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Mouse Mast Cell Proteases: Induction, Molecular Cloning, and Characterization Wei Chu East Tennessee State University

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Mouse Mast Cell Proteases: Induction, Molecular Cloning, and Characterization Wei Chu East Tennessee State University East Tennessee State University Digital Commons @ East Tennessee State University Electronic Theses and Dissertations Student Works May 1991 Mouse Mast Cell Proteases: Induction, Molecular Cloning, and Characterization Wei Chu East Tennessee State University Follow this and additional works at: https://dc.etsu.edu/etd Part of the Biochemistry Commons, Cell Biology Commons, and the Molecular Biology Commons Recommended Citation Chu, Wei, "Mouse Mast Cell Proteases: Induction, Molecular Cloning, and Characterization" (1991). Electronic Theses and Dissertations. Paper 2656. https://dc.etsu.edu/etd/2656 This Dissertation - Open Access is brought to you for free and open access by the Student Works at Digital Commons @ East Tennessee State University. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Digital Commons @ East Tennessee State University. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, white others may be from any type of computer printer. The quality of this reproduction Is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely afreet reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material bad to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor. Ml <18106-1346 USA 313/761-4700 800/521-0600 Order Number 0124275 Mouse mast cell proteases: Induction, molecular cloning, and characterization Chu, Wei, Ph.D. East Tennessee State University, 1091 UMI 300 N. Zccb Rd. Ann Aibor, MI 48106 MOUSE MAST CELL PROTEASES: INDUCTION, MOLECULAR CLONING, AND CHARACTERIZATION A Dissertation Presented to the Faculty of the Department of Biochemistry James H. Quillen College of Medicine East Tennessee State University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in Biomedical Sciences by Wei Chu May, 1991 APPROVAL This is to certify that the Graduate Committee of WET CHU met on the thirteenth day of November. 1990 The committee read and examined his dissertation, supervised his defense of it in an oral examination, and decided to recommend that his study be submitted to the Associate Vice-President for Research and Dean of the Graduate School, in partial fulfillment of the requirements for the degree Doctor of Philosophy in Biomedical Science. /S r l c Signed on behalf of the Graduate Council ii ABSTRACT HOUSE MAST CELL PROTEASES: INDUCTION, MOLECULAR CLONING, AND CHARACTERIZATION By Wei Chu Tryptase, a mast cell-specific serine protease with trypsin-like specificity, has been identified in a mouse mast cell line (ABFTL-6) based on it's enzymatic activity, inhibition properties, and cross-reactivity to a human mast cell tryptase antibody. The effects of fibroblast-conditioned medium and sodium butyrate on ABFTL-6 mast cell differentiation and tryptase expression have been examined. ABFTL-6 mouse mast cells undergo phenotypic changes upon culturing in media supplemented with fibroblast-conditioned media at 50% or 1 mM sodium butyrate. The induced cells increased in size, had larger and more metachromatic cytoplasmic granules, and increased their total cellular protein about four-fold. Tryptase activity increased 13- and 6-fold upon fibroblast- conditioned media and butyrate induction, respectively. However, tryptase antigen levels increased dramatically from 2.3 jxg/106 uninduced cells to 125 (54-fold) and 75 (33-fold) /ig/10* cells induced with fibroblast-conditioned media or butyrate, respectively. A cDNA library was constructed in JLgtlO from ABFTL-6 cell poly (A)+ RNA, and screened with dog mast cell tryptase and rat mast cell chymase cDNAs. Clones encoding two distinct tryptases (mouse tryptases I and II), a chymase (mouse chymase I) and a novel carboxyl terminal chymase (mouse chymase II) were isolated and sequenced. Mouse tryptases I and II have 75% and 70% sequence identity at the nucleotide and amino acid levels, respectively. The deduced amino acid sequence for the mature active enzyme for each mouse tryptase contains 245 residues and all the characteristics of a serine protease. Asp is found in the substrate binding pockets, consistent with a trypsin-like specificity for Arg-X and Lys-X bonds. It is predicted that tryptases are synthesized with prepropeptides, requiring signal peptidase processing and removal of a three amino acid propeptide for activation. Mouse chymase I consists of a 226 amino acid catalytic portion and a 21 amino acid preprosequence. An Asn occurs in the substrate binding pocket, a feature that has not been observed in any other serine protease. iii ACKNOWLEDGMENTS First of all, I wish to express my appreciation to the faculty and staff members of the Biochemistry Department for their support, assistance, and friendship. I would like to thank my thesis committee mombors, Drs. David Chi, Margaret Kougland, David Johnson, and Mitaholl Robinson for their time and valuable advice. My most sincere appreciation goes to my major advisor, Dr. Phillip Musich, who provided mo with the ancouragomont and guidance throughout my graduate years. I also would like to thank those graduate atudants who made my graduate years more memorable and those individuals who have cared and contributed to the completion of this study. Special thanks goes to Mrs. Raymondo Cox for hor final touch of this manuscript. iv CONTENTS Page APPROVAL ......................................... ii ABSTRACT ............................ * ........... iii ACKNOWLEDGEMENTS ................................... iv LIST OF TABLES .................................... ix LIST OF FIGURES .................................. X ABBREVIATIONS ..................................... xii CHAPTER 1. INTRODUCTION ................................... 1 Mast Cell Heterogeneity, Differentiation and Origin ................................. 5 Micro-environmental Regulation of Mast Cell Development and Phenotype ................... 10 Mast Cell Granule-Associated Serine Proteases: Tryptase and Chymase ............ 12 2. MATERIALS AND METHODS ........................ 23 Materials ..................................... 23 M e t h o d s .......................................... 25 Growth of ABFTL-6 Mast Cells and 3T3 Cells ..................................... 25 Preparation of f c m . , ..................... 25 Growth of Mast Cells 3T3 FCM Supplemented Media ....................... 26 Sodium Butyrate Treatment of Mast Cells . 26 Histochemical Staining ..................... 26 Preparation of Mast Cell Extracts .......... 27 Determination of Protein Concentration . 27 v Chapter Page Measurement of Enzymatic Activity .......... 28 Inhibition of Tryptic Activity Assay .... 28 SDS-Polyacrylamide Gel Electrophoresis . 29 t3H] DFP Labeling of Serine Froteases and Fluorography ............................. 30 Immuno-dot and Western Blotting ............ 30 Poly (A)+—RNA I s o l a t i o n ....................... 31 cDNA Synthesis and Library Construction . 34 cDNA Library Screening ..................... 36 Alcohol Precipitation of DNA .............. 37 DNA Isolation ............................... 38 Lambda DNA isolation ..................... 38 Plasmid DNA minipred ..................... 39 Agarose Gel Electrophoresis DNA ............ 40 DNA Restriction Enzyme Digestion .......... 41 Electroelution of DNA Fragments ............ 41 Ligations ................................... 42 Bacterial Transformation ................... 42 DNA Sequencing ............................. 43 Denaturation of double-stranded DNA .... 43 DNA dideoxynucleotide sequencing reactions ............................. 44 DNA sequencing gel electrophoresis .... 44 * Hybridization of Filter-Bonded DNA ........ 45 Radioisotopic labeling of DNA probes . 45 Slot-blotting of nucleic acids .......... 4 6 vi Chapter Page Southern and northern blotting ........ 47 Stripping probe from filters .......... 48 Autoradiography ......................... 48 3. RESULTS ..................................... 50 Morphological Changes of Mouse Mast Cells Induced with FCM and Sodium Butyrate ................................. 50 Effects of FCM and Sodium Butyrate Culture on Mast Cell Tryptase Activity and Antigen Levels ....................... 50 Both FCM and Sodium Butyrate Increased Total DFP-Binding Proteins ............... 61 Molecular Cloning of Mouse Mast Cell' Tryptases ................................. 63 Molecular Cloning of Mouse Mast Cell Chymase ..................................
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