Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
ASF/SF2, a Protein Involved in Multiple RNA and microRNA Processing Pathways, Is a Novel Autoantigen in SLE.
Golding6, Amit, Akhter4, Ehtisham, Hines3, Tonie, Andrade4, Felipe, Sen5, Ranjan, Casciola-Rosen2, Livia, Rosen7, Antony
John Hopkins University, Baltimore, MD
John Hopkins University
Johns Hopkins University School of Medicine, Baltimore, MD
NIA/National Institues of Health
NIAID/National Institutes of Health, Bethesda, MD
The Johns Hopkins University, Baltimore, MD
In an attempt to discover novel lupus autoantigens, we sought to use lymphocytes as a source of antigen. Lymphocytes are likely to be a physiologic source of antigen due to the high frequency of lymphocyte cell death and their proximity to antigen receptors.
For our source of antigen, we used NP40-whole cell lysates of cultured REH cells. REH, is a non-EBV transformed, human ALL line with a combination of features of immature T and B cells. In our initial screen, SLE sera were used in standard Western Blots from 1D SDS-PAGE of REH lysates and a number of sera showed reactivity with a band of molecular weight range 3234 kD (Figure 1).
A repeat 2D gel of REH lysate was coomassie-stained to visualize the proteins and, based on the alignment previously seen between the Western Blot and the 2D Ponceau Stain, the appropriate protein "dots" were sent for peptide sequencing by Mass spectrometry.
The top non-keratin "hit" was SFRS1, also known as ASF/SF2, with 23% sequence coverage. In order to confirm that the SLE sera were in fact identifying ASF/SF2 as a novel autoantigen, the recombinant GST-fusion protein (MW 55kD) was purchased from ABNOVA and tested by immunoblotting using control and SLE patients' sera. We found that 12/57 (21%) of SLE patients' sera reacted with purified ASF/SF2 as compared to 0/25 of control normal sera. A representative blot is shown in [Figure 2B]. Studies to define antibody titers and correlation with disease activity are underway.
ASF/SF2 is a member of the SR family of proteins that are involved in RNA splicing and are regulated by serine phosphorylation by SRPK1. More recently, ASF/SF2 has also been shown to be involved in processing microRNAs separate from its role in RNA splicing. Intriguingly, ASF/SF2, has also been implicated as a proto-oncogene in a number of malignancies. It will be interesting to look for anti-ASF/SF2 antibodies in individuals with certain malignancies and to attempt to correlate these with autoimmune manifestations versus protective immunity. We will also look for anti-ASF/SF2 antibodies in other rheumatologic conditions and investigate the role of phosphorylation and other protein modifications in the creation of autoantibodies.
This protein appeared to be unique from known lupus autoantigens such as anti-Smith bands and anti-Ku70/RNP. Subsequently, a large quantity of REH lysate was prepared in the appropriate buffer and resolved by 2D gel electrophoresis followed by transfer to membrane, scanning of the Ponceau stained blot and Western Blotting with one of the sera showing strong reactivity with the 32 kD band (Figure 2A).
To cite this abstract, please use the following information:
Golding, Amit, Akhter, Ehtisham, Hines, Tonie, Andrade, Felipe, Sen, Ranjan, Casciola-Rosen, Livia, et al; ASF/SF2, a Protein Involved in Multiple RNA and microRNA Processing Pathways, Is a Novel Autoantigen in SLE. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :487