mSiglec-E, a novel mouse CD33-related siglec (sialic acid-binding immunoglobulin-like lectin) that recruits Src homology 2 (SH2)-domain-containing protein tyrosine phosphatases SHP-1 and SHP-2

Abstract

The sialic acid-binding immunoglobulin-like lectins (siglecs) represent a recently defined distinct subset of the immunoglobulin superfamily. By using the Src homology 2 (SH2)-domain-containing protein tyrosine phosphatase SHP-1 as bait in a yeast two-hybrid screen, we have identified a new member of the mouse siglec family, mSiglec-E. The mSiglec-E cDNA encodes a protein of 467 amino acids that contains three extracellular immunoglobulin-like domains, a transmembrane region and a cytoplasmic tail bearing two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). mSiglec-E is highly expressed in mouse spleen, a tissue rich in leucocytes. The ITIMs of mSiglec-E can recruit SHP-1 and SHP-2, two inhibitory regulators of immunoreceptor signal transduction. This suggests that the function of mSiglec-E is probably an involvement in haematopoietic cells and the immune system as an inhibitory receptor. When expressed in COS-7 cells, mSiglec-E was able to mediate sialic acid-dependent binding to human red blood cells, suggesting that mSiglec-E may function through cell-cell interactions. In comparison with the known members of the siglec family, mSiglec-E exhibits a high degree of sequence similarity to both human siglec-7 and siglec-9. The gene encoding mSiglec-E is localized in the same chromosome as that encoding mouse CD33. Phylogenetic analysis reveals that neither mouse mSiglec-E nor CD33 shows a clear relationship with any human siglecs so far identified.

The Membrane-Proximal Immunoreceptor Tyrosine-Based Inhibitory Motif Is Critical for the Inhibitory Signaling Mediated by Siglecs-7 and -9, CD33-Related Siglecs Expressed on Human Monocytes and NK Cells

“…Previous reports on CD33 (22)(23)(24) and mouse Siglec-E (25,26), demonstrated the importance of the membrane-proximal tyrosine motif in recruitment of SHP-1 and SHP-2 by co-immunoprecipitation, but this study on Siglecs-7 and -9 provides the first analysis of the role of tyrosine-based motifs in the inhibitory function for CD33-related Siglecs. Mutation of the membrane-proximal tyrosine motif that conforms to the consensus ITIM led to a complete abrogation of tyrosine-phoshorylation of the receptors, recruitment of the PTPs SHP-1 and SHP-2, and the inhibition of Fc⑀RI-mediated activation of RBL cells.…”

The murine inhibitory receptor mSiglec‐E is expressed broadly on cells of the innate immune system whereas mSiglec‐F is restricted to eosinophils

“…The predicted sequence shares more than 99% identity with those reported previously for MIS/mSiglec-E, the differences likely representing polymorphisms [6,14]. The earlier reports focussed on the inhibitory signaling properties of mSiglec-E and did not investigate its sialic acid binding specificity, nor its expression pattern [6,14]. The latter, in particular, is critical to understanding the potential functions of mSiglec-E in the immune system and was a major goal of the current investigation.…”

“…mSiglec-E is most similar in sequence to human Siglec-7, -8 and -9 [6,14], and at the outset, it was therefore thought likely that mSiglec-E would be expressed on populations of cells that express these three siglecs in humans. However, the results of our study demonstrate that the expression profile of mSiglec-E covers only two of these siglecs, namely hSiglec-7 and -9. hSiglec-8 is expressed specifically on eosinophils, but this cell population expressed mSiglec-E only very weakly.…”

“…These include the 3-Ig-domain siglecs (hSiglec-7, -8 and -9) and the 5-Ig-domain siglecs (hSiglec-10 and hSiglec-11). Within the mouse CD33-related subgroup, there is only one 3-Ig-domain siglec, designated mSiglec-E/MIS [6,14], and one predicted 5-Ig-domain siglec, designated mSiglec-G [13], that are the putative orthologs of hSiglec-9 and hSiglec-10, respectively [13]. Both humans and mice have one 4-Ig-domain siglec, called hSiglec-5 and mSiglec-F, respectively, but it is not clear whether these represent true orthologs or whether they arose independently [13].…”

“…However, the precise nature of these cells was not explored further. In comparison, mSiglec-E was characterized primarily as a myeloid cell inhibitory receptor [6], but studies of cellular expression were limited to Northern blotting and RT-PCR analysis of a few tissues and selected cell lines [6,14].…”