in vivo. More and more data are being collected indicating that MBL can recognize not only pathogens but also “altered self-ligands” on the surface of cells in cases of inflammation and injury. This recognition by MBL causes these cells to be lysed via complement activation [154]. Meprin β KO mice show less damage when subjected to acute renal failure (ARF) via ischemia-reperfusion (IR) injury than their WT meprin counterparts [109]. One potential mechanism for this decreased injury seen in the meprin β KO mice is that since meprin is not sequestered on the kidney brush border cells in meprin β KO mice (the meprin isoform expressed by the kidney brush-border epithelial cells will be secreted meprin A), there will be less complement-mediated damage and thus less inflammatory damage done to the kidney-brush border cells. Some recent published evidence indicates that this is the case [85].

In the case of ARF, a protein inhibitor that masks meprin from MBL-recognition might be a useful therapeutic approach to modulate inflammation. While the role of MBL in intestinal disorders is still being debated and some studies show no link between MBL and IBD, there might be merit in developing inhibitors against meprin to treat IBD [190]. However, Con A

117 cannot be used for treatment of IBD. Con A is a strong inducer of the acute phase response and thus would dramatically increase the amount of IL-6 and other inflammatory cytokines in the intestinal environment, thus having the opposite effect of increasing inflammation rather than curtailing it [191].

4.6 Meprin Involvement in Wound Healing

A damage model that may be of great interest to determine a potential role for meprins in is the wound healing model. Wound healing is a multi-faceted process consisting of three main stages (inflammatory stage, re-epithelialization stage, and the tissue remodeling stage) where proteases play major roles in every stage [192]. There are several rationales for the hypothesis that meprins are involved in wound healing. Meprins are expressed on certain populations of leukocytes and meprin disruption on these leukocytes impairs their movement [92, 193].

Meprins are also expressed in different layers of the skin [90].

A more physiologically revelant rationale for meprin involvement in wound healing is seen with diabetic mice. Diabetic mice display decreased expression of both meprin α and β subunits [113]. Diabetic patients have impaired wound healing and over a 100 physiological factors have been identified as contributing to this deficiency in wound healing. This defect, along with the neuropathy observed with the onset of diabetes, leads to a dramatic increase of lower-limb amputations in diabetic patients [194]. Due to the properties and locations of meprin described previously, pilot studies were undertook to determine if there was a difference with respect to meprins in samples isolated from the wounds of healthy and diabetic patients.

In the studies examining wound healing exudates from human patients, diabetic and non- diabetic, both the meprin α and meprin β subunits were detected, regardless of the diabetic status

118 of the patient. However, these studies are incomplete and several outstanding questions remain.

The activity of the meprin α and meprin β subunits was not evaluated. Attempts to use the BK+ and OCK+ fluorogenic substrates to evaluate the meprin A and meprin B peptidase activity, respectively, of the wound healing exudates failed as all the samples tested showed no change in fluorescent signal. Furthermore, the source of the meprin α and meprin β subunits is unknown.

It is possible that skin cells, which are known to express both meprin α and meprin β [90], filtered through the concentration and centrifugation steps.

The best way to study meprins in wound healing would be to use the meprin KO mice in a wound healing system and compare the dynamics of wound healing between control (healthy) mice, meprin KO mice, and diabetic (db/db) mice. The hypothesis would be that wounds in the diabetic and meprin KO mice would heal more slowly than those in the control mice.

Furthermore, the diabetic and meprin KO mice would be expected to exhibit deficiencies in immune cell migration and collagen breakdown and remodeling.

The wound healing model would also be useful for determining the interplay between meprins, cytokines, and other growth factors at sites of inflammation. For example, IL-6 is needed for proper wound healing. IL-6 KO mice have significantly impaired epithelialization and granulation tissue formation, both of which caused significantly delayed wound healing in the mice. These defects in the KO mice were allievated with the administration of recombinant mouse IL-6 into the mice [135]. Furthermore, this impairment of wound healing was not observed in mice lacking expression of the IL-6R alpha-chain [195]. Therefore, while meprins did not show the extent of IL-6 bioinactivation that the PMN-derived proteases did, this model may further show that meprins fine-tune the activities of inflammatory mediators such as IL-6,

119 but do not completely destroy such activities, leading to a more fine-tuned and appropriate inflammatory response to tissue injury [148].

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Timothy Robert Keiffer Vitae EDUCATION PENNSYLVANIA STATE UNIVERSITY, COLLEGE OF MEDICINE Ph.D. in Integrative Biosciences, Chemical Biology option. Defense Date – Nov. 16, 2011

PENNSYLVANIA STATE UNIVERSITY, State College, PA B.S. in Biochemistry and Molecular Biology, Spring 2005

RESEARCH EXPERIENCE PENNSYLVANIA STATE UNIVERSITY, COLLEGE OF MEDICINE, July 2006-Present Graduate Research Assistant, Principal Investigator: Dr. Judith Bond

My research is focused on ascertaining the role of meprin metalloproteases in inflammatory diseases via evaluating meprin interaction with interleukin-6.

PENNSYLVANIA STATE UNIVERSITY, COLLEGE OF MEDICINE, May – August of 2003, 2004, and 2005 Summer Intern, Principal Investigator: Dr. George Makatadze.

My research was in validating a protein-optimization computer model by expressing and purifying mutants of small model proteins such as ubiquitin, acetylphosphatase, and eglin C and testing their thermodynamic stability by differential scanning calorimetry.

HONORS AND AWARDS ASBMB Graduate Students and Postdocs Travel Award, 2011.

International Proteolysis Society Graduate Student Travel Award, 2011.

PUBLICATIONS Timothy R. Keiffer and Judith S.Bond. Meprins cleave and decrease the bioactivity of Interleukin-6 (Manuscript in Preparation).

Bond JS, Keiffer TR, Sun Q (2011) Pericellular Proteolysis, In Extracellular Matrix Degradation, Biology of Extracellular Matrix (Parks WC, Mecham, RP, eds) Springer- Verlag, Berlin, 2011, pg. 75-94

Gribenko, A. V., Keiffer, T. R., et al. (2006). Amino acid substitutions affecting protein dynamics in eglin C do not affect heat capacity change upon unfolding. Proteins 64(2): 295-300.

Strickler , S.S., Gribenko, A. V., Keiffer, T. R., et al. (2006). Protein stability and surface electrostatics: a charged relationship. Biochemistry Mar 7;45(9):2761-6.