IDENTIFICATION AND CHARACTERIZATION OF RAT 364-KDA GOLGI-ASSOCIATED PROTEIN RECOGNIZED BY AUTOANTIBODIES FROM A PATIENT WITH RHEUMATOID-ARTHRITIS

Autoantibodies from a patient with rheumatoid arthritis recognized an antigen localized in the Golgi complex of various cells tested. The au toantibodies were used as a probe for screening rat NRK cDNA library, resulting in identification of an 11 kbp cDNA. The cDNA contained an o pen reading frame which encodes a 3,187-residue protein with a calcula ted mass of 364 kDa. The predicted protein, GCP364 (for a Golgi comple x-associated protein of 364 kDa), was found to have no NH2-terminal si gnal sequence but a single hydrophobic domain at the COOH terminus and characteristically contain many coiled-coil domains with various size s throughout the entire sequence. The identity of GCP364 with the auto antigen was confirmed by immunofluorescence and immunoblot analysis wi th the autoantibodies and anti-recombinant GCP364 produced in rabbits and by transfection/expression experiments. Search for the protein seq uence data base revealed that GCP364 has 75% identity in amino acid se quence with human GCP372/giantin, indicating that it is a rat homolog of the latter. Immunogold electron microscopy showed that GCP364 was n ot detected on coated vesicles derived from the Golgi membrane, sugges ting no involvement in the formation of transport vesicles. When cells were perforated and incubated with anti-GCP364 serum, the Golgi compl ex localized at perinuclear regions was dispersed into fragment-like s tructures as observed in nocodazole-treated cells. Taken together, the se results suggest that GCP364 is anchored to the membrane by the COOH -terminal hydrophobic domain and has an extremely long cytoplasmic dom ain with coiled-coil structures, which may be involved in the formatio n and/or maintenance of the characteristic Golgi structure.