Human autoantibody to a novel protein of the nuclear coiled body: immunological characterization and cDNA cloning of p80-coilin.

Abstract

SummaryAntibodies producing an unusual immunofluorescent pattern were identified in the sera ofpatients with diverse autoimmune features. This pattern was characterized by the presence of up to six round discrete nuclear bodies in interphase cell nuclei . Immunoblotting analysis showed that these sera recognized an 80-kD nuclear protein, and affinity-purified anti-p80 antibody from the protein band reproduced the fluorescent staining of nuclear bodies . Colloidal gold immunoelectron microscopy showed that the affinity-purified anti-p80 antibody recognized the coiled body, an ultramicroscopic nuclear structure probably first described by the Spanish cytologist Ramon y Cajal. Five cDNA clones were isolated from a MOLT-4 cell Xgt-11 expression library using human antibody and oligonucleotide probes . The longest cDNA insert was 2.1 kb and had an open reading frame of 405 amino acids. A clone encoding a 14-kD COOH-terminal region of the protein was used for expression of a O-galactosidase fusion protein . An epitope was present in this COOH-terminal 14-kD region, which was recognized by 18 of 20 sera with anti-p80 reactivity, and affinity-purified antibody from the recombinant protein also reacted in immunofluorescence to show specific staining of the coiled body. This is the first demonstration and molecular cloning of a protein that appears to have particular identification with the coiled body, and it was designated p80-coilin. Autoantibody to p80-coilin may be useful for the elucidation of the structure and function of the coiled body, and the availability of a cDNA sequence could be helpful in further studies to clarify the clinical significance of this autoantibody response.

Two major autoantigen—Antibody systems of the mitotic spindle apparatus

“…Human HeLa S3, MOLT-4, HepG2, mouse 3T3, rabbit R9ab, and marsupial PtK2 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (FCS) and 2.5 mg/ml gentamicin sulfate at 37°C in 8% CO,. Rat FRTL-5 cells were cultured in Coon's modified Ham's F-12 medium (Sigma, St. Louis, MO) with 5% FCS supplemented with 10 mU/ml of thyrotropin (Sigma), 5 mg/ml of transferrin (Sigma), 10 nM hydrocortisone (Sigma), 10 mg/ml of insulin (Elanco, Indianapolis, IN), 10 ng/ml of glycyl-histidyl-lysine acetate (Calbiochem, La Jolla, CA), and 10 mg/ml somatomedin (Calbiochem). Indian Muntjac deer cells were cultured in Ham's F-10 medium supplemented with 20% FCS under the same conditions.…”

“…As a standard fixation procedure, cells grown to subconfluence on coverslips were washed in phosphate buffered saline (PBS) (pH 7.4), fixed in methanol (-ZOOC) for 10 minutes, followed by acetone (-20°C) for 2 minutes, and air dried. In a second protocol, cells were fixed in 4% formaldehyde in PBS for 20 minutes at room temperature, washed in PBS for 10 minutes with 3 changes, and permeabilized in acetone (-20°C) for 7 minutes.…”

“…IIF was performed as reported previously (10). Human sera were initially diluted 1:40 and were titrated as necessary.…”

“…In indirect immunofluorescence (IIF), autoantibodies are able to provide accurate definition of the topographic distribution of the antigen inside the cell and its relationship to cellular organelles. Some examples are antibodies against CENP polypeptides and their relationship with the centromere (8), antifibrillarin antibodies and the dense fibrillar component of the nucleolus (9), and antibodies to p80-coilin and the coiled body (10).…”

Coilin Shuttles between the Nucleus and Cytoplasm InXenopusOocytes

“…Very little was learned about the composition of these organelles until Raška et al (1991) discovered autoimmune sera that stained them specifically. Using these sera, Andrade et al (1991) cloned a gene encoding a protein, p80-coilin, that occurs in high concentration in coiled bodies. Once coilin became available as a molecular marker, coiled bodies were found to contain many RNA transcription and processing components, including all five of the splicing small nuclear ribonucleoprotein particles (snRNPs), U3 snRNA, U7 snRNA, and several nucleolar proteins, such as fibrillarin and Nopp140 (reviewed by Gall et al, 1995;Lamond and Earnshaw, 1998;Matera, 1998).…”