A Study of the IgM Interaction with Complement Using Mouse IgMlIgG2b Domain-switched Hybrids Frieda Huey Chen A thesis subrnitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Biochemistry University of Toronto O Copyright by Frieda Huey Chen 1998 National Library Bibliothèque nationale l*I of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Canada The author has granted a non- L'auteur a accorde une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan., distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la foxme de rnicrofiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. The further one pursues knowledge, the less one knows. - Lao Tzu Dedicated to those who persevere in spite of it all. ABSTRACT A study of the IgM interaction with complement using mouse IgM/IgGZb domain-switched hybrids Degree of Doctor of Philosophy, 1998. Frieda Huey Chen Graduate Department of Biochemistry, University of Toronto The IgM and IgG classes of immunoglobulins share an important bioiogical function that is distinct fiom that of other classes. When bound to antigen, both can initiate the classical complement pathway by interacting with Clq, a subunit of the fmt component of cornplement (Cl). To study the Clq binding properties of IgM in a form comparable to that of IgG, a 'monomeric' form of mouse IgM was prepared and its function was compared to that of a mouse IgG with the same antigen binding site. Since most of the wildtype IgM is secreted as pentarnen, monomenc IgM was isolated from a mutant (IgM P544G) which is secreted predominantly as monomers. The monomeric form of this mutant was not able to bind Clq or initiate cornpiement-mediated lysis (CML) on haptenated SRBC. Under the same conditions, the positive control (IgG2b) bound Clq and initiated CML in a dose-dependent manner. Because polymeric IgM isolated from diis mutant was able to bind Clq and initiate CML as well as wildtype IgM did, it is unlikely that the P544G mutation is directiy responsible for the inactivity of the mutant monomer. To understand why the monomeric IgM is inactive, mouse IgMlIgG2b hybrid mutants were created and analyzed for their Clq binding properties. Cfl, the intrinsically active C lq binding domain of IgG2b, did not bind C lq or initiate CML when placed in the monomeric IgM background (ppp). This suggests that the activity of an intrinsically active C lq binding domain is hidden by one or both of the neighbouring Cp2 and Cp4 domains. Cp3, the putative Clq binding domain of IgM, rernained inactive when it was placed in the non-inhibitory IgGZb background (yypy). This demonstrates that a pre- formed Clq binding site is not found in the Cp3 domain. Collectively, the results suggest that if the Cp3 contains the entire Clq binding site for IgM, the site is unformed and hidden. To detennine if one or both of the Cp2 and Cp4 domains are responsible for the inactivity of the ppyp hybrid rnonomer two hybrids (pmand lpyy) were created in which these domains were individudy replaced by their corresponding IgG2b dornains (y-hinge and Cy3). Both hybrid monomers were defective in initiating CML whereas only the pyyp hybrid, which has Cp4, was defective in Clq binding. The results show that Cp4 interferes with the interaction between C 1 and the Cy2 domain whereas Cp2 interferes with the subsequent activation of CL Cp2 and Cp4 may therefore have similar inhibitory effects on the Cpdomain in the IgM monomer. The transplantation of Cp3 together with Ck4 hto the IgG background permitted polymer formation. This polymer was able to bind Clq, although both the monomer and the polymer forms were not able to initiate CML;converseiy, all IgM polymers with a transplanted Cy2 domain were active in both Clq binding and CML and demonstrated apparent Kd values for Clq (1-2 x 10-~M) that were similar to that of wild-type IgM. This suggests that the affinity of the C lq binding sites in IgM are comparable to those in IgG2b. The findings reported in this thesis are discussed in relation to the activity of the IgM polymer and are consistent with the view that the 'star' to 'staple' conformational change resulting when IgM binds to antigen is necessary for the expression of the C lq binding site. iii Acknowledgments But there are two expensive forrns of education, either of which a parent rnay procure for his son by sending hirn as solitary pupil to a clergyman: one is, the enjoyment of the reverend gentleman's undivided neglect; the other is, the endurance of the reverend gentleman's undivided attention. -George Eliot frorn The Mill on the Floss Having had the fortune to be a pupil of not one but three supervisors, I am happy to Say that I've enjoyed both their 'undivided attention' in the form of their guidance and support during the critical times, and their 'undivided neglect' in the form of the great freedom with which I was permitted to conduct my studies. Each member of this supervisory team or 'Triumvirate' - Dr. Robert Painter, Dr. David lsenman and Dr. Marc Shulman - has been a vital source of encouragement and direction for which 1 am greatly indebted. Over the years I've grown to appreciate the unique strengths of each. Dr. Painter has never ceased to impress me with his ability 'to see the forest from the trees', a talent that no doubt stems frorn his broad knowledge base - he is as versed in science as he is in Shakespeare. Dr. lsenman has always been tremendously generous with his time and technical expertise, and his tireless enthusiasm as a teacher is infectious. Dr. Shulman has contributed his thoroughness and ability to present alternative interpretations, traits that have made him valuable in maintaining the balance within the triumvirate. In addition to my supervisors. Dr. David Pulleyblank has also made helpful suggestions at my commîttee meetings and during the writing of this thesis. To Say that this work is a result of my efforts alone would be a gross distortion of the truth. Looking back on the whole process it is with humility that I recognize the contributions, both direct and indirect, of so many others and it is to thern that 1 wish to extend my thanks (hopefully without omissions). In the lab which became my home away from home, rny Iabmates becarne my surrogate family. Dr. Sudha Arya, who rnany of us refer to affectionately as 'mom', provided much in the way of emotional support. Her srnile and trademark laughter marked many mernorable days in the lab. Several fellow grad students who became my 'siblings' during this stage of my life and with whom 1 shared many experiences, successes, embarassments, arguments and at times miseries are Roger Ebanks, Nana Lee and John Chan. I thank Roger for sharing his philosophies, Nana for spinning her fairytales and John for being a free-spirit. Thanks go to Myrna Cohen-Doyle for her fabulous cottage parties and for sharing 'His Majesty' with me. I also owe thanks to the following for their friendship (in stream-of-consciousness order): Aiko Taniguchi-Sidle, Amir Khan, Fernando Rock, Maggie Everett, Yuan-Yuan Xu. Ranga Robinson, Navneet Aluwalia, Davinder Chawla, Shelley Hepworth and Owen Rowland. Shirley Furesz, Bhushan Nagar, Alp Oran, Erik Wiersma, Pat Bronskill, Augustin Nguyen, and members of the Dr. Isenman's, Dr. William's, Dr. PulleyblanKs, Dr. Klein's, Dr. Segall's and Dr. Bennick's labs past and present. I would also like to acknowledge the indispensable services provided by the staff in the departmental office - Anna, Hazel, Suzanne, Carol and Farhia and - and by the fifth floor caretaking staff. I must also thank several fnends outside of the gray building known as 'Med Scia for providing me with (too) many pleasant distractions. 1 will always remember jamming till the wee hours of the morning on some broken down piano with Wendy Lau, camping with Sue Choi, and Lucky Palace discussions with Deborah Villeneuve. Thanks to Nancy Ryu and Joseph Nachman for our musical partnerships. Thanks to Alma V. and Sing Hoo Yuen at Fudger House for their words of encouragement ('Why not? You should try!') and for being an 'appreciative audience'. Thanks to Nadia Saracoglu for her calming influence and for her 'spa' night hamrnock. I owe my parents many thanks for their care and concern throughout the years and for enduring my decision to pursue a doctoral degree. Finally 1 would like to express my deepest gratitude to two people who suffered the most at the hands of my true personality: Ann, who has been more friend than younger sister in a11 our years together and whose unwavering belief in me has helped me through the darker moments of self- doubt; and Steve, who has been supportive, patient. and above al1 understanding. This work was supported by the Medical Research Council of Canada. The author also acknowledges the support provided by a Govemment of Ontario Graduate Scholarship.