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Gunnar C. Hansson - One of the best experts on this subject based on the ideXlab platform.
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carbachol induced muc17 endocytosis is concomitant with nhe3 internalization and cftr membrane recruitment in enterocytes
American Journal of Physiology-cell Physiology, 2013Co-Authors: Thaher Pelaseyed, Ida J Gustafsson, Anna Ermund, Jenny K Gustafsson, Gunnar C. HanssonAbstract:We have reported that transmembrane mucin MUC17 binds PDZ protein PDZK1, which retains MUC17 apically in enterocytes. MUC17 and transmembrane mucins MUC3 and MUC12 are suggested to build the entero...
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the c terminus of the transmembrane mucin muc17 binds to the scaffold protein pdzk1 that stably localizes it to the enterocyte apical membrane in the small intestine
Biochemical Journal, 2008Co-Authors: Emily K Malmberg, Thaher Pelaseyed, Asa C Petersson, Ursula Seidler, Hugo R De Jonge, John R Riordan, Gunnar C. HanssonAbstract:The membrane-bound mucins have a heavily O-glycosylated extracellular domain, a single-pass membrane domain and a short cytoplasmic tail. Three of the membrane-bound mucins, MUC3, MUC12 and MUC17, are clustered on chromosome 7 and found in the gastrointestinal tract. These mucins have C-terminal sequences typical of PDZ-domain-binding proteins. To identify PDZ proteins that are able to interact with the mucins, we screened PDZ domain arrays using YFP (yellow fluorescent protein)-tagged proteins. MUC17 exhibited a strong binding to PDZK1 (PDZ domain containing 1), whereas the binding to NHERF1 (Na + /H + -exchanger regulatory factor 1) was weak. Furthermore, we showed weak binding of MUC12 to PDZK1, NHERF1 and NHERF2. GST (glutathione transferase) pull-down experiments confirmed that the C-terminal tail of MUC17 co-precipitates with the scaffold protein PDZK1 as identified by MS. This was mediated through the C-terminal PDZ-interaction site in MUC17, which was capable of binding to three of the four PDZ domains in PDZK1. Immunostaining of wild-type or Pdzk1 −/− mouse jejunum with an antiserum against Muc3(17), the mouse orthologue of human MUC17, revealed strong brush-border membrane staining in the wild-type mice compared with an intracellular Muc3(17) staining in the Pdzk1 −/− mice. This suggests that Pdzk1 plays a specific role in stabilizing Muc3(17) in the apical membrane of small intestinal enterocytes.
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The C-terminus of the transmembrane MUC17 mucin binds to the scaffold protein PDZK1 that stably localizes it to the enterocyte apical membrane in the small intestine
Biochemical Journal, 2008Co-Authors: Emily K Malmberg, Thaher Pelaseyed, John R Riordan, Åsa Petersson, Ursula E. Seidler, Hugo De Jonge, Gunnar C. HanssonAbstract:The membrane bound mucins have a heavily O-glycosylated extracellular domain, a single pass membrane domain and a short cytoplasmic tail. Three of the membrane bound mucins, MUC3, MUC12 and MUC17, are clustered on chromosome 7 and found in the gastrointestinal tract. These mucins have C-terminal sequences typical for PDZ domain binding proteins. To identify PDZ proteins able to interact with the mucins, we screened PDZ domain arrays using YFP-tagged proteins. MUC17 exhibited a strong binding to PDZK1 whereas the binding to NHERF1 was weak. Furthermore, we showed weak binding of MUC12 to PDZK1, NHERF1 and NHERF2. GST pull-down experiments confirmed that the C-terminal tail of MUC17 co-precipitates with the scaffold protein PDZK1 as identified by mass spectrometry. This was mediated through the C-terminal PDZ-interaction site in MUC17 which was capable of binding to three of the four PDZ domains in PDZK1. Immunostaining of wild-type or Pdzk1-/- mouse jejunum with an antiserum against Muc3(17), the mouse orthologue of human MUC17, revealed strong brush border membrane staining in the wild-type mice compared to an intracellular Muc3(17) staining in the Pdzk1-/- mice. This suggests that Pdzk1 plays a specific role in stabilizing Muc3(17) in the apical membrane of small intestinal enterocytes.
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An inventory of mucin genes in the chicken genome shows that the mucin domain of Muc13 is encoded by multiple exons and that ovomucin is part of a locus of related gel-forming mucins
BMC Genomics, 2006Co-Authors: Tiange Lang, Gunnar C. Hansson, Tore SamuelssonAbstract:Background Mucins are large glycoproteins that cover epithelial surfaces of the body. All mucins contain at least one PTS domain, a region rich in proline, threonine and serine. Mucins are also characterized by von Willebrand D (VWD) domains or SEA domains. We have developed computational methods to identify mucin genes and proteins based on these properties of the proteins. Using such methods we are able to characterize different organisms where genome sequence is available with respect to their mucin repertoire. Results We have here made a comprehensive analysis of potential mucins encoded by the chicken ( Gallus gallus ) genome. Three transmembrane mucins (Muc4, Muc13, and Muc16) and four gel-forming mucins (Muc6, Muc2, Muc5ac, and Muc5b) were identified. The gel-forming mucins are encoded within a locus similar to the corresponding human mucins. However, the chicken has an additional gene inserted between Muc2 and Muc5ac that encodes the the α-subunit of ovomucin, a protein similar to Muc2, but it is lacking a PTS domain. We also show that the β-subunit of ovomucin is the orthologue of human MUC6. The transmembrane Muc13 gene is in chicken as well as in mammals adjacent to the HEG (heart of glass) gene. HEG has PTS, EGF and transmembrane domains like Muc13, suggesting that these two proteins are evolutionary related. Unlike previously known mucins, the PTS domain of Muc13 is encoded by multiple exons, where each exon encodes a repeat unit of the PTS domain. Conclusion We report new mucin homologues in chicken and this information will aid in understanding the evolution of mucins in vertebrates. The fact that ovomucin, a protein not found in mammals, was located in the same locus as other gel-forming mucins provides strong support that these proteins are evolutionary related. Furthermore, a relationship of HEG and the transmembrane Muc13 is suggested on the basis of their biochemical properties and their presence in the same locus. Finally, our finding that the chicken Muc13 is distributed between multiple exons raises the interesting possibility that the length of the PTS domain could be controlled by alternative splicing.
Thaher Pelaseyed - One of the best experts on this subject based on the ideXlab platform.
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Study of mucin turnover in the small intestine by in vivo labeling
Scientific Reports, 2018Co-Authors: Hannah Schneider, Thaher Pelaseyed, Frida Svensson, Malin E. V. JohanssonAbstract:Mucins are highly glycosylated proteins which protect the epithelium. In the small intestine, the goblet cell-secreted Muc2 mucin constitutes the main component of the loose mucus layer that traps luminal material. The transmembrane mucin Muc17 forms part of the carbohydrate-rich glycocalyx covering intestinal epithelial cells. Our study aimed at investigating the turnover of these mucins in the small intestine by using in vivo labeling of O-glycans with N-azidoacetylgalactosamine. Mice were injected intraperitoneally and sacrificed every hour up to 12 hours and at 24 hours. Samples were fixed with preservation of the mucus layer and stained for Muc2 and Muc17. Turnover of Muc2 was slower in goblet cells of the crypts compared to goblet cells along the villi. Muc17 showed stable expression over time at the plasma membrane on villi tips, in crypts and at crypt openings. In conclusion, we have identified different subtypes of goblet cells based on their rate of mucin biosynthesis and secretion. In order to protect the intestinal epithelium from chemical and bacterial hazards, fast and frequent renewal of the secreted mucus layer in the villi area is combined with massive secretion of stored Muc2 from goblet cells in the upper crypt.
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the mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system
Immunological Reviews, 2014Co-Authors: Thaher Pelaseyed, Anna Ermund, Joakim H Bergstrom, George M H Birchenough, Andre Schutte, Sjoerd Van Der Post, Ana M Rodriguezpineiro, Jenny K Gustafsson, Frida Svensson, Elisabeth E L NystromAbstract:The gastrointestinal tract is covered by mucus that has different properties in the stomach, small intestine and colon. The large highly glycosylated gel-forming mucins MUC2 and MUC5AC are the major components of the mucus in the intestine and stomach, respectively. In the small intestine mucus limits the number of bacteria that can reach the epithelium and the Peyer’s patches. In the large intestine the inner mucus layer separates the commensal bacteria from the host epithelium. The outer colonic mucus layer is the natural habitat for the commensal bacteria. The intestinal goblet cells not only secrete the MUC2 mucin, but also a number of typical mucus components: CLCA1, FCGBP, AGR2, ZG16, and TFF3. The goblet cells have recently been shown to have a novel gate-keeping role for the presentation of oral antigens to the immune system. Goblet cells deliver small intestinal luminal material to the lamina propria dendritic cells of the tolerogenic CD103+-type. In addition to the gel forming mucins, the transmembrane mucins MUC3, MUC12 and MUC17 form the enterocyte glycocalyx that can reach about a micrometer out from the brush border. The MUC17 mucin can shuttle from a surface to an intracellular vesicle localization suggesting that enterocytes might control and report epithelial microbial challenge. There is not only communication from the epithelial cells to the immune system, but also in the opposite direction. One example of this is IL10 that can affect and improve the properties of the inner colonic mucus layer. The mucus and epithelial cells of the gastrointestinal tract are the primary gate keepers and controllers of bacterial interactions with the host immune system, but our understanding of this relationship is still in its infancy.
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carbachol induced muc17 endocytosis is concomitant with nhe3 internalization and cftr membrane recruitment in enterocytes
American Journal of Physiology-cell Physiology, 2013Co-Authors: Thaher Pelaseyed, Ida J Gustafsson, Anna Ermund, Jenny K Gustafsson, Gunnar C. HanssonAbstract:We have reported that transmembrane mucin MUC17 binds PDZ protein PDZK1, which retains MUC17 apically in enterocytes. MUC17 and transmembrane mucins MUC3 and MUC12 are suggested to build the entero...
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the c terminus of the transmembrane mucin muc17 binds to the scaffold protein pdzk1 that stably localizes it to the enterocyte apical membrane in the small intestine
Biochemical Journal, 2008Co-Authors: Emily K Malmberg, Thaher Pelaseyed, Asa C Petersson, Ursula Seidler, Hugo R De Jonge, John R Riordan, Gunnar C. HanssonAbstract:The membrane-bound mucins have a heavily O-glycosylated extracellular domain, a single-pass membrane domain and a short cytoplasmic tail. Three of the membrane-bound mucins, MUC3, MUC12 and MUC17, are clustered on chromosome 7 and found in the gastrointestinal tract. These mucins have C-terminal sequences typical of PDZ-domain-binding proteins. To identify PDZ proteins that are able to interact with the mucins, we screened PDZ domain arrays using YFP (yellow fluorescent protein)-tagged proteins. MUC17 exhibited a strong binding to PDZK1 (PDZ domain containing 1), whereas the binding to NHERF1 (Na + /H + -exchanger regulatory factor 1) was weak. Furthermore, we showed weak binding of MUC12 to PDZK1, NHERF1 and NHERF2. GST (glutathione transferase) pull-down experiments confirmed that the C-terminal tail of MUC17 co-precipitates with the scaffold protein PDZK1 as identified by MS. This was mediated through the C-terminal PDZ-interaction site in MUC17, which was capable of binding to three of the four PDZ domains in PDZK1. Immunostaining of wild-type or Pdzk1 −/− mouse jejunum with an antiserum against Muc3(17), the mouse orthologue of human MUC17, revealed strong brush-border membrane staining in the wild-type mice compared with an intracellular Muc3(17) staining in the Pdzk1 −/− mice. This suggests that Pdzk1 plays a specific role in stabilizing Muc3(17) in the apical membrane of small intestinal enterocytes.
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The C-terminus of the transmembrane MUC17 mucin binds to the scaffold protein PDZK1 that stably localizes it to the enterocyte apical membrane in the small intestine
Biochemical Journal, 2008Co-Authors: Emily K Malmberg, Thaher Pelaseyed, John R Riordan, Åsa Petersson, Ursula E. Seidler, Hugo De Jonge, Gunnar C. HanssonAbstract:The membrane bound mucins have a heavily O-glycosylated extracellular domain, a single pass membrane domain and a short cytoplasmic tail. Three of the membrane bound mucins, MUC3, MUC12 and MUC17, are clustered on chromosome 7 and found in the gastrointestinal tract. These mucins have C-terminal sequences typical for PDZ domain binding proteins. To identify PDZ proteins able to interact with the mucins, we screened PDZ domain arrays using YFP-tagged proteins. MUC17 exhibited a strong binding to PDZK1 whereas the binding to NHERF1 was weak. Furthermore, we showed weak binding of MUC12 to PDZK1, NHERF1 and NHERF2. GST pull-down experiments confirmed that the C-terminal tail of MUC17 co-precipitates with the scaffold protein PDZK1 as identified by mass spectrometry. This was mediated through the C-terminal PDZ-interaction site in MUC17 which was capable of binding to three of the four PDZ domains in PDZK1. Immunostaining of wild-type or Pdzk1-/- mouse jejunum with an antiserum against Muc3(17), the mouse orthologue of human MUC17, revealed strong brush border membrane staining in the wild-type mice compared to an intracellular Muc3(17) staining in the Pdzk1-/- mice. This suggests that Pdzk1 plays a specific role in stabilizing Muc3(17) in the apical membrane of small intestinal enterocytes.
Ann Harris - One of the best experts on this subject based on the ideXlab platform.
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N-Glycosylation of the MUC1 mucin in epithelial cells and secretions
Glycobiology, 2006Co-Authors: Simon Parry, Shih Hsing Leir, Franz-georg Hanisch, Mark Sutton-smith, Howard R. Morris, Anne Dell, Ann HarrisAbstract:The MUC1 mucin is an important tumor-associated antigen that shows extensive glycosylation in vivo. The O-glycosylation of this molecule, which has been well characterized in many cell types and tissues, is important in conferring the unusual biochemical and biophysical properties on a mucin. N-Glycosylation is crucial to the folding, sorting, membrane trafficking, and secretion of many proteins. Here, we evaluated the N-glycosylation of MUC1 derived from two sources: endogenous MUC1 isolated from human milk and a recombinant epitope-tagged MUC1F overexpressed in Caco2 colon carcinoma cells. N-Glycans on purified MUC1F/MUC1 were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), gas chromatography-mass spectrometry (GC-MS), and CAD-ESI-MS/MS. The spectra indicate that MUC1F N-glycans have compositions consistent with high-mannose structures (Hex 5-9 HexNAc 2 ) and complex/hybrid-type glycans (NeuAc 0-3 Fuc 0-3 Hex 3-8 HexNAc 3-7 ). Many of the N-glycan structures are identical on MUC1F and native MUC1; however, a marked difference is seen between the N-glycans on membrane-bound and secreted forms of the native molecule.
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The role of the SEA (sea urchin sperm protein, enterokinase and agrin) module in cleavage of membrane-tethered mucins.
The FEBS journal, 2005Co-Authors: Timea Palmai-pallag, Michael A Hollingsworth, Naila Khodabukus, Leo Kinarsky, Shih Hsing Leir, Simon Sherman, Ann HarrisAbstract:The membrane-tethered mucins are cell surface-associated dimeric or multimeric molecules with extracellular, transmembrane and cytoplasmic portions, that arise from cleavage of the primary polypeptide chain. Following the first cleavage, which may be cotranslational, the subunits remain closely associated through undefined noncovalent interactions. These mucins all share a common structural motif, the SEA module that is found in many other membrane-associated proteins that are released from the cell surface and has been implicated in both the cleavage events and association of the subunits. Here we examine the SEA modules of three membrane-tethered mucins, MUC1, MUC3 and MUC12, which have significant sequence homology within the SEA domain. We previously identified the primary cleavage site within the MUC1 SEA domain as FRPG/SVVV a sequence that is highly conserved in MUC3 and MUC12. We now show by site-directed mutagenesis that the F, G and S residues are important for the efficiency of the cleavage reaction but not indispensable and that amino acids outside this motif are probably important. These data are consistent with a new model of the MUC1 SEA domain that is based on the solution structure of the MUC16 SEA module, derived by NMR spectroscopy. Further, we demonstrate that cleavage of human MUC3 and MUC12 occurs within the SEA domain. However, the SEA domains of MUC1, MUC3 and MUC12 are not interchangeable, suggesting that either these modules alone are insufficient to mediate efficient cleavage or that the 3D structure of the hybrid molecules does not adequately re-create an accessible cleavage site.
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Identification of MUC1 proteolytic cleavage sites in vivo.
Biochemical and biophysical research communications, 2001Co-Authors: Simon Parry, Michael A Hollingsworth, Howard S. Silverman, Kimberly M. Mcdermott, Anthony C. Willis, Ann HarrisAbstract:Mucins are high molecular weight glycoproteins that provide a protective layer on epithelial surfaces and are involved in cell-cell interactions, signaling, and metastasis. The identification of several membrane-tethered mucins, including MUC1, MUC3, MUC4, and MUC12, has incited interest in the processing of these mucins and the mechanisms that govern their release from the cell surface. MUC1 consists of an extracellular subunit and a membrane-associated subunit. The two moieties are produced from a single precursor polypeptide by an early proteolytic cleavage event but remain associated throughout intracellular processing and transport to the cell surface. We identified the MUC1 proteolytic cleavage site and showed it to be identical in pancreas and colon cell lines and not to be influenced by the presence of heavily glycosylated tandem repeats. The MUC1 cleavage site shows homology with sequences in other cell-surface-associated proteins and may represent a common mechanism for processing of these molecules.
Emily K Malmberg - One of the best experts on this subject based on the ideXlab platform.
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the c terminus of the transmembrane mucin muc17 binds to the scaffold protein pdzk1 that stably localizes it to the enterocyte apical membrane in the small intestine
Biochemical Journal, 2008Co-Authors: Emily K Malmberg, Thaher Pelaseyed, Asa C Petersson, Ursula Seidler, Hugo R De Jonge, John R Riordan, Gunnar C. HanssonAbstract:The membrane-bound mucins have a heavily O-glycosylated extracellular domain, a single-pass membrane domain and a short cytoplasmic tail. Three of the membrane-bound mucins, MUC3, MUC12 and MUC17, are clustered on chromosome 7 and found in the gastrointestinal tract. These mucins have C-terminal sequences typical of PDZ-domain-binding proteins. To identify PDZ proteins that are able to interact with the mucins, we screened PDZ domain arrays using YFP (yellow fluorescent protein)-tagged proteins. MUC17 exhibited a strong binding to PDZK1 (PDZ domain containing 1), whereas the binding to NHERF1 (Na + /H + -exchanger regulatory factor 1) was weak. Furthermore, we showed weak binding of MUC12 to PDZK1, NHERF1 and NHERF2. GST (glutathione transferase) pull-down experiments confirmed that the C-terminal tail of MUC17 co-precipitates with the scaffold protein PDZK1 as identified by MS. This was mediated through the C-terminal PDZ-interaction site in MUC17, which was capable of binding to three of the four PDZ domains in PDZK1. Immunostaining of wild-type or Pdzk1 −/− mouse jejunum with an antiserum against Muc3(17), the mouse orthologue of human MUC17, revealed strong brush-border membrane staining in the wild-type mice compared with an intracellular Muc3(17) staining in the Pdzk1 −/− mice. This suggests that Pdzk1 plays a specific role in stabilizing Muc3(17) in the apical membrane of small intestinal enterocytes.
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The C-terminus of the transmembrane MUC17 mucin binds to the scaffold protein PDZK1 that stably localizes it to the enterocyte apical membrane in the small intestine
Biochemical Journal, 2008Co-Authors: Emily K Malmberg, Thaher Pelaseyed, John R Riordan, Åsa Petersson, Ursula E. Seidler, Hugo De Jonge, Gunnar C. HanssonAbstract:The membrane bound mucins have a heavily O-glycosylated extracellular domain, a single pass membrane domain and a short cytoplasmic tail. Three of the membrane bound mucins, MUC3, MUC12 and MUC17, are clustered on chromosome 7 and found in the gastrointestinal tract. These mucins have C-terminal sequences typical for PDZ domain binding proteins. To identify PDZ proteins able to interact with the mucins, we screened PDZ domain arrays using YFP-tagged proteins. MUC17 exhibited a strong binding to PDZK1 whereas the binding to NHERF1 was weak. Furthermore, we showed weak binding of MUC12 to PDZK1, NHERF1 and NHERF2. GST pull-down experiments confirmed that the C-terminal tail of MUC17 co-precipitates with the scaffold protein PDZK1 as identified by mass spectrometry. This was mediated through the C-terminal PDZ-interaction site in MUC17 which was capable of binding to three of the four PDZ domains in PDZK1. Immunostaining of wild-type or Pdzk1-/- mouse jejunum with an antiserum against Muc3(17), the mouse orthologue of human MUC17, revealed strong brush border membrane staining in the wild-type mice compared to an intracellular Muc3(17) staining in the Pdzk1-/- mice. This suggests that Pdzk1 plays a specific role in stabilizing Muc3(17) in the apical membrane of small intestinal enterocytes.
Michael A Hollingsworth - One of the best experts on this subject based on the ideXlab platform.
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MUC1 regulates cyclin D1 gene expression through p120 catenin and β-catenin.
Oncogenesis, 2014Co-Authors: Thomas Caffrey, Maria M. Steele, Ashley M. Mohr, Pankaj K. Singh, Prakash Radhakrishnan, David Lee Kelly, Michael A HollingsworthAbstract:MUC1 interacts with β-catenin and p120 catenin to modulate WNT signaling. We investigated the effect of overexpressing MUC1 on the regulation of cyclin D1, a downstream target for the WNT/β-catenin signaling pathway, in two human pancreatic cancer cell lines, Panc-1 and S2-013. We observed a significant enhancement in the activation of cyclin D1 promoter-reporter activity in poorly differentiated Panc1.MUC1F cells that overexpress recombinant MUC1 relative to Panc-1.NEO cells, which express very low levels of endogenous MUC1. In stark contrast, cyclin D1 promoter activity was not affected in moderately differentiated S2-013.MUC1F cells that overexpressed recombinant MUC1 relative to S2-013.NEO cells that expressed low levels of endogenous MUC1. The S2-013 cell line was recently shown to be deficient in p120 catenin. MUC1 is known to interact with P120 catenin. We show here that re-expression of different isoforms of p120 catenin restored cyclin D1 promoter activity. Further, MUC1 affected subcellular localization of p120 catenin in association with one of the main effectors of P120 catenin, the transcriptional repressor Kaiso, supporting the hypothesis that p120 catenin relieved transcriptional repression by Kaiso. Thus, full activation of cyclin D1 promoter activity requires β-catenin activation of TCF-lef and stabilization of specific p120 catenin isoforms to relieve the repression of KAISO. Our data show MUC1 enhances the activities of both β-catenin and p120 catenin.
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Abstract 895: Glycopeptides in pancreatic adenocarcinoma
Cancer Research, 2011Co-Authors: Neeley Remmers, Judy M. Anderson, Pankaj Kumar Singh, Michael A HollingsworthAbstract:There were an estimated 43,140 new cases and 36,800 deaths due to pancreatic adenocarcinoma (PA) for 2010, ranking PA as the third leading cause of cancer related death. Virtually all long term survivors are diagnosed early but these survivors only account for 7% of those diagnosed highlighting the need for an early diagnostic test. CA19-9, the only current clinical serum-based assay used to monitor PA, detects an oligosaccharide blood group antigen sialyl Lewisa (SLeA; Neu5Acα2-3Galβ1-3[Fucα1-4]GlcNAc) that can be present as an O-linked oligosaccharide on glycoproteins and is expressed at relatively low levels on normal epithelial cells. Unfortunately, approximately 15% of the population cannot synthesize SLeA due to a lack of expression of the necessary enzyme to add the final fucose residue thus lowering the sensitivity of the assay. We investigated the expression of other O-linked glycans expessed in PA – sialyl Tn (STn; NeuAcα2-6GalNAc), Tn (GalNAc), T (Galβ1-3GalNAc) and sialyl LewisC (SLeC; Neu5Acα2-3Galβ1-3GlcNAc) – and the aberrant expression of mucin core proteins. In an effort to establish an antigenic signature for PA, we have performed immunohistochemical analysis on primary tumor and liver metastatic tumor samples from 28 autopsy patients diagnosed with PA to analyze their mucin and glycosylation expression. STn and Tn were expressed in the majority of patients in both the primary and liver metastatic tumors and SLeC expression mirrored that of SLeA albeit at lower levels. In those patients lacking SLeA expression SLeC could be detected indicating that the CA19-9 test could be enhanced when combined with these glycans. MUC1, MUC4 and MUC6 were detected in ≫70% of the primary tumor samples analyzed. We also investigated the potential of mucin glycopeptides (STn/Tn on MUC1, STn/Tn on MUC4, and ST/T on MUC1) in providing a unique signature for PA. We found that both STn/Tn on MUC1 and STn/Tn on MUC4 were expressed in >90% of the primary tumor samples analyzed while ST/T on MUC1 was predominantly expressed in normal controls. Differences were also observed in the mucin and glycan signatures between primary and liver metastatic tumors. For example, MUC2 and MUC5B were expressed in the liver metastases while MUC17 was restricted to primary tumor. Finally, we analyzed the expression of MUC1, MUC4, STn/Tn on MUC1, STn/Tn on MUC4, STn and Tn in 4 pancreatitis samples. These antigens were observed in 75-100% of these cases indicating that these particular antigens may by induced by the early inflammatory process leading towards a pro-tumorigenic microenvironment. We conclude that glycopeptide structures comprise an expression pattern unique to PA with potential as biomarkers of early disease and warrant further investigation. Supported by grants U01CA111294 and U01CA128437 from the NCI Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 895. doi:10.1158/1538-7445.AM2011-895
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The role of the SEA (sea urchin sperm protein, enterokinase and agrin) module in cleavage of membrane-tethered mucins.
The FEBS journal, 2005Co-Authors: Timea Palmai-pallag, Michael A Hollingsworth, Naila Khodabukus, Leo Kinarsky, Shih Hsing Leir, Simon Sherman, Ann HarrisAbstract:The membrane-tethered mucins are cell surface-associated dimeric or multimeric molecules with extracellular, transmembrane and cytoplasmic portions, that arise from cleavage of the primary polypeptide chain. Following the first cleavage, which may be cotranslational, the subunits remain closely associated through undefined noncovalent interactions. These mucins all share a common structural motif, the SEA module that is found in many other membrane-associated proteins that are released from the cell surface and has been implicated in both the cleavage events and association of the subunits. Here we examine the SEA modules of three membrane-tethered mucins, MUC1, MUC3 and MUC12, which have significant sequence homology within the SEA domain. We previously identified the primary cleavage site within the MUC1 SEA domain as FRPG/SVVV a sequence that is highly conserved in MUC3 and MUC12. We now show by site-directed mutagenesis that the F, G and S residues are important for the efficiency of the cleavage reaction but not indispensable and that amino acids outside this motif are probably important. These data are consistent with a new model of the MUC1 SEA domain that is based on the solution structure of the MUC16 SEA module, derived by NMR spectroscopy. Further, we demonstrate that cleavage of human MUC3 and MUC12 occurs within the SEA domain. However, the SEA domains of MUC1, MUC3 and MUC12 are not interchangeable, suggesting that either these modules alone are insufficient to mediate efficient cleavage or that the 3D structure of the hybrid molecules does not adequately re-create an accessible cleavage site.
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Identification of MUC1 proteolytic cleavage sites in vivo.
Biochemical and biophysical research communications, 2001Co-Authors: Simon Parry, Michael A Hollingsworth, Howard S. Silverman, Kimberly M. Mcdermott, Anthony C. Willis, Ann HarrisAbstract:Mucins are high molecular weight glycoproteins that provide a protective layer on epithelial surfaces and are involved in cell-cell interactions, signaling, and metastasis. The identification of several membrane-tethered mucins, including MUC1, MUC3, MUC4, and MUC12, has incited interest in the processing of these mucins and the mechanisms that govern their release from the cell surface. MUC1 consists of an extracellular subunit and a membrane-associated subunit. The two moieties are produced from a single precursor polypeptide by an early proteolytic cleavage event but remain associated throughout intracellular processing and transport to the cell surface. We identified the MUC1 proteolytic cleavage site and showed it to be identical in pancreas and colon cell lines and not to be influenced by the presence of heavily glycosylated tandem repeats. The MUC1 cleavage site shows homology with sequences in other cell-surface-associated proteins and may represent a common mechanism for processing of these molecules.