Molecular chaperones are targets of autoimmunity in Ro(SS-A) immune mice - PubMed (original) (raw)
Molecular chaperones are targets of autoimmunity in Ro(SS-A) immune mice
G Kinoshita et al. Clin Exp Immunol. 1999 Feb.
Abstract
We have used a murine model of experimental anti-Ro(SS-A) autoimmunity to dissect additional intermolecular interactions between the 52-kD Ro (Ro52) and 60-kD Ro (Ro60) autoantigens and molecular chaperones. Immune responses to members of the heat shock protein hsp70 and hsp90 families were measured by immunoblotting and ELISA in sera from mice immunized and boosted with purified recombinant Ro52, Ro60 and La (SS-B). All Ro52 and Ro60 immune sera immunoblotted the inducible glucose-regulated protein grp78 and hsp70 species but not constitutive hsc70 or hsp90. The kinetics of antibody production and reciprocal affinity purification experiments indicated that the grp78 and hsp70 responses were cross-reactive but distinct from immune responses to the primary Ro52 and Ro60 immunogens and the endoplasmic reticulum (ER)-resident chaperone calreticulin. No responses to molecular chaperones were detected in the La-immunized mice. Control immunizations indicated that the recruited grp78 and hsp70 responses were specific for the Ro proteins and not due to immunization with denatured protein. The rapid spreading of immunity to the inducible grp78 and hsp70 in Ro52- and Ro60-immunized mice suggests that these components may co-localize and physically associate under certain physiological conditions which may promote autoimmunization. The potential importance of the ER-resident chaperones grp78 and calreticulin is further supported by their co-localization with Ro in small apoptotic membrane blebs and the finding of a novel putative grp78 binding motif in the carboxyl-terminal region of Ro52.
Figures
Fig. 1
Representative immunoblots of antisera from Ro52-, Ro60-, La-, hen egg lysozyme (HEL)-, calreticulin (CR)–maltose binding protein (MBP)- and MBP-immunized C3H/HeJ mice with hsp70, hsc70, grp78, hsp90, CR–MBP, MBP control and the primary immunogens. Positions of molecular weight standards are shown on the left. Protein transfer was verified by Ponceau red staining of filters. Reactivity was developed using a peroxidase-coupled second antibody and enhanced chemiluminescence fluorography.
Fig. 2
Kinetics of appearance of antibodies to the heat shock/stress proteins hsp70, grp78 and calreticulin (CR)–maltose binding protein (MBP) in C3H/HeJ mice immunized and boosted twice with 100 μg of Ro52, Ro60 or La. Sera were collected at 7-day intervals and tested by ELISAs at a dilution of 1:500. Each group of sera was tested for reactivity with their respective immunogen (▪) and hsp70 (□), grp78 (•), calreticulin (CR)–MBP (○) and MBP control (▴).
Fig. 3
Recruited antibodies to hsp70 in Ro52- and Ro60-immunized mice cross-react with grp78. Anti-hsp70 antibodies were affinity-purified on hsp70 bound to nitrocellulose (Materials and Methods). The bound antibodies were eluted and tested alongside the untreated serum for immunoblot reactivity with calreticulin (CR)–maltose binding protein (MBP), hsp70, grp78 and Ro52 or Ro60. Anti-grp78 antibodies were affinity-purified on grp78 by the same method and tested accordingly. Reactivity was developed using a peroxidase-coupled second antibody and enhanced chemiluminescence fluorography.
Fig. 4
Predicted grp78 binding score for mouse (m)La (upper panel), mRo52 (middle panel) and mRo60 (lower panel). Grp78 binding was predicted using a computer algorithm which scores overlapping heptamers based on an optimizing scoring matrix. A cut-off score of + 11 was chosen to predict candidate binding sites based on the predictive outcome of a series of synthetic peptides (M.-J. Gething, personal communication). Only Ro52 contained a clustered series of dominant binding sequences (amino acids 378–391). Ro60 showed some potential binding sites whilst La did not (the single peak of binding potential between amino acids 239 and 245 was not considered significant due to the close proximity of unfavourable charged amino acids).
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