Colm J. Reid

Oligosaccharides Expressed on MUC1 Produced by Pancreatic and Colon Tumor Cell Lines

MUC1 is expressed at the apical surface of ductal epithelia of tissues, including breast, pancreas, airway, and the gastrointestinal tract, where its functions include lubrication and protection of the epithelia. In addition, roles for MUC1 have been suggested in both adhesive and antiadhesive properties of tumor cells, and extensive O-glycosylation of the MUC1 tandem repeat domain may contribute to these functions. Little information is available on the specific O-glycosylation of MUC1. One problem in identifying different MUC1 glycoforms has been that monoclonal antibodies raised against the MUC1 core protein recognize epitopes in the tandem repeat domain, which is often glycosylated to an extent that obscures these epitopes. We developed an epitope-tagged form of MUC1 that allowed the detection of multiple MUC1 glycoforms and established the presence of a number of important blood group and tumor-associated carbohydrate antigens on MUC1 expressed by two pancreatic tumor cell lines (Panc-1 and S2-013) and two colon tumor cell lines (Caco-2 and HT-29). Antigens detected include sialyl-Lewisa, sialyl-Lewisc, sialyl-Lewisx, and sialyl-Tn.

Developmental expression of mucin genes in the human gastrointestinal system

Background and aims—Mucin glycoproteins play a key role in the normal function of the epithelium lining the gastrointestinal tract. The expression of mucin genes, MUC 3, 4, 5AC, 5B, 6, 7, and 8 in human fetal tissues was examined to establish the localisation and age of onset of expression of each mucin gene during human development. Methods—Mucin gene expression was assayed by mRNA in situ hybridisation. Results—Expression of MUC3 was detected in the small intestine and colon from 13 weeks gestation onwards and at low levels in the main pancreatic duct at 13 weeks only. MUC4 expression was seen at a low level in the colonic epithelium from 13 weeks of gestation but not elsewhere in the gastrointestinal tract. MUC5AC mRNA was detected in the colon at 17 weeks and at high levels in the stomach at 23 weeks. MUC6transcripts were evident in the pancreatic ducts from 13 weeks of gestation and at high levels in the stomach at 23 weeks.MUC5B, MUC7, and MUC8transcripts were not detected. Conclusions—Mucin genes are expressed from the early mid-trimester of gestation in the developing human fetal gastrointestinal tract.

myo-Inositol catabolism and catabolite regulation in Rhizobium leguminosarum bv. viciae

On the basis of enzyme assays, myo-inositol appears to be catabolized via 2-keto-myo-inositol and D-2,3-diketo-4-deoxy epi-inositol in Rhizobium leguminosarum bv. viciae, as occurs in Klebsiella aerogenes. The first two enzymes of the pathway, myo-inositol dehydrogenase and 2-keto-myo-inositol dehydratase were increased 7- and 77-fold, respectively, after growth of R. leguminosarum on myo-inositol compared to glucose. Cells of R. leguminosarum grown on glucose as the carbon source and then resuspended in myo-inositol, increased myo-inositol-dependent O2 consumption by sixfold in 3 h, confirming this to be an inducible pathway. Succinate, malate and glucose exhibited strong catabolite repression of the myo-inositol catabolic pathway with myo-inositol dehydrogenase and 2-keto-myo-inositol dehydratase being repressed by at least 68% and 80%, respectively, in all cases. A dicarboxylate transport mutant (dctA) was unable to repress either enzyme when grown on myo-inositol and succinate. There was no repression of the first two enzymes of the myo-inositol catabolic pathway in the background of constitutive expression of the dicarboxylate transport system, indicating a dicarboxylate must be present to cause repression. The data imply that dicarboxylates are required intra-cellularly to repress these enzymes. Three transposon mutants were isolated that cannot grow on myo-inositol as the sole carbon source and were unable to induce either myo-inositol dehydrogenase or 2-keto-myo-inositol dehydratase. The mutants were unaffected in the ability to nodulate peas and vetch. Furthermore, vetch plants infected with one mutant (RU360) reduced acetylene at the same rate as plants infected with the wild type. Bacteroids did not oxidize myo-inositol, nor were the catabolic enzymes detected, confirming myo-inositol is not important as an energy source in bacteroids. The possible role of myo-inositol in the rhizosphere is considered.

Identification of chromosomal genes located downstream of dctD that affect the requirement for calcium and the lipopolysaccharide layer of Rhizobium leguminosarum.

In Rhizobium leguminosarum both the C4-dicarboxylate transport system and wild-type lipopolysaccharide layer (LPS) are essential for nitrogen fixation. A Tn5 mutant (RU301) of R. leguminosarum bv. viciae 3841, was isolated that is only able to synthesize LPS II, which lacks the O-antigen. Strain RU301 exhibits a rough colony morphology, flocculates in culture and is unable to swarm in TY agar. It also fails to grow on organic acids, sugars or TY unless the concentration of calcium or magnesium is elevated above that normally required for growth. The defects in the LPS and growth in strain RU301 were complemented by a series of cosmids from a strain 3841 cosmid library (pRU3020-pRU3022) and a cosmid from R. leguminosarum bv. phaseoli 8002 (pIJ1848). The transposon insertion in strain RU301 was shown to be located in a 3 kb EcoRI fragment by Southern blotting and cloning from the chromosome. Sub-cloning of pIJ1848 demonstrated that the gene disrupted by the transposon in strain RU301 is located on a 2.4 kb EcoRI-PstI fragment (pRU74). R. leguminosarum bv. viciae VF39-C86, which is one of four LPS mutants previously isolated by U. B. Priefer (1989, J Bacteriol 171, 6161-6168), was also complemented by sub-clones of pIJ1848 but not by pRU74, suggesting the mutation is in a gene adjacent to that disrupted in strain RU301. Complementation and Southern analysis indicate that the region contained in pIJ1848 does not correspond to any other cloned Ips genes. Two dctA mutants, RU436 and RU437, were also complemented by pIJ1848 and pRU3020. Mapping of pIJ1848 and Southern blotting of plasmid-deleted strains of R. leguminosarum revealed that dctD and the region mutated in strain RU301 are located approximately 10 kb apart on the chromosome. Analysis of homogenotes demonstrated that there is not a large region important in calcium utilization, organic acid metabolism or LPS biosynthesis located between the gene disrupted in strain RU301 and dctD. Strain VF39C-86 also required an elevated concentration of calcium for growth on succinate, while strains mutated in the alpha-chromosomal or beta-plasmid group of Ips genes grew at the same calcium concentrations as the wild type, demonstrating that the additional calcium requirement is not a property of all LPS rough mutants. Strain RU301 nodulates peas, but does not reduce acetylene, demonstrating that the gene mutated in this strain is essential for nitrogen fixation.

Divergent Mechanisms of Interaction of Helicobacter pylori and Campylobacter jejuni with Mucus and Mucins

ABSTRACT Helicobacter pylori and Campylobacter jejuni colonize the stomach and intestinal mucus, respectively. Using a combination of mucus-secreting cells, purified mucins, and a novel mucin microarray platform, we examined the interactions of these two organisms with mucus and mucins. H. pylori and C. jejuni bound to distinctly different mucins. C. jejuni displayed a striking tropism for chicken gastrointestinal mucins compared to mucins from other animals and preferentially bound mucins from specific avian intestinal sites (in order of descending preference: the large intestine, proximal small intestine, and cecum). H. pylori bound to a number of animal mucins, including porcine stomach mucin, but with less avidity than that of C. jejuni for chicken mucin. The strengths of interaction of various wild-type strains of H. pylori with different animal mucins were comparable, even though they did not all express the same adhesins. The production of mucus by HT29-MTX-E12 cells promoted higher levels of infection by C. jejuni and H. pylori than those for the non-mucus-producing parental cell lines. Both C. jejuni and H. pylori bound to HT29-MTX-E12 mucus, and while both organisms bound to glycosylated epitopes in the glycolipid fraction of the mucus, only C. jejuni bound to purified mucin. This study highlights the role of mucus in promoting bacterial infection and emphasizes the potential for even closely related bacteria to interact with mucus in different ways to establish successful infections.

Aspartate transport by the Dct system in Rhizobium leguminosarum negatively affects nitrogen-regulated operons.

Amino acid uptake by the general amino acid permease (Aap) of Rhizobium leguminosarum strain 3841 was severely reduced by the presence of aspartate in the growth medium when glucose was the carbon source. The reduction in transport by the Aap appeared to be caused by inhibition of uptake and not by transcriptional repression. However, as measured with lacZ fusions, the Ntr-regulated gene glnII was repressed by aspartate. The negative regulatory effect on both the Aap and glnII was prevented by mutation of any component of the dicarboxylate transport (Dct) system or by the inclusion of a C4- dicarboxylate in the growth medium, including the non-metabolizable analogue 2-methylsuccinate. As measured by total uptake and with a dctA-lacZ fusion, aspartate was an efficient inducer of the Dct system, but slightly less so than succinate alone or succinate and aspartate together. Thus, aspartate does not cause overexpression of DctA leading to improper regulation of other operons. Transport measurements revealed that the Dct system has an apparent Km for succinate of 5 microM and an apparent Ki for aspartate inhibition of succinate uptake of 5 mM. These data imply that the Dct-mediated accumulation of aspartate causes an unregulated build-up of aspartate or a metabolic product of it in the cell. This accumulation of aspartate is prevented either by mutation of the dct system or by the presence of a higher affinity substrate that will reduce access of aspartate to the carrier protein. Elevation or disruption of the intracellular aspartate pool is predicted to disrupt N-regulated operons and nitrogen fixation.

Glycoproteins and glycosidases of the cervix during the periestrous period in cattle.

The cervix and its secretions undergo biochemical and physical changes under the differential influences of estrogen and progesterone. These include changes in the glycoprotein profile of the endocervix and its secretions. A comprehensive survey of such changes in cervical epithelium and cervical secretions was performed on bovine samples throughout the periestrous period. Cervical tissue samples and swabs were collected from synchronized beef heifers that were slaughtered 1) 12 h after controlled intravaginal progesterone-releasing device (CIDR) removal, 2) 24 h after CIDR removal, 3) at the onset of estrus, 4) 12 h after the onset of estrus, 5) 48 h after the onset of estrus, and 6) 7 d after the onset of estrus. Histological staining with hematoxylin and eosin, periodic acid Schiff, Alcian blue, and high-iron diamine was carried out to map overall patterns of stored glycoproteins and tissue structure. Biotinylated lectins were also used to detect the presence and distribution of a range of saccharide structures. The activities of β-galactosidase, α-L-fucosidase, β-N-acetyl-hexosaminidase, and sialidase were measured in cervical swabs using specific substrates. The epithelial layer of the cervix exhibited dynamic changes in cellular hypertrophy and amounts of stored glycoprotein. The greatest content of neutral and acidic mucins was observed 48 h after onset of estrus (P < 0.05). Sialylated mucins predominated at the bases of cervical folds, whereas sulfated mucins were more abundant (P < 0.05) at their apices. The stained area of core mucin glycans changed (P < 0.05) in association with follicular versus luteal phases, whereas terminal glycans changed (P < 0.05) mainly at the time of estrus and shortly thereafter. The greatest activity of β-galactosidase and sialidase was observed 12 h after onset of estrus, whereas β-hexosaminidase and α-fucosidase peaked at the luteal time point (P < 0.05). Taken together, we suggest that the well-known changes in the endocervix and its secretions that are associated with the physiological modulation of sperm transport and function of the cervical barrier are, in part, driven by glycosylation changes.

Molecular aspects of mucin biosynthesis and mucus formation in the bovine cervix during the periestrous period

Mucus within the cervical canal represents a hormonally regulated barrier that reconciles the need to exclude the vaginal microflora from the uterus during progesterone dominance, while permitting sperm transport at estrus. Its characteristics change during the estrous cycle to facilitate these competing functional requirements. Hydrated mucin glycoproteins synthesized by the endocervical epithelium form the molecular scaffold of this mucus. This study uses the bovine cervix as a model to examine functional groups of genes related to mucin biosynthesis and mucus production over the periestrous period when functional changes in cervical barrier function are most prominent. Cervical tissue samples were collected from 30 estrus synchronized beef heifers. Animals were slaughtered in groups starting 12 h after the withdrawal of intravaginal progesterone releasing devices (controlled internal drug releases) until 7 days postonset of estrus (luteal phase). Subsequent groupings represented proestrus, early estrus, late estrus, metestrus, and finally the early luteal phase. Tissues were submitted to next generation RNA-seq transcriptome analysis. We identified 114 genes associated with biosynthesis and intracellular transport of mucins, and postsecretory modifications of cervical; 53 of these genes showed at least a twofold change in one or more experimental group in relation to onset of estrus, and the differences between groups were significant (P < 0.05). The majority of these genes showed the greatest alteration in their expression in the 48 h postestrus and luteal phase groups.

Microbial interaction with mucus and mucins

Publisher Summary Mucus represents the front-line defensive barrier between the external environment and the tissues of the host. The molecular scaffolding of mucus comprises hydrated mucins, which are very high-molecular mass, glycosylated glycoproteins presenting arrays of O-linked glycans. Mucins are integral to microbial interactions with epithelial surfaces and have potential roles in microbial trophism, the presentation of ligands to block microbial binding or stabilize colonization and the provision of feedstocks for microbial metabolism. The protective physicochemical properties of mucus are attributable to their high carbohydrate content. Microbes are capable of expressing specific lectins and mucin-degrading enzymes to engage with, utilize, and penetrate mucus gels. Normally, the dynamic nature of such gels represents an optimal adaptation through host–microbial crosstalk, enabling the maintenance of barrier function through the coregulation of mucus turnover and microbial ecology. Mucin expression and glycosylation are biologically responsive to changes in the sub- and supra-mucosal environment and are significantly influenced by inflammation and microbial colonization. Impairment of mucus gel turnover may lead to abnormal colonization, inflammatory pathology, and biofilm formation.

Expression of the MUC 6 Mucin Gene in Development of the Human Kidney and Male Genital Ducts

The MUC 6 mucin cDNA was isolated from a human stomach cDNA library and has been shown to be expressed in a number of other tissues in the gastrointestinal tract, including the gallbladder, pancreas, and parts of the ileum and colon. Here we establish that MUC 6 is expressed transiently in the nephrogenic zone of the kidney in the early mid-trimester of development. MUC 6 transcripts were detected in the epithelium of ureteric buds at 13 weeks and at lower levels from 17 to 23 weeks of gestation. Traces of MUC 6 mRNA were seen in the collecting ducts but not elsewhere in the developing kidney, and MUC 6 glycoprotein was detected in the epithelium of ureteric buds and collecting ducts. MUC 6 transcripts were absent from adult kidney. This pattern of expression of MUC 6 in the developing kidney suggests a role in epithelial organogenesis. MUC 6 transcripts were also present at low levels in mid-trimester epididymal epithelium.