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Structural and Functional Studies of the Plant Lectin Peanut Agglutinin

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Structural and Functional Studies of the Plant Lectin Peanut Agglutinin Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 1989 Structural and Functional Studies of the Plant Lectin Peanut Agglutinin Eugene J. Zaluzec Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Chemistry Commons Recommended Citation Zaluzec, Eugene J., "Structural and Functional Studies of the Plant Lectin Peanut Agglutinin" (1989). Dissertations. 2882. https://ecommons.luc.edu/luc_diss/2882 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 1989 Eugene J. Zaluzec ( STRUCTURAL AND FUNCTIONAL STUDIES OF THE PLANT LECTIN PEANUT AGGLUTININ by Eugene J. Zaluzec A Dissertation Submitted to the Faculty of the Graduate School of Loyola University of Chicago in Partial Fulfillment the of the Requirements for the Degree of Doctor of Philosophy September 1989 ACKNOWLEDGMENTS I would like to express my sincere gratitude to Drs. Stephen F. Pavkovic and Kenneth W. Olsen for the training, support, and guidance they provided throughout this thesis project. I also extend my appreciation to Drs. Duarte Mota De Freitas, Albert W. Herlinger, and Mir Shamsuddin for serving in my thesis committee. I am exceedingly thankful to Dr. Mir Shamsuddin, Ghaus Ansari, and Children's Memorial Hospital (Chicago) for their assistance in the amino acid analysis of PNA, Dr. Larry Fox, Salley Dorwin and Abbott Laboratories (Abbott Park, 11.) for the help they provided in the amino acid sequencing of PNA, Dr. Paula M. Fitzgerald (Merck Sharp and Dohme Research Laboratories) for supplying the MERLOT computer programs for the determination of crystal structures by molecular replacement, Dr. Fred L. Suddath (Georgia Institute of Technology) for supplying the pea lectin atomic coordinates, Dr. David Ollis (Northwestern University) for supplying the PROTEIN computer programs for the determination of crystal structures and his help in data analysis, Dr. Bruno Jaselskis (Loyola University of ii Chicago) for the use of his HPLC in the manual sequence analysis of PNA, Drs. Leslie W. M. Fung and Duarte Mota De Freitas (Loyola University of Chicago) for the use of their laboratories and equipment in various aspects of this thesis project, and Marianne Yung (Loyola University of Chicago) for providing crystals of PNA-BAP complex and her help in the PNA binding assays. I would finally like to thankfully acknowledge Loyola University of Chicago for providing financial support, and the faculty, staff1 and students of the chemistry department for making my graduate education an enlightening experience. iii VITA The author, Eugene J. Zaluzec, is the fourth son of John and Nettie Zaluzec. He was born March 25, 1956, in Chicago, Illinois. He received his primary education at Oliver H. Perry Public School in Chicago. His secondary education was from Mendel Catholic High School in Chicago. In August, 1973, Mr. Zaluzec entered Thorton Community College in South Holland, Illinois, from where he received the degree of Associate of Applied Science in electronics technology. From January, 1976, to January, 1981, Mr. Zaluzec was on active duty in the United States Air Force with a primary specialty of airborne avionic communications specialist attaining the rank of E-4 sergeant. In August, 1981, he entered Southern Illinois University, receiving the degree of Bachelor of Science in chemistry in May, 1984. In August, 1984, Mr. Zaluzec was admitted as a Ph.D. candidate to the Chemistry Department of Loyola University of Chicago. This thesis is being submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy. iv TABLE OF CONTENTS Page ACKNOWLEDGMENTS. ii VITA ................. .... iv TABLE OF CONTENTS .. ............. v LIST OF TABLES ..... .. x LIST OF ILLUSTRATIONS .................. .xiii ABBREVIATIONS OF VARIABLE NAMES ......................... xix Chapter Page I. INTRODUCTION ...................................... 1 A. LECTINS ...................................... 1 Historical Background ............ 1 Occurrence and Distribution .. 3 Molecular Properties ......................... 4 Biological Functions ............ ..... 6 Applications and Utilizations ... ..... 8 V B. LEGUMINOUS LECTINS .......................... 11 Primary Structure Homology .................. 11 Species Heterogeneity ....................... 17 Metal Binding Sites ......................... 23 Hydrophobic Binding Sites ................... 28 Glycosylation Site .......................... 33 Three Dimensional Structures ................ 35 C. PEANUT AGGLUTININ ........................... 37 Species Heterogeneity .. •..................... 37 Carbohydrate Specificity .. .... 3 8 Macromolecular Properties. ..... 40 Primary Structure .............. ...... 41 Crystallographic Studies ....... ...... 41 Biological Functions ........................ 44 I I. STATEMENT OF RESEARCH ............................ 45 I I I. MATERIALS AND METHODS ............................ 48 A. MATERIALS ................................... 48 B. PREPARATION OF PEANUT AGGLUTININ ............ 50 Saline Fractionation......... .. 50 Affinity Chromatography...... .. 51 1. Preparation of Lactosyl-Sepharose 6B ... 51 2. Isolation of Peanut Agglutinin ......... 52 vi Gel Electrophoresis ......................... 52 1. Dissociating (SDS) Gels ................ 53 2. Dissociating (Urea) Gels ............... 54 3. Non-dissociating Gels .................. 55 c. AMiNO ACID COMPOSITION ... .56 Acid Hydrolysis .......... .56 Performic Acid Oxidation (Cystine) .......... 57 N-Bromosuccinimide Assay (Tryptophan) ....... 57 Amino Acid Analysis .... ' ..................... 58 D. AMINO ACID SEQUENCING ....................... 58 Chemical Digestion ............ ... 58 1. Cyanogen Bromide Cleavage. ... 58 2. CNBr Peptide Isolation ... .60 3. CNBr Peptide Composition. .60 Enzymatic Digestion ......................... 60 1. Trypsin Cleavage ............ .... 60 2. Tryptic Peptide Isolation ... ... 61 3. Tryptic Peptide Composition. .62 Manual Sequence Analysis ......... .62 1. Edman Degradation ...................... 62 2. Phenylthiohydantoin Chromatography ..... 64 Automated Sequence Analysis ................. 64 E. BINDING ASSAYS .............................. 65 vii Carbohydrate Binding ........................ 6 5 1. Preparation of N-Dansyl Galactosamine .. 65 2. Fluorometric Titrations ................ 66 Hydrophobic Binding..................... .67 1. Fluorometric Titrations .... .67 Gel Filtration .................. .69 F. CRYSTALLOGRAPHY .................. .69 Crystallization of Peanut Agglutinin ........ 69 1. Peanut Agglutinin/Lactose .............. 69 2. Peanut Agglutinin/~6 -Benzylaminopurine.70 Heavy Metal Derivatization ......... 70 1. Crystal Soaking Derivatization. .70 2. Co-crystallization ....... .71 X-Ray Diffraction ............. .71 1. Crystal Mounting ....................... 71 2. Data Collection ........................ 72 IV. RESULTS ......................................... 74 A. ISOLATION OF PEANUT AGGLUTININ .............. 74 B. THE PRIMARY STRUCTURE OF PEANUT AGGLUTININ .. 81 Amino Acid Composition ...................... 81 Cyanogen Bromide Fractionation .. ... 88 Trypsin Fractionation .......... .. 9 3 viii Sequence Analysis ........................... 95 C. THE BINDING OF LIGANDS TO PEANUT AGGLUTININ.116 Preparation of N-Dansylgalactosamine .. 116 Binding of Ligands to PNA ............. .120 1. Binding of DnsGalN and Lactose . .124 2. Binding of TNS and BAP. .128 3. Binding of ANS. .143 4. Effects of BAP on DnsGalN Binding. .143 5. Determination of Ligand Stoichiometry .. 144 D. CRYSTALLOGRAPHY OF PEANUT AGGLUTININ ........ 146 Crystallization of PNA-Lactose ... 146 Crystallization of PNA-BAP ....... 148 Heavy Atom Derivatization of PNA. .148 Diffraction Data Analysis ....... .150 Heavy Atom Derivative Assays ................ 158 V. CONCLUSIONS ...................................... 162 Primary Structure of PNA .... /. .163 Hydrophobic Binding of PNA .. .166 Crystallography of PNA ................. 17 5 REFERENCES .............................................. 17 9 APPENDIX ............................................... 189 ix LIST OF TABLES Table Page I. 1. Blood Type-Specific Lectins ................... 9 I. 2. Characteristics of Legume Lectins ............ 13 I . 3 . Metal Binding Regions of Legume Lectins ...... 27 1.4. Carbohydrate Binding Residues of Legume Lectins Determined by X-ray Diffraction ..... 29 I . 5 . Homology of Legume Lectins to the Subunit Hydrophobic Binding Region of Con A ......... 31 I . 6 . Glycosylation Sites of Legume Lectins ....... 34 I . 7 . Inhibition of Hemagglutinating Activity of Peanut Agglutinin ................ .39 X I. 8. Amino Acid Composition Reported for Peanut Agglutinin .. .... 42 JV.l. Composition of Amino Acids Found in Peanut Agglutinin .. .... 84 IV.2. Amino Acid Composition of Cyanogen Bromide Peptides of Peanut Agglutinin ................ 92 IV.3. Amino Acid Composition of Tryptic Peptides Peanut Agglutinin ....... ...... 9 6 IV.4. Composition of PNA Tryptic Peptides Compared to Non-Digested PNA ........ ...... 96 IV.5. Sequence Analysis of PNA Tryptic Peptides .... 98 IV.6. Total Composition of Segments Not Sequenced in the Primary
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