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 autoantibodies were used as a probe for screening rat NRK cDNA library, resulting in identification of an 11 kbp cDNA. The cDNA contained an open reading frame which encodes a 3,187-residue protein with a calculated mass of 364 kDa. The predicted protein, GCP364 (for a Golgi complex-associated protein of 364 kDa), was found to have no NH2-terminal signal sequence but a single hydrophobic domain at the COOH terminus and characteristically contain many coiled-coil domains with various sizes throughout the entire sequence. The identity of GCP364 with the autoantigen was confirmed by immunofluorescence and immunoblot analysis with the autoantibodies and anti-recombinant GCP364 produced in rabbits and by transfection/expression experiments. Search for the protein sequence data base revealed that GCP364 has 75% identity in amino acid sequence with human GCP372/giantin, indicating that it is a rat homolog of the latter. Immunogold electron microscopy showed that GCP364 was not detected on coated vesicles derived from the Golgi membrane, suggesting no involvement in the formation of transport vesicles. When cells were perforated and incubated with anti-GCP364 serum, the Golgi complex localized at perinuclear regions was dispersed into fragment-like structures as observed in nocodazole-treated cells. Taken together, these results suggest that GCP364 is anchored to the membrane by the COOH-terminal hydrophobic domain and has an extremely long cytoplasmic domain with coiled-coil structures, which may be involved in the formation and/or maintenance of the characteristic Golgi structure.
Toki
C
C
Department of Biochemistry, Fukuoka University School of Medicine.
Fujiwara
T
T
Sohda
M
M
Hong
H S
HS
Misumi
Y
Y
Ikehara
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001172:Arthritis, Rheumatoid / Q000276:immunology*; D001323:Autoantibodies / Q000378:metabolism*; D015152:Blotting, Northern; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000302:isolation & purification; D015723:Gene Library; D006056:Golgi Apparatus / Q000276:immunology / Q000378:metabolism*; D000077614:Golgi Matrix Proteins; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011817:Rabbits; D051381:Rats; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001323:Autoantibodies; D018076:DNA, Complementary; C110393:GCP364 protein, rat; D000077614:Golgi Matrix Proteins; D008565:Membrane Proteins; C087574:macrogolgin