NMR analysis of human salivary mucin (MUC7) derived O-linked model glycopeptides: comparison of structural features and carbohydrate–peptide interactions

Abstract: Two series of glycopeptides with mono- and disaccharides, [GalNAc and Galβ (1–3)GalNAc]O-linked to serine and threonine at one, two or three contiguous sites were synthesized and characterized by ¹H NMR. The conformational effects governed by O-glycosylation were studied and compared with the corresponding non-glycosylated counterparts using NMR, CD and molecular modelling. These model peptides encompassing the aa sequence, PAPPSSSAPPE (series I ) and APPETTAAPPT (series II ) were essentially derived from a 23-aa tandem repeat sequence of low molecular weight human salivary mucin (MUC7). NOEs, chemical shift perturbations and temperature coefficients of amide protons in aqueous and non-aqueous media suggest that carbohydrate moiety in threonine glycosylated peptides (series II ) is in close proximity to the peptide backbone. An intramolecular hydrogen bonding between the amide proton of GalNAc or Galβ (1–3)GalNAc and the carbonyl oxygen of the O-linked threonine residue is found to be the key structure stabilizing element. The carbohydrates in serine glycosylated peptides (series I ), on the other hand, lack such intramolecular hydrogen bonding and assume a more apical position, thus allowing more rotational freedom around the O-glycosidic bond. The effect of O-glycosylation on peptide backbone is clearly reflected from the observed overall differences in sequential NOEs and CD band intensities among the various glycosylated and non-glycosylated analogues. Delineation of solution structure of these (glyco)peptides by NMR and CD revealed largely a poly l -proline type II and/or random coil conformation for the peptide core. Typical peptide fragments of tandem repeat sequence of mucin (MUC7) showing profound glycosylation effects and distinct differences between serine and threonine glycosylation as observed in the present investigation could serve as template for further studies to understand the multifunctional role played by mucin glycoproteins.     

Mucin-negative biopsy in extra-mammary Paget''s disease. A diagnostic problem

A case of extra-mammary Paget's disease with a mucin-negative small biopsy is reported. Study of a small series of vulvar Paget's disease demonstrated that areas devoid of mucin in the mm range are frequently found, creating the conditions for diagnostic problems in small biopsies. In this situation, positive immunoreactions for carcinoembryonic antigen and low molecular weight cytokeratins and a negative reaction for S-100 protein serves to differentiate Paget's disease from other pagetoid lesions.     

Mucin gene expression in cardiac myxoma

Myxoma is the most common benign neoplasm of the heart. This work is the first to present an unusual left atrium and mitral valve cardiac myxoma which cannot be completely resected. This cardiac myxoma was also associated with abundant mucopolysaccharidic matrix, including mucin. Mucin gene expression is cell- and tissue-specific, with variations during cell differentiation and inflammation, and is altered during carcinogenesis. The expression of mucin genes in cardiac myxoma has never been elucidated previously. Detailed immunohistochemical analysis of MUC1, MUC2 and MUC5AC has been performed in this left atrium and mitral valve myxoma. Notably, the expressions of mucins in cardiac myxoma must be further evaluated.     

Stimulation by intragastrically administered E2 prostaglandins of human gastric mucus output

Abstract. Prostaglandin E₂ and 15(R)15 methyl prostaglandin E₂ were instilled intragastrically to study gastric mucus output in healthy male subjects during an infusion of pentagastrin. Mucus was measured by determining total, free, and bound N-acetyl neuraminic acid (NANA) in gastric recoveries. NANA is a sialic acid located at the end terminals on the carbohydrate chains of mucus glycoprotein. It contributes to viscosity and prevents enzymatic degradation of mucus.
The methyl analogue of prostaglandin E₂ increased the gastric output of NANA and inhibited gastric acid secretion in a dose-dependent fashion. NANA produced in response to the analogue was bound to mucus glycoprotein. Prostaglandin E₂ increased the output of NANA without affecting the gastric acid secretion. Both prostaglandin E₂ and its methyl analogue increased volumes, pH, and NANA content of gastric aspirates withdrawn prior to start of the pentagastrin infusion, indicating a stimulation of the alkaline gastric secretion.
The results show that oral E₂ prostaglandins have dual effects on gastric secretion, and that their ability to stimulate mucus production is not secondary to gastric acid inhibition. Further studies are needed to examine whether their effect is mainly to increase the incorporation of NANA into mucus glycoproteins, or to stimulate the release and/or production of gastric mucus, and also to quantitate the gastric nonparietal secretion.
The stimulatory properties of E₂ prostaglandins on gastric mucus and alkaline secretion may be one mechanism by which they protect the gastric mucosa against experimentally induced damage.     

胃癌组织中粘蛋白1和基质金属蛋白酶9的表达与临床病理特征的关系

目的探讨胃癌组织中粘蛋白1（MUC1）和基质金属蛋白酶9（MMP-9）的表达与临床病理特征的关系。方法采用免疫组织化学SP法检测r52例胃癌组织、31例良性胃溃疡组织中MUC1和MMP-9的表达。结果MUC1和MMP-9在胃癌组织中的阳性表达率分别为63．46％、76．92％,在良性胃溃疡组织中均无表达。MUC1和MMP-9在胃癌组织中的表达与组织学分级、临床分期、有无淋巴结转移密切相关。结论MUC1和MMP-9在胃癌组织中高表达与胃癌的侵袭性行为有关,有可能成为判断胃癌预后的标志物及基因治疗的潜在靶点。     

Two distinct pathways of carcinogenesis in primary sclerosing cholangitis

Zen Y, Quaglia A, Heaton N, Rela M & Portmann B
(2011) Histopathology  59 , 1100–1110
Two distinct pathways of carcinogenesis in primary sclerosing cholangitis Aims: To identify clinicopathological characteristics of cholangiocarcinoma and premalignant lesions arising in patients with primary sclerosing cholangitis (PSC). Methods and results: This study consisted of 25 patients with PSC and bile duct neoplasia [16 with cholangiocarcinoma and nine with biliary intra‐epithelial neoplasia (BilIN) equivalent to biliary dysplasia]. Tumour cell morphology, growth patterns, history of inflammatory bowel disease and postoperative survival were recorded. Immunohistochemistry for CK7, CK20, MUC1, MUC2, MUC5AC, MUC6 and CDX2 was performed to characterize cell phenotypes. Cholangiocarcinoma and BilIN were classified into intestinal (n = 14) and non‐intestinal classical (n = 11) types. Intestinal‐type lesions showed histological features resembling intestinal dysplasia or adenocarcinoma. Intestinal‐type cholangiocarcinoma commonly showed intraductal papillary proliferation and mucinous nodule formation. Intestinal‐type lesions often had an intestinal immunophenotype that was not detected in classical‐type lesions: CK20, 50% versus 0% (P = 0.007); MUC2, 86% versus 0% (P < 0.001); CDX2, 54% versus 0% (P = 0.003). Less commonly, intestinal‐type cholangiocarcinoma showed perineural invasion (P = 0.003). Patients with intestinal‐type cholangiocarcinoma had a more favourable cancer‐specific prognosis than those with classical‐type cholangiocarcinoma (P = 0.043). Conclusions: Bile ducts in PSC show two distinct dysplasia–carcinoma sequences as evidenced by differences in cell morphology, growth patterns, immunophenotypes and grade of malignancy.     

(1)H-MRS can detect aberrant glycosylation in tumour cells: a study of the HeLa cell line

Glycosylation is the most abundant and diverse form of post-translational modification of proteins. Two types of glycans exist in glycoproteins: N-glycans and O-glycans often coexisting in the same protein. O-glycosylation is frequently found on secreted or membrane-bound mucins whose overexpression and structure alterations are associated with many types of cancer. Mucins have several cancer-associated structures, including high levels of Lewis antigens characterized by the presence of terminal fucose. The present study deals with the identification of MR signals from N-acetylgalactosamine and from fucose in HeLa cells by detecting a low-field signal in one-dimensional (1D) spectra assigned to the NH of N-acetylgalactosamine and some cross peaks assigned to fucose in two-dimensional (2D) spectra. The increase of Golgi pH by treatment with ammonium chloride allowed the N-acetylgalactosamine signal assignment to be confirmed. Behaviour of MR peak during cell growth and comparison with studies from literature taken together made it possible to have more insight into the relationship between aberrantly processed mucin and the presence of non-processed N-acetylgalactosamine residues in HeLa cells. Fucose signals, tentatively ascribed to residues bound to galactose and to N-acetylglucosamine, are visible in both intact cell and perchloric acid spectra. Signals assigned to fucose bound to galactose are more evident in ammonium chloride-treated cells where structural changes of mucin-related Lewis antigens are expected as a result of the higher Golgi pH. A common origin for the N-acetylgalactosamine and fucose resonances attributing them to aberrantly processed mucin can be inferred from the present results.     

Effects of baicalin and wogonin on mucin release from cultured airway epithelial cells

Scutellaria baicalensis Georgi has been used for the treatment of diverse chronic inflammatory diseases including respiratory disease in oriental medicine and its major components - baicalin, baicalein and wogonin - were reported to have various biological effects. This study investigated whether baicalin, baicalein and wogonin affect basal and ATP-induced mucin release from cultured airway epithelial cells. Confluent primary hamster tracheal surface epithelial (HTSE) cells were metabolically radiolabeled using (3)H-glucosamine for 24 h and chased for 30 min in the presence of varying concentrations of each agent to assess the effects on (3)H-mucin release. The results were as follows: (1) Baicalein did not affect both basal and ATP-induced mucin release significantly. (2) Baicalin and wogonin increased basal mucin release at the highest concentrations (10(-3) m). (3) However, baicalin and wogonin significantly inhibited ATP-induced mucin release. It is concluded that baicalin and wogonin can slightly increase basal mucin release whereas they can inhibit ATP-induced mucin release, by directly acting on airway mucin-secreting cells. It is suggested that baicalin and wogonin be further investigated for the possible use as mucoregulators during the treatment of chronic airway diseases.     

Human MUC4 mucin induces ultra-structural changes and tumorigenicity in pancreatic cancer cells

MUC4 is a type-1 transmembrane glycoprotein and is overexpressed in many carcinomas. It is a heterodimeric protein of 930 kDa, composed of a mucin-type subunit, MUC4alpha, and a membrane-bound growth factor-like subunit, MUC4beta. MUC4 mRNA contains unique 5' and 3' coding sequences along with a large variable number of tandem repeat (VNTR) domain of 7-19 kb. A direct association of MUC4 overexpression has been established with the degree of invasiveness and poor prognosis of pancreatic cancer. To understand the precise role of MUC4 in pancreatic cancer, we engineered a MUC4 complementary DNA construct, mini-MUC4, whose deduced protein (320 kDa) is comparable with that of wild-type MUC4 (930 kDa) but represents only 10% of VNTR. Stable ectopic expression of mini-MUC4 in two human pancreatic cancer cell lines, Panc1 and MiaPaCa, showed that MUC4 minigene expression follows a biosynthesis and localisation pattern similar to the wild-type MUC4. Expression of MUC4 resulted in increased growth, motility, and invasiveness of the pancreatic cancer cells in vitro. Ultra-structural examination of MUC4-transfected cells showed the presence of increased number and size of mitochondria. The MUC4-expressing cells also demonstrated an enhanced tumorigenicity in an orthotopic xenograft nude mice model, further supporting a direct role of MUC4 in inducing the cancer properties. In conclusion, our results suggest that MUC4 promotes tumorigenicity and is directly involved in growth and survival of the cancer cells.