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Effects of variable and constant domains on physicochemical properties and secretion of mouse-chicken chimeric IgY, human IgG1, and IgE antibodies

가변 및 불변 부위가 쥐-닭 키메라 면역글로불린 Y, 인간 면역글로불린 G1, 면역글로불린 E 항체의 물리화학적 특성 및 분비에 미치는 영향

  • 최주호 (Juho Choi, 아주대학교 일반대학원)

초록/요약

The variable (V) and constant (C) regions of antibodies mutually affect their structures, which in turn influence their affinity, and specificity. The consequences of C-region switching between mammalian and evolutionarily distinct avian antibodies and the impact of V-region absence on antibody secretion remain unknown. Most studies have focused on IgG secretion, while other isotypes, particularly IgE, are less understood. In this study, I constructed mouse-human and mouse-chicken chimeric antibodies, as well as V region-deletion or alteration mutants. These antibodies were assessed for their physicochemical properties and secretion process in HEK293 cells. Physicochemical properties of chimeric antibodies depended on the V and C region combination. The absence of VH or VL domain did not affect expression and secretion of mutant IgGs. An aberrant pseudo Vκ (ψVκ) domain hindered antibody secretion, distorted light chain (LC) structure, decreased its stability. In the absence of heavy-light interchain disulfides, Cκ allowed individual secretion of its cognate heavy chain (HC) of IgG. Unlike IgG, the absence of VH or VL domains blocked IgE secretion. V domains are not critical for IgG expression, assembly, and secretion, but are crucial for IgE secretion. An intact VL domain is essential for LC or assembled IgG secretion. These findings provide valuable insights for designing various types of antibody molecules with improved properties and secretion efficiency.

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목차

I. INTRODUCTION 1
II. MATERIALS AND METHODS 20
A. Construction of expression vectors 20
B. Purification of antibody proteins 22
C. Deglycosylation and periodic acid-Schiff (PAS) staining 22
D. Determination of thermostability of purified Abs 23
E. Nano-differential scanning fluorimetry (nanoDSF) 24
F. Measurement of functional stability of Abs under thermal stress 24
G. Determination of aggregation/degradation behavior of Abs under thermal stress 25
H. Size-exclusion chromatography (SEC) 25
I. Transient transfections 25
J. SDS-PAGE and immunoblotting 26
K. Cell culture and immunoprecipitation 26
L. Establishment of BiP-expressing HEK293 cell line 27
M. Sandwich enzyme-linked immunosorbent assay (ELISA) 28
N. In silico modeling of Fab proteins 28
O. Protease susceptibility assay 29
III. RESULT 30
A. The C domain of chicken Abs is compatible with the V domains of mouse Abs. 30
B. V-C combination affects to the antigen binding affinity of chimeric MC IgY. 35
C. V-C combination affects to the structural stability of chimeric MC IgY. 38
D. V-C combination affects to the functional stability of chimeric MC IgY under thermal stress condition. 41
E. V-C combination affects to aggregation/degradation behaviors of Abs 47
F. The presence of Cκ domain allows the secretion of fully assembled Ig fragments regardless of the presence of the VH or VL domains. 50
G. The presence of ψVκ domain does not only inhibit the secretion of HCs but can also lead to the loss of the LC's own secretory ability. 53
H. The presence of ψVκ domain interferes with HC-LC association. 56
I. The conformational change of the LC due to the presence of ψVκ domain may have caused the loss of association with HC and the ability of LC to secrete itself. 59
J. BiP binding to LC/ψVκ impairs its secretion capability. 61
K. The presence of ψVκ domain decreases the structured stability of LC. 64
L. The presence of LC leads the secretion of HC regardless of the formation of H-L interchain disulfides. 67
M. The presence of Cκ domain allows the individual secretion of IgG HC. 71
N. The presence of Cκ domain can facilitate the individual secretion of IgG4 HC. 74
O. The presence of V domains of IgE is prerequisite for the secretion of fully assembled IgE. 79
IV. DISCUSSION 82
V. CONCLUSION 89
REFERENCES 90
국문요약 98

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