Characterization of Rat HSA/CD24 Protein Bearing the B Lineage-Restricted Epitope Recognized by MAb HIS50

Abstract

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.