Biomedical subjects

Paul R Crocker

Publications and source records attributed to Paul R Crocker.

At least 19 recordsLinked to original sources

PubMed2006

SOCS3 targets Siglec 7 for proteasomal degradation and blocks Siglec 7-mediated responses.

CD33-related Siglecs (sialic acid-binding immunoglobulin-like lectins) 5-11 are inhibitory receptors that contain a membrane proximal ITIM (immunoreceptor tyrosine-based inhibitory motif) (I/V/L/)XYXX(L/V), which can recruit SHP-1/2. However, little is known about the regulation of these receptors. SOCS3 (suppressor of cytokine signaling 3) is up-regulated during inflammation and competes with SHP-1/2 for binding to ITIM-like motifs on various cytokine receptors resulting in inhibition of signaling. We show that SOCS3 binds the phosphorylated ITIM of Siglec 7 and targets it for proteasomal-mediated degradation, suggesting that Siglec 7 is a novel SOCS target. Following ligation, the ECS E3 ligase is recruited by SOCS3 to target Siglec 7 for proteasomal degradation, and SOCS3 expression is decreased concomitantly. In addition, we found that SOCS3 expression blocks Siglec 7-mediated inhibition of cytokine-induced proliferation. This is the first time that a SOCS target has been reported to degrade simultaneously with the SOCS protein and that inhibitory receptors have been shown to be degraded in this way. This may be a mechanism by which the inflammatory response is potentiated during infection.

Animals↗

PubMed2006

Plasmacytoid dendritic cells do not migrate in intestinal or hepatic lymph.

Plasmacytoid dendritic cells (pDCs) recognize pathogen-associated molecules, particularly viral, and represent an important mechanism in innate defense. They may however, also have roles in steady-state tolerogenic responses at mucosal sites. pDCs can be isolated from blood, mucosa, and lymph nodes (LNs). Although pDCs can express peripherally derived Ags in LNs and at mucosal sites, it is not clear whether pDCs actually migrate from the periphery in lymph or whether LN pDCs acquire Ags by other mechanisms. To determine whether pDCs migrate in lymph, intestine or liver-draining LNs were removed and thoracic duct leukocytes (TDLs) were collected. TDLs expressing MHC-II and CD45R, but not TCRalphabeta or CD45RA, were then analyzed. These enriched TDLs neither transcribe type I IFNs nor secrete inflammatory cytokines in response to viral stimuli in vitro or after a TLR7/8 stimulus in vivo. In addition, these TDLs do not express CD5, CD90, CD200, or Siglec-H, but do express Ig, and therefore represent B cells, despite their lack of CD45RA expression. Intestinal and hepatic lymph are hence devoid of bona fide pDCs under both steady-state conditions and after TLR7/8 stimulation. This shows that any role for pDCs in Ag-specific T cell activation or tolerance must differ from the roles of classical dendritic cells, because it cannot result from peripheral Ag capture, followed by migration of pDCs via lymph to the LN.

Animals↗

PubMed2006

Crystallographic and in silico analysis of the sialoside-binding characteristics of the Siglec sialoadhesin.

The Siglec family of receptors mediates cell-surface interactions through recognition of sialylated glycoconjugates. Previously reported structures of the N-terminal domain of the Siglec sialoadhesin (SnD1) in complex with various sialic acid analogs revealed the structural template for sialic acid binding. To characterize further the carbohydrate-binding properties, we have determined the crystal structures of SnD1 in the absence of ligand, and in complex with 2-benzyl-Neu5NPro and 2-benzyl-Neu5NAc. These structures reveal that SnD1 undergoes very few structural changes on ligand binding and detail how two novel classes of sialic acid analogs bind, one of which unexpectedly can induce Siglec dimerization. In conjunction with in silico analysis, this set of structures informs us about the design of putative ligands with enhanced binding affinities and specificities to different Siglecs, and provides data with which to test the effectiveness of different computational drug design protocols.

Algorithms↗

PubMed2006

Sialoadhesin deficiency ameliorates myelin degeneration and axonopathic changes in the CNS of PLP overexpressing mice.

PLP overexpressing mice display demyelination and axonopathic changes, accompanied by an elevation of CD8+ T-lymphocytes and CD11b+ macrophages in the CNS. By crossbreeding these mutants with RAG-1-deficient mice lacking mature lymphocytes, we could recently demonstrate a pathogenetic impact of the CD8+ cells. In the present study, we investigated the pathogenetic impact of CD11b+ macrophages by crossbreeding the myelin mutants with knockout mice deficient for the macrophage-restricted adhesion molecule sialoadhesin (Sn). In the wild-type mice, Sn is barely detectable on CD11b+ cells, whereas in the myelin mutants, almost all CD11b+ cells express Sn. In the double mutants, upregulation of CD8+ T-cells and CD11b+ macrophages is reduced and pathological alterations are ameliorated. These data indicate that in a primarily genetically caused myelin disorder of the CNS macrophages expressing Sn partially mediate pathogenesis. These findings may have substantial impact on treatment strategies for leukodystrophic disorders and some forms of multiple sclerosis.

Animals↗

PubMed2006

Sialoadhesin promotes the inflammatory response in experimental autoimmune uveoretinitis.

Macrophages are a prominent component of the effector cell compartment in a number of CD4+ T cell-mediated organ-specific autoimmune diseases. In this study, we investigated the role of the sialic acid binding Ig-like lectin sialoadhesin (Sn, Siglec-1) in a model of interphotoreceptor retinal binding protein peptide-induced experimental autoimmune uveoretinitis in mice with targeted deletion of Sn. Our data show that compared with wild-type mice, experimental autoimmune uveoretinitis is reduced in severity in the initial stages in the Sn knockout (KO) mice. In addition, there is a reduction in the proliferative capacity of T cells from the KO mice draining lymph nodes after immunization with interphotoreceptor retinal binding protein peptides, which is manifest some days before disease onset and persists for the duration of disease. Furthermore, activated T cells from the draining lymph nodes of Sn KO mice secrete lower levels of IFN-gamma. The data suggest a role for Sn in "fine tuning" the immune response to autoantigens by modulating T cell priming.

Amino Acid Sequence↗

PubMed2006

Siglec-7 undergoes a major conformational change when complexed with the alpha(2,8)-disialylganglioside GT1b.

The siglecs are a group of mammalian sialic acid binding receptors expressed predominantly in the immune system. The CD33-related siglecs show complex recognition patterns for sialylated glycans. Siglec-7 shows a preference for alpha(2,8)-disialylated ligands and provides a structural template for studying the key interactions that drive this selectivity. We have co-crystallized Siglec-7 with a synthetic oligosaccharide corresponding to the alpha(2,8)-disialylated ganglioside GT1b. The crystal structure of the complex offers a first glimpse into how this important family of lectins binds the structurally diverse gangliosides. The structure reveals that the C-C' loop, a region implicated in previous studies as driving siglec specificity, undergoes a dramatic conformational shift, allowing it to interact with the underlying neutral glycan core of the ganglioside. The structural data in combination with mutagenesis studies show that binding of the ganglioside is driven by extensive hydrophobic contacts together with key polar interactions and that the binding site structure is complementary to preferred solution conformations of GT1b.

Amino Acid Sequence↗

PubMed2006

Probing the cis interactions of the inhibitory receptor Siglec-7 with alpha2,8-disialylated ligands on natural killer cells and other leukocytes using glycan-specific antibodies and by analysis of alpha2,8-sialyltransferase gene expression.

Siglec-7 is a CD33-related sialic acid-binding Ig-like lectin expressed strongly on NK cells, where it can function as an inhibitory receptor. Its sialic acid-binding activity on NK cells is masked by cis interactions with sialylated glycans, which are likely to be important for regulating the inhibitory function of Siglec-7, which exhibits an unusual preference for alpha2,8-linked disialic acids, a motif found in "b-series" gangliosides and some glycoproteins. To investigate the presence of alpha2,8-linked disialic acids on NK cells, T cells, monocytes, and B cells, we first analyzed their expression of all known alpha2,8-sialyltransferase genes by quantitative PCR. Unlike T cells, B cells, and monocytes, NK cells consistently expressed mRNA encoding ST8Sia VI, which creates alpha2,8-linked disialic acids on O-linked glycans of glycoproteins. All blood leukocytes expressed ST8Sia IV, implicated in polysialic acid synthesis, and NK cells variably expressed high levels of ST8Sia V mRNA required for GT3 expression. Two human IgM antibodies, Ha1 and Pi1, with specificity for the alpha2,8-disialyl motif reacted strongly with NK cells in a sialic acid-dependent manner and less strongly with T cells and monocytes. Antibody-induced clustering of Siglec-7 on NK cells resulted in partial colocalization with anti-Ha1. Finally, MALDI-TOF mass spectrometric analysis of isolated NK cell O-glycans revealed the presence of a peak at mass-to-charge ratio of 1619.4 mass units, corresponding to a putative alpha2,8-disialylated glycan. Together, these results suggest that NK cells are decorated with alpha2,8-disialic acid structures implicated in regulation of cellular activation via interactions with Siglec-7.

Animals↗

PubMed2006

The structure of siglec-7 in complex with sialosides: leads for rational structure-based inhibitor design.

Siglecs (sialic acid binding Ig-like lectins) are transmembrane receptors for sialylated glycoconjugates that modulate cellular interactions and signalling events in the haematopoietic, immune and nervous systems. Siglec-7 is a structural prototype for the recently described family of immune inhibitory CD33-related siglecs and is predominantly expressed on natural killer cells and monocytes, as well as subsets of CD8 T-cells. Siglec-specific inhibitors are desired for the detection of masked and unmasked forms of siglecs, to aid in dissection of signalling pathways and as tools to investigate siglecs as potential therapeutic targets. As a first step towards this end, we present the crystal structure of siglec-7 in complex with a sialylated ligand, the ganglioside analogue DSLc4 [alpha(2,3)/alpha(2,6) disialyl lactotetraosyl 2-(trimethylsilyl)ethyl], which allows for a detailed description of the binding site, required for structure-guided inhibitor design. Mutagenesis and binding assays were used to demonstrate a key structural role for Lys131, a residue that changes conformation upon sialic acid binding. Differences between the binding sites of siglec family members were then exploited using alpha-methyl Neu5Ac (N-acetylneuraminic acid) as a basic scaffold. A co-crystal of siglec-7 in complex with the sialoside inhibitor, oxamido-Neu5Ac [methyl alpha-9-(amino-oxalyl-amino)-9-deoxy-Neu5Ac] and inhibition data for the sialosides gives clear leads for future inhibitor design.

Animals↗

PubMed2006

Analysis of the CD33-related siglec family reveals that Siglec-9 is an endocytic receptor expressed on subsets of acute myeloid leukemia cells and absent from normal hematopoietic progenitors.

CD33 (Siglec-3) is expressed on most acute myeloid leukemia (AML) cells and is currently being exploited as a therapeutic target. The purpose of this study was to investigate the expression pattern and potential utility of the seven recently described CD33-related siglecs as markers in AML. Besides CD33, Siglec-9 was the most highly expressed, particularly on AML cells with features of monocytic differentiation that also expressed Siglecs-5 and -7. Siglec-9 was absent from normal bone marrow myeloid progenitors but present on monocytic precursors. Using primary AML cells or transfected rat basophilic leukemia cells, Siglec-9 mediated rapid endocytosis of anti-Siglec-9 mAb. In contrast to CD33 and Siglec-5, levels of soluble Siglec-9 were low or undetectable in bone marrow plasma from AML patients and serum from normal donors. These features suggest that Siglec-9 provides not only a useful marker for certain subsets of AML, but also a potential therapeutic target.

Acute Disease↗

PubMed2006

The antigen recognized by MOMA-I is sialoadhesin.

The monoclonal antibody MOMA-1 is a widely-used marker for marginal metallophilic macrophages in spleen and some other subsets of macrophages. The antigen recognized by MOMA-1 has yet to be characterized, but its expression pattern is similar to that of sialoadhesin (Sn, CD169, Siglec-1), a member of the sialic acid binding Ig-like lectin (Siglec) family. Using flow cytometry of Sn-transfected cells and staining of lymphoid tissue sections from Sn-deficient mice, we demonstrate here that the antigen recognized by MOMA-1 is Sn.

Animals↗

PubMed2006

Carbohydrate microarrays reveal sulphation as a modulator of siglec binding.

Siglecs are receptors on cells of the immune, haemopoietic, and nervous systems that recognize sialyl-glycans with differing preferences for sialic acid linkage and oligosaccharide backbone sequence. We investigate here siglec binding using microarrays of Lewis(x) (Le(x))- and 3'-sialyl-Le(x)-related probes with different sulphation patterns. These include sulphation at position 3 of the terminal galactose of Le(x), position 6 of the galactose of Le(x) and sialyl-Le(x), position 6 of N-acetylglucosamine of Le(x) and sialyl-Le(x), or both positions of sialyl-Le(x). Recombinant soluble forms of five siglecs have been investigated: human Siglec-7, -8, -9, and murine Siglec-F and CD22 (Siglec-2). Each siglec has a different binding pattern. Unlike two C-type lectins of leukocytes, L-selectin and Langerin, which also bind to sulphated analogues of sialyl-Le(x), the siglecs do not give detectable binding signals with sulphated analogues that are lacking sialic acid. The sulphate groups modulate, however, positively or negatively the siglec binding intensities to the sialyl-Le(x) sequence.

Animals↗

PubMed2006

Characterization of Siglec-H as a novel endocytic receptor expressed on murine plasmacytoid dendritic cell precursors.

We describe the cloning and characterization of Siglec-H, a novel murine CD33-related siglec-like molecule with 2 immunoglobulin domains. Unlike other CD33-related siglecs, Siglec-H lacks tyrosine-based signaling motifs in its cytoplasmic tail. Although Siglec-H has the typical structural features required for sialic acid binding, no evidence for carbohydrate recognition was obtained. Specific monoclonal and polyclonal antibodies (Abs) were raised to Siglec-H and used to define its cellular expression pattern and functional properties. By flow cytometry, Siglec-H was expressed specifically on plasmacytoid dendritic cell (pDC) precursors in bone marrow, spleen, blood, and lymph nodes. Staining of tissue sections showed that Siglec-H was also expressed in a subset of marginal zone macrophages in the spleen and in medullary macrophages in lymph nodes. Using bone marrow-derived pDC precursors that express Siglec-H, addition of Abs did not influence cytokine production, either in the presence or absence of synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG). In comparison, Siglec-H functioned as an endocytic receptor and mediated efficient internalization of anti-Siglec-H Abs. By immunizing mice with ovalbumin-conjugated anti-Siglec-H Ab in the presence of CpG, we demonstrate generation of antigen-specific CD8 T cells in vivo. Targeting Siglec-H may therefore be a useful way of delivering antigens to pDC precursors for cross-presentation.

Amino Acid Sequence↗

PubMed2006

Sialic acid-binding immunoglobulin-like lectin 7 mediates selective recognition of sialylated glycans expressed on Campylobacter jejuni lipooligosaccharides.

siglecs are a family of sialic-acid binding immunoglobulin-like lectins mostly expressed by cells of the immune system that have the potential to interact with sialylated glycans expressed not only on host cells but also on certain pathogens. Campylobacter jejuni is a common pathogen of humans that expresses surface lipooligosaccharides (LOS) that can be modified with ganglioside-like terminal structures in the core oligosaccharides. In this study, we examined the interaction of 10 siglecs with LOS purified from four different C. jejuni isolates expressing GM1-like, GD1a-like, GD3-like, and GT1a-like oligosaccharides. Of all siglecs examined, only Siglec-7 exhibited specific, sialic acid-dependent interactions with C. jejuni LOS in solid-phase binding assays. Binding was especially prominent with LOS from the HS:19(GM1(+) GT1a(+)) isolate, with weaker binding with LOS from the HS:19(GD3(+)) isolate. Binding of Siglec-7 was also observed with intact bacteria expressing these LOS structures. Specific binding of HS:19(GM1(+) GT1a(+)) bacteria was demonstrated with Siglec-7 expressed on transfected Chinese hamster ovary cells and with peripheral blood leukocytes, among which HS:19(GM1(+) GT1a(+)) bacteria bound selectively to both natural killer cells and monocytes which naturally express Siglec-7. These results raise the possibility that, in addition to their role in generating autoimmune antibody responses, C. jejuni LOS could interact with Siglec-7 expressed by leukocytes, modulate the host-pathogen interaction, and contribute to the clinical outcome and the development of secondary complications such as Guillain-Barré syndrome.

Animals↗

PubMed2006

Sialoadhesin-deficient mice exhibit subtle changes in B- and T-cell populations and reduced immunoglobulin M levels.

Sialoadhesin (Sn, also called Siglec-1 or CD169) is a transmembrane receptor and the prototypic member of the Siglec family of sialic acid binding immunoglobulin-like lectins. It is expressed on specialized subsets of resident macrophages in hematopoietic and lymphoid tissues and on inflammatory macrophages. In order to investigate its function, we generated Sn-deficient mice and confirmed that these mice are true nulls by fluorescence-activated cell sorter analysis and immunohistochemistry. Mice deficient in Sn were viable and fertile and showed no developmental abnormalities. Analysis of cell populations revealed no differences in bone marrow, peritoneal cavity, and thymus, but there was a small increase in CD8 T cells and a decrease in B220-positive cells in spleens and lymph nodes of Sn-deficient mice. Furthermore, in spleen there was a slight decrease in follicular B cells with an increase in numbers of marginal zone B cells. B- and T-cell maturation as well as responses to stimulation with thioglycolate were only slightly affected by Sn deficiency. Immunoglobulin titers in Sn-deficient mice were significantly decreased for immunoglobulin M (IgM) but similar for IgG subclasses. These results suggest a role for sialoadhesin in regulating cells of the immune system rather than in influencing steady-state hematopoiesis.

Animals↗

PubMed2005

Mouse Siglec-F and human Siglec-8 are functionally convergent paralogs that are selectively expressed on eosinophils and recognize 6'-sulfo-sialyl Lewis X as a preferred glycan ligand.

Mouse sialic acid-binding immunoglobulin-like lectin F (Siglec-F) is an eosinophil surface receptor, which contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain, implicating it as a regulator of cell signaling as documented for other siglecs. Here, we show that the sialoside sequence 6'-sulfo-sLe(X) (Neu5Acalpha2-3[6-SO4] Galbeta1-4[Fucalpha1-3]GlcNAc) is a preferred ligand for Siglec-F. In glycan array analysis of 172 glycans, recombinant Siglec-F-Fc chimeras bound with the highest avidity to 6'-sulfo-sLe X. Secondary analysis showed that related structures, sialyl-Lewis X (sLe X) and 6-sulfo-sLe X containing 6-GlcNAc-SO4 showed much lower binding avidity, indicating significant contribution of 6-Gal-SO4 on Siglec-F binding to 6'-sulfo-sLe x. The lectin activity of Siglec-F on mouse eosinophils was "masked" by endogenous cis ligands and could be unmasked by treatment with sialidase. Unmasked Siglec-F mediated mouse eosinophil binding and adhesion to multivalent 6'-sulfo-sLe X structure, and these interactions were inhibited by anti-Siglec-F monoclonal antibody (mAb). Although there is no clear-cut human ortholog of Siglec-F, Siglec-8 is encoded by a paralogous gene that is expressed selectively by human eosinophils and has recently been found to recognize 6'-sulfo-sLe X. These observations suggest that mouse Siglec-F and human Siglec-8 have undergone functional convergence during evolution and implicate a role for the interaction of these siglecs with their preferred 6'-sulfo-sLe X ligand in eosinophil biology.

Animals↗

PubMed2005

Siglec-5 (CD170) can mediate inhibitory signaling in the absence of immunoreceptor tyrosine-based inhibitory motif phosphorylation.

Siglec-5 (CD170) is a member of the recently described human CD33-related siglec subgroup of sialic acid binding Ig-like lectins and is expressed on myeloid cells of the hemopoietic system. Similar to other CD33-related siglecs, Siglec-5 contains two tyrosine-based motifs in its cytoplasmic tail implicated in signaling functions. To investigate the role of these motifs in Siglec-5-dependent signaling, we used transfected rat basophil leukemia cells as a model system. Tyrosine phosphorylation of Siglec-5 led to recruitment of the tyrosine phosphatases SHP-1 and SHP-2, as seen in both pull-down assays and microscopy. Siglec-5 could efficiently inhibit FcepsilonRI-mediated calcium fluxing and serotonin release after co-cross-linking. Surprisingly, a double tyrosine to alanine mutant of Siglec-5 could still mediate strong inhibition of serotonin release in the absence of detectable tyrosine phosphorylation, whereas a double tyrosine to phenylalanine mutant lost all inhibitory activity. In comparison, suppression of Siglec-5-dependent adhesion to red blood cells was reversed by either tyrosine to alanine or tyrosine to phenylalanine mutations of the membrane proximal tyrosine-based motif. Using an in vitro phosphatase assay with synthetic and recombinant forms of the cytoplasmic tail, it was shown that a double alanine mutant of Siglec-5 had weak, but significant SHP-1 activating properties similar to those of wild type, non-phosphorylated cytoplasmic tail, whereas a double phenylalanine mutant was inactive. These findings establish that Siglec-5 can be classified as an inhibitory receptor with the potential to mediate SHP-1 and/or SHP-2-dependent signaling in the absence of tyrosine phosphorylation.

Amino Acid Motifs↗

PubMed2005

Siglecs in innate immunity.

Siglecs are sialic acid-binding Ig-like lectins expressed in a highly specific manner, and which are implicated in signaling and adhesive functions. The CD33-related siglecs represent a distinct subgroup that is undergoing rapid evolution within the innate immune system, with the potential to trigger apoptosis and provide inhibitory signals. CD22 is a well-characterised B cell restricted siglec that has been shown to mediate both sialic acid-dependent and -independent signaling functions in B cell regulation. As endocytic receptors, siglecs provide portals of entry for certain viral and bacterial pathogens, as well as therapeutic opportunities for targeting innate immune cells in disease.

Animals↗

PubMed2004

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.

Siglec-7 and Siglec-9 are two members of the recently characterized CD33-related Siglec family of sialic acid binding proteins and are both expressed on human monocytes and NK cells. In addition to their ability to recognize sialic acid residues, these Siglecs display two conserved tyrosine-based motifs in their cytoplasmic region similar to those found in inhibitory receptors of the immune system. In the present study, we use the rat basophilic leukemia (RBL) model to examine the potential of Siglecs-7 and -9 to function as inhibitory receptors and investigate the molecular basis for this. We first demonstrate that Siglecs-7 and -9 are able to inhibit the FcepsilonRI-mediated serotonin release from RBL cells following co-crosslinking. In addition, we show that under these conditions or after pervanadate treatment, Siglecs-7 and -9 associate with the Src homology region 2 domain-containing phosphatases (SHP), SHP-1 and SHP-2, both in immunoprecipitation and in fluorescence microscopy experiments using GFP fusion proteins. We then show by site-directed mutagenesis that the membrane-proximal tyrosine motif is essential for the inhibitory function of both Siglec-7 and -9, and is also required for tyrosine phosphorylation and recruitment of SHP-1 and SHP-2 phosphatases. Finally, mutation of the membrane-proximal motif increased the sialic acid binding activity of Siglecs-7 and -9, raising the possibility that "inside-out" signaling may occur to regulate ligand binding.

Amino Acid Motifs↗