Identity of the RNase MRP- and RNase P-associated Th/To autoantigen.
OBJECTIVE
To characterize the molecular identity of the Th/To autoantigen, which is targeted by autoantibodies in scleroderma and which is associated with the human RNase MRP and RNase P ribonucleoprotein complexes.


METHODS
Proteins immunoprecipitated by anti-Th/To+ patient antisera from biotinylated total HeLa cell extracts were analyzed by immunoblotting. The association of autoantigenic proteins with the RNase MRP complex was analyzed by reconstitution experiments and ultraviolet crosslinking. The reactivity of patient sera with all known RNase MRP/RNase P proteins was analyzed by immunoprecipitation of the individual recombinant proteins.


RESULTS
The previously defined Th40 autoantigen appeared to be identical to the Rpp38 protein. Paradoxically, Rpp38 did not bind to the P3 domain of the RNase MRP RNA, as suggested by previously published data for Th40, and only half of the anti-Th/To+ sera contained anti-Rpp38 reactivity. Two other RNase MRP/RNase P subunits, Rpp20 and Rpp25, were found to interact with the P3 domain. The previously reported 40-kd species associated with this domain appeared to consist of Rpp20 and/or Rpp25 associated with a nuclease-resistant RNA fragment. Finally, we demonstrated that almost all tested anti-Th/To+ patient sera contained autoantibodies to Rpp25 and hPop1, indicating that these proteins harbor the most frequently targeted Th/To determinants.


CONCLUSIONS
Our data unequivocally define the identity of the Th/To autoantigen and demonstrate that Th/To autoepitopes are found on several protein subunits of RNase MRP/RNase P.
Van Eenennaam
Hans
H
University of Nijmegen, Nijmegen, The Netherlands.
Vogelzangs
Judith H P
JH
Lugtenberg
Dorien
D
Van Den Hoogen
Frank H J
FH
Van Venrooij
Walther J
WJ
Pruijn
Ger J M
GJ
D051017:Apoptosis Regulatory Proteins; D001324:Autoantigens / Q000235:genetics* / Q000276:immunology*; D001483:Base Sequence / Q000235:genetics; D001666:Binding Sites, Antibody; D002352:Carrier Proteins; D004722:Endoribonucleases / Q000235:genetics / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits / Q000276:immunology; D016337:RNA, Catalytic / Q000276:immunology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000276:immunology; D043262:Ribonuclease P; D012261:Ribonucleoproteins / Q000276:immunology
D051017:Apoptosis Regulatory Proteins; D001324:Autoantigens; D002352:Carrier Proteins; D000939:Epitopes; C490350:POP1 protein, human; C112706:POP7 protein, human; D021122:Protein Subunits; D016337:RNA, Catalytic; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C455399:RPP25 protein, human; D012261:Ribonucleoproteins; C071036:To autoantigen; D004722:Endoribonucleases; C058330:mitochondrial RNA-processing endoribonuclease; C468368:RPP14 protein, human; C104645:RPP38 protein, human; D043262:Ribonuclease P