RESUMEN

AIMS: To study the expression of mucins in peripheral airways in patients with chronic obstructive pulmonary disease (COPD). METHODS AND RESULTS: Peripheral lung sections from smokers with COPD (n = 9) and age-matched controls including smokers (n = 11) and lifelong non-smokers with normal lung function (n = 6) were stained with alcian blue, periodic acid-Schiff (PAS) and by immunohistochemistry of mucins (MUC): MUC2, MUC4, MUC5AC, MUC5B and MUC6. Histochemical staining and immunoreactivity of bronchiolar epithelium were graded and the presence or absence of stained mucus in the bronchiolar lumen was evaluated. There were no differences in alcian blue and PAS epithelial staining between the three groups. Intraluminal PAS staining was significantly more frequent among COPD subjects (P < 0.05). The expression of MUC5AC was significantly higher in the bronchiolar epithelium of patients with COPD (P < 0.05). Within the bronchiolar lumen, the predominant mucin was MUC5B. Intraluminal MUC5B was significantly more frequent among COPD patients (P < 0.05). CONCLUSIONS: COPD is specifically associated with increased expression of MUC5B in the bronchiolar lumen and of the mucin MUC5AC in the bronchiolar epithelium. These changes in mucin production in the peripheral airways may contribute to the pathophysiology of COPD.

Asunto(s)

Mucinas/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Anciano , Bronquios/metabolismo , Bronquios/patología , Femenino , Humanos , Inmunohistoquímica , Masculino , Mucina 2 , Mucina 5B , Mucinas/genética , Fumar/efectos adversos

RESUMEN

The separation and characterization of salivary mucins is not straightforward because of their large size, heterogeneity, and molecular interactions. The MUC5B and MUC7 mucins are major glycoprotein components of saliva that are thought to play a vital role in maintaining oral health. MUC5B is also a major component of respiratory mucus and is produced by the tracheal and bronchial glands, while MUC7 has a more limited pattern of expression in the bronchial tree. MUC5B is a gel-forming mucin and thus confers viscosity, whereas MUC7 is much smaller. MUC7 has anti-fungal activity, and both mucins interact with bacteria. The aim of this work was to produce new monoclonal antibodies that can be used to quantify and characterize these mucins by standard laboratory procedures. Peptide sequences in non-conserved and non-glycosylated regions were selected and monoclonal antibodies produced by an efficient immunization and cloning strategy, and screening against purified mucins. Three new antibodies-EU-MUC5Ba and EU-MUC5Bb (against MUC5B) and EU-MUC7a (against MUC7)-were isolated that do not show cross-reactivity with other gel-forming mucins. All work on immunohistochemistry can be used for semi-quantitative immunoblotting after agarose gel electrophoresis. These reagents are valuable tools to study changes in these mucins in oral and respiratory disease, and unlike other monoclonal antibodies to these mucins they recognize epitopes that are not affected by glycosylation.

Asunto(s)

Anticuerpos Monoclonales/biosíntesis , Mucinas/inmunología , Animales , Anticuerpos Monoclonales/química , Proteínas Bacterianas/inmunología , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Hibridomas , Inmunohistoquímica , Mucina 5B , Mucinas/química , Mucinas/metabolismo , Conejos , Saliva/química , Glándulas Salivales/metabolismo , Proteínas y Péptidos Salivales , Tráquea/química

RESUMEN

AIMS: The airways of patients with asthma are characterized by chronic inflammatory changes comprising mainly T-cells and eosinophils, and airway remodelling with goblet cell metaplasia and submucosal gland hyperplasia. Mucus hypersecretion is often a marked feature, particularly in status asthmaticus. The matrix of airway sputum consists of high molecular glycoproteins and mucins. In this study, the expression and distribution of the major gel-forming mucins MUC5AC and MUC5B were studied in fatal status asthmaticus tissues and bronchial biopsies of mild asthmatic patients. The effect of inhaled corticosteroids on the expression of these mucins was also investigated. METHODS AND RESULTS: Polyclonal antibodies specific for MUC5AC and MUC5B, and a monoclonal antibody for MUC5B were used to stain lung tissues and airway mucosal biopsies obtained from patients who died of status asthmaticus (n=5) and from mild asthmatics (n=4), respectively. Immunohistochemistry for MUC5AC revealed abundant staining of goblet cells situated in the epithelial surface lining and glandular ducts of tissues from patients with fatal asthma. MUC5B immunoreactivity was restricted to mucous cells of submucosal glands and to epithelial cells. In mild asthmatics, large amounts of MUC5B, but not MUC5AC, positive extracellular mucus was found in the airway lumen as plugs, adjacent to the epithelial lining and in the necks of glandular secretory ducts of mild asthmatics. The distribution of MUC5AC and MUC5B in bronchial biopsies of mild asthmatics was similar before and after inhaled steroid treatment. CONCLUSIONS: The expression of MUC5AC and MUC5B shares a similar distribution to normal airways in different states of asthma. The distribution is not affected by topical corticosteroid therapy.

Asunto(s)

Asma/patología , Mucinas/biosíntesis , Mucosa Respiratoria/patología , Asma/metabolismo , Humanos , Inmunohistoquímica , Mucina 5AC , Mucina 5B , Isoformas de Proteínas/biosíntesis , Mucosa Respiratoria/química , Índice de Severidad de la Enfermedad

RESUMEN

Hypersecretion of airway mucus is a characteristic feature of chronic airway diseases like cystic fibrosis (CF) and leads via impairment of the muco-ciliary clearance and bacterial superinfection to respiratory failure. The major components of the mucus matrix forming family of mucins in the airways are MUC5AC and MUC5B. To investigate the expression of these glycoproteins in CF, immunohistochemistry was carried out on trachea, bronchi and peripheral lung obtained from CF patients and compared to normal lung tissues. MUC5AC immunohistochemistry demonstrated signals in goblet cells of the epithelial lining. Also, goblet cells inside glandular secretory ducts revealed MUC5AC-positive staining. In comparison to those from normal subjects, CF sections were characterized by inflammatory changes and goblet cell hyperplasia, resulting in increased numbers of MUC5AC-positive cells. Immunohistochemical staining for MUC5B showed abundant staining of submucosal glands and epithelial goblet cells. Inside the glands, the immunoreactivity was restricted to glandular mucous cells. MUC5AC and MUC5B are expressed in the same histological pattern in CF compared to normal tissues with an increase of MUC5AC-positive cells due to goblet cell hyper- and metaplasia.

Asunto(s)

Fibrosis Quística/metabolismo , Pulmón/química , Mucinas/análisis , Bronquios/química , Estudios de Casos y Controles , Humanos , Inmunohistoquímica/métodos , Mucina 5AC , Mucina 5B , Tráquea/química

RESUMEN

Sequence similarities between the oligomeric mucins (MUC2, MUC5AC, MUC5B) and the von Willebrand factor suggest that they may be assembled in a similar way. After oligomerization, a fragment corresponding to the D1 and D2 domains is released from the von Willebrand factor. This cleavage does not appear to occur in pig submaxillary mucin, the only mammalian mucin in which this cleavage has been examined thus far, but whether other oligomeric mucins undergo N terminus proteolysis is not known. Antibodies recognizing the D1, D2, D3, and the first Cys domains in MUC5B were established and used to investigate to what extent proteolytic cleavage occurs within the N-terminal part of salivary MUC5B. The antibodies against the D1 and D2 domains identified a polypeptide corresponding in size to a MUC5B fragment generated by cleavage within the D' domain analogously with the von Willebrand factor propolypeptide. The antibodies did not recognize the main mucin population, suggesting that the major part of salivary MUC5B is subjected to this cleavage. An antibody recognizing the D3 domain was used to reveal a second cleavage site in the "soluble" but not in the "insoluble" MUC5B fraction: the first structural difference observed between soluble and insoluble salivary MUC5B. The identification of these cleavage events shows that the N-terminal sites for MUC5B oligomerization are present in the D3 domain and/or in domains located C-terminal to this part of the molecule.

Asunto(s)

Mucinas/química , Mucinas/metabolismo , Péptidos/química , Saliva/metabolismo , Glándulas Salivales/metabolismo , Factor de von Willebrand/metabolismo , Western Blotting , Centrifugación , Centrifugación por Gradiente de Densidad , Cesio/farmacología , Cloruros/farmacología , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Guanidina/farmacología , Humanos , Inmunohistoquímica , Mucina 5B , Unión Proteica , Estructura Terciaria de Proteína , Tráquea/metabolismo

RESUMEN

The four secretory mucin genes clustered on chromosome 11, MUC2, MUC5AC, MUC5B and MUC6, were screened in 37 patients with cancers in the left hemi-colon or rectum and 10 normal rectal controls. The mucin genes were detected by in situ hybridization using oligonucleotide probes to the variable number tandem repeat (VNTR) sequences, while the proteins were stained with non-VNTR (MUC2, MUC5AC and MUC5B) or VNTR (MUC6) antibodies. Low levels of MUC2 mRNA were detected in non-mucinous adenocarcinomas (5/27) while a higher proportion of mucinous carcinomas (4/9) was positive. All 25 cases of adjacent normal tissue expressed MUC2 mRNA. No transcripts for MUC5AC, MUC5B or MUC 6 were detected in any of these specimens. MUC2 protein product was detected immunohistochemically in 34/36 carcinoma specimens, with no change from normal controls. There was de novo expression of MUC5AC in 23/36 carcinomas. No MUC5B or MUC6 protein was detected. No difference in MUC2 and MUC5AC protein was found between mucinous and non-mucinous carcinomas. The level of MUC2 was increased in moderately differentiated cancers compared with normal controls and decreased in the poorly differentiated group. Decreased MUC2 was found in poorly differentiated compared with moderately differentiated tumours. More MUC5AC protein was detected in well and moderately differentiated tumours than in poorly differentiated tumours and in all tumours relative to controls. The pattern of MUC2 staining in cancers was different from control tissue, with strong staining in the perinuclear region and none in goblet cell vesicles. MUC5AC staining was mainly detected in the cytoplasm. Poor detection of MUC2 and MUC5AC mRNA and associated strong staining for the total protein suggests altered biosynthesis and processing, leading to the characteristic subcellular distribution. Hence, change in the synthesis of MUC2 and the de novo appearance of MUC5AC in colorectal carcinomas may be significant events in the adenoma-carcinoma sequence, with possible implications for tumour prognosis.

Asunto(s)

Adenocarcinoma/genética , Cromosomas Humanos Par 11 , Neoplasias Colorrectales/genética , Repeticiones de Minisatélite , Mucinas/genética , Adenocarcinoma Mucinoso/genética , Estudios de Casos y Controles , Marcadores Genéticos , Humanos , Inmunohistoquímica/métodos , Hibridación in Situ , Mucosa Intestinal/metabolismo , Mucina 5AC , Mucina 2 , Mucina 5B , Mucinas/análisis , ARN Mensajero/análisis , Estadísticas no Paramétricas

RESUMEN

Anti-mucin variable number tandem repeat (VNTR) antibodies have been used previously to demonstrate the de novo presence of MUC5AC and MUC6 mucin in colorectal adenomas and increased synthesis of MUC2, the major secreted mucin in normal colorectal mucosa. Here we examined secreted mucins in tubular, tubulovillous and villous adenomas of the rectum using non-VNTR antibodies designed to assess mature mucin. Mucin gene messenger RNAs were detected by in situ hybridization. The anti-MUC2 non-VNTR antibody in the goblet cells of adenomas revealed a staining pattern of increased cytoplasmic, Golgi and membrane staining with no change in goblet vesicle reactivity compared with normal controls. In addition, blank goblet cell vesicle immunostaining for MUC2 was found in the transitional mucosa adjacent to all types of adenoma. Although a trend to overexpression of MUC2 was observed with in situ hybridization this was not detected with immunohistology. De novo synthesis of MUC5AC, but not MUC5B or MUC6 mucin was seen in all adenomas and transitional mucosa using immunohistochemistry. There was no correlation of MUC2 or MUC5AC mucin with polyp size or the grade of dysplasia using the non-VNTR antibodies. This study demonstrates that anti-mucin non-VNTR antibodies reveal a different subcellular-localization in rectal adenomas compared with normal colorectal mucosa. Further, this pattern is in contrast to that reported for anti-mucin VNTR antibodies. Combined use of these reagents may benefit future assessment of these cancers.

Asunto(s)

Adenoma/inmunología , Adenoma/metabolismo , Inmunohistoquímica/métodos , Mucinas/metabolismo , Neoplasias del Recto/inmunología , Neoplasias del Recto/metabolismo , Adenoma/patología , Animales , Anticuerpos/metabolismo , Biomarcadores de Tumor , Mucosa Gástrica/inmunología , Mucosa Gástrica/patología , Humanos , Sueros Inmunes , Repeticiones de Minisatélite/inmunología , Mucina 5AC , Mucina 2 , Mucinas/genética , Mucinas/inmunología , Proteínas de Neoplasias/metabolismo , Péptidos/síntesis química , Péptidos/inmunología , ARN Mensajero/metabolismo , Conejos , Neoplasias del Recto/patología , Fracciones Subcelulares , Transcripción Genética

RESUMEN

Stimulated human submandibular/sublingual (HSMSL) and whole saliva were separated into sol and gel phases and mucins were isolated by density-gradient centrifugation in CsCl/4M guanidinium chloride. MUC5B and MUC7 were identified using anti-peptide antisera raised against sequences within the MUC5B and MUC7 apoproteins respectively. MUC7 was found mainly in the sol phase of both HSMSL and whole saliva, but some MUC7 was consistently present in the gel phase, suggesting that this mucin may interact with the salivary gel matrix. In HSMSL saliva, MUC5B was found in the gel phase; however, most of the material was 'insoluble' in guanidinium chloride and was only brought into solution by reduction. In whole saliva, the MUC5B mucin was present both in the sol and gel phases although some material was again 'insoluble'. Rate-zonal centrifugation of whole saliva showed that MUC5B mucins in the sol phase were smaller than those in the gel phase, suggesting differences in oligomerization and/or degradation. Antibodies against IgA, secretory component, lysozyme and lactoferrin were used to study the distribution of non-gel-forming proteins in the different phases of saliva. The majority of these proteins was found in the sol phase of both HSMSL and whole saliva. However, a significant fraction was present in the gel phase of whole saliva, suggesting a post-secretory interaction with the salivary gel matrix. A monoclonal antibody against a parotid salivary agglutinin was used to show that this protein is present mainly in the gel phase of both whole saliva and parotid secretion.

Asunto(s)

Mucinas/química , Saliva/química , Aglutininas/metabolismo , Centrifugación por Gradiente de Densidad , Cromatografía en Gel , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina A/metabolismo , Inmunohistoquímica , Lactoferrina/metabolismo , Mucina 5B , Mucinas/aislamiento & purificación , Muramidasa/metabolismo , Glándula Parótida/metabolismo , Proteínas y Péptidos Salivales/química , Componente Secretorio/metabolismo

RESUMEN

BACKGROUND: In normal gastric epithelium, MUC5AC is detected in superficial epithelium associated with Lewis type 1 antigens and MUC6 is detected in antral glands with Lewis type 2. Therefore, the stomach constitutes an excellent model to examine the role of glycosyltransferases in determining the specificity of apomucin glycosylation. AIMS: To determine the molecular basis of this association and to examine changes in expression of gastric and intestinal apomucins and their association with Lewis antigens during the gastric carcinogenesis process. METHODS: Fucosyltransferase (FUT1, FUT2, FUT3) and mucin (MUC5AC, MUC6) transcripts were detected using reverse transcription-polymerase chain reaction. Apomucin (MUC2, MUC4, MUC5AC, MUC6) and Lewis antigen (types 1 and 2) expression were analysed using single and double immunohistochemistry and in situ hybridisation. RESULTS: In the normal stomach, FUT1 is exclusively detected associated with MUC6; FUT2 is only detected when MUC5AC is present. This co-regulation is lost in gastric tumours, as is differential expression of MUC5AC and MUC6 in normal gastric epithelial cells. In gastric tumours, especially those with the intestinal phenotype, MUC2 and MUC4 genes are upregulated, and gastric-type and intestinal-type mucins are coexpressed. These changes are early events in the gastric carcinogenesis process, as they are detected in intestinal metaplasia. CONCLUSIONS: The glycosylation pattern found in normal gastric epithelium is dictated by the specific set of fucosyltranferases expressed by the cells rather than by the apomucin sequence. The development of intestinal metaplasia and gastric cancer is associated with the appearance of cellular phenotypes that are absent from normal epithelium.

Asunto(s)

Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Fucosiltransferasas/fisiología , Mucinas Gástricas/metabolismo , Antígeno Lewis X/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Gástricas/enzimología , Antígenos de Carbohidratos Asociados a Tumores/genética , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Glicosilación , Humanos , Inmunohistoquímica , Hibridación in Situ , Antígeno Lewis X/genética , Proteínas de Neoplasias/inmunología , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/inmunología , Regulación hacia Arriba

Asunto(s)

Mucinas/aislamiento & purificación , Geles , Humanos

RESUMEN

To investigate the genetic identities of the mucins secreted in cystic fibrosis (CF) airways, sputum was collected from five individuals. Samples were separated into gel and sol phases by high-speed centrifugation and the gel phase was extracted in 6 M guanidinium chloride. The 'insoluble' residue remaining after extraction of the gel phase was brought into solution by reduction/alkylation. Density-gradient centrifugation in CsCl revealed polydisperse distributions of sialic acid-containing mucins in the gel phase, insoluble residue and sol phase fractions and the degree of variation between the different individuals was low. Antibodies recognizing MUC5AC and MUC5B identified these mucins in each of the fractions. MUC2, however, was present only as a component of the insoluble residue from the gel which accounted for less than 4% by mass of the total mucins. MUC5B and MUC5AC from the gel phase were large oligomeric species composed of disulphide-bond linked subunits and MUC5B was present as two populations with different charge densities which are likely to correspond to MUC5B 'glycoforms'. The sol phase contained, in addition to MUC5AC and MUC5B, mainly smaller mucins which did not react with the antisera and which were probably degraded. MUC5AC appeared to be enriched in the sol, suggesting that this mucin may be more susceptible to proteolytic degradation than MUC5B. The mucins present in sputum remained broadly similar during acute exacerbation and following antibiotic treatment, although the relative amount of an acidic MUC5B glycoform was decreased during infection.

Asunto(s)

Fibrosis Quística/metabolismo , Mucinas/metabolismo , Sistema Respiratorio/metabolismo , Esputo/química , Adulto , Centrifugación , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Electricidad , Geles , Humanos , Peso Molecular , Mucina 5AC , Mucina 2 , Mucina 5B , Solubilidad

RESUMEN

Two proteoglycans differing in size and composition were isolated from human follicular fluid. The larger one of high density had a molecular mass of 3.0x10(6) Da, as determined by laser light-scattering, and was substituted with 15-20 chondroitin sulphate (CS) chains (Mr 60000-65000). Half of the CS disaccharides were 6-sulphated, whereas the remaining ones were non-sulphated. Digestion of the CS proteoglycan with chondroitinase ABC lyase, followed by SDS/PAGE, yielded a protein core of 600 to 700 kDa including substituted oligosaccharides, and a band of 70 kDa that was identified as the heavy-chain component of the inter-alpha-trypsin inhibitor (ITI). Western blotting of the CS proteoglycan showed that this had reactivity with antibodies raised against human versican. Electron microscopy (EM) of the CS proteoglycan also revealed a versican-like structure, with one globular domain at each end of a long extended segment substituted with CS side chains, as well as a structure interpreted as being the heavy chain of ITI attached to CS chains. Laser light-scattering revealed that the smaller proteoglycan had a molecular mass of 1. 1x10(6) Da, and EM demonstrated that it had a globular-protein core structure. The core protein, which showed immunological reactivity with perlecan antibodies, was substituted with approximately seven heparan sulphate (HS) and CS chains of similar size (50-55 kDa), the CS disaccharides being mainly 6-sulphated (68%), with a small proportion being 4-sulphated. The protein core was shown to be heterogeneous, with bands occurring at 215, 330 and 400 kDa after enzymic degradation of the glycosaminoglycan chains followed by SDS/PAGE analysis. The demonstration of intact molecules and fragments obtained after stepwise degradations, as shown by gel chromatography, supported a 'composite' structure of this proteoglycan.

Asunto(s)

Líquido Folicular/química , Proteoglicanos/química , Proteoglicanos/aislamiento & purificación , Álcalis , alfa-Globulinas/análisis , alfa-Globulinas/metabolismo , Aminoácidos/análisis , Western Blotting , Centrifugación por Gradiente de Densidad , Proteoglicanos Tipo Condroitín Sulfato/análisis , Proteoglicanos Tipo Condroitín Sulfato/química , Proteoglicanos Tipo Condroitín Sulfato/aislamiento & purificación , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/ultraestructura , Sulfatos de Condroitina/análisis , Sulfatos de Condroitina/química , Sulfatos de Condroitina/metabolismo , Condroitinasas y Condroitín Liasas/metabolismo , Cromatografía Liquida , Femenino , Proteoglicanos de Heparán Sulfato/química , Proteoglicanos de Heparán Sulfato/aislamiento & purificación , Proteoglicanos de Heparán Sulfato/metabolismo , Heparitina Sulfato/análisis , Humanos , Lectinas Tipo C , Microscopía Electrónica , Peso Molecular , Oligosacáridos/análisis , Oligosacáridos/química , Oligosacáridos/aislamiento & purificación , Oligosacáridos/metabolismo , Proteoglicanos/análisis , Proteoglicanos/metabolismo , Proteoglicanos/ultraestructura , Azufre/análisis , Versicanos

RESUMEN

A microtiter-based assay was developed to study the binding of Helicobacter pylori to pig gastric mucins purified by density-gradient centrifugation in CsCl/4 M guanidinium chloride. Binding of H. pylori was observed over the 'mucin' band as well as with 'low-density' components in the gradients, and binding to the latter was more pronounced when incubations were performed at 37 degrees C as compared to 20 degrees C. At a lower pH, binding of H. pylori (strain SVA 40) to the 'high-density' mucins from pig antrum was increased but binding to the 'low-density' ones was decreased. Binding of the P466 strain (Le(b)-specific) was mainly associated with the 'mucin' band, whereas the MO19 strain reacted preferentially with the 'low-density' components. In summary, H. pylori may bind to gastric mucins and the binding is influenced by temperature, pH and the repertoire of bacterial adhesins.

Asunto(s)

Mucosa Gástrica/microbiología , Helicobacter pylori/metabolismo , Mucinas/metabolismo , Adulto , Animales , Adhesión Bacteriana , Centrifugación por Gradiente de Densidad , Modelos Animales de Enfermedad , Mucosa Gástrica/metabolismo , Infecciones por Helicobacter/microbiología , Helicobacter pylori/aislamiento & purificación , Humanos , Concentración de Iones de Hidrógeno , Masculino , Úlcera Péptica/microbiología , Porcinos

RESUMEN

The "insoluble" glycoprotein complex was isolated from human colonic tissue and mucin subunits were prepared following reduction. Antibodies raised against peptide sequences within MUC2 revealed that virtually all of this mucin occurs in the insoluble glycoprotein complex. In addition, reduction released a 120-kDa C-terminal MUC2 fragment, showing that proteolytic cleavage in this domain may occur and leave the fragment attached to the complex via disulfide bonds. The variable number tandem repeat region and the irregular repeat domain were isolated after trypsin digestion and shown to have molecular weights of 930,000 and 180,000, respectively, suggesting a molecular weight for the entire MUC2 monomer of approximately 1.5 million. Gel chromatography and agarose gel electrophoresis revealed several populations of MUC2 subunits, and analytical ultracentrifugation showed that these have molecular weights on the order of 2 million, 4 million, and 5 million, corresponding to monomers, dimers, and trimers, respectively. Agarose gel electrophoresis of subunits from individuals expressing both a "long" and a "short" MUC2 allele revealed a larger number of populations, consistent with the presence of short and long monomers and oligomers arising from permutations of the two types of monomers. In addition to disulfide bonds, MUC2 monomers are apparently joined by a "novel," reduction-insensitive bond.

Asunto(s)

Colon/química , Mucinas/química , Secuencia de Aminoácidos , Aminoácidos/análisis , Anticuerpos/metabolismo , Disulfuros/química , Glicopéptidos/química , Glicoproteínas/química , Glicoproteínas/inmunología , Humanos , Datos de Secuencia Molecular , Peso Molecular , Monosacáridos/análisis , Mucina 2 , Mucinas/inmunología , Solubilidad , Tripsina/metabolismo

RESUMEN

The O-linked oligosaccharides from three fractions of highly glycosylated mucin glycopeptides obtained from sputum of a patient with cystic fibrosis were characterized and compared regarding size, composition, sequence and when possible linkage positions. Neutral and sialic acid-containing glycans were permethylated and analyzed by high-temperature GC-MS and MALDI-MS, showing more than 60 different oligosaccharides with a size of up to 15 monosaccharide units. Some of the observed oligosaccharides are novel for respiratory secretions, one being a trifucosylated heptasaccharide with the proposed structure: Fuc-Gal-4(Fuc-3)GlcNAc-(Fuc-)Gal-3GalNAcol. The glycosylation of two of the glycopeptide fractions was similar with regard to the neutral and sialylated oligosaccharides despite their different origins from the sol or gel phase. Analysis of the sulfated oligosaccharides by FAB-MS/MS indicated that the gel fraction contained C-6 linked sulfate groups while the two sol fractions also contained C-3 linked sulfate. The results suggest the presence of different glycosylated mucin domains, probably originating from different mucin glycoforms and/or apoproteins in the airway of cystic fibrosis patients.

Asunto(s)

Fibrosis Quística , Glicopéptidos/química , Mucinas/química , Esputo/química , Adolescente , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Femenino , Cromatografía de Gases y Espectrometría de Masas , Glicosilación , Humanos , Datos de Secuencia Molecular , Oligosacáridos/química , Análisis de Secuencia , Espectrometría de Masa Bombardeada por Átomos Veloces , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Ésteres del Ácido Sulfúrico

RESUMEN

Mucins from human whole saliva, as well as from respiratory- and cervical-tract secretions, were subjected to density-gradient centrifugation in CsCl/0.5 M guanidinium chloride. A polydisperse population of MUC5B mucins was demonstrated in all samples using anti-peptide antisera (LUM5B-2, LUM5B-3 and LUM5B-4) raised against sequences within the MUC5B mucin. The sequences recognized by the LUM5B-2 and LUM5B-3 antisera are located within the domains flanking the highly glycosylated regions of MUC5B, and reduction increased the reactivity with these antibodies, suggesting that the epitopes are partially shielded and that these regions are folded and stabilized by disulphide bonds. Rate-zonal centrifugation before and after reduction showed MUC5B to be a large oligomeric mucin composed of disulphide-linked subunits. In saliva and respiratory-tract secretions, populations of MUC5B mucins with different charge densities were identified by ion-exchange HPLC, suggesting the presence of MUC5B 'glycoforms'. In trachea, the F2 monoclonal antibody against the sulpho-Lewis C structure reacted preferentially with the later-to-be-eluted populations. An antibody (LUM5B-4) recognizing a sequence in the C-terminal domain of MUC5B identified, after reduction, the mucin subunits as well as smaller fragments, suggesting that some of the MUC5B mucins are cleaved within the C-terminal domain. Immunohistochemistry revealed that MUC5B is produced by cells dispersed throughout the human submandibular and sublingual glands, in the airway submucosal glands as well as the goblet cells, and in the epithelium and glands of the endocervix. The F2 antibody stained a subpopulation of the MUC5B-producing cells in the airway submucosal glands, suggesting that different cells may produce different glycoforms of MUC5B in this tissue.

Asunto(s)

Cuello del Útero/química , Mucinas/aislamiento & purificación , Sistema Respiratorio/química , Glándulas Salivales/química , Secuencia de Aminoácidos , Animales , Centrifugación por Gradiente de Densidad , Moco del Cuello Uterino/química , Femenino , Geles , Glicosilación , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Mucina 5B , Mucinas/química , Mucinas/genética , Conformación Proteica , Conejos , Saliva/química , Esputo/química

RESUMEN

OBJECTIVE: To test the hypothesis that human cervical mucins affect the motility and hyperactivated motility of human spermatozoa. SETTING: University hospital. PATIENT(S): Healthy donors. INTERVENTION(S): Swim-up sperm fractions of normozoospermic semen samples were incubated in the presence of 0 (control) to 1.3 mg/mL of mucins purified from cervical mucus plugs released during labor. Motility analyses were performed at time 0, and after 0.5, 1, 3, and 7 hours. MAIN OUTCOME MEASURE(S): Sperm kinematic variables recorded by computer-aided sperm analysis. Hyperactivation was defined as linearity <30%, amplitude of lateral head displacement >7.0 microm, and curvilinear velocity >70 microm/s. RESULT(S): A dose-related effect of cervical mucins on sperm motility was found. Mucins at a concentration of 1.3 mg/mL caused an immediate and significant increase in sperm linearity (27%) and straight-line velocity (16%) compared with control samples. During the first 3 hours of incubation, an approximately 25% increase in linearity and straight-line velocity was found; this increase was statistically significant. Effects on the hyperactivation pattern were found as incubation with mucins for 3 and 7 hours significantly reduced the percentage of hyperactivation from 18% to 9%. CONCLUSION(S): Cervical mucins increase the percentage of progressively motile sperm and decrease the percentage of sperm that show hyperactivation.

Asunto(s)

Moco del Cuello Uterino/fisiología , Diagnóstico por Computador , Mucinas/fisiología , Motilidad Espermática/fisiología , Femenino , Humanos , Técnicas In Vitro , Modelos Lineales , Masculino , Embarazo , Valores de Referencia , Donantes de Tejidos

RESUMEN

An antibody (PGM2B) recognizing a pig gastric-mucin apoprotein reacts with the surface epithelium of pig gastric mucosa. Virtually no reactivity was observed over the mucin-producing cells in the glands, which were recognized by the GlcNAc-selective Griffonia simplicifolia II (GSA-II) lectin. Mucins from the glandular tissue of the cardiac region, corpus and antrum were purified using isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. In the cardiac region, two major mucin populations at 1.5 and 1.4 g/ml were identified. The high-density population reacted preferentially with the PGM2B antibody and resembled mucins from the surface epithelium of this region, whereas the low-density population reacted strongly with the GSA-II lectin and appeared to originate from the glands. In the glandular tissue of corpus, a component with strong GSA-II lectin reactivity, which was distinctly different from the surface mucins from this region, was found at 1.4 g/ml, thus resembling the gland component from the cardiac region. Mucins from antrum glandular tissue contained at least two GSA-II lectin-reactive populations banding at 1.5 and 1.4 g/ml, respectively. Gland mucins from all regions were large oligomeric glycoproteins and heterogeneous with respect to charge properties, as shown by using rate-zonal centrifugation and ion-exchange HPLC, respectively. Gel chromatography of mucin glycopeptides showed that gland mucins from antrum and corpus contained significantly longer glycosylated domains than those from the surface mucosa. Thus, mucins from pig gastric glandular tissue comprise a number of large and oligomeric glycoproteins that differ from those from the surface epithelium in buoyant density, apoprotein structure and carbohydrate substitution.

Asunto(s)

Mucosa Gástrica/química , Glicoproteínas/química , Mucinas/química , Animales , Anticuerpos/inmunología , Centrifugación por Gradiente de Densidad , Endopeptidasas/metabolismo , Glicopéptidos/química , Histocitoquímica , Lectinas/metabolismo , Porcinos

RESUMEN

The large glycosylated domains obtained from the rat intestinal mucin Muc2 were isolated from the large and small intestine of the inbred rat strains GOT-W and GOT-BW. The expression of the rat Muc2 in the large intestine was confirmed immunochemically and by Northern blotting. Released oligosaccharides were structurally characterized by gas chromatography-mass spectrometry (neutral and sialylated species) or by tandem mass spectrometry (sulfated species), and a total of 63 structures was assigned. The large intestinal oligosaccharides were found to be identical between the strains, while the small intestinal glycosylation differed. Until now, detailed structural analysis of oligosaccharides isolated from a single mucin core or mucin domain with different origin have not been performed, and the information of different mucin glycoforms has been limited to immunochemistry. Blood group A-determinants (GalNAcalpha1-3(Fucalpha1-2)Galbeta1-, and structures related to the blood group Sda/Cad-related epitope NeuAc/NeuGcalpha1-3(GalNAcbeta1-4)Galbeta1-, were found in GOT-BW small intestine, and also in both large intestines. Blood group H-determinants and NeuAc/NeuGcalpha1-3Galbeta1- were found in all samples. Core 1 (Galbeta1-3GalNAcalpha1-), core 2 (Galbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-), core 3 (GlcNAcbeta1-3GalNAcalpha1-), and core 4 (GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcalpha1- were also found in all the samples. The large intestine were enriched in sulfated oligosaccharides and the small intestine contained higher amounts of sialylated species. Sulfation were found exclusively on C-6 of GlcNAc.

Asunto(s)

Mucosa Intestinal/metabolismo , Intestino Grueso/metabolismo , Intestino Delgado/metabolismo , Mucinas/química , Mucinas/metabolismo , Oligosacáridos/química , Sistema del Grupo Sanguíneo ABO/química , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cromatografía en Gel , Epítopos/análisis , Cromatografía de Gases y Espectrometría de Masas/métodos , Glicopéptidos/química , Glicopéptidos/aislamiento & purificación , Glicosilación , Mucosa Intestinal/química , Datos de Secuencia Molecular , Mucina 2 , Mucinas/biosíntesis , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Oligosacáridos/aislamiento & purificación , Especificidad de Órganos , Ratas , Ácidos Siálicos/análisis

RESUMEN

Pig gastric mucins were isolated from the surface epithelium of the cardiac region, corpus and antrum using density-gradient centrifugation after extraction in 6 M guanidinium chloride. In CsCl/0.5 M guanidinium chloride, mucins solubilized from the cardiac region appeared as a broad unimodal band at 1.52 g/ml whereas those from the corpus and antrum occurred as high- and low-density populations at 1.50 and 1.45 g/ml respectively. High-iron diamine reacted more strongly with the cardiac mucins and the high-density populations from corpus and antrum than with the two low-density ones. In keeping with this, approx. 60% of the oligosaccharides from the former mucins and 20% from the latter contained sulphate. All surface epithelial cells of the cardiac region stained with high-iron diamine, whereas in the corpus only the epithelium in the bottom of the pits reacted, suggesting that the high-density population from this region originates from these cells. Mucins from all regions were composed of subunits, each containing highly glycosylated domains. The mucins from the cardiac region were larger than those from the corpus and antrum, and reduced subunits as well as high-molecular-mass glycopeptides from the cardiac mucins were larger than the corresponding fragments from the other regions. Ion-exchange HPLC showed that reduced subunits from the cardiac mucins and the high-density populations from the corpus and antrum were more 'acidic' than reduced subunits from the two low-density ones. All mucins contained a 'neutral'fraction, in particular those from the antrum. Pig gastric mucus thus contains a number of distinctly different mucin populations varying in buoyant density, size, 'acidity', glycosylation, sulphation and tissue origin.

Asunto(s)

Mucinas Gástricas/análisis , Mucosa Gástrica/metabolismo , Glicoproteínas/análisis , Animales , Centrifugación por Gradiente de Densidad , Cromatografía Líquida de Alta Presión , Epitelio/metabolismo , Mucosa Gástrica/citología , Porcinos