Sialylation Of Glycoproteins Research Articles

ST8Sia2 polysialyltransferase protects against infection by Trypanosoma cruzi.

Glycosylation is one of the most structurally and functionally diverse co- and post-translational modifications in a cell. Addition and removal of glycans, especially to proteins and lipids, characterize this process which has important implications in several biological processes. In mammals, the repeated enzymatic addition of a sialic acid unit to underlying sialic acids (Sia) by polysialyltransferases, including ST8Sia2, leads to the formation of a sugar polymer called polysialic acid (polySia). The functional relevance of polySia has been extensively demonstrated in the nervous system. However, the role of polysialylation in infection is still poorly explored. Previous reports have shown that Trypanosoma cruzi (T. cruzi), a flagellated parasite that causes Chagas disease (CD), changes host sialylation of glycoproteins. To understand the role of host polySia during T. cruzi infection, we used a combination of in silico and experimental tools. We observed that T. cruzi reduces both the expression of the ST8Sia2 and the polysialylation of target substrates. We also found that chemical and genetic inhibition of host ST8Sia2 increased the parasite load in mammalian cells. We found that modulating host polysialylation may induce oxidative stress, creating a microenvironment that favors T. cruzi survival and infection. These findings suggest a novel approach to interfere with parasite infections through modulation of host polysialylation.

Sialyllactose supplementation enhances sialylation of Fc-fusion glycoprotein in recombinant Chinese hamster ovary cell culture

Sialylation during N-glycosylation plays an important role in the half-life of therapeutic glycoproteins in vivo and has sparked interest in the production of therapeutic proteins using recombinant Chinese hamster ovary (rCHO) cells. To improve the sialylation of therapeutic proteins, we examined the effect of sialyllactose supplementation on sialylation of Fc-fusion glycoproteins produced in rCHO cells. Two enzymatically-synthesized sialyllactoses, 3′-sialyllactose (3′-SL) and 6′-sialyllactose (6′-SL), were administered separately to two rCHO cell lines producing the same Fc-fusion glycoprotein derived from DUKX-B11 and DG44, respectively. Two sialyllactoses successfully increased sialylation of Fc-fusion glycoprotein in both cell lines, as evidenced by isoform distribution, sialylated N-glycan formation, and sialic acid content. Increased sialylation by adding sialyllactose was likely the result of increased amount of intracellular CMP-sialic acid (CMP-SA), the direct nucleotide sugar for sialylation. Furthermore, the degree of sialylation enhanced by sialyllactoses was slightly effective or nearly similar compared with the addition of N-acetylmannosamine (ManNAc), a representative nucleotide sugar precursor, to increase sialylation of glycoproteins. The effectiveness of sialyllactose was also confirmed using three commercially available CHO cell culture media. Taken together, these results suggest that enzymatically-synthesized sialyllactose represents a promising candidate for culture media supplementation to increase sialylation of glycoproteins in rCHO cell culture.

Limited impact of cancer-derived gangliosides on anti-tumor immunity in colorectal cancer.

Aberrant glycosylation is a key mechanism employed by cancer cells to evade immune surveillance, induce angiogenesis and metastasis, among other hallmarks of cancer. Sialic acids, distinctive terminal glycan structures located on glycoproteins or glycolipids, are prominently upregulated across various tumor types, including colorectal cancer (CRC). Sialylated glycans modulate anti-tumor immune responses through their interactions with Siglecs, a family of glycan-binding receptors with specificity for sialic acid-containing glycoconjugates, often resulting in immunosuppression. In this paper, we investigated the immunomodulatory function of ST3Gal5, a sialyltransferase that catalyzes the addition of α2-3 sialic acids to glycosphingolipids, since lower expression of ST3Gal5 is associated with better survival of CRC patients. We employed CRISPR/Cas9 to knock out the ST3Gal5 gene in two murine CRC cell lines MC38 and CT26. Glycomics analysis confirmed the removal of sialic acids on glycolipids, with no discernible impact on glycoprotein sialylation. Although knocking out ST3Gal5 in both cell lines did not affect in vivo tumor growth, we observed enhanced levels of regulatory T cells in CT26 tumors lacking ST3Gal5. Moreover, we demonstrate that the absence of ST3Gal5 affected size and blood vessel density only in MC38 tumors. In summary, we ascertain that sialylation of glycosphingolipids has a limited influence on the anti-tumor immune response in CRC, despite detecting alterations in the tumor microenvironment, possibly due to a shift in ganglioside abundance.

Platelets retain function and can be stored following disruption of human leucocyte antigens.

Antibodies to human leucocyte antigen (HLA) Class-I antigens can lead to refractoriness to platelet transfusion. Although this can be overcome by transfusion of HLA-compatible platelets, they are not always available. Disruption of HLA antigens on platelets by acid treatment may be a suitable alternative when no other components are available. The aim of this study was to assess the effect of HLA disruption and subsequent storage of platelet components. Platelet components were treated with 0.9% saline or citric acid solution (pH 3.0), and then stored until expiry (Day 7). HLA and platelet glycoprotein expression, platelet viability, activation and sialylation were measured by flow cytometry. Release of soluble factors was measured by ELISA and metabolism by biochemistry analyser. Reactivity to patient anti-sera containing anti-HLA antibodies was measured using platelet immunofluorescence tests (PIFTs) and monoclonal antibody immobilization of platelet antigen (MAIPA) assays. Platelet function was measured using aggregometry and thromboelastography (TEG). Acid treatment reduced detection of HLA Class-I on platelets by 75%, with significant reductions in reactivity to patient anti-sera. Acid treatment reduced platelet content and viability, increased platelet activation and accelerated metabolism. Glycan cleavage was increased by acid treatment. Treatment reduced platelet activation following agonist stimulation by ADP and TRAP-6, but platelets remained functional, displaying increased aggregation response and reduced time to clot formation by TEG. Although HLA disruption had some detrimental effects, acid-treated platelets remained functional, retaining their capacity to respond to agonists and form clots, and with further development could be used to support refractory patients.

The correlation between human seminal plasma sialoproteins and ejaculate parameters

Sialic acids are negatively charged carbohydrates that are components of saccharide chains covalently linked to macromolecules. Sialylated glycoproteins are important for most biological processes, including reproduction, where they are associated with spermatogenesis, sperm motility, immune responses, and fertilization. Changes in the glycoprotein profile or sialylation in glycoproteins are likely to affect the quality of ejaculate. The aim of this study was to determine differences in the degree of sialylation between normozoospermic ejaculates and ejaculates with a pathological spermiogram using two lectins, Sambucus nigra (SNA) and Maackia amurensis (MAL II/MAA) recognizing α-2,6 or α-2,3 linkage of Sia to galactosyl residues. Our results show a close relationship between seminal plasma (SP) sialoproteins and the presence of anti-sperm antibodies in the ejaculate, apoptotic spermatozoa, and ejaculate quality. Using mass spectrometry, we identified SP sialoproteins such as, semenogelins, glycodelin, prolactin-inducible protein, lactotransferrin, and clusterin that are associated with spermatozoa and contribute to the modulation of the immune response and sperm apoptosis. Our findings suggest a correlation between the degree of SP glycoprotein sialylation and the existence of possible pathological states of spermatozoa and reproductive organs. Glycoproteins sialylation represents a potential parameter reflecting the overall quality of ejaculate and could potentially be utilised in diagnostics.

Effects of autophagy-inhibiting chemicals on sialylation of Fc-fusion glycoprotein in recombinant CHO cells

The occurrence of autophagy in recombinant Chinese hamster ovary (rCHO) cell culture has attracted attention due to its effects on therapeutic protein production. Given the significance of glycosylation in therapeutic proteins, this study examined the effects of autophagy-inhibiting chemicals on sialylation of Fc-fusion glycoproteins in rCHO cells. Three chemical autophagy inhibitors known to inhibit different stages were separately treated with two rCHO cell lines that produce the same Fc-fusion glycoprotein derived from DUKX-B11 and DG44. All autophagy inhibitors significantly decreased the sialylation of Fc-fusion glycoprotein in both cell lines. The decrease in sialylation of Fc-fusion glycoprotein is unlikely to be attributed to the release of intracellular enzymes, given the high cell viability and low activity of extracellular sialidases. Interestingly, the five intracellular nucleotide sugars remained abundant in cells treated with autophagy inhibitors. In the mRNA expression profiles of 27 N-glycosylation-related genes using the NanoString nCounter system, no significant differences in gene expression were noted. With the positive effect of supplementing nucleotide sugar precursors on sialylation, attempts were made to enhance the levels of intracellular nucleotide sugars by supplying these precursors. The addition of nucleotide sugar precursors to cultures treated with inhibitors successfully enhanced the sialylation of Fc-fusion glycoproteins compared to the control culture. This was particularly evident under mild stress conditions and not under relatively severe stress conditions, which were characterized by a high decrease in sialylation. These results suggest that inhibiting autophagy in rCHO cell culture decreases sialylation of Fc-fusion glycoprotein by constraining the availability of intracellular nucleotide sugars.Key points• The autophagy inhibition in rCHO cell culture leads to a significant reduction in the sialylation of Fc-fusion glycoprotein.• The pool of five intracellular nucleotide sugars remained highly abundant in cells treated with autophagy inhibitors.• Supplementation of nucleotide sugar precursors effectively restores decreased sialylation, particularly under mild stress conditions but not in relatively severe stress conditions.Graphical

Severe kidney dysfunction in sialidosis mice reveals an essential role for neuraminidase 1 in reabsorption.

Sialidosis is an ultra-rare multisystemic lysosomal disease caused by mutations in the neuraminidase 1 (NEU1) gene. The severe type II form of the disease manifests with a prenatal/infantile or juvenile onset, bone abnormalities, severe neuropathology, and visceromegaly. A subset of these patients present with nephrosialidosis, characterized by abrupt onset of fulminant glomerular nephropathy. We studied the pathophysiological mechanism of the disease in 2 NEU1-deficient mouse models, a constitutive Neu1-knockout, Neu1ΔEx3, and a conditional phagocyte-specific knockout, Neu1Cx3cr1ΔEx3. Mice of both strains exhibited terminal urinary retention and severe kidney damage with elevated urinary albumin levels, loss of nephrons, renal fibrosis, presence of storage vacuoles, and dysmorphic mitochondria in the intraglomerular and tubular cells. Glycoprotein sialylation in glomeruli, proximal distal tubules, and distal tubules was drastically increased, including that of an endocytic reabsorption receptor megalin. The pool of megalin bearing O-linked glycans with terminal galactose residues, essential for protein targeting and activity, was reduced to below detection levels. Megalin levels were severely reduced, and the protein was directed to lysosomes instead of the apical membrane. Together, our results demonstrated that desialylation by NEU1 plays a crucial role in processing and cellular trafficking of megalin and that NEU1 deficiency in sialidosis impairs megalin-mediated protein reabsorption.

Impact of estrogen on IgG glycosylation and serum protein glycosylation in a murine model of healthy postmenopause.

The glycosylation of immunoglobulin (Ig) G regulates IgG interaction capability with Fc gamma receptors found in all immune cells. In pathogenic conditions, estrogen can impact IgG levels and glycosylation. Following menopause, when estrogen levels decline affecting the immune system and potentially leading to a heightened susceptibility of immune activation. In this study, we aim to determine if estrogen levels can regulate IgG glycosylation in postmenopausal healthy situations. Mice were ovariectomized to simulate an estrogen-deficient postmenopausal status and then treated with 17-beta-estradiol (E2) at different doses and different administration strategies. Using a highly sensitive liquid chromatography-tandem mass spectrometry (MS/MS) glycoproteomic method, we demonstrated that E2 treatment increased the degree of glycosylation on IgG-Fc with both galactosylation and sialylation in the position required for interaction with Fc gamma receptors. We also observed that only long-term estrogen deficiency reduces IgG levels and that estrogen status had no impact on total IgG sialylation on both Fab and Fc domains or general glycoprotein sialylation evaluated by ELISA. Furthermore, E2 status did not affect the total sialic acid content of total cells in lymphoid organs and neither B cells nor plasma cells. The study concluded that E2 treatment does not affect total serum glycoprotein sialylation but alters IgG glycosylation, including IgG sialylation, implying that estrogen functions as an intrinsic modulator of IgG sialylation and could thereby be one pathway by which estrogen modulates immunity.

Linking aberrant glycosylation of plasma glycoproteins with progression of myelodysplastic syndromes: a study based on plasmonic biosensor and lectin array

Aberrant glycosylation of glycoproteins has been linked with various pathologies. Therefore, understanding the relationship between aberrant glycosylation patterns and the onset and progression of the disease is an important research goal that may provide insights into cancer diagnosis and new therapy development. In this study, we use a surface plasmon resonance imaging biosensor and a lectin array to investigate aberrant glycosylation patterns associated with oncohematological disease—myelodysplastic syndromes (MDS). In particular, we detected the interaction between the lectins and glycoproteins present in the blood plasma of patients (three MDS subgroups with different risks of progression to acute myeloid leukemia (AML) and AML patients) and healthy controls. The interaction with lectins from Aleuria aurantia (AAL) and Erythrina cristagalli was more pronounced for plasma samples of the MDS and AML patients, and there was a significant difference between the sensor response to the interaction of AAL with blood plasma from low and medium-risk MDS patients and healthy controls. Our data also suggest that progression from MDS to AML is accompanied by sialylation of glycoproteins and increased levels of truncated O-glycans and that the number of lectins that allow discriminating different stages of disease increases as the disease progresses.

Potential Clinically Relevant Effects of Sialylation on Human Serum AAG-Drug Interactions Assessed by Isothermal Titration Calorimetry: Insight into Pharmacoglycomics?

Human serum alpha-1 acid glycoprotein is an acute-phase plasma protein involved in the binding and transport of many drugs, especially basic and lipophilic substances. It has been reported that the sialic acid groups that terminate the N-glycan chains of alpha-1 acid glycoprotein change in response to certain health conditions and may have a major impact on drug binding to alpha-1 acid glycoprotein. The interaction between native or desialylated alpha-1 acid glycoprotein and four representative drugs-clindamycin, diltiazem, lidocaine, and warfarin-was quantitatively evaluated using isothermal titration calorimetry. The calorimetry assay used here is a convenient and widely used approach to directly measure the amount of heat released or absorbed during the association processes of biomolecules in solution and to quantitatively estimate the thermodynamics of the interaction. The results showed that the binding of drugs with alpha-1 acid glycoprotein were enthalpy-driven exothermic interactions, and the binding affinity was in the range of 10-5-10-6 M. Desialylated alpha-1 acid glycoprotein showed significantly different binding with diltiazem, lidocaine, and warfarin compared with native alpha-1 acid glycoprotein, whereas clindamycin showed no significant difference. Therefore, a different degree of sialylation may result in different binding affinities, and the clinical significance of changes in sialylation or glycosylation of alpha-1 acid glycoprotein in general should not be neglected.

Generation of a ST3GAL3 null mutant induced pluripotent stem cell (iPSC) line (UKWMPi002-A-3) by CRISPR/Cas9 genome editing

Fibroblasts isolated from a skin biopsy of a healthy individual were infected with Sendai virus containing the Yamanaka factors to produce transgene-free human induced pluripotent stem cells (iPSCs). CRISPR/Cas9 was used to generate an isogenic cell line carrying an inactivation of ST3GAL3, a risk gene associated with neurodevelopmental and psychiatric disorders. This ST3GAL3 null mutant (ST3GAL3-/-) iPSC line, which displays the expression of pluripotency-associated markers, the ability to differentiate into cells of the three germ layers in vitro, and a normal karyotype, is a powerful tool to investigate the impact of deficient sialylation of glycoproteins in neural development and plasticity.

Bioinformatics Analyses Identify the Therapeutic Potential of ST8SIA6 for Colon Cancer.

Sialylation of glycoproteins is modified by distinct sialyltransferases such as ST3Gal, ST6Gal, ST6GalNAc, or ST8SIA with α2,3-, α2,6-, or α2,8-linkages. Alteration of these sialyltransferases causing aberrant sialylation is associated with the progression of colon cancer. However, among the ST8- sialyltransferases, the role of ST8SIA6 in colon cancer remains poorly understood. In this study, we explored the involvement of ST8SIA6 in colon cancer using multiple gene databases. The relationship between ST8SIA6 expression and tumor stages/grades was investigated by UALCAN analysis, and Kaplan–Meier Plotter analysis was used to analyze the expression of ST8SIA6 on the survival outcome of colon cancer patients. Moreover, the biological functions of ST8SIA6 in colon cancer were explored using LinkedOmics and cancer cell metabolism gene DB. Finally, TIMER and TISMO analyses were used to delineate ST8SIA6 levels in tumor immunity and immunotherapy responses, respectively. ST8SIA6 downregulation was associated with an advanced stage and poorly differentiated grade; however, ST8SIA6 expression did not affect the survival outcomes in patients with colon cancer. Gene ontology analysis suggested that ST8SIA6 participates in cell surface adhesion, angiogenesis, and membrane vesicle trafficking. In addition, ST8SIA6 levels affected immunocyte infiltration and immunotherapy responses in colon cancer. Collectively, these results suggest that ST8SIA6 may serve as a novel therapeutic target towards personalized medicine for colon cancer.

Comprehensive proteomics and sialiomics of the anti-proliferative activity of safranal on triple negative MDA-MB-231 breast cancer cell lines

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with no efficient treatment. Researchers have indicated the importance of quantitative approaches on proteome and different post-translation modifications studies both in diagnosis and treatment purposes. Sialic acid-containing glycopeptides (the sialiome) is one of these modifications which can be used as a tool in cancer diagnosis or therapeutic strategies since the sialylation is strongly associated with cancer migration and metastasis. Based on our study, safranal, which is a non-toxic compound in orally intakes, exhibits a significant cytotoxic effect on MDA-MB-231 in comparison to normal cells. We conducted a comprehensive proteomics and sialiomics analysis of safranal treated MDA-MB-231 cells by using a combination of TMT labeling and titanium dioxide enrichment of sialylated N-linked glycopeptides to investigate the underlying molecular mechanism behind safranal-induced apoptosis. Safranal has main effect on the inhibition of metabolism and mitochondrial dysfunction. It regulates proteins considered as activator of DNA fragmentation and apoptosis mediators. Moreover, safranal regulates sialylation of glycoproteins involving in cellular adhesion, migration and survival. It suppresses cell survival and metastasis through the alteration of the sialylation level on important signaling receptors. These results highlight the impact of safranal as a potent anticancer compound on TNBCs which also can be strongly used in daily diets. SignificanceIn first step, we evaluated the cell viability of MDA-MB-231 cell lines against the purified saffron components (total crocin, picrocrocin, crocin I and safranal). Safranal was the only compound demonstrated the anti-proliferation effect. In order to obtain an understanding of safranal cytotoxic effect on MDA-MB-231, we designed the three set of treated cell lines in 30 min, 12 h and 24 h time-points in three replicates and a combination of TMT-based labeling quantitative proteomics and titanium dioxide (TiO2)-based enrichment of sialylated N-linked glycopeptides for sialiomics analysis as a strategy to follow the more detailed mechanisms of safranal effect. The results of bioinformatics analysis revealed the multifunction role of safranal on MDA-MB-231 cell lines. Safranal mainly dysregulates mitochondrial function, inhibits metabolism and starts initial signaling of apoptosis which lead to DNA fragmentation. Moreover, safranal caused the majority of down-regulation in sialylation profile in all time-points. Safranal also declines the cell survival, adhesion and migration by dysregulation of the sialylation level in important proteins including integrins, tumor necrosis factor receptor and cell adhesion molecules (CAMs). The results provide a set of therapeutic targets for triple negative breast cancer which can help designing of effective anticancer drugs specially in targeted therapies.

Unexpected Arabinosylation after Humanization of Plant Protein N-Glycosylation.

As biopharmaceuticals, recombinant proteins have become indispensable tools in medicine. An increasing demand, not only in quantity but also in diversity, drives the constant development and improvement of production platforms. The N-glycosylation pattern on biopharmaceuticals plays an important role in activity, serum half-life and immunogenicity. Therefore, production platforms with tailored protein N-glycosylation are of great interest. Plant-based systems have already demonstrated their potential to produce pharmaceutically relevant recombinant proteins, although their N-glycan patterns differ from those in humans. Plants have shown great plasticity towards the manipulation of their glycosylation machinery, and some have already been glyco-engineered in order to avoid the attachment of plant-typical, putatively immunogenic sugar residues. This resulted in complex-type N-glycans with a core structure identical to the human one. Compared to humans, plants lack the ability to elongate these N-glycans with β1,4-linked galactoses and terminal sialic acids. However, these modifications, which require the activity of several mammalian enzymes, have already been achieved for Nicotiana benthamiana and the moss Physcomitrella. Here, we present the first step towards sialylation of recombinant glycoproteins in Physcomitrella, human β1,4-linked terminal N-glycan galactosylation, which was achieved by the introduction of a chimeric β1,4-galactosyltransferase (FTGT). This chimeric enzyme consists of the moss α1,4-fucosyltransferase transmembrane domain, fused to the catalytic domain of the human β1,4-galactosyltransferase. Stable FTGT expression led to the desired β1,4-galactosylation. However, additional pentoses of unknown identity were also observed. The nature of these pentoses was subsequently determined by Western blot and enzymatic digestion followed by mass spectrometric analysis and resulted in their identification as α-linked arabinoses. Since a pentosylation of β1,4-galactosylated N-glycans was reported earlier, e.g., on recombinant human erythropoietin produced in glyco-engineered Nicotiana tabacum, this phenomenon is of a more general importance for plant-based production platforms. Arabinoses, which are absent in humans, may prevent the full humanization of plant-derived products. Therefore, the identification of these pentoses as arabinoses is important as it creates the basis for their abolishment to ensure the production of safe biopharmaceuticals in plant-based systems.

Glycosylation alterations in serum of Alzheimer's disease patients show widespread changes in N-glycosylation of proteins related to immune function, inflammation, and lipoprotein metabolism.

IntroductionThere is an increased need for the development of novel blood‐based biomarkers for early detection, prevention, or intervention in Alzheimer's disease (AD). This study sought to determine whether serum glycopeptide analysis holds potential for identifying novel diagnostics and prognostics of AD.MethodsThe study involved 195 participants, including 96 patients with an AD diagnosis and 99 controls with no cognitive deficit. Utilizing a validated analytical mass spectrometry method, we monitored the site‐specific glycosylation of 52 serum glycoproteins.ResultsPartial least‐squares discriminant analysis revealed that changes in overall sialylation and fucosylation of serum glycoproteins may be indicators of an AD disease state. Loss of fucosylation of immunoglobulin G1 (IgG1) and IgG2 was indicative of AD diagnosis. Individual glycopeptide analysis found separation between the AD patients and controls on complement proteins and apolipoprotein B.DiscussionThe results of this study suggest that serum glycoprofiling may be a promising approach for biomarker discovery.

Characterization of novel interactions with membrane NEU1 highlights new regulatory functions for the Elastin Receptor Complex in monocyte interaction with endothelial cells

BackgroundVascular aging is associated with remodeling of elastin, one of the main extracellular matrix component of the arterial wall, and production of elastin-derived peptides (EDP). These extracellular matrix degradation products have been shown to trigger biological activities through the elastin receptor complex (ERC) and data from the last decade have brought significant insights on the critical role played by its NEU1 subunit in the biological effects mediated by EDP and the ERC in vascular and metabolic diseases.ResultsUsing a proteomic approach, we previously identified new potential interaction partners of membrane NEU1. Here, we validated the interaction between NEU1 and the β2 integrin in human monocytes and show that binding of EDP to the ERC leads to desialylation of β2 integrin through NEU1. A similar action mechanism was identified in human umbilical vein endothelial cells (HUVEC) for intercellular cell adhesion molecule-1 (ICAM-1). Importantly, these effects were associated with a significant increase in monocyte adhesion to endothelial cells and monocyte transendothelial migration.ConclusionsThese results demonstrate that membrane NEU1 sialidase interacts and modulates the sialylation levels of the β2 integrin and ICAM-1 through the ERC in monocytes and endothelial cells, respectively, and suggest that EDP and the ERC, through this newly identified common mode of action governed by NEU1, may be important regulators of circulating monocyte recruitment to inflamed vascular sites. Moreover, by its ability to interact with and to modulate the sialylation of key membrane glycoproteins through NEU1, new biological functions are anticipated for EDP and the ERC in elastin remodeling-associated disorders.

Systematic screening and optimization of single-stranded DNA aptamer specific for N-acetylneuraminic acid: A comparative study

Sialic acids (SAs), particularly its most common derivative N-acetylneuraminic acid (Neu5Ac) are involved in many biological processes in vivo. Thus it is of great significance to detect Neu5Ac and trace the sialylation of glycoproteins and other bio-active molecules. Aptamers are molecular elements for bio-recognition with high affinity, which have been widely used in bioanalysis and imaging. However, the ideal aptamers for saccharides have seldom been reported. Magnetic beads (MB)-based SELEX is a commonly used method for screening of aptamers. Herein, three types of MB-based SELEX with different immobilization manners were carried out to systematically study and compare the screening efficiency, which may provide reference for screening of aptamers specific for low-molecular targets. After screening and sequencing, the obtained aptamer sequences were aligned and analyzed, among which three aptamers were selected as candidates. Then, the aptamer ap 3-1 with the highest affinity was optimized according to the results of molecular simulation docking. An optimal aptamer ap 3-1-a with 21 nucleotides (nt) was eventually obtained, with the Kd value of 55.71 ± 12.29 nM. To verify the practicability of the aptamer, a fluorescent biosensor for Neu5Ac was constructed employing graphene oxide (GO)-based fluorescence quenching and Neu5Ac-induced fluorescence restoration. The biosensor showed a linear response range within 20−1000 nM. The specificity was verified and the interferents-resistance was validated by detecting Neu5Ac in actual samples. This study may inspire the efficient and universal strategy for screening and application of robust and practicable aptamers for low-molecular targets.

Aberrant Sialylation in Cancer: Biomarker and Potential Target for Therapeutic Intervention?

Sialylation is an integral part of cellular function, governing many biological processes including cellular recognition, adhesion, molecular trafficking, signal transduction and endocytosis. Sialylation is controlled by the levels and the activities of sialyltransferases on glycoproteins and lipids. Altered gene expression of these enzymes in cancer yields to cancer-specific alterations of glycoprotein sialylation. Mounting evidence indicate that hypersialylation is closely associated with cancer progression and metastatic spread, and can be of prognostic significance in human cancer. Aberrant sialylation is not only a result of cancer, but also a driver of malignant phenotype, directly impacting key processes such as tumor cell dissociation and invasion, cell-cell and cell-matrix interactions, angiogenesis, resistance to apoptosis, and evasion of immune destruction. In this review we provide insights on the impact of sialylation in tumor progression, and outline the possible application of sialyltransferases as cancer biomarkers. We also summarize the most promising findings on the development of sialyltransferase inhibitors as potential anti-cancer treatments.

The function annotations of ST3GAL4 in human LAMP1 and Lassa virus GP-C interaction from the perspective of systems virology.

Lassa virus (LASV) is a single-stranded RNA virus that has plagued the Sub-Saharan part of Africa, precisely Nigeria where various pathogenic strains with varied genomic isoforms have been identified. The human lysosomal-associated membrane protein 1 (LAMP1) is alternately required for the micropinocytosis of LASV. Therefore, it is of interest to understand the mechanism of action of the host LAMP1 with LASV protein during infection. The role of ST3 beta-galactoside alpha-2, 3-sialyltransferase 4 (ST3GAL4) in the interaction between LASV (glycoprotein) GP-C and the human LAMP1 is relevant in this context. Deposited curated protein sequences of both LAMP1 and LASV GP-C were retrieved for the study. The ST3GAL4 associated data was constructed and analysed from weighted network analysis to infer the function annotations and molecular mediators that characterize the LASV infection. The gene network shows that glycoprotein sialylation, sialyltransferase enzymatic activities and glycosphingolipid biosynthesis are linked with the ST3GAL4 function. However, the physical interaction of FAM 213A, CD8B molecule and proprotein convertase subtilisin/kexin type 1 inhibitor (PCSK1N) with ST3GAL4 is intriguing in this perspective. There are 11 glycosylated asparagine sequons of the human LAMP1 but only nine were assigned a sialylated glycan cap to mediate the LASV GP-C and LAMP1 interaction having exceeded a recommended glycosylation threshold of 0.5. Therefore, the sialylated glycans of the human LAMP1 are a total of nine and these sialylated glycans mediate the molecular recognition between LASV and LAMP1. This study therefore, predicts that there is a cellular interchange between N-linked glycosylation properties of the human LAMP1 and LASV glycoprotein, and sialylation functions of ST3GAL4 in LASV infectivity. Further studies and the clinical trial of this predictive model on the sialylated glycans of LAMP1 will facilitate the understanding of the LASV micropinocytosis process in host cells.

Loss of core fucosylation in both ST6GAL1 and its substrate enhances glycoprotein sialylation in mice.

Fucosyltransferase 8 (FUT8) and β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) are glycosyltransferases that catalyze α1,6-fucosylation and α2,6-sialylation, respectively, in the mammalian N-glycosylation pathway. They are aberrantly expressed in various human diseases. FUT8 is non-glycosylated but is responsible for the fucosylation of ST6GAL1. However, the mechanism for the interaction between these two enzymes is unknown. In this study, we show that serum levels of α2,6-sialylated N-glycans are increased in Fut8-/- mice, whereas the mRNA and protein levels of ST6GAL1 are unchanged in mouse live tissues. The level of α2,6-sialylation on IgG was also enhanced in Fut8-/- mice along with ST6GAL1 catalytic activity increase in both serum and liver. Moreover, it was observed that ST6GAL1 prefers non-fucosylated substrates. Interestingly, increased core fucosylation accompanied by a reduction in α2,6-sialylation, was detected in rheumatoid arthritis patient serum. These findings provide new insight into the interactions between FUT8 and ST6GAL1.