The role of capsulin in the morphogenesis and differentiation of fetal rat gastric mucosa.

The signals that guide the morphogenesis and differentiation of rat fetal gastric mucosa remain largely unknown. We have investigated the role of capsulin in pit/gland formation and epithelial cell differentiation in cultured stomach tissue. Embryonic day 16.5 (E 16.5) stomach tissue cultured for three days in the presence of 1 microM hydrocortisone underwent dramatic transformation, from undifferentiated, stratified cells to differentiated epithelia composed of polarised columnar cells with mucous cells and pit/glands. In the presence of capsulin antisense oligonucleotides directed against capsulin mRNA, tissues do not undergo further development. Significantly, both mucous granules and pit/gland formation were inhibited compared to capsulin sense/scrambled oligonucleotide treated controls. However, in tissues treated with specific anti-rat HGF-antiserum to neutralise secreted HGF, pit/gland formation was inhibited, but the number of mucous granules remained unchanged compared to controls treated with non-specific antiserum (mouse monoclonal cytokeratin 8 antiserum). This data suggests that capsulin may have a role in the morphogenesis of pit/glands and mucin granule formation in the developing rat gastric mucosa. We discuss the possibility that this role of capsulin may be partly mediated through the actions of HGF.     

Purification and characterization of a human pancreatic adenocarcinoma mucin.

Pancreatic mucins consist of core proteins that are decorated with carbohydrate structures. Previous studies have identified at least two physically distinct populations of mucins produced by a pancreatic adenocarcinoma cell line (HPAF); one is the MUC1 core protein, which includes an oligosaccharide structure identified by a monoclonal antibody (MAb) recognizing the DU-PAN-2 epitope. In this study, we purified and characterized a second mucin fraction, which also shows reactivity with the DU-PAN-2 antibody, but which has an amino acid composition that is not consistent with the MUC1 core protein. This new mucin was purified by ammonium sulfate precipitation, molecular sieve chromatography, and density gradient centrifugation. It eluted in the void volume of a Sepharose 4B column together with an associated low molecular weight protein, which could be further resolved. The mucin is highly polyanionic due to numerous sulfated and sialylated saccharide chains. Carbohydrate analyses of the purified mucin showed the presence of galactose, glucosamine, galactosamine, and sialic acid, but no mannose, glucose, or uronic acid. The purified and deglycosylated mucin shows no reactivity with anti-MUC1 apomucin antibody, but reacts with antiserum against deglycosylated tracheal mucins and antiserum against the MUC4 tandem repeat peptide. Analysis of mucin expression in HPAF cells revealed high levels of MUC1 and MUC4 mRNA, and moderate levels of MUC5AC and MUC5B mRNA. The amino acid composition of the purified mucin shows a high degree of similarity to the MUC4 core protein.     

Molecular cloning and analysis of the mouse homologue of the tumor-associated mucin, MUC1, reveals conservation of potential O-glycosylation sites, transmembrane, and cytoplasmic domains and a loss of minisatellite-like polymorphism.

We present here the full-length cDNA sequence and genomic structure of the mouse homologue of the tumor-associated mucin, MUC1. This mucin (previously called polymorphic epithelial mucin) is present at the apical surface of most glandular epithelial cells. The mouse gene, Muc-1, encodes an integral membrane protein with 40% of its coding capacity made up of serine, threonine, and proline, a composition typical of a highly O-glycosylated protein. The mucin core protein consists of an amino-terminal signal sequence, a tandem repeat domain encoding 16 repeats of 20-21 amino acids, and unique sequence containing transmembrane and cytoplasmic domains. Homology with the human protein is only 34% in the tandem repeat domain, mainly showing conservation of serines and threonines, presumed sites of O-linked carbohydrate attachment. Homology rises to 87% in the transmembrane and cytoplasmic domains, suggesting that these regions may be functionally important. The pattern of expression of the mouse mucin is very similar to that of its human counterpart and accordingly the two promoter regions share high homology, 74%, although previously identified potential hormone-responsive elements are not conserved. Interestingly, the mouse homologue, unlike its human counterpart does not exhibit a variable number tandem repeat polymorphism. We present evidence that suggests that the mouse gene was at one time polymorphic but has mutated away from this state.     

Quantitation and localization of acinar cell-specific mucin in submandibular glands of mice during postnatal development

Summary In this study, antiserum to acinar cell-specific mucin was utilized to determine whether mucin could be detected in the mouse submandibular gland prior to cytodifferentiation of acinar cells. Results from radioimmunoassay indicated that mucin occurs in submandibular glands from newborn mice, i.e., before the appearance of mature acinar cells. Additionally, mucin quantitated in various stages of development was found to be antigenically identical to adult mucin. After sections of glands were treated with immunohistochemical reagents, we observed that the mature acinar cell-specific mucin was present in secretory terminal-tubule cells and in proacinar cells of newborn animals. The present findings suggest that in young animals, the proacinar cells are an immediate precursor of acinar cells and that the secretory terminal-tubule cells may represent an earlier stage in development of acinar cells. In adult female glands, mucin was also detected in the granular intercalating-duct cells. This latter observation is consistent with the hypothesis that these cells are an intermediate in the acinar cell replacement process.     

Localization of a mouse submandibular sialomucin by indirect immunofluorescence

Summary A sialomucin from the mouse submandibular gland was localized in the gland by indirect immunofluorescence. Fluorescence was localized over the acinar cells and, to a lesser extent, in the lumen of the ducts. The mucin antiserum did not show cross-reactivity with cells from the sublingual gland or with other mucous-producing cells from the respiratory and gastrointestinal tracts of the mouse, or with salivary gland tissue of the rat. The sialomucin lacks both sulphate andl-fucose. Localization of a mucin with such a composition, within acinar cells of the mouse submandibular gland, is consistent with previous observations from histochemistry and autoradiography.     

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.     

MUC16 is produced in tracheal surface epithelium and submucosal glands and is present in secretions from normal human airway and cultured bronchial epithelial cells

The gel-forming MUC5AC and MUC5B mucins have been identified as major components of human airway mucus but it is not known whether additional mucin species, possibly with other functions, are also present. MUC16 mucin is a well-known serum marker for ovarian cancer, but the molecule has also been found on the ocular surface and in cervical secretions suggesting that it may play a role on the normal mucosal surface. In this investigation, the LUM16-2 antiserum (raised against a sequence in the N-terminal repeat domain) recognized MUC16 in goblet and submucosal gland mucous cells as well as on the epithelial surface of human tracheal tissue suggesting that the mucin originates from secretory cells. MUC16 mucin was present in 'normal' respiratory tract mucus as well as in secretions from normal human bronchial epithelial (NHBE) cells. MUC16 from NHBE cells was a high-molecular-mass, monomeric mucin which gave rise to large glycopeptides after proteolysis. N- and C-terminal fragments of the molecule were separated on gel electrophoresis showing that the MUC16 apoprotein undergoes a cleavage between these domains, possibly in the SEA domain as demonstrated for other transmembrane mucins; MUC1 and MUC3. After metabolic labeling of NHBE cells, most of the secreted monomeric, high-molecular-mass [(35)S]sulphate-labelled molecules were immunoprecipitated with the OC125 antibody indicating that MUC16 is the major [(35)S]sulphate-labelled mucin in NHBE cell secretions.     

Appearance of acinar-cell-specific mucin in prenatal mouse submandibular glands

The appearance of an acinar-cell-specific mucin was studied during fetal mouse submandibular gland development. The mucin was first detected in stage 23 and was quantitated through birth by radioimmunoassay (RIA). Quantitation results showed that the mucin accumulation was biphasic. Results from Western blotting and radioimmunoassay indicated that the mucin from the prenatal glands was similar both antigenically and in size to the mucin isolated from adult mice. Observations from light microscopy revealed a continuing progression of complexity throughout prenatal development, indicative of morphogenesis characteristic of differentiating exocrine tissues. When sections from various stages were compared morphometrically, it became clear that the overall ratio of epithelial cells to mesenchymal cells increased nearly 6-fold throughout the prenatal stages observed. The study suggests that acinar cell development in the mouse submandibular gland passes through a protodifferentiated stage. The proportions of epithelial and mesenchymal cells in the submandibular gland and the sensitivity of the RIA indicate that the mucin per cell actually increased to detectable levels at the onset of protodifferentiation, and this increase does not reflect a change in the relative proportions of epithelial and mesenchymal cells.     

Comparison of the secretory processes in the parotid and sublingual glands of the mouse. 1. Regulation of the secretory processes.

1. Secretion from the mucous sublingual gland of the mouse has been investigated and compared with the serous parotid gland. The influence of acetylcholine, noradrenalin and adrenalin on the secretion of glycoproteins (e.g. mucins) and proteins (e.g. amylase) from these glands in vitro, and the involvement of cyclic AMP and Ca2+ has been studied. 2. Secretion from the parotid gland could be stimulated by both acetylcholine and the catecholamines. It appears that cyclic AMP plays an important role in the adrenergic secretory process, but not in the cholinergic-induced secretion. In the latter case, exogenous Ca2+ strongly increased the secretion. 3. Mucin secretion from the sublingual gland could be affected by acetylcholine in the presence of exogenous Ca2+. Noradrenalin and adrenalin induced only a slow mucin secretion and, for this secretory process, exogenous Ca2+ is also required. Though cyclic AMP is present in the sublingual gland, no influence on its level could be detected in this gland after stimulation of the adrenergic beta-receptor, whereas, in contrast to the parotid gland, dibutyryl cyclic AMP induced only a slow secretion. Because it was observed that the sublingual gland of the mouse is not innervated sympathetically, it seems reasonable to suppose that the catecholamines stimulate the mucin secretion from this gland via hormonal receptors and not via the adrenergic beta-receptor. 4. The protein secretion from the sublingual gland could be stimulated by both acetylcholine and the catecholamines. An involvement of cyclic AMP in this process was not observed. Addition of exogenous Ca2+ is less important, as was found for the mucin secretion. So it has been concluded that protein and mucin secretion from the sublingual gland are regulated via different pathways.     

A novel protein derived from the MUC1 gene by alternative splicing and frameshifting

Genes that have been designated the name "MUC" code for proteins comprising mucin domains. These proteins may be involved in barrier and protective functions. The first such gene to be characterized and sequenced is the MUC1 gene. Here we report a novel small protein derived from the MUC1 gene by alternative splicing that does not contain the hallmark of mucin proteins, the mucin domain. This protein termed MUC1/ZD retains the same N-terminal MUC1 sequences as all of the other known MUC1 protein isoforms. The common N-terminal sequences comprise the signal peptide and a subsequent stretch of 30 amino acids. In contrast, the MUC1/ZD C-terminal 43 amino acids are novel and result from a reading frameshift engendered by a splicing event that forms MUC1/ZD. The expression of MUC1/ZD at the protein level in human tissues is demonstrated by Western blotting, immunohistochemistry, immunoprecipitation, and an ELISA. Utilization was made of affinity-purified MUC1/ZD-specific polyclonal antibodies as well as two different monoclonal antibodies that are monospecific for the MUC1/ZD protein. The MUC1/ZD protein is expressed in tissues as an oligomeric complex composed of monomers linked by disulfide bonds contributed by MUC1/ZD cysteine residues. MUC1/ZD protein expression did not parallel that of the tandem-repeat array-containing MUC1 protein. Results presented here demonstrate for the first time the expression of a novel MUC1 protein isoform MUC1/ZD, which is generated by an alternative splicing event that both deletes the tandem-repeat array and leads to a C-terminal reading frameshift.     

Cloning and sequencing of a human pancreatic tumor mucin cDNA

A monospecific polyclonal antiserum against deglycosylated human pancreatic tumor mucin was used to select human pancreatic mucin cDNA clones from a lambda gt11 cDNA expression library developed from a human pancreatic tumor cell line. The full-length 4.4-kilobase mucin cDNA sequence included a 72-base pair 5'-untranslated region and a 307-base pair 3'-untranslated region. The predicted amino acid sequence for this cDNA revealed a protein of 122,071 daltons containing 1,255 amino acid residues of which greater than 60% were serine, threonine, proline, alanine, and glycine. Approximately two-thirds of the protein sequence consisted of identical 20-amino acid tandem repeats which were flanked by degenerate tandem repeats and nontandem repeat sequences on both the amino-terminal and carboxyl-terminal ends. The amino acid sequence also contained five putative N-linked glycosylation sites, a putative signal sequence and transmembrane domain, and numerous serine and threonine residues (potential O-linked glycosylation sites) outside and within the tandem repeat position. The cDNA and deduced amino acid sequence of the pancreatic mucin sequence was over 99% homologous with a mucin cDNA sequence derived from breast tumor mucin, even though the native forms of these molecules are quite distinct in size and degree of glycosylation.     

A factor in animal tissues that causes sialyl residues of mucoproteins to react as free sialic acid in the Warren assay

1. The mucin of the Cowper's gland of the boar is a sialomucoprotein similar to submaxillary-gland mucin. When a solution of either mucin has been incubated for 5min or less with a particulate fraction from homogenized uterine endometriumplus-myometrium of the rabbit, 10-20% of sialyl residues (N-acetylneuraminic acid) give a positive Warren reaction for free N-acetylneuraminic acid. The particulate fraction is devoid of neuraminidase and no free (diffusible) N-acetylneuraminic acid appears during incubation. The factor that catalyses the formation of directreading non-diffusible N-acetylneuraminic acid occurs also in liver, kidney and intestinal mucosa of the rabbit. The factor is present in very small (;microsomal') particles and has not yet been solubilized. Homogenates of boar Cowper's gland contain both factor and mucin; thus direct-reading non-diffusible N-acetylneuraminic acid appears when such homogenates are stored. 2. Under optimum conditions 1mg of uterine protein catalyses the formation of 0.05-0.1mumol of direct-reading non-diffusible N-acetylneuraminic acid/min. This activity is considerably higher than the neuraminidase activities of comparable homogenates of animal tissues or of liver lysosomes. The factor is thermostable and its activity shows little variation within (i) the pH range 3-10, (ii) the temperature range 20-37 degrees C. Activity is inhibited strongly by 2,2'-bipyridyl and by ammonium pyrrolidine dithiocarbamate but is unaffected by EDTA. Its action can be simulated by low concentrations of Fe(2+). From this it may be inferred that the factor is a protein-bound from of bivalent iron. A number of pure iron-containing proteins and haemoproteins were completely inactive. The following substrates were not sources of direct-reading non-diffusible N-acetylneuraminic acid: methoxyneuraminic acid, sialyl-lactose, brain gangliosides, and sialoproteins in which N-acetylneuraminic acid is linked to galactose residues. 3. It is proposed that the factor (or Fe(2+)) reacts with the mucin in a manner that renders the C-4-C-5 bond of sialyl residues susceptible to periodate oxidation.     

Subunit structure of deglycosylated human and swine trachea and Cowper's gland mucin glycoproteins

Summary The oligosaccharide chains in human and swine trachea and Cowper's gland mucin glycoproteins were completely removed in order to examine the subunit structure and properties of the polypeptide chains of these glycoproteins. The carbohydrate, which constitutes more than 70% of these glycoproteins, was removed by two treatments with trifluoromethanesulfonic acid for 3 h at 3° and periodate oxidation by a modified Smith degradation. All of the sialic acid, fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine present in these glycoproteins was removed by these procedures.The deglycosylated polypeptide chains were purified and characterized. The size of the monomeric forms of all three polypeptide chains were very similar. Data obtained by gel filtration, release of amino acids during hydrolysis with carboxypeptidase B and gel electrophoresis in the presence of 0.1% dodecyl sulfate showed that a major fraction from each of the three mucin glycoproteins had a molecular size of about 67 kDa. All of the deglycosylated chains had a tendency to aggregate. Digestion with carboxypeptidases showed that human and swine trachea mucin glycoproteins had identical carboxyl terminal sequences, -Val-Ala-Phe-Tyr-Leu-Lys-Arg-COOH. Cowper's gland mucin glycoprotein had a similar carboxyl terminal sequence, -Val-Ala-Tyr-Leu-Phe-Arg-Arg-COOH. The yield of amino acids after long periods of hydrolysis with carboxypeptidases showed that at least 85% of the polypeptide chains in each of the deglycosylated preparations have these sequences. These results suggested that the polypeptide chains in these deglycosylated mucin glycoprotein preparations were relatively homogeneous.The deglycosylated polypeptide chains as well as the intact mucin glycoproteins had blocked amino terminii. The purified polypeptide chains were digested with trypsin-TCPK, and S. aureus V8 protease and the resulting peptides were isolated by gel electrophoresis in the presence of 0.1% dodecyl sulfate and by HPLC. Two partial amino acid sequences from swine trachea mucin glycoprotein, two partial sequences from human trachea mucin glycoprotein and three partial sequences from Cowper's gland mucin glycoprotein were determined. The partial amino acid sequences of the peptides isolated from swine trachea mucin glycoprotein showed more than 70% sequence homology to a repeating sequence present in porcine submaxillary mucin glycoprotein. Five to eight immunoprecipitable bands with sizes ranging from about 40 kDa to 46 kDa were seen when the polypeptide chains were digested with S. aureus V8 protease. All of the bands had blocked amino terminii and differed by a constant molecular weight of about 1.5 kDa. These data suggest that the polypeptides were formed by cleavage of glutamic acid residues present at regular intervals in the chains of all three mucin glycoproteins. These large immunoreactive peptides were formed by the removal of smaller peptides from the carboxyl terminal end of the deglycosylated mucin glycoprotein chains. Taken collectively, these findings indicate that the polypeptide chains in these mucin glycoproteins are very similar in subunit structure and that there is a high degree of homology between their polypeptide chains.     

Purification and characterization of a senile amyloid-related antigenic substance (apoSASSAM) from mouse serum. apoSASSAM is an apoA-II apolipoprotein of mouse high density lipoproteins

Two putative serum precursors which cross-react with antiserum against murine senile amyloid protein (ASSAM) were isolated from the high density lipoprotein (HDL) of normal mouse serum. Apolipoproteins designated "apoSASSAM-1" and "apoSASSAM-2" have the same molecular weight as tissue amyloid fibril protein. ApoSASSAM-1 and apoSASSAM-2 migrate to an intermediate position between apoA-I and apoC on alkaline-urea polyacrylamide gel electrophoresis and are present mainly in HDL apoproteins and to a slight extent in very low density lipoprotein apoproteins when compared to apoC. ApoSASSAM-1 and apoSASSAM-2 are polymorphic; there are two apparent isoproteins of apoSASSAM-1 with isoelectric points of 4.72 and 4.79 and two major isoproteins of apo-SASSAM-2. Subunit bands of ASSAM separated by alkaline-urea polyacrylamide gel electrophoresis and that migrated to the same positions as apoSASSAM-1 and apoSASSAM-2 were labeled by anti-apoSASSAM-1 antiserum. The amino acid compositions of apoSASSAM-1 and apoSASSAM-2 were much the same and closely resembled those of ASSAM and mouse apoA-II. Sequence analysis of apoSASSAM and ASSAM revealed a blocked amino terminus. ApoSASSAM is considered to be a mouse apoA-II and probably transforms to amyloid fibril "ASSAM" in tissues through a process yet to be clarified.     

Co-localization of TFF2 with gland mucous cell mucin in gastric mucous cells and in extracellular mucous gel adherent to normal and damaged gastric mucosa

Trefoil factor 2 (TFF2) is mucin associated peptide that has a mucosal barrier function in addition to participating in repair and healing. We examined the localization of TFF2 and gastric mucins in gastric mucous cells, the surface mucous gel layer (SMGL) adherent to normal gastric mucosa, and in the mucoid cap covering gastric erosions. Carnoy’s solution, or formalin/picric acid-fixed paraffin embedded materials from resected stomachs and formalin-fixed paraffin embedded gastric biopsy materials were used. Sections were immunostained for the TFF2 and histochemically stained for gastric mucins. In addition, thick sectioned gastric mucosa fixed in Carnoy’s solution were stained with FITC-labeled GSA-II lectin specific for gland mucous cell mucin and examined for three-dimensional images of the SMGL using a confocal laser scanning microscope. The TFF2 and gland mucous cell mucin were found intermixed together in the gastric gland mucous cells, in the SMGL in laminated layers, and in the mucoid cap. A laminated arrangement of continuous sheets of gland mucous cell mucin in the SMGL was demonstrated in the three-dimensional images. Co-localization of the TFF2 with gland mucous cell mucin suggests a physical interaction between the TFF2 and gland mucous cell mucin. The TFF2 trapped in the adherent mucins may be responsible for mucosal defense, healing, and repair.