Mutant Mouse Lysozyme Carrying a Minimal T Cell Epitope of Hen Egg Lysozyme Evokes High Autoantibody Response

Abstract

Self proteins including foreign T cell epitope induce autoantibodies. We evaluated the relationship between the size of foreign Ag introduced into self protein and the magnitude of autoantibody production. Mouse lysozyme (ML) was used as a model self protein, and we prepared three different ML derivatives carrying T cell epitope of hen egg white lysozyme (HEL) 107–116, i.e., heterodimer of ML and HEL (ML-HEL), chimeric lysozyme that has residue 1–82 of ML and residue 83–130 of HEL in its sequence (chiMH), and mutant ML that has triple mutations rendering the most potent T cell epitope of HEL (sequence 107–116). Immunization of BALB/c mice with these three ML derivatives induced anti-ML autoantibody responses, whereas native ML induced no detectable response. In particular, mutML generated a 104 times higher autoantibody titer than did ML-HEL. Anti-HEL107–116 T cell-priming activities were almost similar among the ML derivatives. The heterodimerization of mutant ML and HEL led to significant reduction of the autoantibody response, whereas the mixture did not. These results show that size of the nonself region in modified self Ag has an important role in determining the magnitude of the autoantibody response, and that decrease in the foreign region in a modified self protein may cause high-titered autoantibody response.

Solution structure and activity of mouse lysozyme M

“…The dissociation constant of the mouse lysozyme M-(GlcNAc) 3 complex was twice as great as that of the hen lysozyme-(GlcNAc) 3 complex. Previously, we prepared Gln35 hen lysozyme, which had no activity [22].…”

“…1 H-15 N HSQC spectra of mouse lysozyme M with or without a large excess of (GlcNAc) 3 , a substrate analogue of lysozyme, were measured at 35°C in 90 % H 2 O/10 % D 2 O (v/v) at a protein concentration of 1.0 mM, and the pH was adjusted to 3.8 as described in our previous report [5]. The distance restraints were obtained from 15 N-edited NOESY (80 and 120 ms mixing time) and 1 H NOESY (120 ms mixing time).…”

“…In the presence of (GlcNAc) 3 where most of the mouse lysozyme M molecules form an enzyme-substrate complex, 15 NT 1 , T 2 , and heteronuclear NOE values of 102 out of 130 residues were obtained. Of the 28 uncharacterized residues, Lys 1, Pro71, and Pro103 were not assigned [5].…”

“…In the house mouse, the closely related mouse lysozymes M and P tend to be expressed in different tissues; mouse lysozyme M is strongly expressed in both macrophages and macrophage-rich tissues, while mouse lysozyme P is mainly expressed in the small intestine [2]. Recently, our group showed that mutant mouse lysozyme M evoked a strong autoantibody response [3], and we obtained autoantibody for native mouse lysozyme M. However, the three-dimensional structure of mouse lysozyme M has not yet been investigated via X-ray crystallography or nuclear magnetic resonance (NMR), meaning that we cannot yet understand the biological significance of the autoantibody on the basis of protein structure.…”

Crystal Structure of Tapes japonica Lysozyme with Substrate Analogue

“…Mutant Proteins-Site-directed mutagenesis was performed as described in our previous report (27). The resulting mutants were purified using the same protocol as used for the wild-type TJL described in our previous paper (16).…”