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

• Published in: Clinical and experimental immunology Vol. 100; no. 3; pp. 489 - 498
• Main Authors: BOIRE, G., GENDRON, M., MONAST, N., BASTIN, B., MÉNARD, H. A.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-06-1995
• Subjects: Animals; Autoantibodies - immunology; Autoantigens - chemistry; Autoantigens - isolation & purification; Base Sequence; biochemical purification; Blotting, Western; Cloning, Molecular; DNA Primers - chemistry; HeLa Cells; Humans; Molecular Sequence Data; Molecular Weight; Precipitin Tests; Rabbits; ribonucleoproteins; Ribonucleoproteins - chemistry; Ribonucleoproteins - immunology; Ribonucleoproteins - isolation & purification; RNA, Small Cytoplasmic; Ro antigen; Ro proteins
• ISSN: 0009-9104; 1365-2249
• DOI: 10.1111/j.1365-2249.1995.tb03728.x

SUMMARY
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 mo no specific 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.