title: Epitope mapping, expression and post-translational modifications of two isoforms of CD33 (CD33M and CD33m) on lymphoid and myeloid human cells.
abstract: We have tested the usefulness of several commercial anti-CD33 monoclonal antibodies (mAb) to determine the expression and localization of the two CD33 isoforms on several hematopoietic cell lines. The expression of the isoform CD33m, a CD33 transmembrane splice variant lacking the ligand-binding V immunoglobulin (Ig)-like domain, was detected by RT-polymerase chain reaction, western blot, confocal microscopy and flow cytometry on the membrane of several human cell types. CD33m was only detected by the anti-CD33 mAb HIM3-4 on the cell surface, whereas WM53, P67.6, 4D3, HIM3-4, WM54, D3HL60.251 or MY9 detected the CD33M isoform, indicating that HIM3-4 is the only mAb recognizing CD33 C(2) Ig domain. Accordingly, HIM3-4 binding to CD33 did not interfere with the binding of other antibodies against the CD33 V-domain. P67.6 mAb interfered with recognition by the rest of antibodies specific for the V domain. HIM3-4 staining could be increased after the sialidase treatment of all CD33(+) cells. However, this increase was stronger in activated T cells, suggesting a CD33 masking state in this cell population. Confocal microscopy analysis of CD33m HEK 293T-transfected cells revealed that this protein is expressed on the cell membrane and also detected in the Golgi compartment. CD33 is constitutively located outside the lipid raft domains, whereas cross-linked CD33 is highly recruited to this signaling platform. The unique ability of HIM3-4 mAb to detect the masking state of CD33 on different cell lineages makes it a good tool to improve the knowledge of the biological role of this sialic acid-binding Ig-like lectin.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Medicine, Murcia, Spain.
authors.forename: Ana B
authors.initials: AB
authors.lastname: Pérez-Oliva
authors.forename: María
authors.initials: M
authors.lastname: Martínez-Esparza
authors.forename: José J
authors.initials: JJ
authors.lastname: Vicente-Fernández
authors.forename: Rubén
authors.initials: R
authors.lastname: Corral-San Miguel
authors.forename: Pilar
authors.initials: P
authors.lastname: García-Peñarrubia
authors.forename: Trinidad
authors.initials: T
authors.lastname: Hernández-Caselles
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D015703:Antigens, CD / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D018604:Epitope Mapping; D018922:HL-60 Cells; D006801:Humans; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism*; D022423:Myeloid Cells / Q000276:immunology / Q000378:metabolism*; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D063268:Sialic Acid Binding Ig-like Lectin 3
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; C571163:CD33 protein, human; D020033:Protein Isoforms; D063268:Sialic Acid Binding Ig-like Lectin 3