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.

Expression and functional characterization of CD33 transcript variants in human acute myeloid leukemia

“…We used tagged versions of the CD33 proteins to distinguish those from endogenously expressed CD33 in some of these cell lines and to be able to track the CD33 ∆E2 and CD33 ∆E2,E7a variants. While previous studies suggested that one CD33 antibody (clone Him3-4) specifically recognizes the C 2 - set Ig-like domain and could be used to detect CD33 variants that lack exon 2 [9], in our experiments Him3- 4 only recognized CD33 FL and CD33 E7a but not CD33 ∆E2 or CD33 ∆E2,E7a when expressed in virally-transduced ALL cell lines that are devoid of endogenous CD33 (Jurkat, RCH-ACV, REH, or RS4;11: Figure 3) and only recognized CD33 FL but not CD33 ∆E2 when expressed in virally-transduced HEK293T cells (Figure 4). As shown in Figure 5, engineered sublines expressed high amounts of CD33 FL and CD33 E7a in all cell lines.…”

GPI-anchor and GPI-anchored protein expression in PMM2-CDG patients

“…We also performed CD14 immunostaining by incubation of cells with FITC-conjugated 61D3, and after a washing step, they were stained with PE conjugated MФP9- mAb. The fluorescence intensity was analyzed by flow cytometry, and compared to that displayed by direct staining with the fluorochrome-conjugated 3G8- PE-Cy5 or MФP9-PE mAb on neutrophils or monocytes, respectively [21]. …”

Microglial carbohydrate-binding receptors for neural repair

“…The expression of siglecs has been demonstrated on various cell types in a restricted pattern. Thus, Siglec-1 is expressed on macrophages, Siglec-2 on B lymphocytes, Siglec-3 on cells of the lymphoid and myeloid cells such as macrophages, monocytes and microglia (Varki and Angata 2006;Perez-Oliva et al 2011) and MAG/Siglec-4 on myelin-forming cells, the Schwann cells and oligodendrocytes (Matani et al 2007). Siglecs are single-pass (type 1) transmembrane proteins with variable numbers (1-16) of Ig-like constant region type 2 (C2-set Ig-like) domains and an amino-terminal Ig-like variable (V-set Ig-like) domain that bears the sialic-acid-binding site (Crocker 2005;Varki and Angata 2006;Crocker et al 2007;Varki 2009).…”