Human ST3Gal II and ST6GalNAc IV genes increase human serum-mediated cytotoxicity to xenogeneic cells.

Carbohydrate-antigens widely existed on glycoproteins and glycosphingolipids of all mammalian cells play a crucial role in self-defense and immunity. Xeno-reactive antibodies included in natural human sera play a protecting role in an acute phase-rejection of xenotransplantation. In this study, we investigated the effect of an alteration of glycosylation-pattern, caused by human sialyltransferases such as hST3Gal II or hST6GalNAc IV, on human serum mediated cytotoxicity in pig kidney PK15 cells. From LDH cytotoxicity assay, cytotoxicity to human serum was significantly increased in hST3Gal II and hST6GalNAc IV-transfected PK15 cells, as compared to the control. In the hST6Gal I-carrying cells, the cytotoxicity to human serum was rather decreased. Moreover, flow cytometry analysis revealed that an alteration of pig glycosylation-pattern by hST3Gal II or hST6GalNAc IV influences on a binding of human IgM or IgG, respectively, in pig kidney cells, regardless of Gal antigen alteration. Finally, we found that hST6GalNAc IV contributed to increase of terminal disialylated tetrasaccharide structure, disialyl T antigen, as evidenced by increase of the MAL II lectin binding capacity in the hST6GalNAc IV-transfected PK15 cells, compared with control. Therefore, our results suggest that carbohydrate antigens, such as disialyl T antigen, newly synthesized by the ST3Gal II- and ST6GalNAc IV are potentially believed to be new xeno-reactive elements.

Up-regulation of inflammatory reactions by MPT32, a secreted protein of Mycobacterium tuberculosis in RAW264.7 macrophages.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and is still one of the global health burdens. The occurrence of various cases and multidrug resistance confirm that TB has not been completely conquered. For these reasons, the present research has been conducted to explore TB vaccine and drug candidate possibility using Mtb-secreted proteins. Among these proteins, MPT32 is known to have antigenicity and immunogenicity. There has not been a report on the host immune responses and regulation in macrophage cells. The present study was conducted with MPT32 in RAW 264.7 murine macrophage cells that control immune responses by sensing pathogen invasion and environmental change. We have found that MPT32 could activate lipopolysaccharide (LPS)-induced gene expression of metalloproteinase-9 (MMP-9) and inflammation in RAW 264.7 cells. After treating cells with MPT32, the increase in pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß (IL-1ß) and IL-6, was observed. In addition, activated macrophages expressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) to generate various inflammatory mediator molecules, such as nitric oxide (NO). The increase in iNOS and COX-2 levels, which are up-regulators of MMP-9 expression, was also confirmed. The biochemical events are involved in the downstream of activated MAPK signaling and translocation of NF-κ B transcription factor. The present results prove the immunomodulatory effect of MPT32 in the RAW 264.7 murine macrophage cells. it claims the possibility of a TB vaccination and drug candidate using MPT32, contributing to the prevention of TB.

T7 phage display reveals NOLC1 as a GM3 binding partner in human breast cancer MCF-7 cells.

Ganglioside GM3 is a simple monosialoganglioside (NeuAc-Gal-Glc-ceramide) that modulates cell adhesion, proliferation, and differentiation. Previously, we reported isolation of GM3-binding vascular endothelial growth factor receptor and transforming growth factor-ß receptor by the T7 phage display method (Chung et al., 2009; Kim et al., 2013). To further identify novel proteins interacting with GM3, we extended the T7 phage display method in this study. After T7 phage display biopanning combined with immobilized biotin-labeled 3'-sialyllactose prepared on a streptavidin-coated microplate, we isolated 100 candidate sequences from the human lung cDNA library. The most frequently detected clones from the blast analysis were the human nucleolar and coiled-body phosphoprotein 1 (NOLC1) sequences. We initially identified NOLC1 as a molecule that possibly binds to GM3 and confirmed this binding ability using the glutathione S-transferase fusion protein. Herein, we report another GM3-interacting protein, NOLC1, that can be isolated by the T7 phage display method. These results are expected to be helpful for elucidating the functional roles of ganglioside GM3 with NOLC1. When human breast cancer MCF-7 cells were examined for subcellular localization of NOLC1, immunofluorescence of NOLC1 was observed in the intracellular region. In addition, NOLC1 expression was increased in the nucleolus after treatment with the anticancer drug doxorubicin. GM3 and NOLC1 levels in the doxorubicin-treated MCF-7 cells were correlated, indicating possible associations between GM3 and NOLC1. Therefore, direct interactions between carbohydrates and cellular proteins can pave the path for new signaling phenomena in biology.

Regulatory mechanism for the human glioblastoma cell-specific expression of the human GD1c/GT1a/GQ1b synthase (hST8Sia V) gene.

In this study we observed that human GD1c/GT1a/GQ1b synthase (hST8Sia V) is particularly expressed in human glioblastoma cells. To address the mechanism regulating human glioblastoma-specific gene expression of the hST8Sia V, after the transcription start site (TSS) was identified by the 5'-rapid amplification of cDNA end with total RNA from human glioblastoma U87MG cells, the 5'-flanking region (2.5 kb) of the hST8Sia V gene was isolated and its promoter activity was examined. By luciferase reporter assay, this 5'-flanking region revealed strong promoter activity in only U-87MG cells, but not in other tissue-derived cancer cells. 5'-deletion mutant analysis showed that the region from -1140 to -494 is crucial for transcription of the hST8Sia V gene in U87MG cells. This region contains the activator protein-1 (AP-1) binding site, the main target of the c-Jun N-terminal kinase (JNK) downstream. The AP-1 binding site at -1043/-1037 was proved to be indispensable for the hST8Sia V gene-specific expression in U87MG cells by site-directed mutagenesis. Moreover, the transcriptional activation of hST8Sia V gene in U87MG cells was strongly inhibited by a specific JNK inhibitor, SP600125. These results suggest that the hST8Sia V gene-specific expression in U87MG cells is controlled by JNK/AP-1 signaling pathway.

Naturally-Occurring Tyrosinase Inhibitors Classified by Enzyme Kinetics and Copper Chelation.

Currently, there are three major assaying methods used to validate in vitro whitening activity from natural products: methods using mushroom tyrosinase, human tyrosinase, and dopachrome tautomerase (or tyrosinase-related protein-2, TRP-2). Whitening agent development consists of two ways, melanin synthesis inhibition in melanocytes and downregulation of melanocyte stimulation. For melanin levels, the melanocyte cell line has been used to examine melanin synthesis with the expression levels of TRP-1 and TRP-2. The proliferation of epidermal surfaced cells and melanocytes is stimulated by cellular signaling receptors, factors, or mediators including endothelin-1, α-melanocyte-stimulating hormone, nitric oxide, histamine, paired box 3, microphthalmia-associated transcription factor, pyrimidine dimer, ceramide, stem cell factors, melanocortin-1 receptor, and cAMP. In addition, the promoter region of melanin synthetic genes including tyrosinase is upregulated by melanocyte-specific transcription factors. Thus, the inhibition of growth and melanin synthesis in gene expression levels represents a whitening research method that serves as an alternative to tyrosinase inhibition. Many researchers have recently presented the bioactivity-guided fractionation, discovery, purification, and identification of whitening agents. Melanogenesis inhibition can be obtained using three different methods: tyrosinase inhibition, copper chelation, and melanin-related protein downregulation. There are currently four different types of inhibitors characterized based on their enzyme inhibition mechanisms: competitive, uncompetitive, competitive/uncompetitive mixed-type, and noncompetitive inhibitors. Reversible inhibitor types act as suicide substrates, where traditional inhibitors are classified as inactivators and reversible inhibitors based on the molecule-recognizing properties of the enzyme. In a minor role, transcription factors can also be downregulated by inhibitors. Currently, the active site copper iron-binding inhibitors such as kojic acid and chalcone exhibit tyrosinase inhibitory activity. Because the tyrosinase catalysis site structure is important for the mechanism determination of tyrosinase inhibitors, understanding the enzyme recognition and inhibitory mechanism of inhibitors is essential for the new development of tyrosinase inhibitors. The present review intends to classify current natural products identified by means of enzyme kinetics and copper chelation to exhibit tyrosinase enzyme inhibition.

Mycobacterium tuberculosis glycolipoprotein LprG inhibits inflammation through NF-κB signaling of ERK1/2 and JNK in LPS-induced murine macrophage cells.

Mycobacterium tuberoculosis (Mtb) is a contagious pathogen that causes human tuberculosis (TB). TB is a major global health threat that causes 9.6 million illnesses and 1.5 million deaths per year. Recent studies have suggested Mtb-secreted proteins as new candidates for therapeutic drugs and vaccines. LprG is a Mtb-secreted surface glycolipoprotein encoded by lprG (Rv1411c), which forms an operon with Rv1410c, where Rv1410c encodes P55, an efflux pump membrane protein. Various in vitro and in vivo studies have reported on the target-binding activity, cell envelope biosynthesis, and mycobacterial virulence of LprG. However, the anti-inflammatory effect of LprG in macrophages has not yet been investigated. In this study, we demonstrated that LprG can suppress lipopolysaccharide (LPS)-induced inflammation in a macrophage model. LprG inhibited LPS-stimulated nitric oxide (NO) production. LprG also suppressed expression of inducible cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) at the transcriptional and protein levels. In addition, LprG decreased mRNA expression of the pro-inflammatory cytokines interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α). Furthermore, LprG attenuated nuclear factor kappa-B (NF-κB) translocation and IκB phosphorylation. Moreover, LprG specifically inhibited phosphorylated kinases such as c-Jun N-terminal kinase (p-JNK) and extracellular signal-regulated kinase 1/2 (p-ERK1/2), but not p-p38. Taken together, these results suggest that LprG inhibits LPS-stimulated inflammation via downregulation of NO, COX-2, iNOS, and pro-inflammatory cytokines through the NF-κB, AP-1, and MAPK signaling pathways. The present study will aid in the development of anti-inflammatory medications using Mtb. The organism, which has long been regarded as a human pathogenic or human health-threating agent, can be utilized as a future medical resource.

Molecular Research of Glycolysis.

Glycolysis represents the process of breaking down monosaccharides, which involves the energy metabolism, homeostasis, and the linkage of various physiological functions such as muscle movement, development, neurotransmission, etc [...].

Systemic Lectin-Glycan Interaction of Pathogenic Enteric Bacteria in the Gastrointestinal Tract.

Microorganisms, such as bacteria, viruses, and fungi, and host cells, such as plants and animals, have carbohydrate chains and lectins that reciprocally recognize one another. In hosts, the defense system is activated upon non-self-pattern recognition of microbial pathogen-associated molecular patterns. These are present in Gram-negative and Gram-positive bacteria and fungi. Glycan-based PAMPs are bound to a class of lectins that are widely distributed among eukaryotes. The first step of bacterial infection in humans is the adhesion of the pathogen's lectin-like proteins to the outer membrane surfaces of host cells, which are composed of glycans. Microbes and hosts binding to each other specifically is of critical importance. The adhesion factors used between pathogens and hosts remain unknown; therefore, research is needed to identify these factors to prevent intestinal infection or treat it in its early stages. This review aims to present a vision for the prevention and treatment of infectious diseases by identifying the role of the host glycans in the immune response against pathogenic intestinal bacteria through studies on the lectin-glycan interaction.

Glycan-Adhering Lectins and Experimental Evaluation of a Lectin FimH Inhibitor in Enterohemorrhagic Escherichia coli (EHEC) O157:H7 Strain EDL933.

In this study, we tried to develop a FimH inhibitor that inhibits adhesion of enterohemorrhagic Escherichia coli (EHEC) on the epithelium of human intestine during the initial stage of infections. Using a T7 phage display method with a reference strain, EHEC EDL933, FimH was selected as an adherent lectin to GM1a and Gb3 glycans. In order to detect the ligand binding domain (LBD) of FimH, we used a docking simulation and found three binding site sequences of FimH, i.e., P1, P2, and P3. Among Gb3 mimic peptides, P2 was found to have the strongest binding strength. Moreover, in vitro treatment with peptide P2 inhibited binding activity in a concentration-dependent manner. Furthermore, we conducted confirmation experiments through several strains isolated from patients in Korea, EHEC NCCP15736, NCCP15737, and NCCP15739. In addition, we analyzed the evolutionary characteristics of the predicted FimH lectin-like adhesins to construct a lectin-glycan interaction (LGI). We selected 70 recently differentiated strains from the phylogenetic tree of 2240 strains with Shiga toxin in their genome. We can infer EHEC strains dynamically evolved but FimH was conserved during the evolution time according to the phylogenetic tree. Furthermore, FimH could be a reliable candidate of drug target in terms of evolution. We examined how pathogen lectins interact with host glycans early in infection in EDL933 as well as several field strains and confirmed that glycan-like peptides worked as an initial infection inhibitor.

Revisited Metabolic Control and Reprogramming Cancers by Means of the Warburg Effect in Tumor Cells.

Aerobic glycolysis is an emerging hallmark of many human cancers, as cancer cells are defined as a "metabolically abnormal system". Carbohydrates are metabolically reprogrammed by its metabolizing and catabolizing enzymes in such abnormal cancer cells. Normal cells acquire their energy from oxidative phosphorylation, while cancer cells acquire their energy from oxidative glycolysis, known as the "Warburg effect". Energy-metabolic differences are easily found in the growth, invasion, immune escape and anti-tumor drug resistance of cancer cells. The glycolysis pathway is carried out in multiple enzymatic steps and yields two pyruvate molecules from one glucose (Glc) molecule by orchestral reaction of enzymes. Uncontrolled glycolysis or abnormally activated glycolysis is easily observed in the metabolism of cancer cells with enhanced levels of glycolytic proteins and enzymatic activities. In the "Warburg effect", tumor cells utilize energy supplied from lactic acid-based fermentative glycolysis operated by glycolysis-specific enzymes of hexokinase (HK), keto-HK-A, Glc-6-phosphate isomerase, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase, phosphofructokinase (PFK), phosphor-Glc isomerase (PGI), fructose-bisphosphate aldolase, phosphoglycerate (PG) kinase (PGK)1, triose phosphate isomerase, PG mutase (PGAM), glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase isozyme type M2 (PKM2), pyruvate dehydrogenase (PDH), PDH kinase and lactate dehydrogenase. They are related to glycolytic flux. The key enzymes involved in glycolysis are directly linked to oncogenesis and drug resistance. Among the metabolic enzymes, PKM2, PGK1, HK, keto-HK-A and nucleoside diphosphate kinase also have protein kinase activities. Because glycolysis-generated energy is not enough, the cancer cell-favored glycolysis to produce low ATP level seems to be non-efficient for cancer growth and self-protection. Thus, the Warburg effect is still an attractive phenomenon to understand the metabolic glycolysis favored in cancer. If the basic properties of the Warburg effect, including genetic mutations and signaling shifts are considered, anti-cancer therapeutic targets can be raised. Specific therapeutics targeting metabolic enzymes in aerobic glycolysis and hypoxic microenvironments have been developed to kill tumor cells. The present review deals with the tumor-specific Warburg effect with the revisited viewpoint of recent progress.

Characterization of a C-Type Lectin Domain-Containing Protein with Antibacterial Activity from Pacific Abalone (Haliotis discus hannai).

Genes that influence the growth of Pacific abalone (Haliotis discus hannai) may improve the productivity of the aquaculture industry. Previous research demonstrated that the differential expression of a gene encoding a C-type lectin domain-containing protein (CTLD) was associated with a faster growth in Pacific abalone. We analyzed this gene and identified an open reading frame that consisted of 145 amino acids. The sequence showed a significant homology to other genes that encode CTLDs in the genus Haliotis. Expression profiling analysis at different developmental stages and from various tissues showed that the gene was first expressed at approximately 50 days after fertilization (shell length of 2.47 ± 0.13 mm). In adult Pacific abalone, the gene was strongly expressed in the epipodium, gill, and mantle. Recombinant Pacific abalone CTLD purified from Escherichia coli exhibited antimicrobial activity against several Gram-positive bacteria (Bacillus subtilis, Streptococcus iniae, and Lactococcus garvieae) and Gram-negative bacteria (Vibrio alginolyticus and Vibrio harveyi). We also performed bacterial agglutination assays in the presence of Ca2+, as well as bacterial binding assays in the presence of the detergent dodecyl maltoside. Incubation with E. coli and B. subtilis cells suggested that the CTLD stimulated Ca2+-dependent bacterial agglutination. Our results suggest that this novel Pacific abalone CTLD is important for the pathogen recognition in the gastropod host defense mechanism.

Application of Metabolic Reprogramming to Cancer Imaging and Diagnosis.

Cellular metabolism governs the signaling that supports physiological mechanisms and homeostasis in an individual, including neuronal transmission, wound healing, and circadian clock manipulation. Various factors have been linked to abnormal metabolic reprogramming, including gene mutations, epigenetic modifications, altered protein epitopes, and their involvement in the development of disease, including cancer. The presence of multiple distinct hallmarks and the resulting cellular reprogramming process have gradually revealed that these metabolism-related molecules may be able to be used to track or prevent the progression of cancer. Consequently, translational medicines have been developed using metabolic substrates, precursors, and other products depending on their biochemical mechanism of action. It is important to note that these metabolic analogs can also be used for imaging and therapeutic purposes in addition to competing for metabolic functions. In particular, due to their isotopic labeling, these compounds may also be used to localize and visualize tumor cells after uptake. In this review, the current development status, applicability, and limitations of compounds targeting metabolic reprogramming are described, as well as the imaging platforms that are most suitable for each compound and the types of cancer to which they are most appropriate.

Current Perspectives on the Physiological Activities of Fermented Soybean-Derived Cheonggukjang.

Cheonggukjang (CGJ, fermented soybean paste), a traditional Korean fermented dish, has recently emerged as a functional food that improves blood circulation and intestinal regulation. Considering that excessive consumption of refined salt is associated with increased incidence of gastric cancer, high blood pressure, and stroke in Koreans, consuming CGJ may be desirable, as it can be made without salt, unlike other pastes. Soybeans in CGJ are fermented by Bacillus strains (B. subtilis or B. licheniformis), Lactobacillus spp., Leuconostoc spp., and Enterococcus faecium, which weaken the activity of putrefactive bacteria in the intestines, act as antibacterial agents against pathogens, and facilitate the excretion of harmful substances. Studies on CGJ have either focused on improving product quality or evaluating the bioactive substances contained in CGJ. The fermentation process of CGJ results in the production of enzymes and various physiologically active substances that are not found in raw soybeans, including dietary fiber, phospholipids, isoflavones (e.g., genistein and daidzein), phenolic acids, saponins, trypsin inhibitors, and phytic acids. These components prevent atherosclerosis, oxidative stress-mediated heart disease and inflammation, obesity, diabetes, senile dementia, cancer (e.g., breast and lung), and osteoporosis. They have also been shown to have thrombolytic, blood pressure-lowering, lipid-lowering, antimutagenic, immunostimulatory, anti-allergic, antibacterial, anti-atopic dermatitis, anti-androgenetic alopecia, and anti-asthmatic activities, as well as skin improvement properties. In this review, we examined the physiological activities of CGJ and confirmed its potential as a functional food.

Beneficial Effects of Soybean-Derived Bioactive Peptides.

Peptides present in foods are involved in nutritional functions by supplying amino acids; sensory functions related to taste or solubility, emulsification, etc.; and bioregulatory functions in various physiological activities. In particular, peptides have a wide range of physiological functions, including as anticancer agents and in lowering blood pressure and serum cholesterol levels, enhancing immunity, and promoting calcium absorption. Soy protein can be partially hydrolyzed enzymatically to physiologically active soy (or soybean) peptides (SPs), which not only exert physiological functions but also help amino acid absorption in the body and reduce bitterness by hydrolyzing hydrophobic amino acids from the C- or N-terminus of soy proteins. They also possess significant gel-forming, emulsifying, and foaming abilities. SPs are expected to be able to prevent and treat atherosclerosis by inhibiting the reabsorption of bile acids in the digestive system, thereby reducing blood cholesterol, low-density lipoprotein, and fat levels. In addition, soy contains blood pressure-lowering peptides that inhibit angiotensin-I converting enzyme activity and antithrombotic peptides that inhibit platelet aggregation, as well as anticancer, antioxidative, antimicrobial, immunoregulatory, opiate-like, hypocholesterolemic, and antihypertensive activities. In animal models, neuroprotective and cognitive capacity as well as cardiovascular activity have been reported. SPs also inhibit chronic kidney disease and tumor cell growth by regulating the expression of genes associated with apoptosis, inflammation, cell cycle arrest, invasion, and metastasis. Recently, various functions of soybeans, including their physiologically active functions, have been applied to health-oriented foods, functional foods, pharmaceuticals, and cosmetics. This review introduces some current results on the role of bioactive peptides found in soybeans related to health functions.

Physiologically Active Molecules and Functional Properties of Soybeans in Human Health-A Current Perspective.

In addition to providing nutrients, food can help prevent and treat certain diseases. In particular, research on soy products has increased dramatically following their emergence as functional foods capable of improving blood circulation and intestinal regulation. In addition to their nutritional value, soybeans contain specific phytochemical substances that promote health and are a source of dietary fiber, phospholipids, isoflavones (e.g., genistein and daidzein), phenolic acids, saponins, and phytic acid, while serving as a trypsin inhibitor. These individual substances have demonstrated effectiveness in preventing chronic diseases, such as arteriosclerosis, cardiac diseases, diabetes, and senile dementia, as well as in treating cancer and suppressing osteoporosis. Furthermore, soybean can affect fibrinolytic activity, control blood pressure, and improve lipid metabolism, while eliciting antimutagenic, anticarcinogenic, and antibacterial effects. In this review, rather than to improve on the established studies on the reported nutritional qualities of soybeans, we intend to examine the physiological activities of soybeans that have recently been studied and confirm their potential as a high-functional, well-being food.

Inhibitory Effect of Avenanthramides (Avn) on Tyrosinase Activity and Melanogenesis in α-MSH-Activated SK-MEL-2 Cells: In Vitro and In Silico Analysis.

Melanin causes melasma, freckles, age spots, and chloasma. Anti-melanogenic agents can prevent disease-related hyperpigmentation. In the present study, the dose-dependent tyrosinase inhibitory activity of Avenanthramide (Avn)-A-B-C was demonstrated, and 100 µM Avn-A-B-C produced the strongest competitive inhibition against inter-cellular tyrosinase and melanin synthesis. Avn-A-B-C inhibits the expression of melanogenesis-related proteins, such as TRP1 and 2. Molecular docking simulation revealed that AvnC (-7.6 kcal/mol) had a higher binding affinity for tyrosinase than AvnA (-7.3 kcal/mol) and AvnB (-6.8 kcal/mol). AvnC was predicted to interact with tyrosinase through two hydrogen bonds at Ser360 (distance: 2.7 Å) and Asn364 (distance: 2.6 Å). In addition, AvnB and AvnC were predicted to be skin non-sensitizers in mammals by the Derek Nexus Quantitative Structure-Activity Relationship system.

Regulation of human ß-galactoside α2,6-sialyltransferase (hST6Gal I) gene expression during differentiation of human osteoblastic MG-63 cells.

In this study, we found that gene expression of the human ß-galactoside α2,6-sialyltransferase (hST6Gal I) was specifically increased during differentiation of human MG-63 osteoblastic cells by serum starvation (SS). In parallel, a distinct increase in binding to SNA, the α2,6-sialyl-specific lectin, was observed in serum-starved cells, as demonstrated by FACS analysis. 5'-Rapid amplification of cDNA ends analysis demonstrated that the increase of hST6Gal I transcript by SS is mediated by P1 promoter. To elucidate transcriptional regulation of hST6Gal I in SS-induced MG-63 cells, we functionally characterized the P1 promoter region of the hST6Gal I gene. The 5'-deletion analysis of P1 promoter region revealed that the 189 bp upstream region of transcription start site is critical for transcriptional activity of hST6Gal I gene in SS-induced MG-63 cells. This region contains the predicted binding sites for several transcription factors, including AREB6, FOXP1, SIX3, HNF1, YY2, and MOK2. The mutagenesis analysis for these sites and chromatin immunoprecipitation assay demonstrated that the YY2 binding site at -98 to -77 was essential for the SS-induced hST6Gal I gene expression during differentiation of MG-63 cells.

3'-sialyllactose targets cell surface protein, SIGLEC-3, and induces megakaryocyte differentiation and apoptosis by lipid raft-dependent endocytosis.

3'-sialyllactose is one of the abundant components in human milk oligosaccharides (HMOs) that protect infants from various viral infections in early stages of immune system development. 3SL is a combination of lactose and sialic acid. Most sialic acids are widely expressed in animal cells and they bind to siglec proteins. In this study, we demonstrate that 3SL specifically binds to CD33. It induces megakaryocyte differentiation and subsequent apoptosis by targeting cell surface protein siglec-3 (CD33) in human chronic myeloid leukemia K562 cells. The 3SL-bound CD33 was internalized to the cytosol via caveolae-dependent endocytosis. At the molecular level, 3SL-bound CD33 recruits the suppressor of cytokine signaling 3 (SOCS3) and SH2 domain-containing protein tyrosine phosphatase 1 (SHP1). SOCS3 is degraded with CD33 by proteasome degradation, while SHP-1 activates extracellular signal-regulated kinase (ERK) to induce megakaryocytic differentiation and subsequent apoptosis. The present study, therefore, suggests that 3SL is a potential anti-leukemia agent affecting differentiation and apoptosis.

SARS-CoV-2 Evolutionary Adaptation toward Host Entry and Recognition of Receptor O-Acetyl Sialylation in Virus-Host Interaction.

The recently emerged SARS-CoV-2 is the cause of the global health crisis of the coronavirus disease 2019 (COVID-19) pandemic. No evidence is yet available for CoV infection into hosts upon zoonotic disease outbreak, although the CoV epidemy resembles influenza viruses, which use sialic acid (SA). Currently, information on SARS-CoV-2 and its receptors is limited. O-acetylated SAs interact with the lectin-like spike glycoprotein of SARS CoV-2 for the initial attachment of viruses to enter into the host cells. SARS-CoV-2 hemagglutinin-esterase (HE) acts as the classical glycan-binding lectin and receptor-degrading enzyme. Most ß-CoVs recognize 9-O-acetyl-SAs but switched to recognizing the 4-O-acetyl-SA form during evolution of CoVs. Type I HE is specific for the 9-O-Ac-SAs and type II HE is specific for 4-O-Ac-SAs. The SA-binding shift proceeds through quasi-synchronous adaptations of the SA-recognition sites of the lectin and esterase domains. The molecular switching of HE acquisition of 4-O-acetyl binding from 9-O-acetyl SA binding is caused by protein-carbohydrate interaction (PCI) or lectin-carbohydrate interaction (LCI). The HE gene was transmitted to a ß-CoV lineage A progenitor by horizontal gene transfer from a 9-O-Ac-SA-specific HEF, as in influenza virus C/D. HE acquisition, and expansion takes place by cross-species transmission over HE evolution. This reflects viral evolutionary adaptation to host SA-containing glycans. Therefore, CoV HE receptor switching precedes virus evolution driven by the SA-glycan diversity of the hosts. The PCI or LCI stereochemistry potentiates the SA-ligand switch by a simple conformational shift of the lectin and esterase domains. Therefore, examination of new emerging viruses can lead to better understanding of virus evolution toward transitional host tropism. A clear example of HE gene transfer is found in the BCoV HE, which prefers 7,9-di-O-Ac-SAs, which is also known to be a target of the bovine torovirus HE. A more exciting case of such a switching event occurs in the murine CoVs, with the example of the ß-CoV lineage A type binding with two different subtypes of the typical 9-O-Ac-SA (type I) and the exclusive 4-O-Ac-SA (type II) attachment factors. The protein structure data for type II HE also imply the virus switching to binding 4-O acetyl SA from 9-O acetyl SA. Principles of the protein-glycan interaction and PCI stereochemistry potentiate the SA-ligand switch via simple conformational shifts of the lectin and esterase domains. Thus, our understanding of natural adaptation can be specified to how carbohydrate/glycan-recognizing proteins/molecules contribute to virus evolution toward host tropism. Under the current circumstances where reliable antiviral therapeutics or vaccination tools are lacking, several trials are underway to examine viral agents. As expected, structural and non-structural proteins of SARS-CoV-2 are currently being targeted for viral therapeutic designation and development. However, the modern global society needs SARS-CoV-2 preventive and therapeutic drugs for infected patients. In this review, the structure and sialobiology of SARS-CoV-2 are discussed in order to encourage and activate public research on glycan-specific interaction-based drug creation in the near future.

Gram-Negative Bacterial Endotoxin LPS Induces NeuGc Loss through Ets1-Dependent Downregulation of Intestine-Specific pcmah Transcript in Porcine Intestinal Cells.

N-glycolylneuraminic acid (NeuGc), a non-human sialic acid derivative synthesized by cytidine-5'-monophospho-N-acetylneuraminic acid hydroxylase (CMAH), plays a crucial role in mediating infections by certain pathogens. Although it has been postulated that NeuGc biosynthesis and CMAH expression are downregulated during microbial infection, the underlying mechanisms remain unclear. The present study showed that exposure to lipopolysaccharide (LPS), a Gram-negative bacterial endotoxin, leads to loss of NeuGc biosynthesis in pig small intestinal I2I-2I cells. This LPS-induced NeuGc loss was accompanied by decreased CMAH transcript levels, especially intestine-specific 5'pcmah-1. Furthermore, LPS suppressed the activity of the Pi promoter responsible for 5'pcmah-1 by inhibiting DNA binding of Est1. These findings provide insight into the regulatory mechanisms of Neu5Gc biosynthesis during pathogenic infectious events, which may represent a host defense mechanism that protects the self against pathogenic bacterial infections even in non-sanitary environments.