Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product

The MG1 population of mucins was isolated from human whole salivas by gel chromatography followed by isopycnic density gradient centrifugation. The reduced and alkylated MG1 mucins, separated by anion exchange chromatography, were of similar size (radius of gyration 55-64 nm) and molecular weight (2.5-2.9 x 10(6) Da). Two differently-charged populations of MG1 subunits were observed which showed different reactivity with monoclonal antibodies to glycan epitopes. Monosaccharide and amino acid compositional analyses indicated that the MG1 subunits had similar glycan structures on the same polypeptide. An antiserum recognizing the MUC5B mucin was reactive across the entire distribution, whereas antisera raised against the MUC2 and MUC5AC mucins showed no reactivity. Western blots of agarose gel electrophoresis of fractions across the anion exchange distribution indicated that the polypeptide underlying the mucins was the product of the MUC5B gene. Amino acid analysis and peptide mapping performed on the fragments produced by trypsin digestion of the two MG1 populations yielded data similar to that obtained for MUC5B mucin subunits prepared from respiratory mucus (Thornton et al., 1997) and confirmed that the MUC5B gene product was the predominant mucin polypeptide present. Isolation of the MG1 mucins from the secretions of the individual salivary glands (palatal, sublingual, and submandibular) indicate that the palatal gland is the source of the highly charged population of the MUC5B mucin.     

Distinct localization of MUC5B glycoforms in the human salivary glands

Salivary mucins, encoded by the MUC5B gene, make up a heterogeneous family of molecules, which are secreted by several glands, including the submandibular, sublingual, and palatine glands. Previous studies have shown that heterogeneity in the salivary mucin population is related to its multiglandular origin. In the present study we address the question to what extent the mucin (MUC5B) population from a single human salivary gland is made up of different glycoforms. Using monoclonal antibodies to defined protein and sulfated carbohydrate epitopes specific to MUC5B, we conduct an immunohistochemical study of different salivary gland types, including submandibular, sublingual, and labial glands. In all tissues studied we found a mosaic expression pattern of sulfo-Lewis a antigen, recognized by mAb F2, which in salivary glands is exclusively present on MUC5B. On the other hand, mucous acini were uniformly labeled by mAb EU-MUC5Bb, evoked against a peptide-stretch of the tandem repeat region of MUC5B. Double staining with both antibodies confirmed the presence of MUC5B-positive/sulfo-Lewis a-positive cells, as well as MUC5B-positive/sulfo-Lewis a-negative cells within one glandular unit. These results indicate that one and the same salivary gland synthesizes different MUC5B glycoforms.     

Microstereological and histochemical studies of the salivary glands of the giant rat (Cricetomys gambianus, waterhouse)

The histology and histochemistry of the parotid, submandibular and sublingual glands were studied. The submandibular gland contained only serous acini as in the guinea pig, but unlike in many other mammals. The parotid gland contained only serous acini while the sublingual gland was mixed, mucous acini being the predominant secretory tissue interspersed by a few serous acini. Serous demilunes also commonly formed caps on the mucous acini. The ducts of the gland contributed over 30% of the volume of the submandibular gland, while those of the parotid and sublingual glands formed about 12 and 10% of the gland, respectively. The secretions of the parotid gland, as judged by histochemical methods, contained neutral mucins and some sialomucins. Neutral mucins, sulphomucins and sialomucins were detected in both the submandibular gland and sublingual gland.     

Reduced levels of insulin-like growth factor-1 receptor (IGF-1R) suppress cellular signaling in experimental autoimmune sialadenitis (EAS).

The nonobese diabetic mouse (NOD) develops destruction and functional impairment of salivary and lachrymal glands, experimental autoimmune sialadenitis (EAS), resembling and representing a model for Sjogren's syndrome (SS). To investigate the mechanisms of tissue destruction in EAS, we analyzed a cell survival promoter insulin-like growth factor-1 receptor (IGF-1R) in the submandibular glands of NOD mice with this disease. We also evaluated the expression of a downstream effector of IGF-1R, BAD. Receptor-binding autoradiography revealed that the IGF-1R levels in submandibular glands from young NOD mice were lower than those in adult NOD mice. Immunofluorescence staining demonstrated that BAD expression in the epithelial cells of the submandibular gland was consistently enhanced throughout the course of EAS in NOD mice. These findings suggest that a reduction in the levels of IGF-1R induces a defective glandular homeostasis in the submandibular gland epithelial cells and triggers EAS.     

Expression and distribution of osteopontin and matrix metalloproteinase (MMP)-3 and -7 in mouse salivary glands

We investigated the expression and distribution of osteopontin in mouse salivary glands. Western blot analysis showed intense positive bands at the predicted molecular mass (about 60 kDa) in mouse parotid and sublingual glands. However, a cross-reacted band around 30 kDa was strongly detected in submandibular glands. Indirect immunofluorescent analysis showed that osteopontin was localized at the luminal (apical) membranes of the acinar cells in parotid and sublingual glands. However, it was not detected in acinar cells of submandibular glands. No expression was found in ductal cells of any glands. We also examined the expression of matrix metalloproteinase (MMP)-3 and -7. In parotid gland, MMP-3 was observed at 57 kDa, indicating a latent form, but MMP-7 was not detected. In contrast, MMP-7 definitely was observed at 28 kDa area in submandibular gland, whereas MMP-3 was not detected. These results suggest that osteopontin localizes at luminal sites of acinar cells and may be associated with saliva secretion in mouse salivary gland. It is also suggested that osteopontin may be cleaved by MMP-7 in mouse submandibular gland.     

The murine sublingual and submandibular mucins their isolation and characterization

From the mouse sublingual and submandibular glands high-molecular weight glycoproteins (mucins) were isolated. These mucins appeared to be homogeneous in polyacrylamide gel electrophoresis and in the analytical ultracentrifuge. S_20,w values of 10.9 and 5.5 were found for the sublingual and submandibular mucin respectively. With sodium dodecyl sulfate or N-acetylcysteine no subunits could be detected.Both mucins consisted for about 1/3 of protein and 2/3 of carbohydrate. Their mucin character was also denoted by the high content of serine plus threonine. Respectively 42 mol% and 34 mol% of the protein core of the sublingual and submandibular mucins consisted of these amino acids. The main sugars in these mucins were sialic acid, galactosamine, galactose, glucosamine and mannose. The molar ratio for the sublingual and submandibular mucin being 1.00 : 1.03 : 1.08 : 0.26 : 0.23 and 1.00 : 0.71 : 1.10 : 0.65 : 0.53, respectively.The sialic acid content of both mucins was about 25%. Fucose and sulfate, on the other hand, were less than 1%. The presence of sulfate was also indicated by preliminary studies in vivo on the incorporation of [³⁵SO₄] sulfate.     

Secreted human conjunctival mucus contains MUC5AC glycoforms.

This study addresses the extent of variation in secreted end-product mucins in human conjunctival mucus. The aim was to determine whether the variety of mucin species found was encompassed by the mucin genes which have been cloned to date. Extraction into guanidine hydrochloride and separation of mucin constituents, by a combination of cesium chloride density gradient centrifugation, size separation on Sepharose CL-2B, MonoQ ion exchange chromatography and agarose gel electrophoresis, demonstrates a complex mixture of mucins. Sample size limitations precluded compositional amino acid analysis. MUC 5AC and MUC1, 2, and 4 are all detected in the buoyant density range 1.3-1.5 g/ml by antibody binding. The mucins vary in size from >40 x 10(6)to <97 x 10(3)Da. A wide range of molecular size was confirmed using rate zonal centrifugation. The presence of smaller species contrasts with other mucous secretions similarly studied. In each size range are low, medium, and high charge mucins. Sialylation predominates in the medium charge and sulfate in the high charge. Only MUC5AC cross-reactivity is maintained throughout the analysis. It is detected in large and medium sized mucins but accounts for only the least mobile mucins within copurified species of similar density, size, and charge resolved using agarose electrophoresis. MUC5AC cross-reactivity is also detected in both medium and high charge species, indicating the presence of glycoforms.     

Localization of amylase and mucins in the major salivary glands of the mouse

Summary Antibodies against murine submandibular and sublingual mucins have been raised in rabbits. Both antisera appeared to be specific. Using these antibodies, the mucins were localized in the acinar cells of the submandibular and sublingual glands respectively.The dyed amylopectin method was used to estimate the activity of amylase in the salivary glands. The enzyme was localized either by a starch-substrate film method or with antibodies against purified parotid amylase. The activity of amylase in parotid homogenates is about 1000-fold higher than that in homogenates of either submandibular or sublingual glands, in which the activity was comparable. Amylase was localized in the acinar cells of the parotid gland with both localization techniques. In the sublingual gland, amylase was found predominantly in the stroma around the acini, and there was some evidence that amylase was present in the demilune cells as well. In the submandibular gland, contradictory results were obtained with both techniques. With the starch-substrate film method, amylase activity was found in the granular convoluted tubular cells, whereas immuno-reactive amylase could only be demonstrated in the acinar cells of this gland. It is concluded that in the submandibular gland amylase and mucin are present in the same cell type.     

Nerve growth factor-like immunoreactivities in rodent salivary glands and testis

Summary A series of polyclonal affinity-purified antibodies against mouse submandibular-gland nerve growth factor (NGF) are described. Using the submandibular gland of the male mouse and indirect immunofluorescence, the specificity and sensitivity of affinity-purified immunoglobulins and various other fractions from the immunized animals have been tested. It will be shown that affinity-purification schemes, including pre-purification of protein A-fractionated immunoglobulins to remove antibodies that bind to unrelated hydrophilic and hydrophobic proteins, significantly enhance the signal-to-noise ratio and specificity of the antibodies. The antibodies effectively detect NGF-like immunoreactivity in both fresh and fixed glandular tissue. Optimal fixation procedures are described. Fluorescence intensities are linearly correlated to log antibody concentration. By use of the best antibody fractions and optimal fixation protocols, the distribution of NGF-like immunoreactivity is described in eight different salivary glands (rat and mouse, male and female, submandibular and sublingual glands). In addition to the well-known large numbers of immunoreactive cells in the submandibular gland of the male mouse, immunoreactive cells were found in the sublingual gland of male mice and in the submandibular and sublingual glands of female mice. One antibody revealed a weak specific fluorescence also in the submandibular gland of the male mouse. In a survey of genital organs of male mice, one antibody revealed fluorescence in the germ cell line. We conclude that several polyclonal affinity-purified antibodies have been characterized that show a strong NGF-dependent binding to the secretory granules of tubular cells in the submandibular gland of male mice. These antibodies should make it possible to locate endogenous and perturbed NGF levels immunocytochemically, e.g., in the peripheral and central nervous system, where NGF concentrations may be several orders of magnitude lower than in the salivary glands.     

IL-17 sequestration via salivary gland gene therapy in a mouse model of Sjogren’s syndrome suppresses disease-associated expression of the putative autoantigen Klk1b22

IntroductionIL-17 has a putative role in the pathophysiology of Sjogren’s syndrome (SS) and has been shown to be upregulated in the salivary glands of affected individuals. Sequestration of IL-17 with Adenoviral-mediated gene therapy has previously shown a benefit upon the SS-like phenotype in the Aec1/Aec2 mouse model. We sought to understand the proteomic consequences of IL-17 sequestration in the salivary gland of this mouse model as a means of illuminating the role of IL-17 in SS-like disease.MethodsUltrasound-assisted gene transfer (UAGT) was utilized to express a fusion protein composed of the extracellular portion of the IL-17 receptor fused to fragment of crystallization (Fc) in the submandibular glands of Aec1/Aec2 mice at 8 weeks of age. After confirming expression of the fusion protein and local and systemic sequestration of IL-17, proteomic profiling was performed on submandibular glands of a treated cohort of Aec1/Aec2 animals relative to the background strain and sham-treated animals.ResultsThe most notable proteomic signatures of IL-17 sequestration on SS-like disease-related proteins were Kallikrein-related peptidases, including the putative autoantigen Klk1b22. IL-17 sequestration also notably led to an isoelectric shift, but not a molecular weight shift, of Kallikrein-1, attributed to phosphorylation.ConclusionNon-viral IL-17 sequestration gene therapy in the salivary gland is feasible and downregulates expression of a putative SS autoantigen in the Aec1/Aec2 mouse.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0714-2) contains supplementary material, which is available to authorized users.     

The murine sublingual and submandibular mucins. Their isolation and characterization.

From the mouse sublingual and submandibular glands high-molecular weight glycoproteins (mucins) were isolated. These mucins appeared to be homogeneous in polyacrylamide gel electrophoresis and in the analytical ultracentrifuge. S20,W values of 10.9 and 5.5 were found for the sublingual and submandibular mucin respectively. With sodium dodecyl sulfate or N-acetylcysteine no subunits could be detected. Both mucins consisted for about 1/3 of protein and 2/3 carbohydrate. Their mucin character was also denoted by the high content of serine plus threonine. Respectively 42 mol% and 34 mol% of the protein core of the sublingual and submandibular mucins consisted of these amino acids. The main sugars in these mucins were sialic acid, galactosamine, galactose, glucosamine and mannose. The molar ratio for the sublingual and submandibular mucin being 1.00 : 1.03 : 1.08 : 0.26 : 0.23 and 1.00 : 0.71 : 1.10 : 0.65 : 0.53, respectively. The sialic acid content of both mucins was about 25%. Fucose and sulfate, on the other hand, were less than 1%. The presence of sulfate was also indicated by preliminary studies in vivo on the incorporation of [35SO4] sulfate.     

Proteomic analysis of secretion from human transplanted submandibular gland replacing lacrimal gland with severe keratoconjunctivitis sicca

Purpose: Proteomic analysis of secretions from transplanted or non-transplanted submandibular glands in patients with severe keratoconjunctivitis sicca and tears from normal eyes. Experimental design: Secretions from submandibular glands transplanted to replace lacrimal glands and non-transplanted submandibular glands were collected at 1 year from 5 patients with severe keratoconjunctivitis sicca undergoing transplantation, and tears were collected from 3 normal subjects. 2-D electrophoresis (2-DE), then mass spectrometry was used to identify proteins. Western blot analysis was used to confirm protein expression. Results: We identified 34 and 11 distinct proteins in the saliva from transplanted submandibular glands and tears, respectively. The saliva from transplanted submandibular glands contained almost all the proteins abundant in tear fluid. The functions of identified proteins in the saliva from transplanted submandibular gland were mainly immune response and anti-bacterial. In total, 7 proteins showed differential expression between the saliva of transplanted and non-transplanted submandibular glands. The upregulation of short palate, lung and nasal epithelium carcinoma-associated protein 2 and carbonic anhydrase VI was confirmed by Western blot analysis. Conclusions: Identified proteins in saliva from transplanted submandibular glands may protect ocular structures. These findings can help in understanding the functional status of transplanted submandibular glands.     

Increased levels of mucins in the cystic fibrosis mouse small intestine, and modulator effects of the Muc1 mucin expression

The mouse model (Cftr(tm1UNC)/Cftr(tm1UNC)) for cystic fibrosis (CF) shows mucus accumulation and increased Muc1 mucin mRNA levels due to altered splicing (Hinojosa-Kurtzberg AM, Johansson MEV, Madsen CS, Hansson GC, and Gendler SJ. Am J Physiol Gastrointest Liver Physiol 284: G853-G862, 2003). However, it is not known whether Muc1 is a major mucin contributing to the increased mucus and why CF/Muc1-/- mice show lower mucus accumulation. To address this, we have purified mucins from the small intestine of CF mice using guanidinium chloride extraction, ultracentrifugation, and gel filtration and analyzed them by slot blot, gel electrophoresis, proteomics, and immunoblotting. Normal and CF mice with wild-type (WT) Muc1 or Muc1-/- or that are transgenic for human MUC1 (MUC1.Tg, on a Muc1-/- background) were analyzed. The total amount of mucins, both soluble and insoluble in guanidinium chloride, increased up to 10-fold in the CF mice compared with non-CF animals, whereas the CF mice lacking Muc1 showed intermediate levels between the CF and non-CF mice. However, the levels of Muc3 (orthologue of human MUC17) were increased in the CF/Muc1-/- mice compared with the CF/MUC1.Tg animals. The amount of MUC1 mucin was increased several magnitudes in the CF mice, but MUC1 did still not appear to be a major mucin. The amount of insoluble mucus of the large intestine was also increased in the CF mice, an effect that was partially restored in the CF/Muc1-/- mice. The thickness of the firmly adherent mucus layer of colon in the Muc1-/- mice was significantly lower than that of WT mice. The results suggest that MUC1 is not a major component in the accumulated mucus of CF mice and that MUC1 can influence the amount of other mucins in a still unknown way.     

Tissue specificities of protease A-like esteroprotease in male mice

An esteroprotease hydrolyzing p-tosyl-L-arginine methyl ester (TAME) has been purified to homogeneity from male mice submandibular glands by the ammonium sulphate precipitation, Sephadex gel chromatography and DEAE-cellulose chromatography. The enzyme was shown as a single chain acidic protein (pI = 5.7) with the molecular weight of 27.5 K and evidence was obtained to reveal that it was similar to protease A. Using this enzyme as antigen we prepared anti-TAMEase antibody. The immunoblotting studies on tissue specificity using 20 different tissues from male mice revealed that cross-reactivities with anti-TAMEase antibody were observed in the crude extract from the sublingual gland, parotid gland and pancreas. The species specificity studies with the submandibular glands of 7 different species indicated that only the crude extract from rat submandibular glands reacted against anti-TAMEase antibody but it exerted a low TAMEase activity.