TY - JOUR
T1 - Translation of the Human C3b/C4b Receptor mRNA in a Cell-Free System and by Xenopus Oocytes
AU - Kumar, V.
AU - Farries, T.
AU - Swierkoz, J.
AU - Atkinson, J. P.
PY - 1989/5
Y1 - 1989/5
N2 - The C3b/C4b complement receptor (CR1) is a large, single-chain integral membrane glycoprotein present on erythrocytes, leukocytes, glomerular podocytes, and splenic dendritic-reticular cells that mediates the binding of complement-coated particles and immune complexes. CR1 is unusual in that it is polymorphic in size with the four allelic variants having molecular weights of 190000, 220000, 250000, and 280000 (SDS-PAGE, reducing conditions). The in vitro translation of the common (Mr 220000) allelic variant CR1 has been achieved by using mRNA in lysates of rabbit reticulocytes and in Xenopus oocytes. HL-60, a promyelocytic human leukemic cell line, was treated with DMSO to induce differentiation and synthesis of CR1. Poly(A+) RNA was purified from these cells by column chromatography on oligo(dT)-cellulose. In the rabbit reticulocyte system, no CR1 was detected unless the translation mixture was denatured. In the presence of methylmercuric hydroxide, the CR1 translation product, unlike most translation products, had the same molecular weight in gel electrophoresis as the high-mannose-containing pro-CRl and was 15-20K larger than nonglycosylated CR1. This suggests that a cotranslational modification of CR1 structure occurs, probably involving a proteolytic cleavage event. When poly(A+) RNA was translated in Xenopus oocytes, CR1 could be detected by treatment of oocytes with anti-CRl monoclonal antibody followed by fluorescein-conjugated goat anti-mouse IgG. CR1 was diffusely distributed but preferentially localized to the vegetal surface. The molecular weight of this product, identified in immunoprecipitates of lysates of [35S]methionine-labeled oocytes, was identical with that of CR1 of HL-60.
AB - The C3b/C4b complement receptor (CR1) is a large, single-chain integral membrane glycoprotein present on erythrocytes, leukocytes, glomerular podocytes, and splenic dendritic-reticular cells that mediates the binding of complement-coated particles and immune complexes. CR1 is unusual in that it is polymorphic in size with the four allelic variants having molecular weights of 190000, 220000, 250000, and 280000 (SDS-PAGE, reducing conditions). The in vitro translation of the common (Mr 220000) allelic variant CR1 has been achieved by using mRNA in lysates of rabbit reticulocytes and in Xenopus oocytes. HL-60, a promyelocytic human leukemic cell line, was treated with DMSO to induce differentiation and synthesis of CR1. Poly(A+) RNA was purified from these cells by column chromatography on oligo(dT)-cellulose. In the rabbit reticulocyte system, no CR1 was detected unless the translation mixture was denatured. In the presence of methylmercuric hydroxide, the CR1 translation product, unlike most translation products, had the same molecular weight in gel electrophoresis as the high-mannose-containing pro-CRl and was 15-20K larger than nonglycosylated CR1. This suggests that a cotranslational modification of CR1 structure occurs, probably involving a proteolytic cleavage event. When poly(A+) RNA was translated in Xenopus oocytes, CR1 could be detected by treatment of oocytes with anti-CRl monoclonal antibody followed by fluorescein-conjugated goat anti-mouse IgG. CR1 was diffusely distributed but preferentially localized to the vegetal surface. The molecular weight of this product, identified in immunoprecipitates of lysates of [35S]methionine-labeled oocytes, was identical with that of CR1 of HL-60.
UR - http://www.scopus.com/inward/record.url?scp=0024535624&partnerID=8YFLogxK
U2 - 10.1021/bi00435a061
DO - 10.1021/bi00435a061
M3 - Article
C2 - 2665812
AN - SCOPUS:0024535624
VL - 28
SP - 4040
EP - 4046
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 9
ER -