Human sialoglycan ligands for immune inhibitory Siglecs.

Most human Siglecs (sialic acid binding immunoglobulin-like lectins) are expressed on the surfaces of overlapping subsets of immune cells, and most carry immunoreceptor tyrosine-based inhibitory domains on their intracellular motifs. When immune inhibitory Siglecs bind to complementary sialoglycans in their local milieu, engagement results in down-regulation of the immune response. Siglecs have come under scrutiny as potential targets of drugs to modify the course of inflammation (and other immune system responses) and as immune checkpoints in cancer. Human Siglecs bind to endogenous human sialoglycans. The identities of these endogenous human sialoglycan immune regulators are beginning to emerge, along with some general principles that may inform future investigations in this area. Among these principles is the finding that a cell type or tissue may express a ligand for a particular Siglec on a single or a very few of its sialoglycoproteins. The selected protein carrier for a particular Siglec may be unique in a certain tissue, but vary tissue-to-tissue. The binding affinity of endogenous Siglec ligands may surpass that of its binding to synthetic sialoglycan determinants by several orders of magnitude. Since most human Siglecs have evolved rapidly and are distinct from those in most other mammals, this review describes endogenous human Siglec ligands for several human immune inhibitory Siglecs. As the identities of these immune regulatory sialoglycan ligands are defined, additional opportunities to target Siglecs therapeutically may emerge.

Human brain sialoglycan ligand for CD33, a microglial inhibitory Siglec implicated in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by accumulation of misfolded proteins. Genetic studies implicate microglia, brain-resident phagocytic immune cells, in AD pathogenesis. As positive effectors, microglia clear toxic proteins, whereas as negative effectors, they release proinflammatory mediators. An imbalance of these functions contributes to AD progression. Polymorphisms of human CD33, an inhibitory microglial receptor, are linked to AD susceptibility; higher CD33 expression correlates with increased AD risk. CD33, also called Siglec-3, is a member of the sialic acid-binding immunoglobulin-type lectin (Siglec) family of immune regulatory receptors. Siglec-mediated inhibition is initiated by binding to complementary sialoglycan ligands in the tissue environment. Here, we identify a single sialoglycoprotein in human cerebral cortex that binds CD33 as well as Siglec-8, the most abundant Siglec on human microglia. The ligand, which we term receptor protein tyrosine phosphatase zeta (RPTPζ)S3L, is composed of sialylated keratan sulfate chains carried on a minor isoform/glycoform of RPTPζ (phosphacan) and is found in the extracellular milieu of the human brain parenchyma. Brains from human AD donors had twofold higher levels of RPTPζS3L than age-matched control donors, raising the possibility that RPTPζS3L overexpression limits misfolded protein clearance contributing to AD pathology. Mice express the same structure, a sialylated keratan sulfate RPTPζ isoform, that binds mouse Siglec-F and crossreacts with human CD33 and Siglec-8. Brains from mice engineered to lack RPTPζ, the sialyltransferase St3gal4, or the keratan sulfate sulfotransferase Chst1 lacked Siglec binding, establishing the ligand structure. The unique CD33 and Siglec-8 ligand, RPTPζS3L, may contribute to AD progression.

Induction of the endogenous sialoglycan ligand for eosinophil death receptor Siglec-8 in chronic rhinosinusitis with hyperplastic nasal polyposis.

Siglec-8, an immune-inhibitory sialoglycan binding lectin (S8), is expressed on the surface of eosinophils and mast cells, which are potent mediators of allergic inflammation. When S8 engages endogenous sialoglycan ligands, eosinophils undergo apoptosis and mast cell mediator release is inhibited. In the human airway, Siglec-8 ligands (S8L) are sialylated keratan sulfate chains carried on isoforms of the protein Deleted in Malignant Brain Tumors-1 (DMBT1), an immunoregulatory protein that we recently identified as the endogenous ligand for S8, DMBT1S8. We herein report that S8L is overexpressed in chronic rhinosinusitis with nasal polyposis (CRSwNP), a prevalent eosinophilic laden airway disease. Quantification and comparison of the degree to which DMBT1 carries the S8L by immunoblot analysis and lectin blot overlay, respectively, from nasal lavage showed that the S8L/DMBT1 ratio was significantly increased in CRSwNP vs. control or CRS patients. We identified the histological sites of S8L and DMBT1 expression in fresh surgically resected human nasal polyps. Histochemistry of diseased polyps and adjacent nondiseased middle turbinate (MT) tissue from CRSwNP demonstrated colocalization of S8L and DMBT1 with highest staining in submucosal glands >> epithelium > stoma. S8L expression was specifically elevated in the submucosal glands and epithelium of polyp tissue compared to MT. We hypothesize that expression of the isoform of DMBT1 carrying the Siglec-8 binding sialoglycan, DMBT1S8, is induced in polyps of CRSwNP specifically at the site of disease, is produced in the submucosal glands of polyps and secreted into the lumen of the sinonasal cavity as a host response to mitigate eosinophil-mediated inflammation.

Isolation, identification, and characterization of the human airway ligand for the eosinophil and mast cell immunoinhibitory receptor Siglec-8.

BACKGROUND: The immunoinhibitory receptor Siglec-8 on the surface of human eosinophils and mast cells binds to sialic acid-containing ligands in the local milieu, resulting in eosinophil apoptosis, inhibition of mast cell degranulation, and suppression of inflammation. Siglec-8 ligands were found on postmortem human trachea and bronchi and on upper airways in 2 compartments, cartilage and submucosal glands, but they were surprisingly absent from the epithelium. We hypothesized that Siglec-8 ligands in submucosal glands and ducts are normally transported to the airway mucus layer, which is lost during tissue preparation. OBJECTIVE: Our aim was to identify the major Siglec-8 sialoglycan ligand on the mucus layer of human airways. METHODS: Human upper airway mucus layer proteins were recovered during presurgical nasal lavage of patients at a sinus clinic. Proteins were resolved by gel electrophoresis and blotted, and Siglec-8 ligands detected. Ligands were purified by size exclusion and affinity chromatography, identified by proteomic mass spectrometry, and validated by electrophoretic and histochemical colocalization. The affinity of Siglec-8 binding to purified human airway ligand was determined by inhibition of glycan binding. RESULTS: A Siglec-8-ligand with a molecular weight of approximately 1000 kDa was found in all patient nasal lavage samples. Purification and identification revealed deleted in malignant brain tumors 1 (DMBT1) (also known by the aliases GP340 and SALSA), a large glycoprotein with multiple O-glycosylation repeats. Immunoblotting, immunohistochemistry, and enzyme treatments confirmed that Siglec-8 ligand on the human airway mucus layer is an isoform of DMBT1 carrying O-linked sialylated keratan sulfate chains (DMBT1S8). Quantitative inhibition revealed that DMBT1S8 has picomolar affinity for Siglec-8. CONCLUSION: A distinct DMBT1 isoform, DMBT1S8, is the major high-avidity ligand for Siglec-8 on human airways.

Molecular Determinants of the Differential Modulation of Cav1.2 and Cav1.3 by Nifedipine and FPL 64176.

Nifedipine and FPL 64176 (FPL), which block and potentiate L-type voltage-gated Ca2+ channels, respectively, modulate Cav1.2 more potently than Cav1.3. To identify potential strategies for developing subtype-selective inhibitors, we investigated the role of divergent amino acid residues in transmembrane domains IIIS5 and the extracellular IIIS5-3P loop region in modulation of these channels by nifedipine and FPL. Insertion of the extracellular IIIS5-3P loop from Cav1.2 into Cav1.3 (Cav1.3+) reduced the IC50 of nifedipine from 289 to 101 nM, and substitution of S1100 with an A residue, as in Cav1.2, accounted for this difference. Substituting M1030 in IIIS5 to V in Cav1.3+ (Cav1.3+V) further reduced the IC50 of nifedipine to 42 nM. FPL increased current amplitude with an EC50 of 854 nM in Cav1.3, 103 nM in Cav1.2, and 99 nM in Cav1.3+V. In contrast to nifedipine block, substitution of M1030 to V in Cav1.3 had no effect on potency of FPL potentiation of current amplitude, but slowed deactivation in the presence and absence of 10 µM FPL. FPL had no effect on deactivation of Cav1.3/dihydropyridine-insensitive (DHPi), a channel with very low sensitivity to nifedipine block (IC50 ∼93 µM), but did shift the voltage-dependence of activation by ∼-10 mV. We conclude that the M/V variation in IIIS5 and the S/A variation in the IIIS5-3P loop of Cav1.2 and Cav1.3 largely determine the difference in nifedipine potency between these two channels, but the difference in FPL potency is determined by divergent amino acids in the IIIS5-3P loop.

Congenital Disorders of Ganglioside Biosynthesis.

Gangliosides are expressed on all vertebrate cells and tissues, but are particularly abundant in the mammalian brain, where they constitute major cell-surface determinants on all nerve cells. The same four ganglioside structures, GM1, GD1a, GD1b, and GT1b, constitute the great majority of brain gangliosides in all mammals. Biosynthesis of these major brain gangliosides starts with addition of glucose to the ceramide lipid carrier followed by stepwise addition of up to six additional monosaccharides. This primarily involves the sequential action of seven glycosyltransferases, many of which appear to act specifically on glycolipid (rather than glycoprotein) acceptors. Congenital human disorders of ganglioside biosynthesis are exceedingly rare, but provide a glimpse into the functions of these major nerve cell surface components. To date, less than 100 individuals from 36 family pedigrees have been confirmed to carry deleterious mutations in ganglioside biosynthetic genes. Mutations in ST3GAL5 (coding GM3 synthase) were discovered as the basis for severe congenital infantile seizures, whereas mutations in B4GALNT1 (coding GM2/GD2 synthase) are the basis of hereditary spastic paraplegia accompanied by intellectual disability. In this review, we compile and summarize the findings of these studies, compare human disorders with mouse genetic models, and reflect on the implications for understanding ganglioside function and dysfunction in the nervous system.