Purification of antigenically intact Ro ribonucleoproteins; biochemical and immunological evidence that the 52-kD protein is not a Ro protein.
Anti-Ro sera immunoprecipitate Ro ribonucleoproteins (RNPs) from human cell extracts. Ro RNPs are biochemically heterogeneous particles whose functions are unknown and whose exact composition remains controversial. In addition to 60-kD Ro and to La proteins, a 52-kD polypeptide (p52) has been proposed to be a stable component of the Ro RNPs. To confirm the immunological studies supporting this hypothesis, we have biochemically purified Ro RNPs from HeLa cells using non-denaturing conditions. Ro RNPs segregated into three distinct populations, one of which only contained hY5 RNA (RohY5 RNPs). No p52 co-purified with Ro RNPs. Despite the absence of p52, purified Ro RNPs had biochemical and immunological properties identical to those of unfractionated Ro RNPs. Many anti-Ro sera only recognize p52 in immunoblots, and are said to be monospecific anti-p52. Preincubation with purified RohY5 RNPs (free of p52) of all human anti-Ro (including so-called monospecific anti-p52) sera abolished their capacity to immunoprecipitate Ro RNPs from unfractionated HeLa cell extracts. Conversely, preincubation of anti-Ro sera with purified p52 protein specifically inhibited recognition of p52 in immunoblots, but did not interfere with immunoprecipitation of Ro RNPs. Our data demonstrate that anti-p52 antibodies do not target intact Ro RNPs, nor do they target the native 60-kD Ro protein. Contrary to previous reports, p52 protein is not a stable component of antigenically intact Ro RNPs.
Boire
G
G
Département de Médecine, Faculté de Médecine, Université de Sherbrooke, Québec, Canada.
Gendron
M
M
Monast
N
N
Bastin
B
B
Ménard
H A
HA
D000818:Animals; D001323:Autoantibodies / Q000276:immunology; D001324:Autoantigens / Q000737:chemistry* / Q000302:isolation & purification; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011233:Precipitin Tests; D020733:RNA, Small Cytoplasmic; D011817:Rabbits; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D000099207:SS-A Antigen
D001323:Autoantibodies; D001324:Autoantigens; D017931:DNA Primers; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; D000099207:SS-A Antigen; C116006:RO60 protein, human