CD164 monoclonal antibodies that block hemopoietic progenitor cell adhesion and proliferation interact with the first mucin domain of the CD164 receptor

The novel sialomucin, CD164, functions as both an adhesion receptor on huma n CD34(+) cell subsets in bone marrow and as a potent negative regulator of CD34(+) hemopoietic progenitor cell proliferation. These diverse effects a re mediated by at least two functional epitopes defined by the mAbs, 103B2/ 9E10 and 105A5, We report here the precise epitope mapping of these mAbs to gether with that of two other CD164 mAbs, N6B6 and 67D2, Using newly define d CD164 splice variants and a set of soluble recombinant chimeric proteins encoded by exons 1-6 of the CD164 gene, we demonstrate that the 105A5 and 1 03B2/9E10 functional epitopes map to distinct glycosylated regions within t he first mucin domain of CD164, The N6B6 and 67D2 mAbs, in contrast, recogn ize closely associated and complex epitopes that rely on the conformational integrity of the CD164 molecule and encompass the cysteine-rich regions en coded by exons 2 and 3. On the basis of their sensitivities to reducing age nts and to sialidase, O-sialoglycoprotease, and N-glycanase treatments, we have characterized CD164 epitopes and grouped them into three classes by an alogy with CD34 epitope classification, The class I 105A5 epitope is sialid ase, O-glycosidase, and O-sialoglycoprotease sensitive; the class II 103B2/ 9E10 epitope is N-glycanase, O-glycosidase, and O-sialoglycoprotease sensit ive; and the class III N6B6 and 67D2 epitopes are not removed by such enzym e treatments. Collectively, this study indicates that the previously observ ed differential expression of CD164 epitopes in adult tissues is linked wit h cell type specific post-translational modifications and suggests a role f or epitope-associated carbohydrate structures in CD164 function.