Characterization of rat HSA/CD24 protein bearing the B lineage-restricted epitope recognized by MAb HIS50.
HIS50 MAb recognizes a GPI-linked molecule on the surface of rat cells committed to the B lineage but is absent from cells of other hematopoietic lineages. Eukaryotic expression cloning of the cDNA has revealed that HIS50 recognizes a rat homologue of human CD24 and murine HSA. CD24/HSA exists in multiple glycoforms and plays an important role in hematopoietic and neural cell development. HIS50 is significant in that it is the only, presently available, anti-rat CD24/HSA MAb. Furthermore it is the only reported MAb with specificity restricted to B cell forms of CD24. Here we report Western blot analysis of HIS50 antigen (ag). In rat bone marrow HIS50 MAb recognized MW species in the range of 35-70 kDa. These species were shown by immunomagnetic cell sorting to be all derived from the HIS50+ cell subset. In other tissues with varying HIS50 levels as judged by immunostaining, apparent levels of HIS50 ag in the 35-70 kDa range varied accordingly. Thus, Western blot data corresponded to the immunostaining data of HIS50 ag, and the size distribution of B-restricted forms of rat CD24 appeared as wide as that reported for the more extensively expressed forms of human and murine CD24. N-Deglycosylation of cell lysates from lymphoid organs reduced the signal in the 35-70 kDa range, without appearance of lower MW HIS50-reactive species. Thus, unlike certain epitopes on human and murine CD24, HIS50 epitopes appeared (partially) N-linked carbohydrate dependent. The data reported here provide a basis for the further use of HIS50 MAb in studying the role of the highly heterogeneous CD24 molecules in cell development.
Pearce
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Department of Biochemistry, University of Hong Kong.
Ammerlaan
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Opstelten
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D000581:Amidohydrolases; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D015703:Antigens, CD / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology*; D015153:Blotting, Western; D001854:Bone Marrow Cells; D051927:CD24 Antigen; D002454:Cell Differentiation / Q000276:immunology; D002469:Cell Separation; D003902:Detergents; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D006031:Glycosylation; D008297:Male; D008562:Membrane Glycoproteins; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D017830:Octoxynol; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011092:Polyethylene Glycols; D051381:Rats; D011916:Rats, Inbred F344; D012972:Sodium Hydroxide
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D051927:CD24 Antigen; C497890:Cd24a protein, mouse; D003902:Detergents; D000939:Epitopes; D008562:Membrane Glycoproteins; D011092:Polyethylene Glycols; D012972:Sodium Hydroxide; D017830:Octoxynol; C010615:Nonidet P-40; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase