Cellular translocation and secretion of sialidases.

Sialidases (or neuraminidases) catalyze the hydrolysis of sialic acid (Sia)-containing molecules, mostly the removal of the terminal Sia on glycans (desialylation) of either glycoproteins or glycolipids. Therefore, sialidases can modulate the functionality of the target glycoprotein or glycolipid and are involved in various biological pathways in health and disease. In mammalian cells, there are four kinds of sialidase, which are Neu1, Neu2, Neu3, and Neu4, based on their subcellular locations and substrate specificities. Neu1 is the lysosomal sialidase, Neu2 is the cytosolic sialidase, Neu3 is the plasma membrane-associated sialidase, and Neu4 is found in the lysosome, mitochondria, and endoplasmic reticulum. In addition to specific subcellular locations, sialidases can translocate to different subcellular localizations within particular cell conditions and stimuli, thereby participating in different cellular functions depending on their loci. Lysosomal sialidase Neu1 can translocate to the cell surface upon cell activation in several cell types, including immune cells, platelets, endothelial cells, and epithelial cells, where it desialylates receptors and thus impacts receptor activation and signaling. On the other hand, cells secrete sialidases upon activation. Secreted sialidases can serve as extracellular sialidases and cause the desialylation of both extracellular glycoproteins or glycolipids and cell surface glycoproteins or glycolipids on their own and other cells, thus playing roles in various biological pathways as well. This review discusses the recent advances and understanding of sialidase translocation in different cells and secretion from different cells under different conditions and their involvement in physiological and pathological pathways.

Solvent Isotope Effects on the Creation of Fluorescent Quantum Defects in Carbon Nanotubes by Aryl Diazonium Chemistry.

The integration of aryl diazonium and carbon nanotube chemistries has offered rich and versatile tools for creating nanomaterials of unique optical and electronic properties in a controllable fashion. The diazonium reaction with single-wall carbon nanotubes (SWCNTs) is known to proceed through a radical or carbocation mechanism in aqueous solutions, with deuterated water (D2O) being the frequently used solvent. Here, we show strong water solvent isotope effects on the aryl diazonium reaction with SWCNTs for creating fluorescent quantum defects using water (H2O) and D2O. We found a deduced reaction constant of ∼18.2 times larger value in D2O than in H2O, potentially due to their different chemical properties. We also observed the generation of new defect photoluminescence over a broad concentration range of diazonium reactants in H2O, as opposed to a narrow window of reaction conditions in D2O under UV excitation. Without UV light, the physical adsorption of diazonium on the surface of SWCNTs led to the fluorescence quenching of nanotubes. These findings provide important insights into the aryl diazonium chemistry with carbon nanotubes for creating promising material platforms for optical sensing, imaging, and quantum communication technologies.

Investigation of cofactor activities of endothelial microparticle-thrombomodulin with liposomal surrogate.

Thrombomodulin (TM) is a type I transmembrane glycoprotein mainly expressed on the endothelial cells, where it binds thrombin to form the thrombin-TM complex that can activate protein C and thrombin-activable fibrinolysis inhibitor (TAFI) and induce anticoagulant and anti-fibrinolytic reactions, respectively. Cell activation and injury often sheds microparticles that contain membrane TM, which circulate in biofluids like blood. However, the biological function of circulating microparticle-TM is still unknown even though it has been recognized as a biomarker of endothelial cell injury and damage. In comparison with cell membrane, different phospholipids are exposed on the microparticle surface due to cell membrane ''flip-flop'' upon cell activation and injury. Liposomes can be used as a microparticle mimetics. In this report, we prepared TM-containing liposomes with different phospholipids as surrogates of endothelial microparticle-TM and investigated their cofactor activities. We found that liposomal TM with phosphatidylethanolamine (PtEtn) showed increased protein C activation but decreased TAFI activation in comparison to liposomal TM with phosphatidylcholine (PtCho). In addition, we investigated whether protein C and TAFI compete for the thrombin/TM complex on the liposomes. We found that protein C and TAFI did not compete for the thrombin/TM complex on the liposomes with PtCho alone and with low concentration (5%) of PtEtn and phosphatidylserine (PtSer), but competed each other on the liposomes with higher concentration (10%) of PtEtn and PtSer. These results indicate that membrane lipids affect protein C and TAFI activation and microparticle-TM may have different cofactor activities in comparison to cell membrane TM.

The role of cell surface sialic acids for SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a new virus that has higher contagious capacity than any other previous human coronaviruses (HCoVs) and causes the current coronavirus disease 2019 pandemic. Sialic acids are a group of nine-carbon acidic α-keto sugars, usually located at the end of glycans of cell surface glycoconjugates and serve as attachment sites for previous HCoVs. It is therefore speculated that sialic acids on the host cell surface could serve as co-receptors or attachment factors for SARS-CoV-2 cell entry as well. Recent in silico modeling, molecular modeling predictions and microscopy studies indicate potential sialic acid binding by SARS-CoV-2 upon cell entry. In particular, a flat sialic acid-binding domain was proposed at the N-terminal domain of the spike protein, which may lead to the initial contact and interaction of the virus on the epithelium followed by higher affinity binding to angiotensin-converting enzyme 2 (ACE2) receptor, likely a two-step attachment fashion. However, recent in vitro and ex vivo studies of sialic acids on ACE2 receptor confirmed an opposite role for SARS-CoV-2 binding. In particular, neuraminidase treatment of epithelial cells and ACE2-expressing 293T cells increased SARS-CoV-2 binding. Furthermore, the ACE2 glycosylation inhibition studies indicate that sialic acids on ACE2 receptor prevent ACE2-spike protein interaction. On the other hand, a most recent study indicates that gangliosides could serve as ligands for receptor-binding domain of SARS-CoV-2 spike protein. This mini-review discusses what has been predicted and known so far about the role of sialic acid for SARS-CoV-2 infection and future research perspective.

High-throughput multiplex assays with mouse macrophages on pillar plate platforms.

It is challenging to rapidly identify immune responses that reflect the state and capability of immune cells due to complex heterogeneity of immune cells and their plasticity to pathogens and modulating molecules. Thus, high-throughput and easy-to-use cell culture and analysis platforms are highly desired for characterizing complex immune responses and elucidating their underlying mechanisms as well. In response to this need, we have developed a micropillar chip and a 384-pillar plate, printed mouse macrophage, RAW 264.7 cell line in alginate on the pillar plate platforms, and established multiplex cell-based assays to rapidly measure cell viability, expression of cell surface markers, and secretion of cytokines upon stimulation with model compound, lipopolysaccharide (LPS), as well as synthetic N-glycan polymers that mimic native glycoconjugates and could bind to lectin receptors on RAW 264.7 cells. Interestingly, changes in RAW 264.7 cell viability, expression levels of cell surface makers, and release of cytokines measured from the pillar plate platforms in the presence and absence of LPS were well correlated with those obtained from their counterpart, the 96-well plate with 2D-cultured macrophages. With this approach, we identified that α2,3-linked N-sialyllactose polymer has significant macrophage modulation activity among the N-glycan polymers tested. Therefore, we successfully demonstrated that our pillar plate platforms with 3D-cultured macrophages can streamline immune cell imaging and analysis in high throughput in response to compound stimulation. We envision that the pillar plate platforms could potentially be used for rapid characterization of immune cell responses and for screening immune cell-modulating molecules.

Investigation of substrate specificity of sialidases with membrane mimetic glycoconjugates.

Sialidases or neuraminidases play important roles in various physiological and pathological processes by cleaving terminal sialic acids (Sias) (desialylation) from the glycans of both glycoproteins and glycolipids. To understand the biological significance of desialylation by sialidases, it is important to investigate enzyme specificity with native substrate in biological membrane of cells. Herein, we report a membrane-mimicking system with liposome ganglioside conjugates containing different lipids for evaluating substrate specificity of sialidase and the lipid effect on the enzyme activity. Briefly, liposomes of phosphatidylcholine (PC) and cholesterol with ganglioside (GM3 or GM1) along with different percentage of phosphatidylserine (PS) or phosphatidylethanolamine (PE) were prepared and characterized. Their desialylation profiles with Arthrobacter ureafaciens (bacterial) sialidase and H1N1 (influenza viral) sialidase were quantified by HPLC method. A diversity of substrate preference was found for both bacterial and viral sialidase to the liposome ganglioside conjugate platform. The apparent Km and Vmax were dependent on the type of lipid. These results indicate that the intrinsic characteristics of the membrane-like system affect the sialidase specificity and activity. This biomimetic substrate provides a better tool for unravelling the substrate specificity and the biological function of sialidases and for screening of functional sialidase inhibitors as well.

Sialidase substrates for Sialdiase assays - activity, specificity, quantification and inhibition.

Sialidases are glycosidases responsible for the removal of sialic acid (Sia) residues (desialylation) from glycan portions of either glycoproteins or glycolipids. By desialylation, sialidases are able to modulate the functionality and stability of the Sia-containing molecules and are involved in both physiological and pathological pathways. Therefore, evaluation of sialidase activity and specificity is important for understanding the biological significance of desialylation by sialidases and its function and the related molecular mechanisms of the physiological and pathological pathways. In addition, it is essential for developing novel mechanisms and approaches for disease treatment and diagnosis and pathogen detection as well. This review summarizes the most recent sialidase substrates for evaluating sialidase activity and specificity and screening sialidase inhibitors, including (i) general sialidase substrates, (ii) specific sialidase substrates, (iii) native sialidase substrates and (iv) cellular sialidase substrates. This review also provides a brief introduction of recent instrumental methods for quantifying the sialidase activity, such as UV, fluorescence, HPLC and LC-MS methods.

Carbohydrate- and Chain Length-Controlled Complexation of Carbon Nanotubes by Glycopolymers.

Stable dispersions of single-wall carbon nanotubes (SWCNTs) by biopolymers in an aqueous environment facilitate their potential biological and biomedical applications. In this report, we investigated a small library of precision synthesized glycopolymers with monosaccharide and disaccharide groups for stabilizing SWCNTs via noncovalent complexation in aqueous conditions. Among the glycopolymers tested, disaccharide lactose-containing glycopolymers demonstrate effective stabilization of SWCNTs in water, which strongly depends on carbohydrate density and polymer chain length as well. The introduction of disaccharide lactose potentially makes glycopolymers less flexible as compared to those containing monosaccharide and facilitates the wrapping conformation of polymers on the surface of SWCNTs while preserving intrinsic photoluminescence of nanotubes in the near-infrared region. This work demonstrates the synergistic effects of the identity of carbohydrate pendant groups and polymer chain length of glycopolymers on stabilizing SWCNTs in water, which has not been achieved previously.

Sialylation status and mechanical properties of THP-1 macrophages upon LPS stimulation.

Cell surface receptors are the key contributors of macrophage function. Most macrophage cell surface receptors are glycoproteins with sialic acids at the terminal of their glycans. It is well recognized that lipopolysaccharide (LPS) induces cell surface sialylation changes that may in turn contribute to macrophage functions. In addition, cellular mechanics such as elasticity is also a major determinant of macrophage function, which in turn is modulated by LPS. In this report, we characterized the sialylation status of macrophages upon LPS stimulation and assessed the changes in its mechanical properties and function. Specifically, we confirmed that sialylation status is closely related to macrophage biomechanical characteristics (elastic modulus, tether force, tether radius, adhesion force, and membrane tension) and thus directly involved in macrophage function. Further, we modulated macrophage sialylation status by feeding the cell with exogenous free sialic acid (Neu5Ac, Neu5Gc) and sialidase inhibitors, and examined the resulting effects on cellular mechanics and function. A systematic recognition of sialylation status related to cellular mechanics of macrophages will contribute to defining their phenotypes and elucidate macrophage functional diversity.

Design and synthesis of biotinylated cardiac glycosides for probing Nur77 protein inducting pathway.

The orphan nuclear receptor Nur77 (also known as TR3 or nerve growth factor-induced clone B NGFI-B) functions as a nuclear transcription factor in the regulation of target gene expression and plays a critical role in the regulation of differentiation, proliferation, apoptosis, and survival of many different cell types. Recent studies demonstrate that Nur77 also involves many important physiological and pathological processes including cancer, inflammation and immunity, cardiovascular diseases, and bone diseases. Our previous studies showed that cardiac glycosides could induce the expression of Nur77 protein and its translocation from the nucleus to the cytoplasm and subsequent targeting to mitochondria, leading to apoptosis of cancer cells. In order to probe the Nur77 protein inducting pathway, we designed and synthesized a series of novel biotinylated cardiac glycosides from ß-Antiarin and α-Antiarin, two typical cardiac glycosides from the plant of Antiaris toxicaria. The induction of Nur77 protein expression of these biotinylated cardiac glycosides and their inhibitory effects on NIH-H460 cancer cell proliferation were evaluated. Results displayed that some biotinylated cardiac glycosides could significantly induce the expression of Nur77 protein comparable with their parent compounds ß-Antiarin and α-Antiarin. Also, their streptavidin binding activities were evaluated. Among them, biotinylated cardiac glycosides P4b and P5a exhibited significant effect on the induction of Nur77 expression along with high binding capacity with streptavidin, suggesting that they can be used as probes for probing Nur77 protein inducting pathway.

Recent approaches for directly profiling cell surface sialoform.

Sialic acids (SAs) are nine-carbon monosaccharides existing at the terminal location of glycan structures on the cell surface and secreted glycoconjugates. The expression levels and linkages of SAs on cells and tissues, collectively known as sialoform, present the hallmark of the cells and tissues of different systems and conditions. Accordingly, detecting or profiling cell surface sialoforms is very critical for understanding the function of cell surface glycans and glycoconjugates and even the molecular mechanisms of their underlying biological processes. Further, it may provide therapeutic and diagnostic applications for different diseases. In the past decades, several kinds of SA-specific binding molecules have been developed for detecting and profiling specific sialoforms of cells and tissues; the experimental materials have expanded from frozen tissue to living cells; and the analytical technologies have advanced from histochemistry to fluorescent imaging, flow cytometry and microarrays. This review summarizes the recent bioaffinity approaches for directly detecting and profiling specific SAs or sialylglycans, and their modifications of different cells and tissues.

Sialyltransferase inhibition and recent advances.

Sialic acids, existing as terminal sugars of glycoconjugates, play important roles in various physiological and pathological processes, such as cell-cell adhesion, immune defense, tumor cell metastasis, and inflammation. Sialyltransferases (STs) catalyze the transfer of sialic acid residues to non-reducing oligosaccharide chains of proteins and lipids, using cytidine monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) as the donor. Elevated sialyltransferase activity leads to overexpression of cell surface sialic acids and contributes to many disease developments, such as cancer and inflammation. Therefore, sialyltransferases are considered as potential drug targets for disease treatment. Inhibitors of sialyltransferases thus are of medicinal interest, especially for the cancer therapy. In addition, sialyltransferase inhibitors are useful tool to study sialyltransferase function and related mechanisms. This review highlights recent development of inhibitors of sialyltransferases reported since 2004. The inhibitors are summarized as eight groups: 1) sialic acid analogs, 2) CMP-sialic acid analogs, 3) cytidine analogs, 4) oligosaccharide derivatives, 5) aromatic compounds, 6) flavonoids, 7) lithocholic acid analogs, and 8) others. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions.

Synthesis of MeON-neoglycosides of digoxigenin with 6-deoxy- and 2,6-dideoxy-d-glucose derivatives and their anticancer activity.

Cardiac glycosides show anticancer activities and their deoxy-sugar chains are vital for their anticancer effects. In order to study the structure-activity relationship (SAR) of cardiac glycosides toward cancers and get more potent anticancer agents, a series of MeON-neoglycosides of digoxigenin was synthesized and evaluated. First, ten 6-deoxy- and 2,6-dideoxy-d-glucopyranosyl donors were synthesized starting from methyl α-d-glucopyranoside and 2-deoxy-d-glucose. Meanwhile, the digoxigenin was obtained by acidic hydrolysis of commercially available digoxin as glycosyl acceptor. Then, a 22-member MeON-neoglycoside library of digoxigenin was successfully synthesized by neoglycosylation method. Finally, the induction of Nur77 expression and its translocation from the nucleus to cytoplasm together with cytotoxicity of these MeON-neoglycosides were evaluated. The SAR analysis revealed that C3 glycosylation is required for their induction of Nur77 expression. Moreover, some MeON-neoglycosides (2b and 8b) could significant induce the expression of Nur77 and its translocation from the nucleus to cytoplasm. However, these compounds showed no inhibitory effects on the proliferation of cancer cells, suggesting that they may not induce apoptosis of NIH-H460 cancer cells and their underlying potential and application toward cancer cells deserves future study.

Sialylation and desialylation dynamics of monocytes upon differentiation and polarization to macrophages.

Sialic acids (SAs) often exist as the terminal sugars of glycan structures of cell surface glycoproteins and glycolipids. The level and linkages of cell surface SAs, which are controlled by both sialylation and desialylation processes and environment cues, can dramatically impact cell properties and represent different cellular status. In this study, we systematically examined the sialylation and desialylation profiles of THP-1 monocytes after differentiation to M0 macrophages, and polarization to M1 and M2 macrophages by the combination of LC-MS/MS, flow cytometry and confocal microscopy. Interestingly, both α2-3- and α2-6-linked SAs on the cell surface decreased after monocytes were differentiated to macrophages, which was in accordance with the increased level of free SA in the cell culture medium and the elevated activity of endogenous Neu1 sialidase. Meanwhile, the siaoglycoconjugates inside the cells increased as confirmed by confocal microscopy and the total SA inside the cells increased as determined by LC-MS/MS. Western blot analysis showed higher expression levels of sialyltransferases, including ST3Gal-I, ST3Gal-V, ST6Gal-I and ST6GalNAc-II. Further, upon polarization, the cell surface sialylation levels of M1 and M2 macrophages remained the same as M0 macrophages, while a slight decrease of cellular SAs in the M1 macrophages but increase in the M2 macrophages were confirmed by LC-MS/MS.

Preparation of chain-end clickable recombinant protein and its bio-orthogonal modification.

Introducing unique functional group into protein is an attractive approach for site-selective protein modification applications. In this report, we systemically investigated four site-selective strategies to introduce azide functionality into recombinant thrombomodulin (TM456), via direct recombinant expression with unnatural amino acid, chemical, and enzymatic modification for its bio-orthogonal modification application. First, a straightforward recombinant method to express TM456 with azide functionality near C-terminus by replacing methionine with azidohomoanlanine from methionine auxotroph Escherichia coli cell was investigated. Next, a sortase-mediated ligation (SML) method to incorporate azide functionality into the C-terminus of recombinant TM456 was demonstrated. The third is to add azide functionality to the N-terminal amine of recombinant TM456via amidation chemistry, and the fourth is tyrosine selective three-component Mannich reaction to introduce azide functionality to recombinant TM456. Overall, SML of recombinant protein affords the highest overall yield for incorporating azide functionality into the C-terminus recombinant TM456 since the key protein expression step uses natural amino acids. Also, single site modification facilitates the highest TM456 activity.

Globally profiling sialylation status of macrophages upon statin treatment.

Sialic acids (SAs) are widely expressed on immune cells and their levels and linkages named as sialylation status vary upon cellular environment changes related to both physiological and pathological processes. In this study, we performed a global profiling of the sialylation status of macrophages and their release of SAs in the cell culture medium by using flow cytometry, confocal microscopy and liquid chromatography tandem mass spectrometry (LC-MS/MS). Both flow cytometry and confocal microscopy results showed that cell surface α-2,3-linked SAs were predominant in the normal culture condition and changed slightly upon treatment with atorvastatin for 24 h, whereas α-2,6-linked SAs were negligible in the normal culture condition but significantly increased after treatment. Meanwhile, the amount of total cellular SAs increased about three times (from 369 ± 29 to 1080 ± 50 ng/mL) upon treatment as determined by the LC-MS/MS method. On the other hand, there was no significant change for secreted free SAs and conjugated SAs in the medium. These results indicated that the cell surface α-2,6 sialylation status of macrophages changes distinctly upon atorvastatin stimulation, which may reflect on the biological functions of the cells.

Multi-dimensional glycan microarrays with glyco-macroligands.

Glycan microarray has become a powerful high-throughput tool for examining binding interactions of carbohydrates with the carbohydrate binding biomolecules like proteins, enzymes, antibodies etc. It has shown great potential for biomedical research and applications, such as antibody detection and profiling, vaccine development, biomarker discovery, and drug screening. Most glycan microarrays were made with monovalent glycans immobilized directly onto the array surface via either covalent or non-covalent bond, which afford a multivalent glycans in two dimensional (2D) displaying. A variety of glyco-macroligands have been developed to mimic multivalent carbohydrate-protein interactions for studying carbohydrate-protein interactions and biomedical research and applications. Recently, a number of glyco-macroligands have been explored for glycan microarray fabrication, in particular to mimick the three dimensional (3D) multivalent display of cell surface carbohydrates. This review highlights these recent developments of glyco-macroligand-based microarrays, predominantly, novel glycan microarrays with glyco-macroligands like glycodendrimers, glycopolymers, glycoliposomes, neoglycoproteins, and glyconanoparticles with the effort in controlling the density and orientation of glycans on the array surface, which facilitate both their binding specificity and affinity and thus the high performance of glycan microarrays.

Recombinant thrombomodulin of different domains for pharmaceutical, biomedical, and cell transplantation applications.

Thrombomodulin (TM) is a membrane glycoprotein mainly expressed by vascular endothelial cells and is involved in many physiological and pathological processes, such as coagulation, inflammation, cancer development, and embryogenesis. Human TM consists of 557 amino acids divided into five distinct domains: N-terminal lectin-like domain (designated as TMD1); six epidermal growth factor (EGF)-like domain (TMD2); Ser/Thr-rich domain (TMD3); transmembrane domain (TMD4); and cytoplasmic tail domain (TMD5). The different domains are responsible for different biological functions of TM. In the past decades, various domains of TM have been cloned and expressed for TM structural and functional study. Further, recombinant TMs of different domains show promising antithrombotic and anti-inflammatory activity in both rodents and primates and a recombinant soluble TM has been approved for therapeutic application. This review highlights recombinant TMs of diverse structures and their biological functions, as well as the complex interactions of TM with factors involved in the related biological processes. Particularly, recent advances in exploring recombinant TM of different domains for pharmaceutical, biomedical, and cell transplantation applications are summarized.

BSA-boronic acid conjugate as lectin mimetics.

We report bovine serum albumin (BSA)-boronic acid (BA) conjugates as lectin mimetics and their glyco-capturing capacity. The BSA-BA conjugates were synthesized by amidation of carboxylic acid groups in BSA with aminophenyl boronic acid in the presence of EDC, and were characterized by Alizarin Red S (ARS) assay and SDS-PAGE gel. The BSA-BA conjugates were immobilized onto maleimide-functionalized silica beads and their sugar capturing capacity and specificity were confirmed by ARS displacement assay. Further, surface plasmon resonance (SPR) analysis of the glyco-capturing activity of the BSA-BA conjugates was conducted by immobilizing BSA-BA onto SPR gold chip. Overall, we demonstrated a BSA-BA-based lectin mimetics for glyco-capturing applications. These lectin mimetics are expected to provide an important tool for glycomics and biosensor research and applications.

Chemoenzymatic bio-orthogonal chemistry for site-specific double modification of recombinant thrombomodulin.

Best of both worlds: A one-pot strategy for site-specific PEGylation through strain-promoted alkyne-azide cycloaddition (SPAAC) and fluorescent labeling through sortase A-mediated ligation (SML) of recombinant thrombomodulin without prior chemical modification and without diminishing the protein activity has been developed.