The Myeloid-specific Sialic Acid-binding Receptor, CD33, Associates with the Protein-tyrosine Phosphatases, SHP-1 and SHP-2

Abstract

The myeloid restricted membrane glycoprotein, CD33, is a member of the recently characterized "sialic acidbinding immunoglobulin-related lectin" family. Although CD33 can mediate sialic acid-dependent cell interactions as a recombinant protein, its function in myeloid cells has yet to be determined. Since CD33 contains two potential immunoreceptor tyrosine-based inhibition motifs in its cytoplasmic tail, we investigated whether it might act as a signaling receptor in myeloid cells. Tyrosine phosphorylation of CD33 in myeloid cell lines was stimulated by cell surface cross-linking or by pervanadate, and inhibited by PP2, a specific inhibitor of Src family tyrosine kinases. Phosphorylated CD33 recruited both the protein-tyrosine phosphatases, SHP-1 and SHP-2. CD33 was dephosphorylated in vitro by the co-immunoprecipitated tyrosine phosphatases, suggesting that it might also be an in vivo substrate. The first CD33 phosphotyrosine motif is dominant in CD33-SHP-1/SHP-2 interactions, since mutating tyrosine 340 in a CD33-cytoplasmic tail fusion protein significantly reduced binding to SHP-1 and SHP-2 in THP-1 lysates, while mutation of tyrosine 358 had no effect. Furthermore, the NH 2 -terminal Src homology 2 domain of SHP-1 and SHP-2, believed to be essential for phosphatase activation, selectively bound a CD33 phosphopeptide containing tyrosine 340 but not one containing tyrosine 358. Finally, mutation of tyrosine 340 increased red blood cell binding by CD33 expressed in COS cells. Hence, CD33 signaling through selective recruitment of SHP-1/ SHP-2 may modulate its ligand(s) binding activity.Over the last few years, a novel family of sialic acid-dependent recognition molecules has emerged. This family, recently designated "siglecs" (sialic acid-binding Ig-related lectins), is a structurally related subgroup of the immunoglobulin superfamily that includes CD22 (siglec-2), sialoadhesin (siglec-1), MAG (siglec-4), CD33 (siglec-3), and the newest member of the family, siglec-5 (1). All siglecs have an NH 2 -terminal V-set Ig-like domain that contains the sialic acid binding site, followed by varying numbers of C2-set domains. In addition to common structural features that would appear to adapt these molecules for functional protein-carbohydrate cellular interactions, each member exhibits a very specific pattern of tissue distribution. While CD22 is restricted to B cells, sialoadhesin to macrophages, and myelin-associated glycoprotein (MAG) to myelinating oligodendrocytes and Schwann cells, CD33 and siglec 5 are expressed only on cells of the myelomonocytic lineage.CD22 is perhaps the best characterized member of the siglec family. In addition to being an adhesion receptor for sialic-acid bearing ligands on leukocytes and erythrocytes, CD22 has an important regulatory role as a signal transduction molecule in B cells (2, 3). The cytoplasmic tail of CD22 has six tyrosines, two of which are encompassed within sequences which conform with immunoreceptor tyrosine-based activation motifs (ITAM), 1 while the other four form po...

Sialoside Specificity of the Siglec Family Assessed Using Novel Multivalent Probes

“…However, the additional galactose in the branched ␣2-6 sialylated T antigen (17) resulted in decreased affinity for Siglecs-2, -9, and -10 and increased affinity for Siglecs-1, -4, -5, -7, and -8. Most remarkable is the 100-fold increase in affinity observed for MAG (Siglec-4) for the ␣2-6 sialylated-T antigen (17). Equally remarkable are the affinities of MAG for the ␣2-3 sialylated (8) and ␣2-3/␣2-6 disialylated (18) T antigens with inhibitory potencies 300 and 1,500 higher than the reference compound (5), respectively.…”

Cytotoxic activity of gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukemia correlates with the expression of protein kinase Syk

“…3 The cytoplasmic tail of CD33 contains two immune tyrosine-based inhibitory motifs (ITIMs) and therefore, may serve as a potential inhibitory receptor. [4][5][6] Engagement of CD33 induced apoptosis and inhibition of proliferation in leukemia cells from AML and chronic myeloid leukemia patients. [7][8][9][10] However, little is known about the molecular mechanisms of the CD33 signaling events leading to inhibition of cell growth and apoptosis.…”

Siglec-5 (CD170) Can Mediate Inhibitory Signaling in the Absence of Immunoreceptor Tyrosine-based Inhibitory Motif Phosphorylation

“…Some CD33-related siglecs can also inhibit activatory pathways when crosslinked with activating receptors (7,8,12,13). In addition, CD33 and Siglecs-7 and -9 have been shown to exhibit enhanced siglec-dependent adhesion after tyrosine mutation of the proximal ITIM (6,14).…”