Synergistic Properties of Arabinogalactan (AG) and Hyaluronic Acid (HA) Sodium Salt Mixtures

The properties of mixtures of two polysaccharides, arabinogalactan (AG) and hyaluronic acid (HA), were investigated in solution by the measurement of diffusion coefficients D of water protons by DOSY (Diffusion Ordered SpectroscopY), by the determination of viscosity and by the investigation of the affinity of a small molecule molecular probe versus AG/HA mixtures in the presence of bovine submaxillary mucin (BSM) by 1HNMR spectroscopy. Enhanced mucoadhesive properties, decreased mobility of water and decreased viscosity were observed at the increase of AG/HA ratio and of total concentration of AG. This unusual combination of properties can lead to more effective and long-lasting hydration of certain tissues (inflamed skin, dry eye corneal surface, etc.) and can be useful in the preparation of new formulations of cosmetics and of drug release systems, with the advantage of reducing the viscosity of the solutions.

N-Glycan Modifications with Negative Charge in a Natural Polymer Mucin from Bovine Submaxillary Glands, and Their Structural Role

Bovine submaxillary mucin (BSM) is a natural polymer used in biomaterial applications for its viscoelasticity, lubricity, biocompatibility, and biodegradability. N-glycans are important for mucin stability and function, but their structures have not been fully characterized, unlike that of O-glycans. In this study, BSM N-glycans were investigated using liquid chromatography-tandem mass spectrometry. The microheterogeneous structures of 32 N-glycans were identified, and the quantities (%) of each N-glycan relative to total N-glycans (100%) were obtained. The terminal N-acetylgalactosamines in 12 N-glycans (sum of relative quantities; 27.9%) were modified with mono- (10 glycans) and disulfations (2 glycans). Total concentration of all sulfated N-glycans was 6.1 pmol in BSM (20 µg), corresponding to 25.3% of all negatively charged glycans (sum of present N-glycans and reported O-glycans). No N-glycans with sialylated or phosphorylated forms were identified, and sulfate modification ions were the only negative charges in BSM N-glycans. Mucin structures, including sulfated N-glycans located in the hydrophobic terminal regions, were indicated. This is the first study to identify the structures and quantities of 12 sulfated N-glycans in natural mucins. These sulfations play important structural roles in hydration, viscoelasticity control, protection from bacterial sialidases, and polymer stabilization to support the functionality of BSM via electrostatic interactions.

Human Neuraminidases Have Reduced Activity Towards Modified Sialic Acids on Glycoproteins

This work investigated the substrate specificity of hNEU enzymes for a glycoprotein substrate (bovine submaxillary mucin) containing 9-<i>O</i>-acetylated and Neu5Gc residues. Using this model substrate, we observe a general trend for hNEU tolerance of Neu5Ac>Neu5Gc>>>Neu5,9Ac₂, consistent with our previous results with glycolipid substrates. These results expand our understanding of hNEU enzyme specificity and suggest that naturally occurring modifications of sialic acids can play a role in regulating hNEU activity.

Human Neuraminidases Have Reduced Activity Towards Modified Sialic Acids on Glycoproteins

This work investigated the substrate specificity of hNEU enzymes for a glycoprotein substrate (bovine submaxillary mucin) containing 9-<i>O</i>-acetylated and Neu5Gc residues. Using this model substrate, we observe a general trend for hNEU tolerance of Neu5Ac>Neu5Gc>>>Neu5,9Ac₂, consistent with our previous results with glycolipid substrates. These results expand our understanding of hNEU enzyme specificity and suggest that naturally occurring modifications of sialic acids can play a role in regulating hNEU activity.

Human Neuraminidases Have Reduced Activity Towards Modified Sialic Acids on Glycoproteins

This work investigated the substrate specificity of hNEU enzymes for a glycoprotein substrate (bovine submaxillary mucin) containing 9-<i>O</i>-acetylated and Neu5Gc residues. Using this model substrate, we observe a general trend for hNEU tolerance of Neu5Ac>Neu5Gc>>>Neu5,9Ac₂, consistent with our previous results with glycolipid substrates. These results expand our understanding of hNEU enzyme specificity and suggest that naturally occurring modifications of sialic acids can play a role in regulating hNEU activity.

Structural and Quantitative Characterization of Mucin-Type O-Glycans and the Identification of O-Glycosylation Sites in Bovine Submaxillary Mucin

Bovine submaxillary mucin (BSM) is a gel-forming glycoprotein polymer, and Ser/Thr-linked glycans (O-glycans) are important in regulating BSM’s viscoelasticity and polymerization. However, details of O-glycosylation have not been reported. This study investigates the structural and quantitative characteristics of O-glycans and identifies O-glycosylation sites in BSM using liquid chromatography–tandem mass spectrometry. The O-glycans (consisting of di- to octa-saccharides) and their quantities (%) relative to total O-glycans (100%; 1.1 pmol per 1 μg of BSM) were identified with 14 major (>1.0%), 12 minor (0.1%–1.0%), and eight trace (<0.1%) O-glycans, which were characterized based on their constituents (sialylation (14 O-glycans; 81.9%, sum of relative quantities of each glycan), non-sialylation (20; 18.1%), fucosylation (20; 17.5%), and terminal-galactosylation (6; 3.6%)) and six core structure types [Gal-GalNAc, Gal-(GlcNAc)GalNAc, GlcNAc-GalNAc, GlcNAc-(GlcNAc)GalNAc, and GalNAc-GalNAc]. O-glycosylation sites were identified using O-glycopeptides (bold underlined; 56SGETRTSVI, 259SHSSSGRSRTI, 272GSPSSVSSAEQI, 307RPSYGAL, 625QTLGPL, 728TMTTRTSVVV, and 1080RPEDNTAVA) obtained from proteolytic BSM; these sites are in the four domains of BSM. The gel-forming mucins share common domain structures and glycosylation patterns; these results could provide useful information on mucin-type O-glycans. This is the first study to characterize O-glycans and identify O-glycosylation sites in BSM.

Silver Nanoparticles Complexed with Bovine Submaxillary Mucin Possess Strong Antibacterial Activity and Protect against Seedling Infection

ABSTRACT A simple method for the synthesis of nanoparticles (NPs) of silver (Ag) in a matrix of bovine submaxillary mucin (BSM) was reported previously by some of the authors of this study. Based on mucin characteristics such as long-lasting stability, water solubility, and surfactant and adhesive characteristics, we hypothesized that these compounds, named BSM-Ag NPs, may possess favorable properties as potent antimicrobial agents. The goal of this study was to assess whether BSM-Ag NPs possess antibacterial activity, focusing on important plant-pathogenic bacterial strains representing both Gram-negative ( Acidovorax and Xanthomonas ) and Gram-positive ( Clavibacter ) genera. Growth inhibition and bactericidal assays, as well as electron microscopic observations, demonstrate that BSM-Ag NPs, at relatively low concentrations of silver, exert strong antimicrobial effects. Moreover, we show that treatment of melon seeds with BSM-Ag NPs effectively prevents seed-to-seedling transmission of Acidovorax citrulli , one of the most threatening pathogens of cucurbit production worldwide. Overall, our findings demonstrate strong antimicrobial activity of BSM-Ag NPs and their potential application for reducing the spread and establishment of devastating bacterial plant diseases in agriculture. IMPORTANCE Bacterial plant diseases challenge agricultural production, and the means available to manage them are limited. Importantly, many plant-pathogenic bacteria have the ability to colonize seeds, and seed-to-seedling transmission is a critical route by which bacterial plant diseases spread to new regions and countries. The significance of our study resides in the following aspects: (i) the simplicity of the method of BSM-Ag NP synthesis, (ii) the advantageous chemical properties of BSM-Ag NPs, (iii) the strong antibacterial activity of BSM-Ag NPs at relatively low concentrations of silver, and (iv) the fact that, in contrast to most studies on the effects of metal NPs on plant pathogens, the proof of concept for the novel compound is supported by in planta assays. Application of this technology is not limited to agriculture; BSM-Ag NPs potentially could be exploited as a potent antimicrobial agent in a wide range of industrial areas, including medicine, veterinary medicine, cosmetics, textiles, and household products.