CENP-B autoantigen is a conserved protein from humans to higher plants: identification of the aminoterminal domain in Phaseolus vulgaris.
Centromeres are critical structures in cell division, and CENP-B is the most important protein of the centromeric complex recognized by autoantibodies from patients with scleroderma. Our major aim was to demonstrate whether CENP-B is a conserved protein along the phylogenic scale including the higher plants. Vegetal and human cell proteins were extracted from Phaseolus vulgaris and HEp-2 cells and were characterized by PAGE, Western blot, and human autoimmune sera containing anti-CENP-B autoantibodies. The aminoterminus of the gene encoding for CENP-B from HEp-2 cells and Phaseolus vulgaris was isolated by reverse transcriptase-PCR using complementary oligonucleotides to the human CENP-B gene. Also, in situ hybridization was performed on vegetal tissues and HEp-2 cells using human CENP-B box probes. Our main results were as follows: 1) Autoimmune sera were reactive to a vegetal protein of 80 kDa. 2) Affinity-purified anti-CENP-B antibodies recognized a protein from Phaseolus vulgaris with molecular mass similar to that found in human cells. Vegetal and HEp-2 cells CENP-B proteins were immunologically identical. 3) Using RT-PCR, we were able to amplify a cDNA encoding for the aminoterminus domain of CENP-B from Phaseolus vulgaris that had the same molecular behaviour as the cDNA from HEp-2 cells. 4) Complementary oligonucleotides for human CENP-B box hybridized a DNA sequence from Phaseolus vulgaris. In conclusion, CENP-B protein is a conserved protein along the phylogenic scale from humans to higher plants.
Barbosa-Cisneros
O
O
Centro de Biología Experimental, Universidad Autónoma de Zacatecas, Guadalupe, México.
Fraire-Velázquez
S
S
Moreno
J
J
Herrera-Esparza
R
R
D000918:Antibody Specificity; D001323:Autoantibodies / Q000276:immunology / Q000302:isolation & purification; D001324:Autoantigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D002503:Centromere / Q000276:immunology; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D015342:DNA Probes; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D000939:Epitopes / Q000032:analysis; D006801:Humans; D018386:Kinetochores / Q000276:immunology; D010835:Phytohemagglutinins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D010940:Plant Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D012595:Scleroderma, Systemic / Q000276:immunology
D001323:Autoantibodies; D001324:Autoantigens; C497926:CENPB protein, human; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone; D015342:DNA Probes; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D000939:Epitopes; D010835:Phytohemagglutinins; D010940:Plant Proteins