STRUCTURE AND FUNCTION OF ER CLASS 1 ALPHA MANNOSIDASE by KHANITA KARAVEG (Under the Direction of Kelley W. Moremem) ABSTRACT Mammalian class 1 α1,2-mannosidases play critical roles in the maturation of Asn-linked glycoproteins in the endoplasmic reticulum (ER) and Golgi complex as well as influencing the timing and recognition for disposal of terminally misfolded glycoproteins during ER-associated degradation. Despite several recent reports of X-ray structures of class 1 mannosidases, the proposed catalytic mechanism has not yet been experimentally investigated. As a potential target for therapeutic intervention in ER storage disorders, human ER mannosidase I was chosen to investigate the impacts of single and double mutants of three putative catalytic and two glycone binding residues. The kinetics of binding for a D463N mutant to Man9GlcNAc2 was analyzed by surface plasmon resonance indicating that this residue is mainly responsible for substrate binding, but not catalysis. The optimum pH and pKa shift observed in the E330Q/A mutants strongly indicate that E330 is the general acid catalyst. A proton inventory study gave a DIE of 1.8±0.2, but did not resolve the involvement of a second water residue previously proposed from X-ray structure studies. The presumed general base catalyst is E599 based on X-ray structure determination of a co-complex between an α1,2-mannobiose thiodisaccharide substrate analog and human ER mannosidase I resolved to 1.4 Å. The uncleaved thiodisaccharide co-complex 3 bridges the enzyme +1 and -1 subsites and reveals a unique S1 sugar ring conformation for the - 1 subsite residue. This information, in combination with prior X-ray structure data of human ER mannosidase I in a co-complex with the glycone mimic, 1-deoxymannojirimycin, suggests that 3 the class 1 mannosidases employ a novel H4 sugar conformation in the -1 subsite at the catalytic transition state. Potential roles for additional residues adjacent to the catalytic carboxyl side chains are also proposed to influence the ionization state during acid/base catalysis. INDEX WORDS: α-mannosidase, glycosylhydrolase, conformation itinerary, thiodisaccharide, crystallography, mechanism. STRUCTURE AND FUNCTION OF ER CLASS 1 ALPHA-MANNOSIDASE by KHANITA KARAVEG B.S., Chulalongkorn University, Thailand, 1995 M.S. University of Louisville, 1997 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2005 © 2005 Khanita Karaveg All Rights Reserved STRUCTURE AND FUNCTION OF ER CLASS 1 ALPHA-MANNOSIDASE by KHANITA KARAVEG Major Professor: Kelley W. Moremen Committee: Michael Pierce Bi-Cheng Wang Marcus Fechheimer William N. Lanzilotta Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May, 2005 DEDICATION To Mrs. Thanom Karaveg, my mother and Mr. Jinda Karaveg, my father, who gave me the talent and allowed me to choose my own path and have been very supportive. To my sisters, Ms. Sopid Karaveg and Mrs. Mayuree Soitong, who have been so understanding and cheerful from afar for many years. To my niece and nephew who always look up to her/his aunt. Most of all, I wishfully grant my achievement to my beloved daughter Cheyenne R. Webb. iv ACKNOWLEDGEMENTS Firstly, I would like to express my gratitude and appreciation to my major professor, Dr. Kelley W. Moremen for his support, guidance, encouragement, patience and understanding, especially for giving me the opportunity and believing in my ability during the difficult time and through out. I am also truly grateful for his kind effort to ensure the quality of my dissertation during writing and revision process. Secondly, I would like to thank my committee, Dr. Michael Pierce, Dr. Bi-Cheng Wang, Dr. Marcus Fechheimer, and Dr. William N. Lanzilotta for their great insightful direction and much enthusiasm. Thirdly, I would like to thank many colleagues who have contributed their time and effort directly and indirectly to the work presented in this dissertation. Their names and their affiliates are listed as following: Dr. Zhi-Jie Liu, Dr. Wolfram Tempel, Dr. Lirong Chen and Ms Doowon Lee, all are the members of Dr.Wang's crystallography lab; Dr. Aloysius Siriwardena, Department of Chemistry and Biochemistry,University of Mississippi; Dr. Kumar Kolli and Dr. John Glushka, members of complex carbohydrate research center, University of Georgia, and Dr. Eric Roush, Research scientist, BIAcore AB, Inc. Fourthly, I would also like to thank all the members of the Moremen lab, especially, Ms. Trisha Sheahan, Dr. Alison V. Narin, Dr. Lu Meng, and Dr. Steve Mast for their kind friendship and assistance. My acknowledgement also extends to the former members of the Moremen lab, Dr. Daniel S. Gonzalez and Dr. Anita Lal. Lastly, I would like to thank Dr. Srinivas Kidambi for his personal assistance for the past few years, especially for providing me the security and stability with love and care. iv TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.............................................................................................................v LIST OF TABLES......................................................................................................................... vi LIST OF FIGURES ..................................................................................................................... viii CHAPTER 1 INTRODUCTION .........................................................................................................1 2 MATERIALS AND METHODS.................................................................................80 3 RESULTS ..................................................................................................................103 4 DISCUSSION............................................................................................................209 REFERENCES ............................................................................................................................231 v LIST OF TABLES Page Table 1: Nomenclature for the oligosaccharide structures produced by the sugar processing enzymes of the N-linked glycoprotein maturation and biosynthesis pathway..............37 Table 2: Select ERAD substrates of medical relevance.................................................................47 Table 3: Selected glycoside hydrolase and the ligand in -1 subsite...............................................58 Table 4: Crystal structures of Class 1 mannosidases.....................................................................61 Table 5: The effect of dMNJ on the relative activity of HsERMan1 and MmGMan1A in a mixed enzyme reaction...........................................................................................................133 Table 6: Purification of recombinant HsERMan1 from P. pastoris expression culture..............141 Table 7: The inhibition of human class 1 α1,2-mannosidase. .....................................................152 Table 8: The R2 of the proton-inventory curve fits to a polynomial function. ............................157 Table 9: Summary of the mathematical models used to investigate the proton inventory effect.160 Table 10: Kinetic constants for wild type and mutant HsERManI using Man9GlcNAc2 as substrate.......................................................................................................................163 Table 11: Summary of binding kinetic parameters......................................................................169 Table 12: X-ray diffraction data collection and refinement statistics..........................................178 Table 13: Thermodynamic activation parameters for HsERManI; wild type, E330Q and T688A at 25 ºC ........................................................................................................................190 Table 14: Summary of the thermodynamic parameters for the HsERManI and Man9GlcNAc2- glycopeptide at 25 ºC from the van’t Hoff analysis in Figure 51................................197 Table 15: Theoretical activation parameters determined for Man9GlcNAc2-glycopeptide interactions with HsERManI.......................................................................................200 vi Table 16: Summary of binding analysis of HsERManI and oligosaccharide-PA interactions using E330Q mutant as a model for binding studies. ...........................................................205 Table 17: Calculation of the binding contribution of the oligosaccharide...................................206 vii LIST OF FIGURES Page Figure 1: The project outline..........................................................................................................31 Figure 2: The main pathway of N-linked oligosaccharide modification in mammalian glycoprotein biosynthesis..............................................................................................33 Figure 3: The N-linked oligosaccharide attached to nascent polypeptides and the enzymes that modify the glycan..........................................................................................................35 Figure 4: The oligosaccharide structures recognized by the ER quality control machinery and ERAD. ...........................................................................................................................38 Figure 5: Comparison of Man9GlcNAc2 and Man8GlcNAc2 isomer B trimming by mammalian ER and Golgi α1,2-mannosidases .................................................................................40