Comprehensive Characterization of the Viscoelastic Properties of Bovine Submaxillary Mucin (BSM) Hydrogels and the Effect of Additives.

This study presents a comprehensive characterization of the viscoelastic and structural properties of bovine submaxillary mucin (BSM), which is widely used as a commercial source to conduct mucus-related research. We conducted concentration studies of BSM and examined the effects of various additives, NaCl, CaCl2, MgCl2, lysozyme, and DNA, on its rheological behavior. A notable connection between BSM concentration and viscoelastic properties was observed, particularly under varying ionic conditions. The rheological spectra could be well described by a fractional Kelvin-Voigt model with a minimum of model parameters. A detailed proteomics analysis provided insight into the protein, especially mucin composition within BSM, showing MUC19 as the main component. Cryo-scanning electron microscopy enabled the visualization of the porous BSM network structure. These investigations give us a more profound comprehension of the BSM properties, especially those pertaining to viscoelasticity, and how they are influenced by concentration and environmental conditions, aspects relevant to the field of mucus research.

Comparison of proteins with anti-influenza virus effects in parotid and submandibular-sublingual saliva in humans.

BACKGROUND: Saliva possesses antiviral activity, with submandibular-sublingual (SMSL) saliva having higher antiviral activity than parotid saliva. Various salivary proteins have inactivating effects on influenza A virus (IAV), but the detailed relationship between antiviral proteins and salivary anti-IAV activities in the parotid and SMSL glands is unknown. Here, to identify salivary proteins with anti-IAV activity, salivary proteins from parotid and SMSL glands were identified, quantified, and compared using liquid chromatography-mass spectrometry. METHODS: Twelve healthy male volunteers participated in the study. Parotid and SMSL saliva was collected by suction and collection devices. We assessed anti-IAV activities, protein concentrations, and protein-bound sialic acid concentrations in parotid and SMSL saliva. RESULTS: SMSL had significantly higher anti-IAV activity than parotid saliva. SMSL also had higher concentrations of glycoproteins, such as mucin 5B and mucin 7, protein-bound sialic acid, cystatins, and lysozyme C, compared with parotid saliva. Salivary mucin 5B and mucin 7 concentrations significantly positively correlated with the salivary protein-bound sialic acid concentration. Salivary anti-IAV activity significantly positively correlated with protein-bound sialic acid, mucin 5B, mucin 7, cystatin-C, -S, and -SN concentrations. CONCLUSION: Salivary mucins, cystatins, and lysozyme C contribute to the high anti-IAV activity of SMSL saliva.

Improved online LC-MS/MS identification of O-glycosites by EThcD fragmentation, chemoenzymatic reaction, and SPE enrichment.

O-glycosylation is a highly diverse and complex form of protein post-translational modification. Mucin-type O-glycosylation is initiated by the transfer of N-acetyl-galactosamine (GalNAc) to the hydroxyl group of serine, threonine and tyrosine residues through catalysis by a family of glycosyltransferases, the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (E.C. 2.4.1.41) that are conserved across metazoans. In the last decade, structural characterization of glycosylation has substantially advanced due to the development of analytical methods and advances in mass spectrometry. However, O-glycosite mapping remains challenging since mucin-type O-glycans are densely packed, often protecting proteins from cleavage by proteases. Adding to the complexity is the fact that a given glycosite can be modified by different glycans, resulting in an array of glycoforms rising from one glycosite. In this study, we investigated conditions of solid phase extraction (SPE) enrichment, protease digestion, and Electron-transfer/Higher Energy Collision Dissociation (EThcD) fragmentation to optimize identification of O-glycosites in densely glycosylated proteins. Our results revealed that anion-exchange stationary phase is sufficient for glycopeptide enrichment; however, the use of a hydrophobic-containing sorbent was detrimental to the binding of polar-hydrophilic glycopeptides. Different proteases can be employed for enhancing coverage of O-glycosites, while derivatization of negatively charged amino acids or sialic acids would enhance the identification of a short O-glycopeptides. Using a longer than normal electron transfer dissociation (ETD) reaction time, we obtained enhanced coverage of peptide bonds that facilitated the localization of O-glycosites. O-glycosite mapping strategy via proteases, cut-off filtration and solid-phase chemoenzymatic processing. Glycopeptides are enriched by SPE column, followed by release of N-glycans, collection of higher MW O-glycopeptides via MW cut-off filter, O-glycopeptide release via O-protease, and finally detected by LC-MS/MS using EThcD.

Larrea divaricata Cav. aqueous extract and nordihydroguariaretic acid modulate oxidative stress in submandibular glands of diabetic rats: a buccal protective in diabetes.

BACKGROUND: Oxidative stress is an imbalance between the levels of reactive oxygen species (ROS), reactive nitrogen species (RNS) and endogenous antioxidants. The aetiology and pathogenesis of several oral diseases are attributed to this process. The antioxidant enzymes secreted in the saliva by submandibular glands maintain oral health through the scavenging of ROS. The objective of this work was to study the capacity of an aqueous extract of L. divaricata (AE), and its majority compound, nordihydroguariaretic acid (NDGA), to modulate the pro-oxidant/antioxidant status in submandibular glands in a model of oxidative stress induced by streptozotocin (STZ) in rats. METHODS: To induce oxidative stress with STZ, a group of animals was treated i.p. with 1 X PBS (control group) and other group was injected i.p. once with STZ (60 mg/kg). Ten days after the treatment, blood samples were taken from the tail vain to determine the glucose levels. Animals with glucose values ≥300 mg/ml were selected. The submandibular glands of control and STZ treated animals were incubated with either the AE (500 µg/ml) or with NDGA (1.5 µg/ml), and the content of malondialdehyde (MDA), protein carbonyl groups, ROS and RNS, and the activity and expression of peroxidase (Px), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) were assayed. RESULTS: AE decreased the levels of MDA (##P < 0.01) and protein carbonyl groups (#P < 0.05), and modulated the levels of ROS such as hydrogen peroxide (H2O2)(##P < 0.01), superoxide anion (O2.-) (#P < 0.05) and nitric oxide (NO) (#P < 0.05) in relation to the modulation of Px and iNOS expression. NDGA was found to be involved in these effects. CONCLUSIONS: The antioxidant activity of the AE in the submandibular glands would allow the maintenance of the antioxidant pool to prevent oral oxidative diseases.

Evaluating the Role of Drone-Produced Chemical Signals in Mediating Social Interactions in Honey Bees (Apis mellifera).

Pheromones play a critical role in shaping societies of social insects, including honey bees, Apis mellifera. While diverse functions have been ascribed to queen- and worker-produced compounds, few studies have explored the identity and function of male-produced (drone) compounds. However, several lines of evidence suggest that drones engage in a variety of social interactions inside and outside of the colony. Here we elucidate the chemical composition of extracts of the drone mandibular gland, and test the hypothesis that compounds produced in these glands, or a synthetic blend consisting of the six main compounds, mediate drone social interactions in and out of the colony. Drone mandibular glands primarily produce a blend of saturated, unsaturated and methyl branched fatty acids ranging in chain length from nonanoic to docosanoic acids, and both gland extracts and synthetic blends of these chemicals serve to attract drones outside of the hive, but do not attract workers inside the hive. These studies shed light on the role drones and drone-produced chemicals have on mediating social interactions with other drones and highlight their potential importance in communicating with other castes.

Human postmortem lacrimal and submandibular glands stored in RNAlater are suitable for molecular, biochemical, and cell biological studies.

PURPOSE: Gene expression and protein analysis studies require high-quality human tissue which is a challenge and difficult to obtain through live human biopsies. Human postmortem lacrimal gland (LG) and submandibular gland (SMG) tissues have the potential to provide an invaluable source for studying the mechanisms involved in LG and SMG dysfunction. Therefore, we aimed to test the suitability of post-mortem LG and SMG for molecular, biochemical, and cell biological studies. METHODS: LG and SMG tissue from healthy donors was collected and immediately placed in RNAlater solution and then shipped overnight at 4 °C. After receipt, each gland was divided into three pieces for RNA, protein, and histological analysis, respectively. Total RNA isolated from each LG and SMG was analyzed for RNA integrity using an Agilent Bioanalyzer and reverse transcription-PCR (RT-PCR). For histology, tissues were embedded in paraffin and stained with hematoxylin and eosin. For protein analysis, lysates were prepared and processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. RESULTS: When the LG and SMG samples were preserved in RNAlater, the RNA integrity number (RIN) values from the LG and SMG were >7.0 from all three donors, while the RNAs from tissue not preserved in RNAlater were of poorer quality. The gene and/or protein expression of E-cadherin, aquaporin 5, alpha-smooth muscle actin (α-SMA), ß-actin, and GAPDH was preserved in all samples. In addition, histological analyses showed normal tubuloacinar structures of all glands with serous and mucous producing acini within lobules interspersed with adipose fat. CONCLUSIONS: In this study, we determined that RNA, protein, and histological sections obtained from postmortem human LG and SMG tissue preserved in RNAlater were of high quality. This would provide a viable source of human LG and SMG tissue suitable for studies of diseases that affect these glands, such as Sjögren's syndrome.

Structural and Mechanical Properties of Thin Films of Bovine Submaxillary Mucin versus Porcine Gastric Mucin on a Hydrophobic Surface in Aqueous Solutions.

The structural and mechanical properties of thin films generated from two types of mucins, namely, bovine submaxillary mucin (BSM) and porcine gastric mucin (PGM) in aqueous environment were investigated with several bulk and surface analytical techniques. Both mucins generated hydrated films on hydrophobic polydimethylsiloxane (PDMS) surfaces from spontaneous adsorption arising from their amphiphilic characteristic. However, BSM formed more elastic films than PGM at neutral pH condition. This structural difference was manifested from the initial film formation processes to the responses to shear stresses applied to the films. Acidification of environmental pH led to strengthening the elastic character of BSM films with increased adsorbed mass, whereas an opposite trend was observed for PGM films. We propose that this contrast originates from that negatively charged motifs are present for both the central and terminal regions of BSM molecule, whereas a similar magnitude of negative charges is localized at the termini of PGM molecule. Given that hydrophobic motifs acting as an anchor are also localized in the terminal region, electrostatic repulsion between anchoring units of PGM molecules on a nonpolar PDMS surface leads to weakening of the mechanical integrity of the films.

Selective localization of diacylglycerol kinase (DGK)ζ in the terminal tubule cells in the submandibular glands of early postnatal mice.

The present immunohistochemical study was attempted to localize in the submandibular glands of mice at various postnatal stages a diacylglycerol kinase (DGK) isoform termed DGKζ which is characterized by a nuclear localization signal and a nuclear export signal. This attempt was based on following facts: the continuous postnatal differentiation of glandular cells in the rodent submandibular gland, the regulatory role of DGK in the activity of protein kinase C (PKC) through attenuation of diacylglycerol (DAG), and the possible involvement of PKC in various cellular activities including the saliva secretion as well as the cell differentiation. As a result, a selective localization of immunoreactivity for DGKζ was detected in terminal tubule (TT) cells which comprise a majority of the newborn acinar structure and differentiate into the intercalated duct cells and/or the acinar cells. The immunoreactivity was deposited in portions of the cytoplasm lateral and basal to the nucleus, but not in the nuclei themselves. Although the immunoreactive TT cells remained until later stages in female specimen than in male, they eventually disappeared in both sexes by young adult stages. The present finding suggests that the regulatory involvement of DGKζ in PKC functions via control of DAG is exerted in the differentiation of the TT cells. In addition, another possible involvement of DGKζ in the regulation of secretion of the TT cells as well as its functional significance of its nuclear localization in the submandibular ganglion cells was also discussed.

Dopamine, vesicular transporters, and dopamine receptor expression in rat major salivary glands.

The localization of dopamine stores and the expression and localization of dopamine (DAT) and vesicular monoamine transporters (VMAT) type-1 and -2 and of dopamine D1-like and D2-like receptor subtypes were investigated in rat submandibular, sublingual, and parotid salivary glands by HPLC with electrochemical detection, as well as immunochemical and immunohistochemical techniques. Male Wistar rats of 2 mo of age were used. The highest dopamine levels were measured in the parotid gland, followed by the submandibular and sublingual glands. Western blot analysis revealed DAT, VMAT-1, VMAT-2, and dopamine receptors immunoreactivity in membrane preparations obtained from the three glands investigated. Immunostaining for dopamine and transporters was developed within striated ducts. Salivary glands processed for dopamine receptors immunohistochemistry developed an immunoreaction primarily in striated and excretory ducts. In the submandibular gland, acinar cells displayed strong immunoreactivity for the D2 receptor, while cells of the convoluted granular tubules were negative for both D1-like and D2-like receptors. Parotid glands acinar cells displayed the highest immunoreactivity for both D1 and D2 receptors compared with other salivary glands. The above localization of dopamine and dopaminergic markers investigated did not correspond closely with neuron-specific enolase (NSE) localization. This indicates that at least in part, catecholamine stores and dopaminergic markers are independent from glandular innervation. These findings suggest that rat major salivary glands express a dopaminergic system probably involved in salivary secretion. The stronger immunoreactivity for dopamine transporters and receptors in striated duct cells suggests that the dopaminergic system could regulate not only quality, but also volume and ionic concentration of saliva.

Male-specific submaxillary gland protein, a lipocalin allergen of the golden hamster, differs from the lipocalin allergens of Siberian and Roborovski dwarf hamsters.

BACKGROUND: An increasing number of asthma cases upon exposure to hamsters and anaphylactic reactions following hamster bites are being reported, but the allergens responsible are still poorly characterized. In the Golden hamster, male-specific submaxillary gland protein (MSP), a lipocalin expressed in a sex- and tissue-specific manner in the submaxillary and lacrimal glands, is secreted in the saliva, tears and urine. The purpose of this study was to determine if MSP is an allergen, to identify IgE-reactive proteins of different hamster species and to analyse potential cross-reactivities. METHODS: Fur extracts were prepared from four hamster species. Hamster-allergic patients were selected based on a history of positive IgE-test to hamster epithelium. The IgE-reactivity of patients' sera was investigated by means of immunoblot and ELISA. IgE-reactive proteins in fur extracts and the submaxillary gland were identified using anti-MSP antibodies, Edman sequencing or mass spectrometry. MSP was purified from Golden hamster and recombinant MSP was expressed in E. coli. RESULTS: Four patients had IgE-antibodies against 20.5-kDa and 24-kDa proteins of Golden hamster fur extract, which were identified as MSP. IgE-reactive MSP-like proteins were detected in European hamster fur extract. Three patient sera showed IgE-reactive bands at 17-21 kDa in Siberian and Roborovski hamster fur extracts. These proteins were identified as two closely related lipocalins. Immunoblot inhibition experiments showed that they are cross-reactive and are different from MSP. CONCLUSION: MSP lipocalin of the Golden hamster was identified as an allergen, and it is different from the cross-reactive lipocalin allergens of Siberian and Roborovski hamsters. Our findings highlight the need for specific tools for the in vitro and in vivo diagnosis of allergy to different hamster species.

Localization of cystic fibrosis transmembrane conductance regulator signaling complexes in human salivary gland striated duct cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent protein kinase (PKA)-regulated Cl(-) channel, crucial for epithelial cell regulation of salt and water transport. Previous studies showed that ezrin, an actin binding and A-kinase anchoring protein (AKAP), facilitates association of PKA with CFTR. We used immunohistochemistry and immunogold transmission electron microscopy to localize CFTR, ezrin, and PKA type II regulatory (RII) and catalytic (C) subunits in striated duct cells of human parotid and submandibular glands. Immunohistochemistry localized the four proteins mainly to the apical membrane and the apical cytoplasm of striated duct cells. In acinar cells, ezrin localized to the luminal membrane, and PKA RII subunits were present in secretory granules, as previously described. Immunogold labeling showed that CFTR and PKA RII and C subunits were localized to the luminal membrane and associated with apical granules and vesicles of striated duct cells. Ezrin was present along the luminal membrane, on microvilli and along the junctional complexes between cells. Double labeling showed specific protein associations with apical granules and vesicles and along the luminal membrane. Ezrin, CFTR, and PKA RII and C subunits are co-localized in striated duct cells, suggesting the presence of signaling complexes that serve to regulate CFTR activity.

Two-step chromatography purification of IgGs possessing sialidase activity from human blood serum.

Sialation of cell surface is known to be tightly connected with tumorigenicity, invasiveness, metastatic potential and clearance of aged cells, while sialation of immunoglobulin G (IgG) molecules determines their anti-inflammatory properties. Recently, we have found for the first time IgG-antibodies possessing sialidase-like activity (sialylic abzyme) in blood serum of multiple myeloma and systemic lupus erythematosis patients. This abzyme was detected in a pool of IgGs purified by a typical procedure including immunoglobulin's precipitation with ammonium sulfate and following chromatography on protein G-Sepharose column. Here we describe a novel matrix for affinity purification of sialylic abzyme that is based on using bovine submandibular gland mucin conjugated to Sepharose matrix (mucin-Sepharose). This matrix preferentially binds sialidase-like IgGs from a pool of sialidase-active fraction of proteins precipitated with 50% ammonium sulfate from blood serum of the systemic lupus erythematosis patients. That allowed us to develop a new scheme of double-step chromatography purification of sialidase-like IgGs from human blood serum.

[Injection of submandibular gland with recombinant Exendin-4 and adeno-associated virus for the treatment of diabetic rats].

OBJECTIVE: To evaluate the therapeutic effects of recombinant Exendin-4 and double-stranded adeno-associated virus (Exendin-4/dsAAV) on SD rats with type 2 diabetes (T2DM) through injecting it into submandibular gland (SG).
 METHODS: The Exendin-4/dsAAV was injected into submandibular gland of diabetic rat. The insulin and α-amylase were detected by real-time PCR at the 2nd, 4th and 8th weeks. The immunohistochemisty was used to detect the insulin contents in SG at the 8th week. The concentration of blood glucose and levels of insulin secretion were detected after pancreatectomy.
 RESULTS: The SG gland was bigger in Exendin-4/dsAAV group than that in the control group, but the changes in α-amylase were not significant. The Exendin-4 and insulin gene expression was increased in the Exendin-4/dsAAV group (P<0.05). The Exendin-4 and insulin were positive in the SG. The blood glucose was lower and insulin concentration was higher in the Exendin-4/dsAAV group than those in the control group after pancreatectomy (P<0.05), and the insulin content was also increased in the dsAAV groups.
 CONCLUSION: Continuous expression of Exendin-4 in SG may improve glucose control and insulin secretion in T2DM rats through inducing expression of insulin.

Activation of TLR9-dependent p38MAPK pathway in the pathogenesis of primary Sjögren's syndrome in NOD/Ltj mouse.

OBJECTIVE: The objective of this study was to investigate the potential role of Toll-like receptor 9-dependent p38 MAPK signaling pathway in the pathogenesis of primary Sjögren's syndrome (pSS) in NOD/Ltj mouse, aiming to identify an ideal target therapy model for human pSS. METHODS: NOD/Ltj mice were chosen as a model of pSS. The Toll-like receptor 9 and p-p38 MAPK double-positive peripheral blood mononuclear cells (PBMCs) of 4-, 5-, 8-, 10-, and 15-week-old NOD/Ltj mouse were analyzed by flow cytometry. The expressions of Toll-like receptor 9 and p-p38 MAPK in the submandibular gland (SMG) were also examined by immunohistochemistry. The change of stimulated salivary flow rate was dynamically measured, and the histopathology of SMG was evaluated by hematoxylin and eosin stain. RESULTS: The stimulated salivary flow rate in NOD/Ltj was reduced to 50-60% of the flow rate of control mice since the fifth week onwards. The Toll-like receptor 9 and p-p38 MAPK double-positive PBMCs in both groups increased gradually from 5 weeks, peaked at 8 weeks and then gradually decreased at 10 weeks, yet the percentage of Toll-like receptor 9 and p-p38MAPK double-positive PBMCs in 5-, 8-, and 10-week-old NOD/Ltj mouse was significantly increased compared with those in control subjects. After the 10th week onwards, there were no significant differences in the Toll-like receptor 9 and p-p38 MAPK double-positive PBMCs between NOD/Ltj mice and controls. Immunohistochemical staining showed that Toll-like receptor 9 was positive in the acinar epithelium cells and infiltrating lymphocytes in NOD/Ltj mice. p-p38 MAPK was detected in infiltrating lymphocytes and few ductal or acinar epithelium cells adjacent to infiltrating lymphocytes in NOD/Ltj mice. CONCLUSIONS: From the fifth week till the tenth week, Toll-like receptor 9 and p-p38 MAPK double-positive PBMCs were significantly increased in NOD/Ltj mice, accompanied with reduced stimulated salivary flow rate and Toll-like receptor 9 or p-p38 MAPK positive infiltrating lymphocytes observed in the SMG of NOD/Ltj mouse. Our results indicated that activation of Toll-like receptor 9-depended p38 MAPK signal pathway in PBMCs was an early event in pSS which made NOD/Ltj as an ideal therapy model to test the treatment effects of p38 MAPK or Toll-like receptor 9 inhibitors on pSS.

Cholera toxin B subunit-binding and ganglioside GM1 immuno-expression are not necessarily correlated in human salivary glands.

OBJECTIVE: To determine and compare the presence and in situ localization of the glycosphingolipid ganglioside GM1 in human salivary glands using the biomarkers for GM1: cholera toxin and antibodies against GM1. MATERIALS AND METHODS: Immunohistochemical analyses were performed on sections of adult human submandibular, parotid and palatinal glands using cholera toxin sub-unit B and two polyclonal antibodies against ganglioside GM1 as biomarkers. RESULTS: Immunofluorescence microscopy showed that the toxin and antibodies were co-localized in some acini but not in others. The cholera toxin mainly reacted with the cell membranes of the mucous acini in the submandibular gland, while incubation with the antibody against GM1 gave rise to a staining of the cytoplasm. The cytoplasm in some secretory acinar cells in the parotid gland was stained by the cholera toxin, whereas only small spots on the plasma membranes reacted with anti-GM1. The plasma membranes in the parotid excretory ducts appeared to react to anti-GM1, but not to cholera toxin. CONCLUSIONS: Cholera toxin induces the expression of ion channels and carriers in the small intestine and increases the production of secretory mucins. Although their mutual immunohistochemical localization may differ, both cholera toxin and ganglioside GM1 are present in the mucin-producing acini from salivary glands. This could point to a relationship between ganglioside expression and production of salivary mucins.

mRNA expression and localization of LPS-induced ß-defensin isoforms in rat salivary glands.

ß-defensins are small, cationic peptides with broad-spectrum antimicrobial activity that are produced by mucosal epithelia. However, little is known about the expression of ß-defensins in the major salivary glands. The purpose of this study was to characterize expression of rat ß-defensin-1 (RBD-1) and -2 (RBD-2) mRNA within the major salivary glands together with the effect of injection of intraductal lipopolysaccharide (LPS) on that expression. ß-defensin mRNA expression was quantitated by RT-PCR in salivary gland tissues and salivary acinar and striated duct cells collected by laser captured microdissection. RBD-1 and -2 were expressed in the parotid gland, the submandibular gland, and the sublingual gland. ß-defensins were expressed in both the acinar and striated duct cells of the major salivary glands. Intraductal injection of LPS increased expression of RBD-1 and -2 mRNA, which peaked at 12 hrs. These results suggest that salivary cells (acinar and striated duct cells) have the potential to produce ß-defensins.

Preparation of Soluble Mucin Solutions from the Salivary Glands.

Studying salivary gland mucins is important for elucidating the pathogenesis of salivary gland diseases, including tumors and xerostomia, and developing diagnostic methods for them. Classic methods for isolating mucins from salivary glands require sacrificing several animals to obtain sufficient quantities of mucin and are time-consuming. Supported molecular matrix electrophoresis (SMME) was used to characterize mucins and their glycans. With this method, mucins can be analyzed within 2 days using less than 100 mg of tissue and without using expensive equipment, such as an ultracentrifuge. This chapter describes a method for preparing mucin solutions for SMME analysis of salivary gland mucins.

Mass spectrometric O-glycan analysis after combined O-glycan release by beta-elimination and 1-phenyl-3-methyl-5-pyrazolone labeling.

BACKGROUND: Analysis of protein glycosylation is an important first step towards establishing the functions of glycans in health and disease. In contrast to N-glycans which are generally enzymatically released for analysis, there is no corresponding enzyme for O-glycan liberation. Therefore, O-glycans are generally released by chemical methods involving tedious procedures. METHODS: Here, a straightforward method for the combined release and labeling of O-linked glycans from glycoproteins is described. Dimethylamine serves as the releasing agent, and 1-phenyl-3-methyl-5-pyrazolone (PMP) is employed for a prompt reaction with the reducing end of the freshly released O-glycan structures via an aldol condensation followed by a Michael-type addition resulting in a 2:1 stoichiometry of PMP per glycan. Samples are analyzed by nanoLC coupled to mass spectrometry. RESULTS: Mucin from bovine submaxillary gland was used as a model protein to evaluate and optimize the approach that was further applied to bile salt stimulated lipase (BSSL) isolated from human milk. Next to previously reported O-glycan structures two additional oligosaccharides could be detected for BSSL. GENERAL SIGNIFICANCE: In conclusion, the facile protocol established is suitable for the analysis of complex O-linked oligosaccharides from various biological samples. This article is part of a Special Issue entitled Glycoproteomics.

Top-down HPLC-ESI-MS characterization of rat gliadoralin A, a new member of the family of rat submandibular gland glutamine-rich proteins and potential substrate of transglutaminase.

During HPLC-ESI-MS/MS analysis of rat submandibular saliva secreted under isoprenaline stimulation, a protein with an experimental [M+H](1+) = 10,544.24 m/z was detected (17.5 ± 0.7 min). The MS/MS fragmentation pattern, manually investigated, allowed establishing an internal sequence in agreement with a DNA-derived sequence of an unknown rat protein coded D3Z9M3 (Swiss-Prot). To match the experimental MS/MS fragmentation pattern and protein mass with theoretical data, the removal from the N terminus of the signal peptide and from the C terminus of three amino acid (a.a.) residues (Arg-Ala-Val) and the cyclization of the N-terminal glutamine in pyroglutamic had to be supposed, resulting in a mature protein of 90 a.a. HPLC-ESI-MS/MS of the trypsin digest ensured 100% sequence coverage. For the high glutamine content (34/90 = 37.8%) we propose to name this protein rat gliadoralin A 1-90. Low amounts of five different isoforms were sporadically detected, which did not significantly change their relative amounts after stimulation. Gliadoralin A is substrate for transglutaminase-2, having Lys 60 and different Gln residues as major determinants for enzyme recognition. In silico investigation of superior structures evidenced that a small part of the protein adopts an α-helical fold, whereas large segments are unfolded, suggesting an unordered conformation.

Enhanced self-association of mucins possessing the T and Tn carbohydrate cancer antigens at the single-molecule level.

Mucins are linear O-glycosylated glycoproteins involved in inflammation, cell adhesion, and tumorigenesis. Cancer-associated mucins often possess increased expression of the T (Galß1,3GalNAcαThr/Ser) and Tn (GalNAcαThr/Ser) cancer antigens, which are diagnostic markers for several cancers, including colon cancer. We have used AFM based single-molecule forced unbinding under near physiological conditions to investigate the self-interactions between porcine submaxillary mucin (PSM) as well as between PSM analogs possessing various carbohydrates including the T- and Tn-antigen. Distributions of unbinding forces and corresponding force loading rates were determined for force loading rates from 0.18 nN/s to 39 nN/s, and processed to yield most probable unbinding forces f* and lifetimes of the interactions. Parameter f* varied in the range 27 to 50 pN at force loading rates of about 2 nN/s among the various mucins. All mucin samples investigated showed self-interaction, but the tendency was greatest for PSM displaying only the Tn-antigen (Tn-PSM) or a mixture of Tn-, T-antigen, and the trisaccharide Fucα1,2Galß1,3GalNAc (Tri-PSM). Weaker self-interactions were observed for native PSM (Fd-PSM), which consists of a nearly equal mixture of the longer core 1 blood group A tetrasaccharide (GalNAcα1,3(Fucα1,2)Galß1,3GalNAcαSer/Thr) and Tn-antigen. The data are consistent with the truncated Tn and T glycans enhancing self-interaction of the mucins. These carbohydrate cancer antigens may, thus, play an active role in the disease by constitutively activating mucin and mucin-type receptors by self-association on cells.