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Cleavage Specificity and Biochemical Characterization of Mast Cell Serine Proteases

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Cleavage Specificity and Biochemical Characterization of Mast Cell Serine Proteases Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 870 Cutting Edge – Cleavage Specificity and Biochemical Characterization of Mast Cell Serine Proteases BY ULRIKA KARLSON ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2003 !" # $ $ % & " ' & & (" ") !" * + '") , ) $) ' +' - ' . & " " / & 0 . ( ) 1 ) 23) ) ) 4.5 %6768%%6 4 * " " 90: * " ' " ' " " * " & ') 0 " ; & ' ' & 0 & ) !* ; ' & 0 " < " *"" ' & & *"" & & " ' ) " " / & " & * ' " 0 & ) # & " " " " ' & & * 0 " 90(:67 0(6) !" & * / ' " ' " = *"" ' & ' ) !" " 0(67 ' & * " ' & & * = 56 & " ' ) !" " "& " ' & & " " ) " ' " ' = & ' ' & 0(67 " " >?@444 " !A>B ) !" " ' " = * / " & & 0(6) 0(6 " " = " " ) 4 ' 0(6 " " * " * " / & " & ) 0(6 " " '" "& " && ' & ) !" & ' & & " " && & & * & * ' ' & ) !" 0 " 6 & & " 0) ! " = & & 0(68 0(68 * ) 1 * C 9D8): * ' " ' ' )') " * " * " ) E " " " ' * & " " 0(68 " ) 4 " " ' * " * "'" && " ) !" " ' '" ' & " ' " ) " & " ' ! " # ! #$ %&'! #"! ! ()*%+,- ! F , $ 4..5 76$$G 4.5 %6768%%6 6$% 9" HH ) )H I J 6$%: Veni, vidi, vici Julius Caesar 47 B.C. LIST OF PUBLICATIONS This thesis is based on the following articles, which are referred to in the text by their Roman numerals: I. Karlson U., Pejler G., Fröman G., and Hellman L. “Rat mast cell protease 4 is a E-chymase with unusually stringent substrate recognition profile” The Journal of Biological Chemistry. (2002) 277:18579-85 II. Karlson U., Pejler G., Tomasini-Johansson B., and Hellman L. “Extended substrate specificity of rat mast cell protease 5, a rodent D-chymase with elastase-like primary specificity” Accepted for publication in The Journal of Biological Chemistry. III. Hallgren J., Karlson U., Poorafshar M., Hellman L., and Pejler G. “Mechanism for activation of mouse mast cell tryptase: dependence on heparin and acidic pH for formation of active tetramers of mouse mast cell protease 6.” Biochemistry. (2000) 39:13068-77. IV. Hallgren J., Estrada S., Karlson U., Alving K., and Pejler G. “Heparin antagonists are potent inhibitors of mast cell tryptase.” Biochemistry. (2001) 40:7342-9. Reprints were made with permission from the publishers. CONTENTS ABBREVIATIONS........................................................................... 6 INTRODUCTION.............................................................................. 7 GENERAL OVERVIEW ................................................................. 7 MAST CELLS................................................................................. 8 Mast cell heterogeneity and granular content............................ 8 Mast cell activation .................................................................. 14 Mast cells in host defense ....................................................... 15 SERINE PROTEASES................................................................. 17 Catalytic mechanism ............................................................... 18 Substrate binding and specificity of chymotrypsin-like serine proteases...................................................................... 18 Mast cell serine proteases....................................................... 20 What is left to learn about mast cell serine proteases? ........... 28 SUMMARY OF PRESENT STUDIES......................................... 30 AIM............................................................................................... 30 RESULTS AND DISCUSSION .................................................... 30 Paper I..................................................................................... 30 Paper II.................................................................................... 32 Paper III................................................................................... 34 Paper IV................................................................................... 35 CONCLUDING REMARKS .......................................................... 36 ACKNOWLEDGEMENT............................................................... 40 REFERENCES............................................................................... 42 ABBREVIATIONS aa amino acid(s) Ang angiotensin BMMC bone marrow-derived mast cells CPA carboxypeptidase A CS chondroitin sulfate CTMC connective tissue mast cells DFP diisopropyl fluorophosphate HC human chymase Ig immunoglobulin IL interleukin LPS lipopolysaccharide LT leukotriene MC mast cell MCs mast cells MMC mucosal mast cells mMCP mouse mast cell protease MMP matrix metallo-protease PG prostaglandin rMCP rat mast cell protease TGF-E transforming growth factor-E TLR Toll-like receptor TNF-D tumor necrosis factor-D INTRODUCTION GENERAL OVERVIEW Every day we encounter bacteria, viruses and other potentially harmful microbes. The physical barriers, such as the skin and mucosal surfaces, prevent the bacteria from entering the body. However, microbes sometimes penetrate the physical barriers and then our immune system is activated. During an immune response, a battery of cells and effector molecules act to remove the invading microbes by various mechanisms. These mechanisms induce localized inflammatory responses that eliminate the infectious agent with minimal tissue damage to the host. Inflammation involves recruitment of immune cells, such as neutrophils, monocytes, lymphocytes and basophils to the tissue. The resident mast cells are thought to play an important role in the initiation and augmentation of the inflammatory process. Under certain conditions, the immune system can initiate an inappropriate response to a specific antigen, either by responding too vigorously, or by activating inappropriate cells. Allergy is an example of such misdirected actions of the immune system. The antigens in allergic reactions are called allergens. Examples of allergens are proteins from house dust mites, pollen, mold spores, and also fur, saliva and urine derived from different animals. The allergen induces an allergic response by activating mast cells, which react by releasing their pharmacologically potent mediators. The mediators are responsible for the symptoms seen in allergic individuals. Although the symptoms are most often not lethal, they do influence the quality of life of the affected person with manifestations such as sneezing, increased mucus production in the nose and runny eyes. Rodents can be used to investigate aspects of the mast cells and their role in allergy, inflammation and host defense. In the present study we have focused on rodent mast cell serine proteases, which are the major protein constituent of the mast cell granules. Serine proteases are enzymes that are released upon activation of the mast cell and due to their high abundance they are thought to have a central role in mast cell function. Much effort has also been devoted to studies of the biological targets of these proteases. However, there are still many important questions to be answered, and the 7 purpose of this thesis is to shed some light on the biochemical characteristics of these enzymes to increase the understanding of their function in mast cell biology. MAST CELLS Mast cells (MCs) originate from hematopoietic stem cells located in the bone marrow. They circulate in the blood as precursors before homing to the tissue where they finally complete their maturation. Mature MCs are found scattered in the skin and mucosal layers, where they are dormant until activated by environmental stimuli. A general feature of MCs is the staining of their cytoplasmic granules with basic dyes. The acidic granules are storage vesicles for biologically active substances such as histamine, proteoglycans, cytokines and proteases. When the MC is activated, these stored mediators are released by a process called degranulation (Figure 1). Upon activation, the MC also initiates a de novo production of lipid mediators, e.g. leukotrienes and prostaglandins, as well as a cascade of cytokines, that are released by exocytosis minutes to hours after degranulation. The biological effects of these substances are vascular leakage, bronchoconstriction or intestinal hypermotility, increased mucus production and inflammation. These effects may be seen during allergic reactions, in which MCs are the main effector cells. Any allergic response must start with a sensitization phase, where the allergen induces B-lymphocytes to produce allergen-specific immunoglobulin E (IgE). IgE is secreted and binds with high affinity to FcHRI, located on the MC surface. Upon a second encounter with the allergen, the FcHRI-bound IgE molecules bind the allergen, and the MC starts an effector phase by releasing its mediators. In allergic asthma, the mediator release from MCs causes bronchoconstriction as well as mucus production and edema, which contribute to bronchial obstruction. Mast cell heterogeneity and granular content Both rodent and human MCs display a high degree of heterogeneity, revealed most clearly by the content of their granules and their tissue location. Two main subtypes have been
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