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The novel sialomucin, CD164, functions as both an adhesion receptor on human CD34+ cell subsets in bone marrow and as a potent negative regulator of CD34+ hemopoietic progenitor cell proliferation. These diverse effects are 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 together with that of two other CD164 mAbs, N6B6 and 67D2. Using newly defined 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 103B2/9E10 functional epitopes map to distinct glycosylated regions within the first mucin domain of CD164. The N6B6 and 67D2 mAbs, in contrast, recognize closely associated and complex epitopes that rely on the conformational integrity of the CD164 molecule and encompass the cysteine-rich regions encoded by exons 2 and 3. On the basis of their sensitivities to reducing agents and to sialidase, O-sialoglycoprotease, and N-glycanase treatments, we have characterized CD164 epitopes and grouped them into three classes by analogy with CD34 epitope classification. The class I 105A5 epitope is sialidase, O-glycosidase, and O-sialoglycoprotease sensitive; the class II 103B2/9E10 epitope is N-glycanase, O-glycosidase, and O-sialoglycoprotease sensitive; and the class III N6B6 and 67D2 epitopes are not removed by such enzyme treatments. Collectively, this study indicates that the previously observed differential expression of CD164 epitopes in adult tissues is linked with cell type specific post-translational modifications and suggests a role for epitope-associated carbohydrate structures in CD164 function.

Original publication

DOI

10.4049/jimmunol.165.2.840

Type

Journal article

Journal

Journal of immunology (Baltimore, Md. : 1950)

Publication Date

07/2000

Volume

165

Pages

840 - 851

Addresses

Medical Research Council Molecular Haematology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, United Kingdom.

Keywords

Hematopoietic Stem Cells, Humans, Neuraminidase, Metalloendopeptidases, Growth Inhibitors, Peptide Fragments, Membrane Glycoproteins, Neural Cell Adhesion Molecules, Mucins, Receptors, Cell Surface, Protein Isoforms, Antigens, CD, Antibodies, Monoclonal, Binding Sites, Antibody, Epitopes, Cell Adhesion, Antigen-Antibody Reactions, Alternative Splicing, Amino Acid Sequence, Base Sequence, Carbohydrate Conformation, Protein Conformation, Protein Structure, Tertiary, Glycosylation, Molecular Sequence Data, Sialomucins, CD146 Antigen, Endolyn