The upstream region of the FOX3 gene encoding peroxisomal 3-oxoacyl-coenzyme A thiolase in Saccharomyces cerevisiae contains ABF1- and replication protein A-binding sites that participate in its regulation by glucose repression.

Expression of the FOX3 gene, which encodes yeast peroxisomal 3-oxoacyl-coenzyme A thiolase, can be induced by oleate and repressed by glucose. Previously, we have shown that induction was mediated by an oleate response element. Just upstream of this element a negatively acting control region that mediated glucose repression was found. In order to study this negative control region, we carried out DNA-binding assays and analyzed phenotypes of mutations in this region and in the trans-acting factor CAR80, which is identical to UME6. DNA-binding assays showed that two multifunctional yeast proteins, ABF1 and RP-A, interacted with the negative control element independently of the transcriptional activity of the FOX3 gene. ABF1 and RP-A, the latter being identical to BUF, were able to bind to DNA independently of one another but also simultaneously. The phenotypes of mutations in either DNA-binding sites of ABF1, RP-A, or both, which affected the DNA binding of these factors in vitro, indicated that these sites and the proteins that interact with them participate in glucose repression. The involvement of the RP-A site in glucose repression was further supported by our observation that the CAR80 gene product, which is required for repression mediated by the RP-A site, was essential for maintenance of glucose repression. In addition to the RP-A site in the FOX3 promoter, similar sequences were observed in other genes involved in peroxisomal function. RP-A proved to bind to all of these sequences, albeit with various affinities. From these results it is concluded that the ABF1 and RP-A sites are being required in concert to mediate glucose repression of the FOX3 gene. In addition, coordinated regulation of expression of genes involved in peroxisomal function in response to glucose is mediated by proteins associated with the RP-A site, probably RP-A and CAR80.

Intestinal carbamoyl phosphate synthase I in human and rat. Expression during development shows species differences and mosaic expression in duodenum of both species.

The clinical importance of carbamoyl phosphate synthase I (CPSI) relates to its capacity to metabolize ammonia, because CPSI deficiencies cause lethal serum ammonia levels. Although some metabolic parameters concerning liver and intestinal CPSI have been reported, the extent to which enterocytes contribute to ammonia conversion remains unclear without a detailed description of its developmental and spatial expression patterns. Therefore, we determined the patterns of enterocytic CPSI mRNA and protein expression in human and rat intestine during embryonic and postnatal development, using in situ hybridization and immunohistochemistry. CPSI protein appeared during human embryogenesis in liver at 31-35 e. d. (embryonic days) before intestine (59 e.d.), whereas in rat CPSI detection in intestine (at 16 e.d.) preceded liver (20 e.d.). During all stages of development there was a good correlation between the expression of CPSI protein and mRNA in the intestinal epithelium. Strikingly, duodenal enterocytes in both species exhibited mosaic CPSI protein expression despite uniform CPSI mRNA expression in the epithelium and the presence of functional mitochondria in all epithelial cells. Unlike rat, CPSI in human embryos was expressed in liver before intestine. Although CPSI was primarily regulated at the transcriptional level, CPSI protein appeared mosaic in the duodenum of both species, possibly due to post-transcriptional regulation.

MUC4 is increased in high grade intraepithelial neoplasia in Barrett's oesophagus and is associated with a proapoptotic Bax to Bcl-2 ratio.

BACKGROUND: Patients with Barrett's oesophagus (BO) are at risk of oesophageal adenocarcinoma. Because the pattern of mucosal mucins changes during neoplastic progression, it may serve as a marker of intraepithelial neoplasia. AIMS: To determine the expression pattern of mucins in neoplastic BO epithelium (high grade dysplasia) and correlate it with the expression of apoptosis markers Bax and Bcl-2. METHODS: Thirty seven patients with BO were studied: 16 without intraepithelial neoplasia, six with high grade intraepithelial neoplasia (HGN), and 15 with infiltrating adenocarcinoma. Biopsies were obtained from squamous epithelium, Barrett's epithelium, and (when present) foci of suspected HGN or adenocarcinoma. MUC1-4, MUC5AC, MUC5B, MUC6, Bax, and Bcl-2 mRNA were determined by semiquantitative RT-PCR. MUC2, MUC5AC, and MUC6 protein was determined by immunoblotting. RESULTS: Mucin expression varied between neoplastic progression stages in BO. Mucin mRNA levels were low in squamous epithelium, except for MUC4, and were at least four times higher in BO and HGN (p<0.001), but less so in adenocarcinoma. MUC4 expression was significantly lower in BO than in normal squamous epithelium, whereas in HGN and adenocarcinoma, levels were significantly higher than in BO (p = 0.037). The Bax:Bcl-2 ratio was increased in HGN compared with BO (p = 0.04). MUC2, MUC5AC, and MUC6 protein values correlated with mRNA data. CONCLUSIONS: Mucin expression varies during the development of oesophageal adenocarcinoma in BO. MUC4 could serve as a tumour marker in this process. In contrast to animal studies, upregulation of MUC4 in HGN is associated with increased apoptosis, suggesting that MUC4 plays a minor role in apoptosis regulation in BO.

Strategic biochemical analysis of mucins.

MUC-type mucins comprise a family of structurally related molecules, which are expressed in epithelia of the body that are in close contact with the milieu. Because of their large sizes and very complex structures, containing very extensive O-glycosylation, MUC-type mucins are difficult to study by conventional techniques. Many see MUC-type mucins as protective molecules; however, functional studies on the individual MUC-type mucins are very scarce. At present, essential steps in MUC research are to characterize the specific expression patterns of each MUC-type mucin in the body and to find methods to reliably quantify these MUC-type mucins. These aims can only be met at the level of the primary sequences of the MUC-type mucins, as the O-glycosylation even within one species of MUC-type mucin is not only very complex, but may also vary among individuals, organs, and cell types. We will discuss some recent advances in mucin research, particularly the identification of MUC precursor molecules in metabolic labeling experiments. We will try to define some strategic considerations in the study of the expression patterns of MUC-type mucins, which circumvent the complications caused by the very complex and heterogeneous O-glycosylation of the molecules.

Characterization of a transcriptional control element involved in proliferation of peroxisomes in yeast in response to oleate.

Oleate induces the transcription of genes involved in peroxisome biogenesis and stimulates the proliferation of these organelles in Saccharomyces cerevisiae. Previously, we have reported the identification of a region containing a positive regulatory element in the 5' flanking region of the FOX3 gene encoding the peroxisomal enzyme 3-oxoacyl-CoA thiolase. This region contains a 23-bp imperfect inverted-repeat sequence. Full induction, in response to oleate, is mediated by the intact dyad. However, one half-site of the inverted repeat is also able to mediate induction of transcription in response to oleate, albeit to a small extent. Furthermore, the weak binding of protein to each part of the inverted repeat proved to be correlated with the weak activation of transcription, in support of oleate. A DNase-I footprint covered the entire dyad and DNA band-shift experiments indicated that one or more trans-acting factors bind to the imperfect palindrome. The binding of protein to this element seems to be correlated with transcriptional activation, since mutations in both halves of the inverted dyad affected both transcriptional activation and protein binding in vitro. Similar oleate-responsive elements are commonly found in the 5' flanking regions of genes encoding proteins involved in peroxisome biogenesis and the factor(s) binding to oleate-responsive element(s) could therefore be involved in coordination of the expression of oleate-inducible genes and the proliferation of peroxisomes.

Mucin gene structure and expression: protection vs. adhesion.

The cloning of mucin cDNAs brought about by the application of molecular biology and molecular analyses constitutes a major step in understanding mucin structure and function. Here two classes of mucins are described: epithelium-associated and endothelium/leukocyte-associated mucins, which have thus far been described separately in the literature. The epithelial mucins are generally believed to play a role in cytoprotection. The endothelial and leukocyte class of mucins are adhesion molecules involved in lymphocyte homing and lymphocyte activation or are part of the adhesion cascade that plays a role in the initiation of inflammation. Mucins in general contain many threonine and serine residues, which are extensively O-glycosylated. Due to this profound glycosylation, mucins have a filamentous conformation. By virtue of their extended filamentous, and often negatively charged, structure, mucins can act as a barrier protecting the cell. However, when an opposing cell has specific receptors for mucins, adhesion can override the barrier function. Therefore, mucins may be powerful two-edged swords: they are both protective and adhesive.

The oligomerization of a family of four genetically clustered human gastrointestinal mucins.

Mucins are synthesized and secreted by many epithelia. They are complex glycoproteins that offer cytoprotection. In their functional configuration, mucins form oligomers by a biosynthetic process that is poorly understood. A family of four human gastrointestinal mucin genes (MUC2, MUC5AC, MUC5B, and MUC6) is clustered to chromosome 11p15.5. To study oligomerization of these related mucins, we performed metabolic labeling experiments with [35S]amino acids in LS174T cells, and isolated mucin precursors by specific immunoprecipitations that were analyzed on SDS-PAGE. Each of the precursors of MUC2, MUC5AC, MUC5B, and MUC6 formed a single species of disulfide-linked homo-oligomer within 1 h after pulse labeling. Based on apparent molecular masses, these oligomeric precursors were most likely dimers. Inhibition of vesicular RER-to-Golgi transport, with brefeldin A and CCCP, did not affect the dimerization of MUC2 precursors, localizing dimerization to the RER. O-Glycosylation of MUC2 followed dimerization. Inhibition of N-glycosylation by tunicamycin retarded, but did not inhibit, dimerization, indicating that N-glycans play a role in efficient dimerization of MUC2 precursors. Based on sequence homology, the ability of MUC2, MUC5AC, MUC5B and MUC6 to dimerize most likely resides in their C-terminal domains. Thus, the RER-localized dimerization of secretory mucins likely proceeds by similar mechanisms, which is an essential step in the formation of the human gastrointestinal mucus-gels.

MUC2 is the prominent colonic mucin expressed in ulcerative colitis.

BACKGROUND: It has been shown that MUC2 is the prominent mucin synthesised in healthy colon. AIM: To identify the predominant mucins in ulcerative colitis (UC) and to study their biosynthesis. METHODS AND RESULTS: Mucin was purified from UC resection specimens. This mucin on sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) presented as one, high molecular weight, periodic acid/Schiff's reagent (PAS) stainable band. Amino acid composition showed a close resemblance to that of MUC2. Immunoprecipitation with a specific anti-MUC2 antiserum confirmed that this mucin was MUC2. In addition, on the mRNA level MUC2 was also the most prominent mucin expressed in UC. Polyclonal antiserum was elicited, mainly recognising mucin peptide epitopes of UC and normal colonic mucin. Biosynthetic studies with [35S]amino acids showed that the MUC2-precursor in UC displayed a molecular mass on SDS-PAGE of approximately 600 kDa. This precursor was converted into a mature MUC2 with anomalous mobility on SDS-PAGE of 550 kDa and was secreted. Only this 550 kDa band could be labelled with [35S]sulphate and stained by PAS. CONCLUSIONS: This study shows that in parallel with the mucin expression in healthy controls, MUC2 is the major mucin expressed in UC. Qualitatively, MUC2 biosynthesis seems unchanged in UC.

Regulation of transcription of the gene coding for peroxisomal 3-oxoacyl-CoA thiolase of Saccharomyces cerevisiae.

Transferring Saccharomyces cerevisiae cells from glucose- to oleate-containing growth media results in a significant increase in the number and volume of peroxisomes. To investigate this proliferation process we studied the transcriptional regulation of the gene coding for peroxisomal 3-oxoacyl-CoA thiolase (EC 2.3.1.16) in response to the switch in carbon source. Expression was proved to be repressed during growth on glucose, derepressed during growth on glycerol, and induced during growth on oleate as the sole carbon source. By deletion and mutational analysis of sequences upstream of this gene, we have identified a region which is involved in the regulation of transcription. It is contained within a 52-base-pair sequence, UAST52 (upstream activation sequence thiolase 52), located between 203 and 151 nucleotides upstream of the translational initiation codon. This sequence proved to be required for repression, derepression and induction of transcription, and was able to activate transcription from the truncated version of the heterologous iso-1-cytochrome-c (CYC1) promoter in a similar way as in the wild-type promoter context. Sequence comparison revealed that the UAST52 contained a sequence motif ('beta-oxidation box') that is very similar to sequences located in the 5'-upstream regions of the genes coding for two other beta-oxidation enzymes of S. cerevisiae: the peroxisomal acyl-CoA oxidase and the peroxisomal trifunctional beta-oxidation enzyme of S. cerevisiae. Mutational analysis of the 'beta-oxidation box' indicates that this sequence motif acts as a UAS in vivo. Sequence comparison also revealed that just upstream of the 'beta-oxidation box', between positions -213 and -201, a potential binding site occurred for the yeast multifunctional autonomously replicating sequence binding factor ABF1. Gel-retardation-competition experiments indicate that ABF1 binds specifically to this sequence.

Biosynthesis of rat MUC2 in colon and its analogy with human MUC2.

In order to identify the mucins synthesized and secreted in the rat colon, we studied their biochemical characteristics and biosynthesis and evaluated their analogy to human colonic mucins. Purified mucin from both species appeared similar with respect to composition, buoyant density and mobility on SDS/PAGE. Isolated rat colonic mucin (RCM) was used to elicit a polyclonal antiserum, which was used in metabolic labelling studies to identify mucins and mucin precursors. RCM is synthesized as a 600 kDa precursor protein, which oligomerizes before O-glycosylation. The mature, high-molecular mass mucin is secreted and displays an anomalous molecular mass on SDS/PAGE of approximately 650 kDa. Polymorphism in precursor size was found among different rats, suggesting genetic heterogeneity. Molecular mass, biosynthesis and secretion of RCM appeared similar to human MUC2. Moreover, RCM precursor could be immunoprecipitated using specific anti-(human MUC2) antisera, indicating that the RCM can be designated rat MUC2. This study describes the biosynthesis of two homologous mucins in two different species. The high degree of similarity suggests functional analogy.

Biosynthesis of human colonic mucin: Muc2 is the prominent secretory mucin.

BACKGROUND/AIMS: Human colonic epithelium produces large amounts of mucin. The aim of this study was to examine mucin biosynthesis in the human colon. METHODS: Human colonic mucin was isolated using CsCl density gradients, and polyclonal antiserum was raised. Biosynthesis of colonic mucins was studied by labeling colonic explants with 35S-labeled amino acids or [35S]sulfate and subsequent immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The polyclonal antiserum specifically recognized colonic mucin, primarily reacting with peptide epitopes. Biosynthetic pulse/chase experiments showed a 35S-amino acid-labeled mucin precursor of about 600 kilodaltons, which was converted into a mature, glycosylated, and sulfated mucin and subsequently secreted into the medium. This mature mucin comigrated with isolated colonic mucin with an apparent molecular weight of 550 kilodaltons on SDS-PAGE, whereas gel filtration indicated that the molecular weight is actually much larger. Independent immunoprecipitation with an anti-Muc2 antiserum showed cross-reactivity with the 600-kilodalton precursor. CONCLUSIONS: These results show the biosynthesis of a secretory colonic mucin for the first time. This mucin is synthesized as a precursor protein of approximately 600 kilodaltons, which, after glycosylation, is secreted as a glycoprotein with an apparent molecular weight of 550 kilodaltons on SDS-PAGE. It is very likely that this mucin is Muc2.

Biosynthesis of mucins (MUC2-6) along the longitudinal axis of the human gastrointestinal tract.

Little is known about the biosynthesis of mucin molecules in humans. Our aim was to examine the mucin biosynthesis (MUC2-6) along the longitudinal axis of the healthy human gastrointestinal tract. Biopsies of human stomach and small and large intestine were metabolically labeled with 35S-labeled amino acids, [35S]sulfate, or[3H]galactose, immunoprecipitated with antibodies against MUC2-6, and analyzed by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), MUC5AC [apparent molecular weight (M(r)) 500,000] and MUC6 (apparent M(r) 400,000) were detected in the stomach but not in the small or large intestine, MUC3 (apparent M(r) 550,000) was detected in duodenum and jejunum, MUC2 (apparent M(r)600,000) was detected throughout the small and large intestine, and MUC4 (apparent M(r) > 900,000) was detected predominantly in the large intestine. Interestingly, some individuals displayed double bands of MUC2 and MUC3 precursors, suggesting allelic variation within the respective genes. Between small and large intestine mature secreted MUC2 showed differences in mobility on SDS-PAGE, suggesting differences in glycosylation. Each of the MUC2, MUC3, MUC4, MUC5AC, and MUC6 precursors could be distinguished electrophoretically, and each showed region-specific expression along the gastrointestinal tract.

Sulphation and secretion of the predominant secretory human colonic mucin MUC2 in ulcerative colitis.

BACKGROUND: Decreased synthesis of the predominant secretory human colonic mucin (MUC2) occurs during active ulcerative colitis. AIMS: To study possible alterations in mucin sulphation and mucin secretion, which could be the cause of decreased mucosal protection in ulcerative colitis. METHODS: Colonic biopsy specimens from patients with active ulcerative colitis, ulcerative colitis in remission, and controls were metabolically labelled with [35S]-amino acids or [35S]-sulphate, chase incubated and analysed by SDS-PAGE, followed by quantitation of mature [35S]-labelled MUC2. For quantitation of total MUC2, which includes non-radiolabelled and radiolabelled MUC2, dot blotting was performed, using a MUC2 monoclonal antibody. RESULTS: Between patient groups, no significant differences were found in [35S]-sulphate content of secreted MUC2 or in the secreted percentage of either [35S]-amino acid labelled MUC2 or total MUC2. During active ulcerative colitis, secretion of [35S]-sulphate labelled MUC2 was significantly increased twofold, whereas [35S]-sulphate incorporation into MUC2 was significantly reduced to half. CONCLUSIONS: During active ulcerative colitis, less MUC2 is secreted, because MUC2 synthesis is decreased while the secreted percentage of MUC2 is unaltered. Furthermore, sulphate content of secreted MUC2 is unaltered by a specific compensatory mechanism, because sulphated MUC2 is preferentially secreted while sulphate incorporation into MUC2 is reduced.

Intestinal brush border glycohydrolases: structure, function, and development.

The hydrolytic enzymes of the intestinal brush border membrane are essential for the degradation of nutrients to absorbable units. Particularly, the brush border glycohydrolases are responsible for the degradation of di- and oligosaccharides into monosaccharides, and are thus crucial for the energy-intake of humans and other mammals. This review will critically discuss all that is known in the literature about intestinal brush border glycohydrolases. First, we will assess the importance of these enzymes in degradation of dietary carbohydrates. Then, we will closely examine the relevant features of the intestinal epithelium which harbors these glycohydrolases. Each of the glycohydrolytic brush border enzymes will be reviewed with respect to structure, biosynthesis, substrate specificity, hydrolytic mechanism, gene regulation and developmental expression. Finally, intestinal disorders will be discussed that affect the expression of the brush border glycohydrolases. The clinical consequences of these enzyme deficiency disorders will be discussed. Concomitantly, these disorders may provide us with important details regarding the functions and gene expression of these enzymes under specific (pathogenic) circumstances.