THE EXTRACELLULAR PROCESSING OF AGGRECAN AGGREGATE AND ITS EFFECT ON CD44 MEDIATED INTERNALIZATION OF HYALURONAN by Ben Danielson April, 2015 Director of Dissertation: Dr. Warren Knudson Department: Anatomy and Cell Biology In many cells the hyaluronan receptor CD44 mediates the endocytosis of hyaluronan and its delivery to endosomes / lysosomes. The regulation of this process remains largely unknown. In most extracellular matrices hyaluronan is not present as a free polysaccharide but often in complex with other small proteins and macromolecules such as proteoglycans. This is especially true in cartilage where hyaluronan assembles into an aggregate structure with the large proteoglycan termed aggrecan. In this study, when purified aggrecan was added to FITC- conjugated hyaluronan, no internalization of hyaluronan was detected. This suggested that the overall size of the aggregate prevented hyaluronan endocytosis and furthermore that proteolysis of the aggrecan was a required prerequisite for local, cell-based turnover of hyaluronan. To test this hypothesis, limited C-terminal digestion of aggrecan was performed to determine if a size range of aggrecan exists that was permissive of hyaluronan endocytosis. The data demonstrate that only limited degradation of the aggrecan monomer was required to allow for hyaluronan internalization. When hyaluronan was combined with partially degraded, dansyl chloride- labeled aggrecan, blue fluorescent aggrecan was also visualized within intracellular vesicles. It was also determined that sonicated hyaluronan that is of smaller molecular size was internalized more readily than high molecular mass hyaluronan. However, the addition of intact aggrecan to 5 and 10 second sonicated hyaluronan chains re-blocked their endocytosis while aggregates containing 15 second sonicated hyaluronan were internalized. These data suggest that hyaluronan endocytosis is regulated in large part by the extracellular proteolytic processing of hyaluronan-bound proteoglycan. THE EXTRACELLULAR PROCESSING OF AGGRECAN AGGREGATE AND ITS EFFECT ON CD44 MEDIATED INTERNALIZATION OF HYALURONAN A Dissertation Presented To the Faculty of the Department of Anatomy and Cell Biology East Carolina University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in Anatomy and Cell Biology by Ben Danielson April, 2015 © Ben Danielson, 2015 THE EXTRACELLULAR PROCESSING OF AGGRECAN AGGREGATE AND ITS EFFECT ON CD44 MEDIATED INTERNALIZATION OF HYALURONAN by Ben Danielson APPROVED BY: DIRECTOR OF DISSERTATION: _______________________________________________________________ Warren Knudson, Ph.D. COMMITTEE MEMBER: ________________________________________________________ Cheryl B. Knudson, Ph.D. COMMITTEE MEMBER: _______________________________________________________ Ann O. Sperry, Ph.D. COMMITTEE MEMBER: Maria J. Ruiz-Echevarria, Ph.D. CHAIR OF THE DEPARTMENT OF ANATOMY AND CELL BIOLOGY: ____________________________________________ Cheryl B. Knudson, Ph.D. DEAN OF THE GRADUATE SCHOOL: _________________________________________________________ Paul J. Gemperline, PhD ACKNOWLEDGEMENTS First and foremost, I would like to thank my wonderful family for always being there to support, encourage, and motivate me. My parents, Brent and Donna Danielson, have always been a source of comfort and motivation throughout my life, I would not be where I am today without them. I have learned what it means to work hard, and achieve my goals by following their example. My brother Brent and sister Ilsa have done more than they realize (for better or worse) to shape me into the person I am today. I am incredibly grateful to be part of a family that has only grown closer over time and will always make themselves available to support and encourage me throughout my life. I would also like to thank my mentor, Dr. Warren Knudson and co-mentor Dr. Cheryl Knudson, for welcoming me into their lab. Their support, guidance, and patience have been a model for what a mentor should be and I will be forever grateful for my experience with them. I hope to carry their enthusiasm and dedication with me throughout the rest of my professional and personal life. I would also like to thank my committee members Dr. Ann Sperry and Dr. Maria Ruiz-Echevarria for providing valuable and much needed help throughout my graduate studies. I would like to express my sincerest gratitude to Dr. Emily Askew for always making time to lend a helping hand and being a voice of hope and comfort throughout the ups and downs of the whole graduate student experience. I would also like to acknowledge my fellow lab members: Dr. Shinya Ishizuka, Dr. Naoko Ishizuka, Joani Zary-Oswald, Michelle Cobb, Elaine Huang, and Samantha Sellers for all the help and assistance they have given me over the years. I would also like to acknowledge Dr. Joseph Chalovich and Dr. Doug Weidner for their training and expertise. TABLE OF CONTENTS LIST OF FIGURES ............................................................................................................ viii LIST OF ABBREVIATIONS .............................................................................................. x SPECIFIC AIMS ….. ............................................................................................................ xiii CHAPTER 1: BACKGROUND AND SIGNIFICANCE ................................................. 1 1. Osteoarthritis ......................................................................................................... 1 2. Articular cartilage ................................................................................................. 3 2.1. The cellular component of articular cartilage ........................................ 4 2.2. The extracellular matrix of articular cartilage ....................................... 5 3. Aggrecan… ........................................................................................................... 6 3.1. Aggrecan synthesis ................................................................................ 6 3.2. Aggrecan turnover ................................................................................. 8 4. Hyaluronan ............................................................................................................ 9 4.1. Hyaluronan synthesis ............................................................................. 9 4.2. Hyaluronan turnover .............................................................................. 11 4.3. Neutral pH hyaluronidase activity ......................................................... 14 5. CD44…….. ........................................................................................................... 16 6. Summary ............................................................................................................ 18 CHAPTER 2: MATERIALS AND METHODS .............................................................. 32 1. Purifying aggrecan monomer ................................................................................ 32 1.1. Cesium chloride gradient ultracentrifugation ........................................ 32 1.2. DEAE ion exchange chromatography ................................................... 32 1.3. HA affinity chromatography .................................................................. 33 2. Visualizing HA and aggrecan ............................................................................... 34 2.1. Preparation of fluorescein conjugated HA ............................................. 34 2.2. Preparation of biotin-HA ....................................................................... 35 2.3. Preparation of dansylated proteoglycan ................................................. 36 2.4. Agarose gel electrophoresis ................................................................... 36 2.5. DMMB assay ......................................................................................... 37 3. Cell culture ............................................................................................................ 37 3.1. Rat chondrosarcoma cell culture ............................................................ 37 3.2. Hyaluronidase treatment to remove pericellular matrix ........................ 38 3.3. Chloroquine efficacy assay .................................................................... 38 4. Particle exclusion assay ........................................................................................ 39 5. Visualizing FITC-HA binding and internalization ............................................... 39 5.1. Fluorescent microscopy ......................................................................... 39 5.2. Flow cytometry ...................................................................................... 40 5.3. BCA assay for protein concentration determination .............................. 41 5.4. Western blots ......................................................................................... 41 5.5. Morphometric analysis of fluorescent microscopy images ................... 42 5.6. Statistical analysis .................................................................................. 42 6. Producing HA and proteoglycan of decreasing size ............................................. 43 6.1. Sonication of HA ................................................................................... 43 6.2. Clostripain digestion of aggrecan .......................................................... 43 7. Co-immunoprecipitation