Functional and structural investigation of a broadly neutralizing SARS-CoV-2 antibody.

Since its emergence, SARS-CoV-2 has been continuously evolving, hampering the effectiveness of current vaccines against COVID-19. mAbs can be used to treat patients at risk of severe COVID-19. Thus, the development of broadly protective mAbs and an understanding of the underlying protective mechanisms are of great importance. Here, we isolated mAbs from donors with breakthrough infection with Omicron subvariants using a single-B cell screening platform. We identified a mAb, O5C2, which possesses broad-spectrum neutralization and antibody-dependent cell-mediated cytotoxic activities against SARS-CoV-2 variants, including EG.5.1. Single-particle analysis by cryo-electron microscopy revealed that O5C2 targeted an unusually large epitope within the receptor-binding domain of spike protein that overlapped with the angiotensin-converting enzyme 2 binding interface. Furthermore, O5C2 effectively protected against BA.5 Omicron infection in vivo by mediating changes in transcriptomes enriched in genes involved in apoptosis and interferon responses. Our findings provide insights into the development of pan-protective mAbs against SARS-CoV-2.

Bacterial pseudaminic acid binding to Siglec-10 induces a macrophage interleukin-10 response and suppresses phagocytosis.

Pseudaminic acid (Pse) on pathogenic bacteria exopolysaccharide engages with the sialic acid-binding immunoglobulin-type lectin (Siglec)-10 receptor on macrophages via the critical 7-N-acetyl group. This binding stimulates macrophages to secrete interleukin 10 that suppresses phagocytosis against bacteria, but can be reverted by blocking Pse-Siglec-10 interaction with Pse-binding protein as a promising therapy.

Quantitative Analysis of Siglec Ligands by Flow Cytometry.

Siglecs (sialic acid-binding, immunoglobulin superfamily, lectins) are a family of transmembrane receptor-type glycan recognition proteins in vertebrates that are primarily expressed on leukocytes and regulate immune responses. Siglecs are involved in several diseases, such as cancer and neurodegenerative diseases. Most Siglecs suppress the activation of leukocytes by recognizing ligands containing sialic acid, a group of acidic sugars commonly found in vertebrate glycans, but rare among microbes. Siglec ligands are critical in the interaction between leukocytes and target cells. The abundance of the Siglec ligand is influenced by both the abundance of the glycoconjugate carrier (glycoprotein or glycolipid) and that of the terminal glycan epitope directly recognized by the Siglec. Therefore, a direct approach to evaluate the expression level of a Siglec ligand on cells of interest is to analyze the binding of recombinant Siglec protein to these cells. In this article, we describe a protocol for semi-quantitatively analyzing the expression level of Siglec ligands via flow cytometry using recombinant Siglec-Fc fusion protein. Support protocols describe how to remove sialic acids from the cell surface with sialidase under mild conditions to demonstrate the sialic acid dependence of Siglec binding, and the preparation of recombinant Siglec-Fc fusion proteins by transient transfection of mammalian cells. © 2023 Wiley Periodicals LLC. Basic Protocol: Quantitative analysis of Siglec ligands on mammalian cells via flow cytometry with recombinant Siglec-Fc fusion protein Support Protocol 1: Sialidase treatment of mammalian cells Support Protocol 2: Preparation of recombinant Siglec-Fc fusion protein via transient transfection of mammalian cells.

High Density of N- and O-Glycosylation Shields and Defines the Structural Dynamics of the Intrinsically Disordered Ectodomain of Receptor-type Protein Tyrosine Phosphatase Alpha.

The intracellular phosphatase domain of the receptor-type protein tyrosine phosphatase alpha (PTPRA) is known to regulate various signaling pathways related to cell adhesion through c-Src kinase activation. In contrast, the functional significance of its relatively short, intrinsically disordered, and heavily glycosylated ectodomain remains unclear. Through detailed mass spectrometry analyses of a combination of protease and glycosidase digests, we now provide the first experimental evidence for its site-specific glycosylation pattern. This includes the occurrence of O-glycan at the N-glycosylation sequon among the more than 30 O-glycosylation sites confidently identified beside the 7 N-glycosylation sites. The closely spaced N- and O-glycans appear to have mutually limited the extent of further galactosylation and sialylation. An immature smaller form of full-length PTPRA was found to be deficient in O-glycosylation, most likely due to failure to transit the Golgi. N-glycosylation, on the other hand, is dispensable for cell surface expression and contributes less than the extensive O-glycosylation to the overall solution structure of the ectodomain. The glycosylation information is combined with the overall structural features of the ectodomain derived from small-angle X-ray scattering and high-speed atomic force microscopy monitoring to establish a dynamic structural model of the densely glycosylated PTPRA ectodomain. The observed high structural flexibility, as manifested by continuous transitioning from fully to partially extended and fold-back conformations, suggests that the receptor-type phosphatase is anchored to the membrane and kept mostly at a monomeric state through an ectodomain shaped and fully shielded by glycosylation.

Roles of Siglecs in neurodegenerative diseases.

Microglia are resident myeloid cells in the central nervous system (CNS) with a unique developmental origin, playing essential roles in developing and maintaining the CNS environment. Recent studies have revealed the involvement of microglia in neurodegenerative diseases, such as Alzheimer's disease, through the modulation of neuroinflammation. Several members of the Siglec family of sialic acid recognition proteins are expressed on microglia. Since the discovery of the genetic association between a polymorphism in the CD33 gene and late-onset Alzheimer's disease, significant efforts have been made to elucidate the molecular mechanism underlying the association between the polymorphism and Alzheimer's disease. Furthermore, recent studies have revealed additional potential associations between Siglecs and Alzheimer's disease, implying that the reduced signal from inhibitory Siglec may have an overall protective effect in lowering the disease risk. Evidences suggesting the involvement of Siglecs in other neurodegenerative diseases are also emerging. These findings could help us predict the roles of Siglecs in other neurodegenerative diseases. However, little is known about the functionally relevant Siglec ligands in the brain, which represents a new frontier. Understanding how microglial Siglecs and their ligands in CNS contribute to the regulation of CNS homeostasis and pathogenesis of neurodegenerative diseases may provide us with a new avenue for disease prevention and intervention.

Discovery, classification, evolution and diversity of Siglecs.

Immunoglobulin (Ig) superfamily proteins play diverse roles in vertebrates, including regulation of cellular responses by sensing endogenous or exogenous ligands. Siglecs are a family of glycan-recognizing proteins belonging to the Ig superfamily (i.e., I-type lectins). Siglecs are expressed on various leukocyte types and are involved in diverse aspects of immunity, including the regulation of inflammatory responses, leukocyte proliferation, host-microbe interaction, and cancer immunity. Sialoadhesin/Siglec-1, CD22/Siglec-2, and myelin-associated glycoprotein/Siglec-4 were among the first to be characterized as members of the Siglec family, and along with Siglec-15, they are relatively well-conserved among tetrapods. Conversely, CD33/Siglec-3-related Siglecs (CD33rSiglecs, so named as they show high sequence similarity with CD33/Siglec-3) are encoded in a gene cluster with many interspecies variations and even intraspecies variations within some lineages such as humans. The rapid evolution of CD33rSiglecs expressed on leukocytes involved in innate immunity likely reflects the selective pressure by pathogens that interact and possibly exploit these Siglecs. Human Siglecs have several additional unique and/or polymorphic properties as compared with closely related great apes, changes possibly related to the loss of the sialic acid Neu5Gc, another distinctly human event in sialobiology. Multiple changes in human CD33rSiglecs compared to great apes include many examples of human-specific expression in non-immune cells, coinciding with human-specific diseases involving such cell types. Some Siglec gene polymorphisms have dual consequences-beneficial in a situation but detrimental in another. The association of human Siglec gene polymorphisms with several infectious and non-infectious diseases likely reflects the ongoing competition between the host and microbial pathogens.

Molecular Basis and Role of Siglec-7 Ligand Expression on Chronic Lymphocytic Leukemia B Cells.

Siglec-7 (sialic acid-binding immunoglobulin-like lectin 7) is an immune checkpoint-like glycan recognition protein on natural killer (NK) cells. Cancer cells often upregulate Siglec ligands to subvert immunosurveillance, but the molecular basis of Siglec ligands has been elusive. In this study, we investigated Siglec-7 ligands on chronic lymphocytic leukemia (CLL) B cells. CLL B cells express higher levels of Siglec-7 ligands compared with healthy donor B cells, and enzymatic removal of sialic acids or sialomucins makes them more sensitive to NK cell cytotoxicity. Gene knockout experiments have revealed that the sialyltransferase ST6GalNAc-IV is responsible for the biosynthesis of disialyl-T (Neu5Acα2-3Galß1-3[Neu5Acα2-6]GalNAcα1-), which is the glycotope recognized by Siglec-7, and that CD162 and CD45 are the major carriers of this glycotope on CLL B cells. Analysis of public transcriptomic datasets indicated that the low expression of GCNT1 (encoding core 2 GlcNAc transferase, an enzyme that competes against ST6GalNAc-IV) and high expression of ST6GALNAC4 (encoding ST6GalNAc-IV) in CLL B cells, together enhancing the expression of the disialyl-T glycotope, are associated with poor patient prognosis. Taken together, our results determined the molecular basis of Siglec-7 ligand overexpression that protects CLL B cells from NK cell cytotoxicity and identified disialyl-T as a potential prognostic marker of CLL.

Phosphoproteomics Reveals the Role of Constitutive KAP1 Phosphorylation by B-cell Receptor Signaling in Chronic Lymphocytic Leukemia.

Application of B-cell receptor (BCR) pathway inhibitor ibrutinib for chronic lymphocytic leukemia (CLL) is a major breakthrough, yet the downstream effects following inhibition of BCR signaling and during relapse await further clarification. By comparative phosphoproteomic profiling of B cells from patients with CLL and healthy donors, as well as CLL B cells collected at multiple time points during the course of ibrutinib treatment, we provided the landscape of dysregulated phosphoproteome in CLL and its dynamic alterations associated with ibrutinib treatment. Particularly, differential phosphorylation events associated with several signaling pathways, including BCR pathway, were enriched in patient CLL cells. A constitutively elevated phosphorylation level of KAP1 at serine 473 (S473) was found in the majority of CLL samples prior to treatment. Further verification showed that BCR activation promoted KAP1 S473 phosphorylation, whereas ibrutinib treatment abolished it. Depletion of KAP1 in primary CLL cells decelerated cell-cycle progression and ectopic expression of a KAP1 S473 phospho-mimicking mutant accelerated G2-M cell-cycle transition of CLL cells. Moreover, temporal phosphoproteomic profiles using a series of CLL cells isolated from one patient during the ibrutinib treatment revealed the dynamic changes of several molecules associated with BCR signaling in the ibrutinib responsive and recurrent stages. IMPLICATIONS: This phosphoproteomic analysis and functional validation illuminated the phosphorylation of KAP1 at S473 as an important downstream BCR signaling event and a potential indicator for the success of ibrutinib treatment in CLL.

Combining CuAAC reaction enables sialylated Bi- and triantennary pseudo mannose N-glycans for investigating Siglec-7 interactions.

Naturally occurring N-glycans display much diversity in modifications, linkages, and peripheral presentation of the oligosaccharide chain. Despite continued advancements in oligosaccharide synthesis, synthetic access to these natural glycans remains challenging. Biologically relevant complex N-glycan mimetics with various natural and unnatural modifications are an alternate way for investigating glycan-protein interactions. Further supporting this pattern, we report here a new class of sialylated bi- and triantennary pseudo mannose N-glycans reproducing orientation of the underlying glycan chain and branching patterns and replacing the two inner mannopyranosyl units with 1,2,3-triazole rings. Such mimetics are straightforwardly generated by implementing multiple intermolecular Cu(I)-catalyzed azide-alkyne cycloaddition between chemoenzymatically synthesized azido sialosides and rationally designed C-3 and C-6 di-O- or C-2, C-3, and C-6 tri-O-alkynylated mannoside. Human recombinant Siglec-7-Fc fusion protein recognizes almost all sialylated pseudo mannose N-glycans in the microarray. However, a differential Sia-binding pattern was also observed. Given the library size, comparison of pairwise mannose N-glycan combinations showed that biantennary linear α(2,3)α(2,8)- and α(2,6)α(2,8)- or branched α(2,3)α(2,6)-, and triantennary branched α(2,3)α(2,6)-sialyl pseudo N-glycans possess similar binding capabilities and affinity to recombinant Siglec-7-Fc. While the full range of topological mannose arms remain elusive, the bi- and triantennary mimics are simpler structures for interrogating Siglec interactions.

Correction: Boronate affinity-based photoactivatable magnetic nanoparticles for the oriented and irreversible conjugation of Fc-fused lectins and antibodies.

[This corrects the article DOI: 10.1039/C9SC01613A.].

Sugar nucleotide regeneration system for the synthesis of Bi- and triantennary N-glycans and exploring their activities against siglecs.

Enzymatic synthesis that is commenced by the sugar nucleotide regeneration system (SNRS) protocol can minimize 1) the consumption of exorbitant sugar nucleotides, 2) the amount of transferases required, and 3) byproduct feedback inhibition. In this study, LacNAc extensions/modifications of the N-linked mannose core were carried out efficiently with SNRS with high yields and purities on all branches in a uniform manner. In addition, we demonstrate that with SNRS, bacterial glycosyltransferases exhibit a wide acceptor tolerance for bi- and triantennary mannose core structures as substrates for target oligosaccharides. The synthesized small library of mannose core-based glycans and linear O-glycans were screened for their binding affinity against h-Siglecs 2, 4, 7, 9, 14, 15, and m-Siglec-15 to explore their structure-based binding preferences. Microarray data revealed that each Siglec showed few distinct yet overlapping specificities. An increase in branching from mono to di or tri antennary did not necessarily lead to increasing affinity. Glycans with the disialoside sequence α(2,3)α(2,8)/α(2,6)α(2,8) showed high specificity and affinity for Siglec-7, and sLex α(2,3) exhibited a strong affinity for Siglec-9. Explicit recognition of α(2,6)α(2,3)- linear and α(2,3)α(2,6)-branched glycans by Siglecs-2, 4, and 15, respectively, suggests that these structures can act as potential candidates for the further development of high-affinity ligands.

Streamlined single-cell proteomics by an integrated microfluidic chip and data-independent acquisition mass spectrometry.

Single-cell proteomics can reveal cellular phenotypic heterogeneity and cell-specific functional networks underlying biological processes. Here, we present a streamlined workflow combining microfluidic chips for all-in-one proteomic sample preparation and data-independent acquisition (DIA) mass spectrometry (MS) for proteomic analysis down to the single-cell level. The proteomics chips enable multiplexed and automated cell isolation/counting/imaging and sample processing in a single device. Combining chip-based sample handling with DIA-MS using project-specific mass spectral libraries, we profile on average ~1,500 protein groups across 20 single mammalian cells. Applying the chip-DIA workflow to profile the proteomes of adherent and non-adherent malignant cells, we cover a dynamic range of 5 orders of magnitude with good reproducibility and <16% missing values between runs. Taken together, the chip-DIA workflow offers all-in-one cell characterization, analytical sensitivity and robustness, and the option to add additional functionalities in the future, thus providing a basis for advanced single-cell proteomics applications.

Recent Progress in the Methodologies to Identify Physiological Ligands of Siglecs.

Siglecs, a family of receptor-like lectins, recognize glycoproteins and/or glycolipids containing sialic acid in the extracellular space and transduce intracellular signaling. Recently, researchers uncovered significant contributions of Siglecs in cancer immunity, renewing interest in this family of proteins. Previous extensive studies have defined how Siglecs recognize glycan epitopes (glycotopes). Nevertheless, the biological role of these glycotopes has not been fully evaluated. Recent studies using live cells have begun unraveling the constituents of Siglec ligands. These studies demonstrated that glycoprotein scaffolds (counter-receptors) displaying glycotopes are sometimes just as important as the glycotope itself. These new insights may guide future efforts to develop therapeutic agents to target the Siglec - ligand axis.

Identification and functional characterization of a Siglec-7 counter-receptor on K562 cells.

Sialic acid (Sia)-binding immunoglobulin-like lectin 7 (Siglec-7) is an inhibitory receptor primarily expressed on natural killer (NK) cells and monocytes. Siglec-7 is known to negatively regulate the innate immune system through Sia binding to distinguish self and nonself; however, a counter-receptor bearing its natural ligand remains largely unclear. Here, we identified a counter-receptor of Siglec-7 using K562 hematopoietic carcinoma cells presenting cell surface ligands for Siglec-7. We affinity-purified the ligands using Fc-ligated recombinant Siglec-7 and diSia-dextran polymer, a strong inhibitor for Siglec-7. We then confirmed the counter-receptor for Siglec-7 as leukosialin (CD43) through mass spectrometry, immunoprecipitation, and proximity labeling. Additionally, we demonstrated that the cytotoxicity of NK cells toward K562 cells was suppressed by overexpression of leukosialin in a Siglec-7-dependent manner. Taken together, our data suggest that leukosialin on K562 is a counter-receptor for Siglec-7 on NK cells and that a cluster of the Sia-containing glycan epitope on leukosialin is key as trans-ligand for unmasking the cis-ligand.

Siglec-E retards atherosclerosis by inhibiting CD36-mediated foam cell formation.

BACKGROUND: The accumulation of lipid-laden macrophages, foam cells, within sub-endothelial intima is a key feature of early atherosclerosis. Siglec-E, a mouse orthologue of human Siglec-9, is a sialic acid binding lectin predominantly expressed on the surface of myeloid cells to transduce inhibitory signal via recruitment of SH2-domain containing protein tyrosine phosphatase SHP-1/2 upon binding to its sialoglycan ligands. Whether Siglec-E expression on macrophages impacts foam cell formation and atherosclerosis remains to be established. METHODS: ApoE-deficient (apoE-/-) and apoE/Siglec-E-double deficient (apoE-/-/Siglec-E-/-) mice were placed on high fat diet for 3 months and their lipid profiles and severities of atherosclerosis were assessed. Modified low-density lipoprotein (LDL) uptake and foam cell formation in wild type (WT) and Siglec-E-/-- peritoneal macrophages were examined in vitro. Potential Siglec-E-interacting proteins were identified by proximity labeling in conjunction with proteomic analysis and confirmed by coimmunoprecipitation experiment. Impacts of Siglec-E expression and cell surface sialic acid status on oxidized LDL uptake and signaling involved were examined by biochemical assays. RESULTS: Here we show that genetic deletion of Siglec-E accelerated atherosclerosis without affecting lipid profile in apoE-/- mice. Siglec-E deficiency promotes foam cell formation by enhancing acetylated and oxidized LDL uptake without affecting cholesterol efflux in macrophages in vitro. By performing proximity labeling and proteomic analysis, we identified scavenger receptor CD36 as a cell surface protein interacting with Siglec-E. Further experiments performed in HEK293T cells transiently overexpressing Siglec-E and CD36 and peritoneal macrophages demonstrated that depletion of cell surface sialic acids by treatment with sialyltransferase inhibitor or sialidase did not affect interaction between Siglec-E and CD36 but retarded Siglec-E-mediated inhibition on oxidized LDL uptake. Subsequent experiments revealed that oxidized LDL induced transient Siglec-E tyrosine phosphorylation and recruitment of SHP-1 phosphatase in macrophages. VAV, a downstream effector implicated in CD36-mediated oxidized LDL uptake, was shown to interact with SHP-1 following oxidized LDL treatment. Moreover, oxidized LDL-induced VAV phosphorylation was substantially lower in WT macrophages comparing to Siglec-E-/- counterparts. CONCLUSIONS: These data support the protective role of Siglec-E in atherosclerosis. Mechanistically, Siglec-E interacts with CD36 to suppress downstream VAV signaling involved in modified LDL uptake.

A Guillain-Barré syndrome-associated SIGLEC10 rare variant impairs its recognition of gangliosides.

Guillain-Barré syndrome (GBS), including its variant Miller Fisher syndrome (MFS), is an acute peripheral neuropathy that involves autoimmune mechanisms leading to the production of autoantibodies to gangliosides; sialic acid-containing glycosphingolipids. Although association with various genetic polymorphisms in the major histocompatibility complex (MHC) is shown in other autoimmune diseases, GBS is an exception, showing no such link. No significant association was found by genome wide association studies, suggesting that GBS is not associated with common variants. To address the involvement of rare variants in GBS, we analyzed Siglec-10, a sialic acid-recognizing inhibitory receptor expressed on B cells. Here we demonstrate that two rare variants encoding R47Q and A108V substitutions in the ligand-binding domain are significantly accumulated in patients with GBS. Because of strong linkage disequilibrium, there was no patient carrying only one of them. Recombinant Siglec-10 protein containing R47Q but not A108V shows impaired binding to gangliosides. Homology modeling revealed that the R47Q substitution causes marked alteration in the ligand-binding site. Thus, GBS is associated with a rare variant of the SIGLEC10 gene that impairs ligand binding of Siglec-10. Because Siglec-10 regulates antibody production to sialylated antigens, our finding suggests that Siglec-10 regulates development of GBS by suppressing antibody production to gangliosides, with defects in its function predisposing to disease.

Preparation of Recombinant Siglecs and Identification of Their Ligands.

Siglecs are transmembrane receptor-like vertebrate lectins that recognize glycans containing sialic acid. Most Siglecs also interact with intracellular signal transduction molecules, and modulate immune responses. Recombinant soluble Siglecs fused with the fragment crystallizable (Fc) region of immunoglobulin G (Siglec-Fc) are a versatile tool for the investigation of Siglec functions. We describe protocols for the production of recombinant Siglec-Fc, the analysis of expression of Siglec ligands by flow cytometry, and the identification of the Siglec ligand candidates based on proximity labeling.

Siglecs that Associate with DAP12.

Siglecs are a family of transmembrane receptor-like glycan-recognition proteins expressed primarily on leukocytes. Majority of Siglecs have an intracellular sequence motif called immunoreceptor tyrosine-based inhibitory motif (ITIM) and associate with Src homology region 2 domain-containing tyrosine phosphatase-1 (SHP-1), and negatively regulate tyrosine phosphorylation-mediated intracellular signaling events. On the other hand, some Siglecs have a positively charged amino acid residue in the transmembrane domain and associate with DNAX activation protein of 12 kDa (DAP12), which in turn recruits spleen tyrosine kinase (Syk). These DAP12-associated Siglecs play diverse functions. For example, Siglec-15 is conserved throughout vertebrate evolution and plays a role in bone homeostasis by regulating osteoclast development and function. Human Siglec-14 and -16 have inhibitory counterparts (Siglec-5 and -11, respectively), which show extremely high sequence similarity with them at the extracellular domain but interact with SHP-1. The DAP12-associated Siglec in such "paired receptor" configuration counteracts the pathogens that exploit the inhibitory counterpart. Polymorphisms (mutations) that render DAP12-associated inactive Siglecs are found in humans, and some of these appear to be associated with sensitivity or resistance of human hosts to bacterially induced conditions. Studies of mouse Siglec-H have revealed complex and intriguing functions it plays in regulating adaptive immunity. Many questions remain unanswered, and further molecular and genetic studies of DAP12-associated Siglecs will yield valuable insights with translational relevance.

Siglec-15: a potential regulator of osteoporosis, cancer, and infectious diseases.

Siglec-15 is a member of the Siglec family of glycan-recognition proteins, primarily expressed on a subset of myeloid cells. Siglec-15 has been known to be involved in osteoclast differentiation, and is considered to be a potential therapeutic target for osteoporosis. Recent studies revealed unexpected roles of Siglec-15 in microbial infection and the cancer microenvironment, expanding the potential pathophysiological roles of Siglec-15. Chemical biology has advanced our understanding of the nature of Siglec-15 ligands, but the exact nature of Siglec-15 ligand depends on the biological context, leaving plenty of room for further exploration.

Boronate affinity-based photoactivatable magnetic nanoparticles for the oriented and irreversible conjugation of Fc-fused lectins and antibodies.

The utilization of immuno-magnetic nanoparticles (MNPs) for the selective capture, enrichment, and separation of specific glycoproteins from complicated biological samples is appealing for the discovery of disease biomarkers. Herein, MNPs were designed and anchored with abundant boronic acid (BA) and photoreactive alkyl diazirine (Diaz) functional groups to obtain permanently tethered Fc-fused Siglec-2 and antiserum amyloid A (SAA) mAb with the assistance of reversible boronate affinity and UV light activation in an orientation-controlled manner. The Siglec-2-Fc-functionalized MNPs showed excellent stability in fetal bovine serum (FBS) and excellent efficiency in the extraction of cell membrane glycoproteins. The anti-SAA mAb-functionalized MNPs maintained active Ab orientation and preserved antigen recognition capability in biological samples. Thus, the BA-Diaz-based strategy holds promise for the immobilization of glycoproteins, such as antibodies, with the original protein binding activity maintained, which can provide better enrichment for the sensitive detection of target proteins.