Citation
Hardy, Richard Randolph (1981) Studies of the Specificities and Function of Immunoglobulins. I. Relationship Between Structures and Specificity in Dinitrophenyl-Binding Mouse Myeloma Immunoglobulins. II. Effector Function Triggering in Immunoglobulins. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/wcn5-3w02. https://resolver.caltech.edu/CaltechTHESIS:05082018-102024886
Abstract
Chapter 1:
This chapter presents the background to part I (chapters one through six) in which the structure of the antibody binding site and the specific interactions that occur between the site and antigen are considered.
Chapter 2:
The relation between structure and specificity of antibodies has been explored by 19F NMR studies of the binding of trifluoromethyl analogues of nitrophenyl haptens to the three mouse myeloma immunoglobulins M315, M460, and X25. Haptens were used with trifluoromethyl groups located at the ortho or para positions of the phenyl ring or attached to the side chain, two atoms removed from the ring (i.e., -NHCH2CF3). The changes in chemical shift between hapten free in solution and bound to antibody are sensitive to microenvironment and range from 1.7-ppm downfield to 1-ppm upfield. The shifts of p-trifluoromethylnitrophenyl haptens bound to M315 and M460 are both large downfield shifts, which are likely caused by van der Waals interaction and ring-current effects, particularly from tyrosine-34 (L); these haptens do not show similar shifts when bound to X25 which has a deletion of tyrosine-34 (L). Other differences in the binding of the aromatic rings of haptens by M315, M460, and X25 are observed and their origins considered. The importance of hydrogen bonding in the thermodynamic affinity of antibody for hapten has been estimated by comparisons of binding affinities for haptens with trifluoromethyl groups in place of nitro groups.
Chapter 3:
A protein modification reagent, tetranitromethane, has been employed to specifically nitrate a binding site tyrosine residue in three nitrophenyl-specific myeloma proteins. The nitration does not significantly alter the affinity for hapten, which rules out a hydrogen bond from this tyrosine to the hapten. In the case of M315 and X25, 19F resonances on labelled haptens are specifically perturbed from the native protein position, which is interpreted to indicate proximity of the reporter group and the modified tyrosine. A method is described for the introduction of reporter groups into the protein by a reduction of the nitrotyrosine to aminotyrosine.
Chapter 4:
Proton nuclear magnetic resonance has been employed to observe perturbations of methylated nitrophenyl haptens that take place on binding to three nitrophenyl-specific myeloma proteins. A comparison of the previously observed 19F shifts from fluorinated haptens with these proton shifts permits the separation of the proton ring current shifts from the fluorine paramagnetic component. The paramagnetic component, which is large and different for each protein, is ascribed either to van der Waals crowding or to charge transfer interaction or a combination of the two.
Chapter 5:
The contribution of charge transfer to the 19F paramagnetic shift has been examined by the use of model complexes. Hapten with tryptophan methyl ester model complexes allows determination of the total shift by comparison of methyl and trifluoromethyl shifts. This demonstrated that the para-substituted shift was much larger than the ortho-substituted shift, which is similar to the result found in the protein study. A computer program was employed to determine the intensity and position of the charge transfer absorption band in these model complexes and with the three proteins. The intensity of the charge transfer interaction appears to correlate with the size of the 19F paramagnetic shift. The difference in shift between the ortho- and para-substituted positions is attributed to the particular resonance structures that are important in binding. The association constants of the model complexes were employed to decompose the binding energies of several haptens for the three proteins, and this analysis indicated a hydrogen bond between hapten and antibody for M315 but not M460 or X25.
Chapter 6:
The nitration study (Chapter 3) indicated a particular tyrosine (Tyr-33H) was near the chain reporter group of the hapten when bound to M315. This result, in combination with the charge transfer data, was used to modify a model of the M315 binding site. A computer graphics system was employed to assemble the first heavy chain hypervariable loop with the tyrosine reoriented so as to fit next to the hapten. The resulting structure, when analyzed by a Diamond refinement energy minimization program, was more precisely fit to standard X-ray coordinates than the original structure.
Chapter 7:
This chapter presents background on effector function and effector function triggering, which is the subject of the final two chapters. The importance of the integration of antigen binding with cellular effector function is described and several effector functions are mentioned. The two major theories of effector triggering are outlined.
Chapter 8:
Complement fixation studies on the nitrophenyl-specific myeloma protein A22 (and IgM) have shown that both mono- and poly-substituted antigens trigger fixation. This result contrasts with previous work on IgG that demonstrated that only antigens capable of forming complexes could trigger complement fixation. This apparent discord is resolved when one considers that the IgM is already aggregated (as compared to IgG) and so does not require further aggregation after the binding of antigen in order to fix complement. The binding of a single monosubstituted antigen per IgM was sufficient for triggering and the triggering could be competitively inhibited by hapten. The fixation did not occur through the alternate pathway. A DNP-specific hybridoma IgG2a protein acted as a control such that, over the entire dilution range employed in this study, no complement was fixed with the monosubstituted antigen, while with polyvalent antigen considerable fixation occurred.
Chapter 9:
A cell culture line of MOPC 460 has been established by alternate growth in animals and in culture. A cell culture line of MOPC 315 has been established by somatic cell fusion of the MOPC 315 cells with a nonsecreting myeloma that can grow in culture. Secreting cells were selected by a solid phase radioimmunoassay. Two hybridoma lines secreting DNP-specific IgG were also employed in this study. 13C-enriched tryptophan was incorporated into large-scale cultures of these cells and their secreted immunoglobulin was thereby enriched in 13C tryptophan. The various proteins were isolated by affinity chromatography of the spent culture fluid and then studied by 13C NMR. NMR of the isolated protein revealed an envelope of resonances near the position of free tryptophan, but shifted over a 10 parts-per-million range. Observations of the proteins with and without monovalent antigen showed large changes in the envelope for the IgG2a protein (a complement fixing class), but only slight changes for the IgG1 (a non-fixing class). Reduction of the IgG2a produced large changes in the envelope as did papain fragmentation. The results generally support the concept that a conformational change is associated with antigen binding (at least in the IgG2a subclass) and may constitute a major part of the triggering of effector function.
Item Type: | Thesis (Dissertation (Ph.D.)) | ||||||||
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Subject Keywords: | Chemistry | ||||||||
Degree Grantor: | California Institute of Technology | ||||||||
Division: | Chemistry and Chemical Engineering | ||||||||
Major Option: | Chemistry | ||||||||
Thesis Availability: | Public (worldwide access) | ||||||||
Research Advisor(s): |
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Thesis Committee: |
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Defense Date: | 23 March 1981 | ||||||||
Funders: |
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Record Number: | CaltechTHESIS:05082018-102024886 | ||||||||
Persistent URL: | https://resolver.caltech.edu/CaltechTHESIS:05082018-102024886 | ||||||||
DOI: | 10.7907/wcn5-3w02 | ||||||||
Default Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | ||||||||
ID Code: | 10858 | ||||||||
Collection: | CaltechTHESIS | ||||||||
Deposited By: | Mel Ray | ||||||||
Deposited On: | 08 May 2018 21:07 | ||||||||
Last Modified: | 16 Apr 2021 22:18 |
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