Purification of antigenically intact Ro ribonucleoproteins; biochemical and Immunological evidence that the 52-kD protein is not a Ro protein

Abstract

SUMMARYAnti-Ro sera immutioprecipitate Ro dboaucleoproteins (RNPs) from human cell extracts. Ro RNPs are biochemically heterogeneous particles whose functiotis 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 {Ro''^^ 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 Ro""^^ 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.

Clinical and Pathological Roles of Ro/SSA Autoantibody System

“…In humans, the Ro60 gene is approximately 32 kb in size, located on chromosome 19, while the Ro52 gene is 8.8 kb in size, located on chromosome 11. Although the Ro52 protein was initially suggested to be part of the Ro-hY-RNA complex with Ro60 [49, 52, 53], subsequent studies failed to confirm a direct interaction of the proteins [54, 55]. Recent studies provided evidence that Ro52 and Ro60 are localized to different cell compartments and that anti-Ro52 and anti-Ro60 antibodies have different clinical associations [15].…”

Nucleic acid-associated autoantigens: Pathogenic involvement and therapeutic potential

“…Ro52 possesses multiple N-terminal zinc-finger motifs, a central leucine zipper, a potential N-glycosylation site [236], but does not bind to nucleic acids. It remains questionable if Ro52 is physically associated with the Ro60/Y RNA complex in the cell [237,238]. However, Ro52 and the chaperone calreticulin appear to colocalize with Ro60 in apoptotic blebs [239].…”

B cell apotopes of the 60-kDa Ro/SSA and La/SSB autoantigens

“…The 48-kDa La protein is transiently associated with the Ro/La RNP particle through binding with the Y RNAs and potentially Ro60 [3][4][5]. Although most patients with anti-Ro60 also produce antibodies against a structurally unrelated 52-kDa Ro (Ro52) [6], there is no evidence that Ro52 is physically associated with the Ro60/Y RNA complex [7].…”