The autoantigen La/SSB is a calmodulin-binding protein.
The work reported here has been directed to the identification of new nuclear calmodulin-binding proteins. To achieve this goal, nuclei from rat hepatocytes were purified and a fraction enriched in DNA- and RNA-binding proteins was extracted using DNase I and RNase A. Calmodulin-binding proteins present in this nuclear subfraction were purified by chromatography using first a DEAE-Sephacel column and subsequently a calmodulin-Sepharose column. Four major polypeptides of 118, 107, 48 and 45 kDa were found to bind to the calmodulin column in a Ca(2+)-dependent way. [125I]-calmodulin overlay analysis confirmed that the proteins of 118, 48 and 45 kDa are calmodulin-binding proteins. These proteins bind single-stranded and also double-stranded DNA. A partial amino acid sequence obtained from the 48 kDa protein revealed a 100% identity with the La/SSB protein, an autoantigen implicated in several autoimmune diseases, such as lupus erythematosus and Sjögren's syndrome. Two-dimensional gel electrophoresis, Western blot analysis and experiments of binding to poly(U), also supports the identity of p48 as La/SSB. CaM and La/SSB protein colocalize in the heterochromatinic regions within the nucleus of rat hepatocytes. Preincubation of La/SSB with calmodulin in the presence of Ca2+ resulted in an increase in the binding of ssDNA to La/SSB, suggesting that calmodulin can play a role in the regulation of the association of La/SSB with DNA.
Castro
A
A
Department de Biologia Cellular, Facultat de Medicina, Universitat de Barcelona, Spain.
Faura
M
M
Agell
N
N
Renau-Piqueras
J
J
Bachs
O
O
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001324:Autoantigens / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism; D008099:Liver / Q000166:cytology; D008297:Male; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012261:Ribonucleoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014529:Uridine / Q000378:metabolism; D000097585:SS-B Antigen
D000906:Antibodies; D001324:Autoantigens; D002147:Calmodulin; D004268:DNA-Binding Proteins; D012261:Ribonucleoproteins; D014157:Transcription Factors; D014529:Uridine; D000097585:SS-B Antigen