Carbohydrate Sulfation As a Mechanism for Fine-Tuning Siglec Ligands.

The immunomodulatory family of Siglecs recognizes sialic acid-containing glycans as "self", which is exploited in cancer for immune evasion. The biochemical nature of Siglec ligands remains incompletely understood, with emerging evidence suggesting the importance of carbohydrate sulfation. Here, we investigate how specific sulfate modifications affect Siglec ligands by overexpressing eight carbohydrate sulfotransferases (CHSTs) in five cell lines. Overexpression of three CHSTs─CHST1, CHST2, or CHST4─significantly enhance the binding of numerous Siglecs. Unexpectedly, two other CHSTs (Gal3ST2 and Gal3ST3) diminish Siglec binding, suggesting a new mode to modulate Siglec ligands via sulfation. Results are cell type dependent, indicating that the context in which sulfated glycans are presented is important. Moreover, a pharmacological blockade of N- and O-glycan maturation reveals a cell-type-specific pattern of importance for either class of glycan. Production of a highly homogeneous Siglec-3 (CD33) fragment enabled a mass-spectrometry-based binding assay to determine ≥8-fold and ≥2-fold enhanced affinity for Neu5Acα2-3(6-O-sulfo)Galß1-4GlcNAc and Neu5Acα2-3Galß1-4(6-O-sulfo)GlcNAc, respectively, over Neu5Acα2-3Galß1-4GlcNAc. CD33 shows significant additivity in affinity (≥28-fold) for the disulfated ligand, Neu5Acα2-3(6-O-sulfo)Galß1-4(6-O-sulfo)GlcNAc. Moreover, joint overexpression of CHST1 with CHST2 in cells greatly enhanced the binding of CD33 and several other Siglecs. Finally, we reveal that CHST1 is upregulated in numerous cancers, correlating with poorer survival rates and sodium chlorate sensitivity for the binding of Siglecs to cancer cell lines. These results provide new insights into carbohydrate sulfation as a general mechanism for tuning Siglec ligands on cells, including in cancer.

Increasing phagocytosis of microglia by targeting CD33 with liposomes displaying glycan ligands.

CD33 is an immunomodulatory receptor expressed by microglia and genetically linked to Alzheimer's disease (AD) susceptibility. While antibodies targeting CD33 have entered clinical trials to treat neurodegeneration, it is unknown whether the glycan-binding properties of CD33 can be exploited to modulate microglia. Here, we use liposomes that multivalently display glycan ligands of CD33 (CD33L liposomes) to engage CD33. We find that CD33L liposomes increase phagocytosis of cultured monocytic cells and microglia in a CD33-dependent manner. Enhanced phagocytosis strongly correlates with loss of CD33 from the cell surface and internalization of liposomes. Increased phagocytosis by treatment with CD33L liposomes is dependent on a key intracellular signaling motif on CD33 as well as the glycan-binding ability of CD33. These effects are specific to trans engagement of CD33 by CD33L liposomes, as cis engagement through insertion of lipid-linked CD33L into cells produces the opposite effect on phagocytosis. Moreover, intracerebroventricular injection of CD33L liposomes into transgenic mice expressing human CD33 in the microglial cell lineage enhances phagocytosis of microglia in a CD33-dependent manner. These results demonstrate that multivalent engagement of CD33 with glycan ligands can modulate microglial cell function.

The CD33 short isoform is a gain-of-function variant that enhances Aß1-42 phagocytosis in microglia.

BACKGROUND: CD33 is genetically linked to Alzheimer's disease (AD) susceptibility through differential expression of isoforms in microglia. The role of the human CD33 short isoform (hCD33m), preferentially encoded by an AD-protective CD33 allele (rs12459419T), is unknown. Here, we test whether hCD33m represents a loss-of-function or gain-of-function variant. METHODS: We have developed two models to test the role of hCD33m. The first is a new strain of transgenic mice expressing hCD33m in the microglial cell lineage. The second is U937 cells where the CD33 gene was disrupted by CRISPR/Cas9 and complemented with different variants of hCD33. Primary microglia and U937 cells were tested in phagocytosis assays and single cell RNA sequencing (scRNAseq) was carried out on the primary microglia. Furthermore, a new monoclonal antibody was developed to detect hCD33m more efficiently. RESULTS: In both primary microglia and U937 cells, we find that hCD33m enhances phagocytosis. This contrasts with the human CD33 long isoform (hCD33M) that represses phagocytosis, as previously demonstrated. As revealed by scRNAseq, hCD33m+ microglia are enriched in a cluster of cells defined by an upregulated expression and gene regulatory network of immediate early genes, which was further validated within microglia in situ. Using a new hCD33m-specific antibody enabled hCD33m expression to be examined, demonstrating a preference for an intracellular location. Moreover, this newly discovered gain-of-function role for hCD33m is dependent on its cytoplasmic signaling motifs, dominant over hCD33M, and not due to loss of glycan ligand binding. CONCLUSIONS: These results provide strong support that hCD33m represents a gain-of-function isoform and offers insight into what it may take to therapeutically capture the AD-protective CD33 allele.

Erratum: Author Correction: Repression of phagocytosis by human CD33 is not conserved with mouse CD33.

[This corrects the article DOI: 10.1038/s42003-019-0698-6.].

The effect of amino acids on non-enzymatic browning of glucosamine: Generation of butterscotch aromatic and bioactive health compounds without detectable levels of neo-formed alkylimidazoles.

A mixture of glucosamine (GlcN, 15% w/v) and different amino acids in 1:1 M ratio was incubated at 70 °C for 12 h. The resulting GlcN-amino acid caramels were analysed for α-dicarbonyl compounds, polyhydroxyalkyl pyrazines, heterocyclic compound and alkylimidazoles. All the analyses were performed by using HPLC-MS/MS followed by pooling the variables with principal component analysis (PCA). GlcN-Gly caramels generated the greatest amount of butterscotch aromatic compound diacetyl and polyhydroxyalkyl pyrazines (fructosazine and deoxyfructosazine). The potentially toxic heterocyclic compound, 5-hydroxymethylfurfural (HMF) was generated in greater amounts with the GlcN-Arg caramels. However, the toxic alkylimidazoles (4-MEI and THI) were not present in any of the GlcN-amino acid caramels. The results suggest that caramel with butterscotch aroma and bioactivity can be produced with GlcN-amino acid at 70 °C. The PCA performed discriminated the majority of the GlcN-amino acid combinations; GlcN-Gly and GlcN-Ser were best discriminated.

Repression of phagocytosis by human CD33 is not conserved with mouse CD33.

CD33 is an immunomodulatory receptor linked to Alzheimer's disease (AD) susceptibility via regulation of phagocytosis in microglia. Divergent features between human CD33 (hCD33) and murine CD33 (mCD33) include a unique transmembrane lysine in mCD33 and cytoplasmic tyrosine in hCD33. The functional consequences of these differences in restraining phagocytosis remains poorly understood. Using a new αmCD33 monoclonal antibody, we show that mCD33 is expressed at high levels on neutrophils and low levels on microglia. Notably, cell surface expression of mCD33 is entirely dependent on Dap12 due to an interaction with the transmembrane lysine in mCD33. In RAW264.7 cultured macrophages, BV-2 cultured microglia, primary neonatal and adult microglia, uptake of cargo - including aggregated Aß1-42 - is not altered upon genetic ablation of mCD33. Alternatively, deletion of hCD33 in monocytic cell lines increased cargo uptake. Moreover, transgenic mice expressing hCD33 in the microglial cell lineage showed repressed cargo uptake in primary microglia. Therefore, mCD33 and hCD33 have divergent roles in regulating phagocytosis, highlighting the importance of studying hCD33 in AD susceptibility.

Improved bactericidal capacity of UV-B radiation against E. coli strains by photosensitizing bacteria with fructosazine - An advanced Maillard reaction product.

This study investigated the effect of UV-B irradiation and the combinational effect with glucosamine caramel, fructosazine and riboflavin on the antimicrobial activities against Bacillus subtilis (ATCC 6633) and two strains of Escherichia coli (AW 1.7 and ATCC 25922). The quantum yield of fructosazine was two times less than that of tryptophan, indicating its ability to emit fluorescent light but less efficiently than tryptophan. UV-B treatment alone was efficient to achieve a bactericidal effect for both E. coli stains tested, however no effect was found for Bacillus subtilis for up to 80 mJ/cm2 UV-B. The combination of UV-B with photosensitizers fructosazine, glucosamine caramel and riboflavin enhanced the UV-B efficacy against E. coli strains at lower UV-B doses, while Bacillus subtilis ATCC 6633 was more resistant to the treatment combinations. High-performance liquid chromatography showed the production of different fructosazine reaction products occurred during irradiation, including the possible formation of endoperoxides.

Argpyrimidine-tagged rutin-encapsulated biocompatible (ethylene glycol dimers) nanoparticles: Application for targeted drug delivery in experimental diabetes (Part 2).

Diabetes mellitus is characterized by hyperglycemia and associated complications. However, long-term diabetes control is not often sustained by currently available therapeutic approaches. Research on nanoparticle-mediated drug delivery systems is in progress. Here we have tested a ligand (argpyrimidine)-tagged drug (rutin)-encapsulated biocompatible (ethylene glycol dimers) nanoparticle for targeted drug delivery in streptozotocin-induced diabetic rats. Argpyrimidine, being an advanced glycation end product (AGE), directs the nanoparticles to interact with cell surface receptors of AGEs (RAGE) and delivers the drug into the cells. The bioflavonoid rutin possesses antihyperglycemic property, and has been used for nanocapsulation. Two doses of nanoparticles containing 20mg rutin/kg body weight were administered (i.v. at 7days interval) into streptozotocin-induced diabetic rats. Compared to free rutin, nanoparticle treatment appears to be significantly more effective in controlling the diabetogenic effects - hyperglycemia, hyperlipidemia, oxidative stress etc, including heart-associated complications. This approach may thus be explored for drug delivery in the treatment of diabetes mellitus.

Bimolecular interaction of argpyrimidine (a Maillard reaction product) in in vitro non-enzymatic protein glycation model and its potential role as an antiglycating agent.

Non- enzymatic glycation, also known as Maillard reaction, is one of the most important and investigated reactions in biochemistry. Maillard reaction products (MRPs) like protein-derived advanced glycation end products (AGEs) are often referred to cause pathophysiological complications in human systems. On contrary, several MRPs are exogenously used as antioxidant, antimicrobial and flavouring agents. In the preset study, we have shown that argpyrimidine, a well-established AGE, interacts with bovine serum albumin (BSA) and glucose individually in standard BSA-glucose model system and successfully inhibits glycation of the protein. Bimolecular interaction of argpyrimidine with glucose or BSA has been studied independently. Chromatographic purification, different spectroscopic studies and molecular modeling have been used to evaluate the nature and pattern of interactions. Binding of argpyrimidine with BSA prevents incorporation of glucose inside the native protein. Argpyrimidine can also directly entrap glucose. Both these interactions may be associated with the antiglycation potential of argpyrimidine, indicating a beneficial function of an AGE.

Transport of the Glucosamine-Derived Browning Product Fructosazine (Polyhydroxyalkylpyrazine) Across the Human Intestinal Caco-2 Cell Monolayer: Role of the Hexose Transporters.

The transport mechanism of fructosazine, a glucosamine self-condensation product, was investigated using a Caco-2 cell model. Fructosazine transport was assessed by measuring the bidirectional permeability coefficient across Caco-2 cells. The mechanism of transport was evaluated using phlorizin, an inhibitor of sodium-dependent glucose cotransporters (SGLT) 1 and 2, phloretin and quercetin, inhibitors of glucose transporters (GLUT) 1 and 2, transcytosis inhibitor wortmannin, and gap junction disruptor cytochalasin D. The role of hexose transporters was further studied using downregulated or overexpressed cell lines. The apparent permeability (Pa,b) of fructosazine was 1.30 ± 0.02 × 10-6 cm/s. No significant (p > 0.05) effect was observed in fructosazine transport by adding wortmannin and cytochalasin D. The presence of phlorizin, phloretin, and quercetin decreased fructosazine transport. The downregulated GLUT cells line was unable to transport fructosazine. In human intestinal epithelial Caco-2 cells, GLUT1 or GLUT2 and SGLT are mainly responsible for fructosazine transport.

Fructosazine, a Polyhydroxyalkylpyrazine with Antimicrobial Activity: Mechanism of Inhibition against Extremely Heat Resistant Escherichia coli.

Fructosazine is a polyhydroxyalkylpyrazine recently reported to have antimicrobial activity against heat-resistant Escherichia coli AW 1.7. This study investigated fructosazine's antimicrobial mechanism of action and compared it to that of riboflavin. Fructosazine-acetic acid was effective in permeabilizing the outer membrane based on an evaluation of bacterial membrane integrity using 1-N-phenyl-1-naphthylamine and propidium iodide. The uptake of fructosazine by E. coli was pH-dependent with a greater uptake at pH 5 compared to pH 7 for all times throughout 16 h, except 2, 3, and 10 h. Fructosazine generates 1O2, which is partially why it damages E. coli. DNA fragmentation was confirmed by fluorescence microscopy, and the fructosazine-acetic acid was the second most intense treatment after riboflavin-acetic acid. Electron microscopy revealed membrane structural damage by fructosazine at pH 5 and 7. This study provides evidence that fructosazine exerts antimicrobial action by permeabilizing the cell membrane, damaging membrane integrity, and fragmenting DNA.

Argpyrimidine-tagged rutin-encapsulated biocompatible (ethylene glycol dimers) nanoparticles: Synthesis, characterization and evaluation for targeted drug delivery.

Diabetes mellitus represents a major metabolic disorder affecting millions of people all over the world. Currently available therapeutic treatments are not good enough to control the long-term complications of diabetes. Active targeting via inclusion of a specific ligand on the nanoparticles provides effective therapeutic approach in different diseases. However, such specific drug delivery systems have not been explored much in diabetes due to lack of suitable biological targets in this disorder. Our objective is to synthesize a ligand-tagged drug-loaded nanoparticle for delivery of the drug at specific sites to enhance its therapeutic efficiency in diabetic condition. The nanoparticles have been prepared by using biocompatible ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester) dimers. Although advanced glycation end products (AGEs) are the root causes of diabetic complications, argpyrimidine, an AGE, possesses antioxidant and reducing activities. AGE interacts selectively with its cell surface receptors (RAGE), which are significantly increased in diabetic condition. We have selected RAGE as the target of argpyrimidine, which is tagged on the nanoparticles as a ligand. Rutin, having anti-hyperglycemic and anti-glycating activities, has been used for nanoencapsulation. Rutin-loaded argpyrimidine-tagged nanoparticles have been synthesized and characterized. We have demonstrated the drug releasing capacity and target specificity of the synthesised drug delivery system under ex vivo and in vivo conditions.

Iron (Fe(2+))-Catalyzed Glucosamine Browning at 50 °C: Identification and Quantification of Major Flavor Compounds for Antibacterial Activity.

Glucosamine browning at 50 °C with (GlcN/Fe(2+)) or without iron (GlcN) was studied over time from 0 to 48 h. Generation of reactive oxygen species (ROS), H2O2, and (1)O2, along with α-dicarbonyls, fructosazine, and deoxyfructosazine, was evaluated. Singlet oxygen generation increased over time and was greater in GlcN/Fe(2+) caramel solution. The presence of iron significantly increased the concentration of α-dicarbonyls at an early incubation time (3 h). Fructosazine and deoxyfructosazine were the major degradation products at 48 h comprising together up to 37 and 49% in GlcN and GlcN/Fe(2+), respectively. GlcN/Fe(2+) (48 h) exhibited a MIC50 against highly heat-resistant Escherichia coli AW 1.7 at pH 5, but not at pH 7. Despite several antimicrobial compounds being produced during browning, GlcN/Fe(2+) created a synergistic environment for the fructosazine-organic acids to confer their antimicrobial activity. GlcN caramel solutions have the potential to serve as both flavoring compounds and antimicrobial agents in formulated food systems.

Isolation and characterisation of methanol-soluble fraction of Alternanthera philoxeroides (Mart.) - evaluation of their antioxidant, α-glucosidase inhibitory and antimicrobial activity in in vitro systems.

Alternanthera philoxeroides (Mart.) is a tropical weed commonly known as alligator weed. It grows rapidly within a small span of time and easily available all over the world. The objective of this work was to isolate and characterise the major phenolic components present in the methanol-soluble fraction (fraction X) of A. philoxeroides leaves and to explore the biological activity (antioxidant, α-glucosidase inhibition and antimicrobial) of the fraction in in vitro system. Chromatographic (HPLC) and spectroscopic (MALDI-TOF, ¹H NMR) techniques were used to purify and characterise the phenolics present in fraction X. Five major phenolics (kaempferol, ferulic acid, salicylic acid, syringic acid and chlorogenic acid) were found in fraction X. The fraction showed anti-oxidant property, dose-dependent inhibition of α-glucosidase activity and anti-microbial activity. Hence fraction X from the weed has therapeutic potential in pathophysiological condition.

Methylglyoxal-induced modifications of hemoglobin: structural and functional characteristics.

Methylglyoxal (MG) reacts with proteins to form advanced glycation end products (AGEs). Although hemoglobin modification by MG is known, the modified protein is not yet characterized. We have studied the nature of AGE formed by MG on human hemoglobin (HbA(0)) and its effect on structure and function of the protein. After reaction of HbA(0) with MG, the modified protein (MG-Hb) was separated and its properties were compared with those of the unmodified protein HbA(0). As shown by MALDI-mass spectrometry, MG converted Arg-92α and Arg-104ß to hydroimidazolones in MG-Hb. Compared to HbA(0), MG-Hb exhibited decreased absorbance around 280nm, reduced tryptophan fluorescence (excitation 285nm) and increased α-helix content. However, MG modification did not change the quaternary structure of the heme protein. MG-Hb appeared to be more thermolabile than HbA(0). The modified protein was found to be more effective than HbA(0) in H(2)O(2)-mediated iron release and oxidative damages involving Fenton reaction. MG-Hb exhibited less peroxidase activity and more esterase activity than HbA(0). MG-induced structural and functional changes of hemoglobin may enhance oxidative stress and associated complications, particularly in diabetes mellitus with increased level of MG.

Inhibitory effect of Piper betle Linn. leaf extract on protein glycation--quantification and characterization of the antiglycation components.

Piper betle Linn. is a Pan-Asiatic plant having several beneficial properties. Protein glycation and advanced glycation end products (AGEs) formation are associated with different pathophysiological conditions, including diabetes mellitus. Our study aims to find the effect of methanolic extract of P. betle leaves on in vitro protein glycation in bovine serum albumin (BSA)-glucose model. The extract inhibits glucose-induced glycation, thiol group modification and carbonyl formation in BSA in dose-dependent manner. It inhibits different stages of protein glycation, as demonstrated by using glycation models: hemoglobin-delta-gluconolactone (for early stage, Amadori product formation), BSA-methylglyoxal (for middle stage, formation of oxidative cleavage products) and BSA-glucose (for last stage, formation of AGEs) systems. Several phenolic compounds are isolated from the extract. Considering their relative amounts present in the extract, rutin appears to be the most active antiglycating agent. The extract of P. betle leaf may thus have beneficial effect in preventing protein glycation and associated complications in pathological conditions.

Fructose-induced modifications of myoglobin: Change of structure from met (Fe3+) to oxy (Fe2+) form.

We studied structural modifications of metmyoglobin (Mb) after short-term (6 days) and long-term (30 days) glycation by fructose (fructation). Fructation caused gradual changes in the structure of the protein with respect to increased absorbance at 280 nm, enhanced fluorescence emission (with excitation at 285 nm), increased surface accessible tryptophan residues and reduced α-helix content and change in tertiary structure. However, long-term fructation changed Mb to oxymyoglobin (MbO2), as demonstrated by different spectroscopic (absorption, fluorescence, circular dichroic and electron paramagnetic resonance) studies and trifluoperazine-induced oxygen release experiment. Fructation appeared to modify Arg139 to arg-pyrimidine, which exhibited antioxidative activity and might be involved in the conversion of met (Fe3+) to oxy (Fe2+) form of myoglobin.