Determination of the structure of a novel acidic oligosaccharide with blood-group activity isolated from bovine submaxillary-gland mucin.
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ABSTRACT:
SUBMITTER: D'Arcy SM
PROVIDER: S-EPMC1138681 | biostudies-other | 1989 Jun
REPOSITORIES: biostudies-other
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Project description:In this study we investigated the structures of 11 neutral oligosaccharides released from bovine submaxillary mucin by alkaline borohydride treatment and isolated by h.p.l.c. One hexa-, one penta-, three tetra-, four tri- and two di-saccharides containing core types 1, 2, 3 or 4 were obtained. We report their structures, determined by a combination of one- and two-dimensional 1H n.m.r. spectroscopy at 270 MHz and methylation analysis involving g.l.c.-m.s., along with their approximate molar ratios. Only three of these oligosaccharides have previously been reported in this source. Of the new oligosaccharides, one contains the blood-group-A antigenic determinant, two contain the blood-group-H type 2 determinant, while another contains the blood-group-H type 3 determinant. The oligosaccharide GlcNAc beta (1----6)[GlcNAc beta (1----3)]GalNAcol, although previously found as a core structure, has been isolated here as a novel trisaccharide.
| S-EPMC1151551 | biostudies-other
Project description:Rapid separation and independent analysis of isomeric species are needed for the structural characterization of carbohydrates in glycomics research. Ion mobility-mass spectrometry techniques were used to examine a series of isomeric neutral oligosaccharide-alditols derived from bovine submaxillary mucin. Several analytical techniques were employed: (1) off line separation of the oligosaccharide-alditol mixture by HPLC; (2) direct and rapid evaluation of isomeric heterogeneity of oligosaccharides by electrospray ionization-ion mobility-time of flight mass spectrometry; and (3) mobility-selected MS2 and MS3 to evaluate isomeric mobility peaks by dual gate ion mobility-tandem mass spectrometry. Multiple isomeric ion mobility peaks were observed for the majority of oligosaccharide-alditols, which was achieved on the millisecond time scale after LC separation. Fragmentation spectra obtained from the collision-induced dissociation of isomeric precursor ions could be essentially identical, or dramatically different for a given precursor m/z using the dual-gate ion mobility quadrupole ion trap mass spectrometer. This further confirmed the need for rapid physical resolution of isomeric precursor species prior to their tandem mass spectral analysis.
| S-EPMC3949761 | biostudies-literature
Project description: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.
| S-EPMC5795074 | biostudies-literature
Project description:A lambda gt11 cDNA library prepared from bovine submaxillary gland mRNA was screened with polyclonal anti-apo-bovine submaxillary mucin antibodies with the aim of obtaining the deduced amino acid sequence of the mucin core protein. One of the positive clones had a 1.8 kilobase (kb) cDNA insert and coded for an incomplete protein. A 2.0-kb cDNA clone was isolated by rescreening the library with the 1.8-kb cDNA. Nucleotide sequencing of the full-length 2.0-kb cDNA revealed an open reading frame that coded for a 563-amino acid protein. A striking feature of the cloned protein is the skewed distribution of the amino acids, most notably that of the hydroxy amino acids and cysteine. The amino-terminal domain of 339 residues is very rich in threonine, serine, and glycine and poor in cysteine, aspartic acid, tyrosine, phenylalanine, and tryptophan. In contrast, the carboxyl-terminal domain of 224 residues is rich in cysteine, aspartic acid, tyrosine, lysine, and asparagine and relatively poor in threonine, serine, and glycine. A search of the protein data bank for homologies to the deduced amino acid sequence revealed statistically significant matches to several proteins, including the porcine submaxillary apomucin fragment. The cysteine-rich domain by itself was not statistically homologous with any of the registered polypeptide sequences. RNA blot analysis using DNA probes corresponding to the mucin-like and cysteine-rich regions detected a nearly identical pattern of transcripts, demonstrating that the characterized clones are not artifacts of cDNA library construction. The blots also showed the presence of polydisperse transcripts in bovine submaxillary gland but no detectable hybridization signals in liver or brain RNA.
| S-EPMC54625 | biostudies-other
Project description: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 ?g 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.
| S-EPMC7226346 | biostudies-literature
Project description: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.
| S-EPMC7796149 | biostudies-literature
Project description:A number of cDNA fragments coding for bovine submaxillary mucin (BSM) were cloned, and the nucleotide sequence of the largest clone, BSM421, was determined. Two peptide sequences determined from the purified apoBSM were found near the N-terminus of the mucin-coding region of BSM421. This clone does not contain a start or stop codon, but its 3' end overlaps with the 5' end of a previously isolated clone, lambdaBSM10. The composite sequence of 1589 amino acid residues consists of five distinct protein domains, which are numbered from the C-terminus. The cysteine-rich domain I can be further divided into a von Willebrand factor type C repeat and a cystine knot. Domains III and V consist of similar repeated peptide sequences with an average of 47 residues. Domains II and IV do not contain such sequences but are similar to domains III and V in being rich in serine and threonine, many of which are predicted to be potential O-glycosylation sites. Domain III also contains two sequences that match the ATP/GTP-binding site motif A (P-loop). Only beta-strands and no alpha-helices are predicted for the partial deduced amino acid sequence. Northern analysis of submaxillary gland RNA with the BSM421 probe detected multiple messages of BSM with sizes from 1.1 to over 10 kb. The tandemly repeated, non-identical peptide sequences of approx. 47 residues in domains III and V of BSM differ from the tandemly repeated, identical 81-residue sequences of pig submaxillary mucin (PSM), although both BSM and PSM contain similar C-terminal domains. In contrast, two peptide sequences of ovine submaxillary mucin are highly similar (86% and 65% identical respectively) to the corresponding sequences in domain V of BSM.
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Project description:High-pressure liquid chromatography was used to identify the sialo-oligosaccharide products obtained after sialylation of [14C]Gal-GalNAc-protein in vitro by an ovine submaxillary-gland microsomal fraction. Among other products, two isomeric trisaccharides could be identified. NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAcol and Gal beta 1 leads to 3-(NeuAc alpha 2 leads to 6)GalNAcol respectively, indicating that ovine submaxillary gland contains two sialyltransferases acting on mucin-type acceptors, a beta-galactoside alpha 2 leads to 3 sialyltransferase and a N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase. This conclusion was fully supported by methylation analysis of the two trisaccharide products.
| S-EPMC1163658 | biostudies-other
Project description:This study presents a comprehensive characterization of the viscoelastic and structural properties of Bovine Submaxillary Mucin (BSM), a good biomaterial model system for mucins and mucus in general. We conducted concentration studies of BSM and examined the effects of various additives - sodium chloride, calcium chloride, lysozyme, and DNA - on its rheological behavior. Through oscillatory and shear flow macrorheological experiments, we established a detailed correlation between the concentration of BSM and its viscoelastic properties, uncovering changes in the rheological moduli with the addition of sodium and calcium chloride, particularly at higher concentrations. This highlights the mucins responsiveness to ionic strength and composition variations. The presence of lysozyme and DNA further influenced the rheological characteristics of BSM, adding a new dimension to our understanding of mucin behavior. The rheological spectra could be well-described by a Fractional Kelvin-Voigt Model (FKVM), which allows to describe the complete behavior with a minimum of model parameters. A detailed proteomics analysis provided insight into the molecular interactions within BSM, enhancing our understanding of its structural intricacies. Complementing this, cryo-scanning electron microscopy (cryo-SEM) was employed to visualize the microstructural alterations and network properties in relation to the rheological data. By elucidating the complex interplay between mucin concentration, environmental conditions, and viscoelastic properties, this research significantly contributes to the field of mucus research and lays an important basis for future studies and advanced applications of numerous mucin-based biomaterials.
2024-07-25 | PXD048381 | Pride
Project description:Mucins, the major components of salivary mucus, are large glycoproteins abundantly modified with O-glycans. Mucins present on the surface of oral tissues contribute greatly to the maintenance of oral hygiene by selectively adhering to the surfaces of microbes via mucin O-glycans. However, due to the complex physicochemical properties of mucins, there have been relatively few detailed analyses of the mechanisms controlling the expression of mucin genes and the glycosyltransferase genes involved in glycosylation. Analysis performed using supported molecular matrix electrophoresis, a methodology developed for mucin analysis, and knockout mice without the polycomb group protein Bmi-1 revealed that Bmi-1 regulates mucin levels in the submandibular gland by suppressing the expression of the mucin Smgc gene, and that Bmi-1 also regulates mucin O-glycosylation via suppression of the glycosyltransferase Gcnt3 gene in the submandibular gland.
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