Physiological properties of CD33

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CD33 is a 67kD member of the sialic-acid-binding immunoglobulin-like lectins (Siglecs), a discrete subset of the immunoglobulin (Ig) superfamily molecules (7-10). The human CD33 (Siglec-3) gene, located on chromosome 19q13.3, encodes a single pass, type I transmembrane glycoprotein consisting of an amino-terminal V-set Ig-like domain that mediates sialic-acid recognition, a C2-set Ig-like domain, a transmembrane domain, and an intracellular domain (11-14) (Figure 1). It may be expressed as a homodimer in its physiological state (15). At least in myeloid cell lines – expression in primary cells has not been studied – a shorter isoform lacking exon 2, which encodes the V-set domain, has been identified, but it is unknown whether this splice isoform is expressed on the cell surface (16). The cytoplasmic tail contains 2 conserved tyrosine-based signaling motifs, comprising a membrane-proximal immunoreceptor tyrosine-based inhibitory motif (ITIM) at position 340 and a membrane-distal ITIM-like motif at position 358. Upon phosphorylation, likely by Src family kinases, these tyrosine motifs provide docking sites for the recruitment and activation of the Src homology-2 (SH2) domain-containing tyrosine phosphatases, SHP-1 and SHP-2 (15, 17, 18). While both SHP-1 and SHP-2 are recruited to Y340, Y358 primarily functions to recruit SHP-2. In turn, these tyrosine phosphatases may dephosphorylate CD33 as part of a potential negative feedback control of CD33 signaling (17, 18) or may dephosphorylate and negatively regulate nearby receptors (15). The SH2 domain-containing suppressor of cytokine signaling 3 (SOCS3) can competer with SHP-1/2 for binding to phosphorylated CD33, leading to recruitment of the ECS (Elongin B/C-Cul2/Cul5-SOCS-box protein) E3 ubiquitin ligase complex and concomitant accelerated proteosomal degradation of both CD33 and SOCS3 (19). Indeed, CD33 can become ubiquitylated on several lysine residues located in the cytoplasmic domain. CD33 mono-ubiquitylation or poly-ubiquitylation, which requires intact ITIMs and is enhanced by tyrosine phosphorylation, involves both the lysine cluster around amino acid residues 309-315 and the 352 residue, whereas the first 2 intracellular lysines (residues 283 and 288) contribute little, if at all, to overall ubiquitylation of CD33 (20). Besides SOCS3, the Cbl family of E3 ubiquitin ligases can also bind to CD33 in an ITIM-dependent manner, and ubiquitylation of CD33 by Cbl proteins has been demonstrated experimentally (20).

In addition to tyrosine phosphorylation, CD33 is also rapidly phosphorylated on serine residues as a consequence of protein kinase C activation, with S307 being the strongest putative phosphorylation site. It has been speculated that this may occur as a consequence of cytokine signaling and may regulate its sialic acid-dependent binding activity, but the biological significance of serine phosphorylation of CD33 has not been elucidated in detail (21). Equally little is known about downstream signaling events, although cross-linking of CD33 can induce tyrosine phosphorylation of the proto-oncogenes Cbl and Vav in normal monocytes (22). Likewise, several signaling intermediates (Cbl, Vav, Syk, CrkL, and Plc-γ1) have been shown to form complexes with CD33, at least upon pharmacological tyrosine phosphorylation (22, 23), but the physiological significance of these interactions is unknown.

CD33 exhibits a high degree of sequence similarity with 9 other Siglecs that, together, encompass the rapidly evolving subset of “CD33-related Siglecs” (8). These are mainly expressed on leukocytes in a cell type-specific manner. In healthy individuals, CD33 is primarily found on multipotent myeloid precursors, unipotent colony-forming cells, and maturing granulocytes and monocytes but not outside the hematopoietic system; it is down-regulated to low levels on peripheral granulocytes and resident macrophages while it is retained on circulating monocytes as well as dendritic cells (24-28). Besides expression in the myeloid cell lineage, CD33 may be found on subsets of B lymphocytes and activated human T and natural killer cells (16, 29-34). In vitro studies of normal bone marrow indicated that CD33 is not expressed on pluripotent hematopoietic stem cells (26, 27, 35) (Figure 2). Consistently, clinical studies demonstrated delayed but durable multilineage engraftment after transplantation of CD33-depleted autografts in patients with AML (36, 37), providing further evidence that normal hematopoietic stem cells lack CD33. The putative promoter sequence of CD33 contains a critical PU.1 site (38) but the regulation of CD33 expression has so far not been studied in detail; nevertheless, down-regulation of CD33 has been observed on monocytes by activation via T-cell contact, Fcg receptor cross-linking, or pharmacological stimulation with phorbol myristate acetate or lipopolysaccharide (39).

The physiological function of CD33 is poorly understood. Similar to other CD33-related Siglecs, sialic acid-dependent cell adhesion with preference for a2-6 over a2-3 sialyllactosamines has been demonstrated (13, 17, 40). The adhesive properties of CD33 are modulated by a1-3 linked fucose (“fucosylation”), which reduces binding to a2-3 sialyllactosamines (40), as well as by endogenous sialoglycoconjugates that are present on the cell surface as cis ligands (13). Intrinsically, CD33-mediated cell adhesion is regulated by the proximal ITIM motif (13, 17) as well as glycosylation of the extracellular domains; in fact, mutation of a single N-linked glycosylation site in the V-set Ig-like domain can unmask CD33’s ligand binding function (41). Because of the ITIM and ITIM-like motifs, CD33 is thought to function as an inhibitory receptor by reducing the activity of tyrosine kinase-driven signaling pathways (10). In support of this notion, early studies demonstrated that cross-linking of CD33 with CD64 (FCGR1A, the high-affinity Fcγ receptor 1a), limits CD64-mediated tyrosine phosphorylation and Ca⁺⁺ mobilization through SHP-1 (15, 18). Increasing evidence suggests that the primary function of CD33-related Siglecs may involve dampening of host immune responses and setting of appropriate activation thresholds for the regulation of cellular growth, survival, and the production of soluble mediators (9). Consistently, CD33 constitutively suppresses the production of several pro-inflammatory cytokines (IL-1β, TNF-α, and IL-8) by human monocytes in a sialic-acid ligand-dependent and SOCS3-dependent manner (39). Conversely, reduction of cell surface CD33, or interruption of sialic acid binding, leads to activation of p38 mitogen-activated protein kinase (MAPK) and enhances cytokine secretion (39). Likewise, SOCS3 activity reduces CD33-mediated repression of cytokine signaling and enhances cytokine-induced cellular proliferation (19). On the other hand, antibody-engagement of CD33, along with CD33 phosphorylation and recruitment of SHP-1, reduces the syntheses of pro-inflammatory cytokines (TNF- α, IL-6, IL-1β) and chemokines (RANTES, MCP-1, IL-8), in macrophages in vitro (42).

While limited, emerging data suggest a role of CD33 in the pathophysiology of several human diseases. Specifically, CD33 expression was found to be significantly reduced on monocytes of patients with type 2 diabetes relative to healthy individuals while secretion of several cytokines (TNF-α, IL-8, IL-12p70) was increased; consistently, high glucose conditions in vitro decreased CD33 transcription and protein expression, whereas TNF-α secretion and SOCS3 expression were increased, suggesting a role of CD33 in the generation of the pro-inflammatory milieu characteristic of diabetes (43). Furthermore, although no genetic disorders have been associated so far with mutations in CD33, a single nucleotide polymorphism (SNP) within the CD33 gene (rs3865444) has been associated with the development of Alzheimer’s disease (44-46).