Multivalent Interactions of Human Primary Amine Oxidase with the V and C22 Domains of Sialic Acid-Binding Immunoglobulin-Like Lectin-9 Regulate Its Binding and Amine Oxidase Activity.
ABSTRACT: Sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) on leukocyte surface is a counter-receptor for endothelial cell surface adhesin, human primary amine oxidase (hAOC3), a target protein for anti-inflammatory agents. This interaction can be used to detect inflammation and cancer in vivo, since the labeled peptides derived from the second C2 domain (C22) of Siglec-9 specifically bind to the inflammation-inducible hAOC3. As limited knowledge on the interaction between Siglec-9 and hAOC3 has hampered both hAOC3-targeted drug design and in vivo imaging applications, we have now produced and purified the extracellular region of Siglec-9 (Siglec-9-EC) consisting of the V, C21 and C22 domains, modeled its 3D structure and characterized the hAOC3-Siglec-9 interactions using biophysical methods and activity/inhibition assays. Our results assign individual, previously unknown roles for the V and C22 domains. The V domain is responsible for the unusually tight Siglec-9-hAOC3 interactions whereas the intact C22 domain of Siglec-9 is required for modulating the enzymatic activity of hAOC3, crucial for the hAOC3-mediated leukocyte trafficking. By characterizing the Siglec-9-EC mutants, we could conclude that R120 in the V domain likely interacts with the terminal sialic acids of hAOC3 attached glycans whereas residues R284 and R290 in C22 are involved in the interactions with the active site channel of hAOC3. Furthermore, the C22 domain binding enhances the enzymatic activity of hAOC3 although the sialic acid-binding capacity of the V domain of Siglec-9 is abolished by the R120S mutation. To conclude, our results prove that the V and C22 domains of Siglec-9-EC interact with hAOC3 in a multifaceted and unique way, forming both glycan-mediated and direct protein-protein interactions, respectively. The reported results on the mechanism of the Siglec-9-hAOC3 interaction are valuable for the development of hAOC3-targeted therapeutics and diagnostic tools.
Project description:Human primary amine oxidase (hAOC3), also known as vascular adhesion protein 1, mediates leukocyte rolling and trafficking to sites of inflammation by a multistep adhesion cascade. hAOC3 is absent on the endothelium of normal tissues and is kept upregulated during inflammatory conditions, which is an applicable advantage for imaging inflammatory diseases. Sialic acid binding immunoglobulin like-lectin 9 (Siglec-9) is a leukocyte ligand for hAOC3. The peptide (CARLSLSWRGLTLCPSK) based on the region of Siglec-9 that interacts with hAOC3, can be used as a specific tracer for hAOC3-targeted imaging of inflammation using Positron Emission Tomography (PET). In the present study, we show that the Siglec-9 peptide binds to hAOC3 and triggers its amine oxidase activity towards benzylamine. Furthermore, the hAOC3 inhibitors semicarbazide and imidazole reduce the binding of wild type and Arg/Ala mutated Siglec-9 peptides to hAOC3. Molecular docking of the Siglec-9 peptide is in accordance with the experimental results and predicts that the R3 residue in the peptide interacts in the catalytic site of hAOC3 when the topaquinone cofactor is in the non-catalytic on-copper conformation. The predicted binding mode of Siglec-9 peptide to hAOC3 is supported by the PET studies using rodent, rabbit and pig AOC3 proteins.
Project description:Interactions between endothelial selectins and the leukocyte counter-receptor PSGL1 mediates leukocyte recruitment to inflammation sites. PSGL1 is highly sialylated, making it a potential ligand for Siglec-5, a leukocyte-receptor that recognizes sialic acid structures. Binding assays using soluble Siglec-5 variants (sSiglec-5/C4BP and sSiglec-5/Fc) revealed a dose- and calcium-dependent binding to PSGL1. Pre-treatment of PSGL1 with sialidase reduced Siglec-5 binding by 79?±?4%. In confocal immune-fluorescence assays, we observed that 50% of Peripheral Blood Mononuclear Cells (PBMCs) simultaneously express PSGL1 and Siglec-5. Duolink-proximity ligation analysis demonstrated that PSGL1 and Siglec-5 are in close proximity (<40?nm) in 31?±?4% of PBMCs. In vitro perfusion assays revealed that leukocyte-rolling over E- and P-selectin was inhibited by sSiglec-5/Fc or sSiglec-5/C4BP, while adhesion onto VCAM1 was unaffected. When applied to healthy mice (0.8?mg/kg), sSiglec-5/C4BP significantly reduced the number of rolling leukocytes under basal conditions (10.9?±?3.7 versus 23.5?±?9.3 leukocytes/field/min for sSiglec-5/C4BP-treated and control mice, respectively; p?=?0.0093). Moreover, leukocyte recruitment was inhibited over a 5-h observation period in an in vivo model of TNFalpha-induced inflammation following injection sSiglec-5/C4BP (0.8?mg/kg). Our data identify PSGL1 as a ligand for Siglec-5, and soluble Siglec-5 variants appear efficient in blocking PSGL1-mediated leukocyte rolling and the inflammatory response in general.
Project description:Sialic acid-binding immunoglobulin-like lectins (Siglec)-like domains of streptococcal serine-rich repeat (SRR) adhesins recognize sialylated glycans on human salivary, platelet, and plasma glycoproteins via a YTRY sequence motif. The SRR adhesin from Streptococcus sanguinis strain SK1 has tandem sialoglycan-binding domains and has previously been shown to bind sialoglycans with high affinity. However, both domains contain substitutions within the canonical YTRY motif, making it unclear how they interact with host receptors. To identify how the S. sanguinis strain SK1 SRR adhesin affects interactions with sialylated glycans and glycoproteins, we determined high-resolution crystal structures of the binding domains alone and with purified trisaccharides. These structural studies determined that the ligands still bind at the noncanonical binding motif, but with fewer hydrogen-bonding interactions to the protein than is observed in structures of other Siglec-like adhesins. Complementary biochemical studies identified that each of the two binding domains has a different selectivity profile. Interestingly, the binding of SK1 to platelets and plasma glycoproteins identified that the interaction to some host targets is dominated by the contribution of one binding domain, whereas the binding to other host receptors is mediated by both binding domains. These results provide insight into outstanding questions concerning the roles of tandem domains in targeting host receptors and suggest mechanisms for how pathogens can adapt to the availability of a range of related but nonidentical host receptors. They further suggest that the definition of the YTRY motif should be changed to ?TRX, a more rigorous description of this sialic acid-recognition motif given recent findings.
Project description: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.
Project description:Sialic acid (Sia) Ig-like binding lectins are important mediators of recognition and signaling events among myeloid cells. To investigate the molecular mechanism underlying sialic acid Ig-like lectin (Siglec) functions, we determined the crystal structure of the two N-terminal extracellular domains of human myeloid cell inhibitory receptor Siglec-5 (CD170) and its complexes with two sialylated carbohydrates. The native structure revealed an unusual conformation of the CC' ligand specificity loop and a unique interdomain disulfide bond. The alpha(2,3)- and alpha(2,6)-sialyllactose complexed structures showed a conserved Sia recognition motif that involves both Arg124 and a portion of the G-strand in the V-set domain forming beta-sheet-like hydrogen bonds with the glycerol side chain of the Sia. Only few protein contacts to the subterminal sugars are observed and mediated by the highly variable GG' linker and CC' loop. These structural observations, in conjunction with surface plasmon resonance binding assays, provide mechanistic insights into linkage-dependent Siglec carbohydrate recognition and suggest that Siglec-5 and other CD33-related Siglec receptors are more promiscuous in sialoglycan recognition than previously understood.
Project description:BACKGROUND:Leishmania donovani, belonging to a unicellular protozoan parasite, display the differential level of linkage-specific sialic acids on their surface. Sialic acids binding immunoglobulin-like lectins (siglecs) are a class of membrane-bound receptors present in the haematopoetic cell lineages interact with the linkage-specific sialic acids. Here we aimed to explore the utilization of sialic acids by Leishmania donovani for siglec-mediated binding, phagocytosis, modulation of innate immune response and signaling pathways for establishment of successful infection in the host. METHODOLOGY/PRINCIPLE FINDINGS:We have found enhanced binding of high sialic acids containing virulent strains (AG83+Sias) with siglec-1 and siglec-5 present on macrophages compared to sialidase treated AG83+Sias (AG83-Sias) and low sialic acids-containing avirulent strain (UR6) by flow cytometry. This specific receptor-ligand interaction between sialic acids and siglecs were further confirmed by confocal microscopy. Sialic acids-siglec-1-mediated interaction of AG83+Sias with macrophages induced enhanced phagocytosis. Additionally, sialic acids-siglec-5 interaction demonstrated reduced ROS, NO generation and Th2 dominant cytokine response upon infection with AG83+Sias in contrast to AG83-Sias and UR6. Sialic acids-siglecs binding also facilitated multiplication of intracellular amastigotes. Moreover, AG83+Sias induced sialic acids-siglec-5-mediated upregulation of host phosphatase SHP-1. Such sialic acids-siglec interaction was responsible for further downregulation of MAPKs (p38, ERK and JNK) and PI3K/Akt pathways followed by the reduced translocation of p65 subunit of NF-?? to the nucleus from cytosol in the downstream signaling pathways. This sequence of events was reversed in AG83-Sias and UR6-infected macrophages. Besides, siglec-knockdown macrophages also showed the reversal of AG83+Sias infection-induced effector functions and downstream signaling events. CONCLUSIONS/SIGNIFICANCES:Taken together, this study demonstrated that virulent parasite (AG83+Sias) establish a unique sialic acids-mediated binding and subsequent phagocytosis in the host cell through the selective exploitation of siglec-1. Additionally, sialic acids-siglec-5 interaction altered the downstream signaling pathways which contributed impairment of immune effector functions of macrophages. To the best of our knowledge, this is a comprehensive report describing sialic acids-siglec interactions and their role in facilitating uptake of the virulent parasite within the host.
Project description:Siglecs are sialic acid-binding Ig-like lectins that recognize sialoglycans via amino-terminal V-set domains. CD33-related Siglecs (CD33rSiglecs) on innate immune cells recognize endogenous sialoglycans as "self-associated molecular patterns" (SAMPs), dampening immune responses via cytosolic immunoreceptor tyrosine-based inhibition motifs that recruit tyrosine phosphatases. However, sialic acid-expressing pathogens subvert this mechanism through molecular mimicry. Meanwhile, endogenous host SAMPs must continually evolve to evade other pathogens that exploit sialic acids as invasion targets. We hypothesized that these opposing selection forces have accelerated CD33rSiglec evolution. We address this by comparative analysis of major CD33rSiglec (Siglec-3, Siglec-5, and Siglec-9) orthologs in humans, chimpanzees, and baboons. Recombinant soluble molecules displaying ligand-binding domains show marked quantitative and qualitative interspecies differences in interactions with strains of the sialylated pathogen, group B Streptococcus, and with sialoglycans presented as gangliosides or in the form of sialoglycan microarrays, including variations such as N-glycolyl and O-acetyl groups. Primate Siglecs also show quantitative and qualitative intra- and interspecies variations in expression patterns on leukocytes, both in circulation and in tissues. Taken together our data explain why the CD33rSiglec-encoding gene cluster is undergoing rapid evolution via multiple mechanisms, driven by the need to maintain self-recognition by innate immune cells, while escaping 2 distinct mechanisms of pathogen subversion.
Project description:Human neutrophil Siglec-9 is a lectin that recognizes sialic acids (Sias) via an amino-terminal V-set Ig domain and possesses tyrosine-based inhibitory motifs in its cytoplasmic tail. We hypothesized that Siglec-9 recognizes host Sias as "self," including in cis interactions with Sias on the neutrophil's own surface, thereby dampening unwanted neutrophil reactivity. Here we show that neutrophils presented with immobilized multimerized Siaalpha2-3Galbeta1-4GlcNAc units engage them in trans via Siglec-9. The sialylated capsular polysaccharide of group B Streptococcus (GBS) also presents terminal Siaalpha2-3Galbeta1-4GlcNAc units, and similarly engages neutrophil Siglec-9, dampening neutrophil responses in a Sia- and Siglec-9-dependent manner. Reduction in the neutrophil oxidative burst, diminished formation of neutrophil extracellular DNA traps, and increased bacterial survival are also facilitated by GBS sialylated capsular polysaccharide interactions with Siglec-9. Thus, GBS can impair neutrophil defense functions by coopting a host inhibitory receptor via sialoglycan molecular mimicry, a novel mechanism of bacterial immune evasion.
Project description:Pseudomonas aeruginosa (PA) is commonly associated with nosocomial and chronic infections of lungs. We have earlier demonstrated that an acidic sugar, sialic acid, is present in PA which is recognized and bound by sialic acid binding immunoglobulin type lectins (siglecs) expressed on neutrophils. Here, we have tried to gain a detailed insight into the immunosuppressive role of sialic acid-siglec interactions in macrophage-mediated clearance of sialylated PA (PA+Sia). We have demonstrated that PA+Sia shows enhanced binding (~1.5-fold) to macrophages due to additional interactions between sialic acids and siglec-E and exhibited more phagocytosis. However, internalization of PA+Sia is associated with a reduction in respiratory burst and increase in anti-inflammatory cytokines secretion which is reversed upon desialylation of the bacteria. Phagocytosis of PA+Sia is also associated with reduced intracellular calcium ion concentrations and altered calcium-dependent signaling which negatively affects phagosome maturation. Consequently, although more PA+Sia was localized in early phagosomes (Rab5 compartment), only fewer bacteria reach into the late phagosomal compartment (Rab7). Possibly, this leads to reduced phagosome lysosome fusion where reduced numbers of PA+Sia are trafficked into lysosomes, compared to PA-Sia. Thus, internalized PA+Sia remain viable and replicates intracellularly in macrophages. We have also demonstrated that such siglec-E-sialic acid interaction recruited SHP-1/SHP-2 phosphatases which modulate MAPK and NF-?B signaling pathways. Disrupting sialic acid-siglec-E interaction by silencing siglec-E in macrophages results in improved bactericidal response against PA+Sia characterized by robust respiratory burst, enhanced intracellular calcium levels and nuclear translocation of p65 component of NF-?B complex leading to increased pro-inflammatory cytokine secretion. Taken together, we have identified that sialic acid-siglec-E interactions is another pathway utilized by PA in order to suppress macrophage antimicrobial responses and inhibit phagosome maturation, thereby persisting as an intracellular pathogen in macrophages.
Project description:Here we characterize Siglec-10 as a new member of the Siglec family of sialic acid-binding Ig-like lectins. A full-length cDNA was isolated from a human spleen library and the corresponding gene identified. Siglec-10 is predicted to contain five extracellular Ig-like domains and a cytoplasmic tail containing three putative tyrosine-based signalling motifs. Siglec-10 exhibited a high degree of sequence similarity to CD33-related Siglecs and mapped to the same region, on chromosome 19q13.3. The expressed protein was able to mediate sialic acid-dependent binding to human erythrocytes and soluble sialoglycoconjugates. Using specific antibodies, Siglec-10 was detected on subsets of human leucocytes including eosinophils, monocytes and a minor population of natural killer-like cells. The molecular properties and expression pattern suggest that Siglec-10 may function as an inhibitory receptor within the innate immune system.