OB-BP1/Siglec-6

Abstract

We report the expression cloning of a novel leptinbinding protein of the immunoglobulin superfamily (OB-BP1) and a cross-hybridizing clone (OB-BP2) that is identical to a recently described sialic acid-binding Itype lectin called Siglec-5. Comparisons to other known Siglec family members (CD22, CD33, myelin-associated glycoprotein, and sialoadhesin) show that OB-BP1, OB-BP2/Siglec-5, and CD33/Siglec-3 constitute a unique related subgroup with a high level of overall amino acid identity: OB-BP1 versus Siglec-5 (59%), OB-BP1 versus CD33 (63%), and OB-BP2/Siglec-5 versus CD33 (56%). The cytoplasmic domains are not as highly conserved, but display novel motifs which are putative sites of tyrosine phosphorylation, including an immunoreceptor tyrosine kinase inhibitory motif and a motif found in SLAM and SLAM-like proteins. Human tissues showed high levels of OB-BP1 mRNA in placenta and moderate expression in spleen, peripheral blood leukocytes, and small intestine. OB-BP2/Siglec-5 mRNA was detected in peripheral blood leukocytes, lung, spleen, and placenta. A monoclonal antibody specific for OB-BP1 confirmed high expression in the cyto-and syncytiotrophoblasts of the placenta. Using this antibody on peripheral blood leukocytes showed an almost exclusive expression pattern on B cells. Recombinant forms of the extracellular domains of OB-BP1, OB-BP2/Siglec-5, and CD33/Siglec-3 were assayed for specific binding of leptin. While OB-BP1 exhibited tight binding (K d 91 nM), the other two showed weak binding with K d values in the 1-2 M range. Studies with sialylated ligands indicated that OB-BP1 selectively bound Neu5Ac␣2-6GalNAc␣ (sialylTn) allowing its formal designation as Siglec-6. The identification of OB-BP1/Siglec-6 as a Siglec family member, coupled with its restricted expression pattern, suggests that it may mediate cell-cell recognition events by interacting with sialylated glycoprotein ligands expressed on specific cell populations. We also propose a role for OB-BP1 in leptin physiology, as a molecular sink to regulate leptin serum levels.Sialic acid-binding immunoglobulin superfamily member lectins (Siglecs), 1 a recently designated family of cell surface molecules (1), are a subset of the I-type lectins (2), which in turn belong to the larger immunoglobulin superfamily. Siglecs share conserved cysteine residues which form two characteristic disulfide bonds: an intra-␤-sheet bond within the NH 2 -terminal V-set immunoglobulin (Ig) domain, the other between the V-set domain and the proximal C2-set domain (3). Family members each have a single V-set NH 2 -terminal Ig domain followed by a variable number of C2-set Ig domains, as many as 16 for sialoadhesin (4), or as few as one for CD33 (5). While the family members are notable for structural similarities within their extracellular domains, overall primary amino acid sequence identities among them is relatively low (ϳ30%). In contrast to the majority of immunoglobulin superfamily members which recognize protein ligands, Siglec family members have all been shown to...

Cloning and Characterization of a Novel Mouse Siglec, mSiglec-F

“…7). However, in view of the fact that, unlike hSiglec-6, mSiglec-F is not expressed on B lymphoid cells but restricted to cells of myeloid lineage, we consider that hSiglec-5 is more likely to be the ortholog of mSiglec-F. Further support comes from the finding that mSiglec-F is not expressed in the mouse placenta (data not shown), whereas hSiglec-6 is expressed in this fetal organ (19).…”

“…Sialic Acid Binding Specificity of mSiglec-F-Sialic acid linkage specificity was analyzed by a standard ELISA assay as previously described (16,18,19). Briefly, protein A (5 g/ml in 50 mM sodium bicarbonate buffer, pH 9.5) was immobilized to the wells of 96-well plates (Nunc, 269620) overnight at 4°C.…”

“…mSiglec-F-Fc Protein Preparation-The mSiglec-F cDNA fragment encoding the first two Ig-like domains was amplified by PCR using Pwo polymerase (Roche Molecular Biochemicals) with primers mSL5 5Ј-Expr and mSL5 3Ј-Chi2D (5Ј-ATCTCCCCAGGACACCCTGATGGTC-3Ј), digested with XbaI and cloned to XbaI-EcoRV sites of EK-Fc/pEDdC vector (16,18,19). Upon mammalian cell transfection, the resulting construct (mSiglec-F-EK-Fc/pEDdC) expressed a recombinant soluble mSiglec-F-Fc protein (a fusion protein of the first two Ig-like domains of mSiglec-F and human IgG Fc fragment, with an enterokinase cleavage site/FLAG tag (DYKDDDDK) between them).…”

“…2). To determine a more detailed ligand binding specificity, a recombinant fusion protein mSiglec-F-Fc was prepared and used in an ELISA assay, with biotinylated polyacrylamide arrays carrying multiple copies of sialylated oligosaccharides as ligands (16,18,19). As shown in Fig.…”

Siglec-7 Undergoes a Major Conformational Change When Complexed with the α(2,8)-Disialylganglioside GT1b

“…With the exceptions of myelin-associated glycoprotein (MAG, Siglec-4), found in the nervous system (9,10), and Siglec-6, expressed on placental trophoblast cells (11), siglec expression appears to be restricted to cells of the hemopoietic and immune systems. Eleven siglecs have been described in humans; of these, eight belong to the recently characterized CD33-related siglec sub-family (CD33/Siglec-3 and Siglec-5-11) (12).…”