CD33/Siglec-3 binding specificity, expression pattern, and consequences of gene deletion in mice - PubMed (original) (raw)

CD33/Siglec-3 binding specificity, expression pattern, and consequences of gene deletion in mice

Els C M Brinkman-Van der Linden et al. Mol Cell Biol. 2003 Jun.

Abstract

Mouse CD33/Siglec-3 (mCD33) is the apparent ortholog of human CD33/Siglec-3 (hCD33), a member of the Siglec (sialic acid-binding Ig superfamily lectin) family of sialic acid-recognizing cell-surface lectins. We examined the binding specificity and expression pattern of mCD33 and explored its functions by generating mice deficient in this molecule. Like hCD33, mCD33 is expressed on myeloid precursors in the bone marrow, albeit mostly in the more mature stages of the granulocytic lineage. Moreover, unlike hCD33, mCD33 in peripheral blood is primarily expressed on granulocytes. Also, unlike hCD33, mCD33 did not bind to alpha2-3- or alpha2-6-linked sialic acids on lactosamine units. Instead, it showed distinctive sialic acid-dependent binding only to the short O-linked glycans of certain mucins and weak binding to the sialyl-Tn epitope. Binding was enhanced by removal of 9-O-acetyl groups and attenuated by truncation of the glycerol-like side chain of sialic acids. Mice deficient in CD33 were viable and fertile in a controlled-access specific-pathogen-free vivarium, showed no major morphological or histological abnormalities, had no changes in bone marrow or peripheral leukocyte subpopulations, and had very minor differences in biochemical and erythrocyte parameters. Cellular responses to intraperitoneally injected proinflammatory stimulants, as well as subsequent interleukin-6 secretion, were also apparently unaffected. These results indicate substantial species differences in CD33 expression patterns and ligand recognition and suggest functional degeneracy between mCD33 and the other CD33-related Siglec proteins expressed on cells of the myeloid lineage.

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Figures

FIG. 1.

FIG. 1.

Binding of mCD33-Fc to BSM: effect of 9-O-acetylation and side chain modification of sialic acid. Biotinylated BSM was treated with base or with CHE-Fc to remove 9-O-acetyl esters from its sialic acids. ELISA was used to assay the interaction between mCD33-Fc and BSM as described in Materials and Methods. In some instances, the BSM was also treated with mild periodate to truncate sialic acid side chains. The mean _A_405 values of triplicate wells are plotted for each sample. The error bars indicate standard deviations.

FIG. 2.

FIG. 2.

CD33 expression on mouse hematopoietic cells. (A) Hematopoietic cells in bone marrow from wild-type (top panel) and CD33-deficient (bottom panel) mice were stained with the anti-CD33 antibody and analyzed by flow cytometry. The majority of Mac-1-positive cells stained weakly. (B) CD33 expression on leukocytes from various tissues. Solid bars, wild-type cells; open bars, CD33-deficient cells. MFI, median fluorescence intensity. (C) Examples of the light microscopic appearance of Giemsa-stained CD33-positive cells obtained by cell sorting from peripheral blood or bone marrow. The dominant morphology in both compartments is that of mature granulocytes.

FIG. 3.

FIG. 3.

Targeted deletion of the mCD33 gene. A CD33 gene-targeting construct was prepared by replacing the 3.8-kb DNA fragment containing exons 1 to 5 of the CD33 gene with the pPGK-neo-bpA cassette, with flanking 1.3- and 7.2-kb arms to mediate homologous recombination. A, _Apa_I; E, _Eco_RI; H, _Hin_dIII; S, _Sac_I.

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