Immunochemical characterization of the human blood cell membrane glycoprotein recognized by the monoclonal antibody 12E7

Abstract

The 12E7 murine monoclonal antibody recognizes a protease-sensitive component of human red cells, platelets and lymphocytes which could not be detected on granulocytes. Scatchard analyses indicated that the 125I-labelled antibody binds to 1000, 4000 and 27,000 antigen sites on each red cell, platelet and lymphocyte respectively, with a binding constant ranging from 4 x 10(7) to 9 x 10(7) M-1. The membrane components recognized by the monoclonal antibody were characterized by immunostaining on nitrocellulose sheets. A 28 kDa sialoglycoprotein was visualized following electrophoretic transfer of the red cell and lymphocyte membrane proteins separated by SDS/polyacrylamide-gel electrophoresis. Another component of 25 kDa was also clearly identified in the lymphocyte and platelet lysates, but was barely detectable in the red cell membrane preparations. Enzyme treatment of intact platelets, as well as analysis of the membrane and cytosolic preparations from these cells, have shown that the 25 kDa component was of cytoplasmic origin. The mobility of the 28 kDa membrane component is decreased following neuraminidase treatment of intact blood cells, but these cells still react normally with the monoclonal antibody, indicating that sialic acids are not required for binding. The 28 kDa component is present on red cell membranes prepared from S-s-U-, En(a-) and Gerbich(-) individuals, demonstrating that it is a new sialoglycoprotein not derived from glycophorins A, B, C or D. The 28 kDa component was totally solubilized with 0.1% Triton X-100 from red cell membranes and behaves like the other red cell membrane sialoglycoproteins since it was extracted in the aqueous phase following chloroform/methanol/water or butanol/water partitionings. The 28 kDa component could be partially purified by h.p.l.c. gel permeation chromatography and preparative SDS/polyacrylamide-gel electrophoresis. The material finally obtained strongly inhibits the 12E7 monoclonal as well as human anti-Xga antibodies, suggesting either that the 28 kDa glycoprotein carries both antigens or that the 12E7 and Xga-active molecules copurified.

Partial Characterisation of the Human Erythrocyte Antigen Pt^a

“…In this report, we have also shown that the protein carrying the Pta antigen is not sialoglycoprotein y. Furthermore, the structure carrying the Pta antigen does not appear to be the same as the 28-kilodalton erythrocyte membrane component recog nised by the monoclonal antibody 12E7 [33]. This com ponent also migrates between sialoglycoprotein ß and sialoglycoprotein y but, in contrast to the Pta protein, its mobility is altered following neuraminidase treatment of intact erythrocytes and it is totally solubilised from red cell membranes with 0.1 % Triton X-100.…”

Xg Blood Group System

“…CD99 is destroyed by the proteases papain, pronase, trypsin, and chymotrypsin [46,50,51] . CD99 is destroyed by the proteases papain, pronase, trypsin, and chymotrypsin [46,50,51] .…”

“…It is generally sialidaseresistant [46,50] , but one exceptional CD99 -like antibody did not react with sialidase -treated cells [51] . Sialidase treatment of the cells resulted in a reduction in apparent MW of the CD99 structure [26,27,50,53] . Sialidase treatment of the cells resulted in a reduction in apparent MW of the CD99 structure [26,27,50,53] .…”

Production of a New Murine Monoclonal Antibody with Fy6 Specificity and Characterization of the Immunopurified N-Glycosylated Duffy-Active Molecule

“…Washed red cells were treated with DCC-treated trypsin (55 U/ mg; Serva), Chymotrypsin (43 U/mg; Serva) and neuraminidase from Vibrio cholerae (0.1 U/ml; Behringwerke) as previously described [12,13]. Treatments with papain (from Carica papaya; 0.98 U/mg; Serva), ficin (1-2 U/mg, Sigma) and pronase (8 U/mg, Sigma) were perform ed as described [14,15].…”