Molecular cloning of cDNAs derived from a novel human intestinal mucin gene

A human small intestinal lambda gt11 cDNA library was screened with antibodies to deglycosylated small intestinal mucin. Four partial cDNA clones were isolated that define a novel human mucin gene. These include two partial cDNA clones, SIB 124 and SIB 139, that contain 51 nucleotide tandem repeats which encode a seventeen amino acid repetitive peptide with a consensus sequence of HSTPSFTSSITTTETTS. SIB 139 hybridized to messages produced by small intestine, colon, colonic tumors and also by high mucin variant LS174T colon cancer cells. The gene from which cDNAs SIB 124 and SIB 139 are derived (proposed name MUC 3) maps to chromosome 7, distinct from other known human mucin genes.     

cDNA for the carboxyl-terminal region of a rat intestinal mucin-like peptide.

When subjected to thiol reduction, purified intestinal mucins have been shown to undergo a decrease in molecular mass and to liberate a 118-kDa glycopeptide (Roberton, A. M., Mantle, M., Fahim, R. E. F., Specian, R., Bennick, A., Kawagishi, S., Sherman, P., and Forstner, J. F. (1989) Biochem. J. 261, 637-647). The latter has been called a putative "link" component because it is assumed to be important for disulfide bond-mediated mucin polymerization. Controversy exists as to whether the putative link is an integral mucin component or a separate mucin-associated glycopeptide. In the present study both NH2-terminal and internal amino acid sequences of the 118-kDa glycopeptide of rat intestinal mucin were used to generate opposing oligonucleotide primers for polymerase chain reaction. A specific 1.2-kilobase (kb) product was obtained, from which a 0.5-kb HindIII fragment was used as a probe to screen a lambda ZAP II cDNA library of rat intestine. A 2.6-kb cDNA (designated MLP 2677) was sequenced and revealed an open reading frame of 2.5 kb encoding 837 amino acids. The deduced amino acid sequence showed that the putative link peptide is equivalent to the carboxyl-terminal 689 amino acids of a larger peptide. Northern blots revealed a mRNA size of approximately 9 kb. Computer searches revealed no sequence homology with other proteins, but similarities were seen in the alignment of cysteine residues in the link and in several domains of human von Willebrand factor, as well as cysteine-rich areas of bovine and porcine submaxillary mucins and a frog skin mucin designated FIM-B.1. In keeping with earlier demonstrations of the presence of mannose in the 118-kDa glycopeptide, there were several (13) consensus sequences for attachment of N-linked oligosaccharides within the link domain. Further sequencing of MLP 2677 in a direction 5' to the codon specifying the NH2-terminal proline of the link has revealed a coding region for 148 amino acids, including a unique 75-amino acid domain rich in cysteine and proline, and a region containing 4.5-variable tandem repeats (each 11-12 amino acids) rich in serine, threonine, and proline. The presence of mucin-like tandem repeats suggests that the entire cysteine-rich link peptide represents the carboxyl-terminal region (75.5 kDa) of a mucin-like peptide (MLP). The latter is estimated to have a molecular mass of approximately 300 kDa.     

家蚕MLP基因的克隆及其结构分析

利用生物信息学的方法快速获得家蚕MLP (Muscle LIM protein, MLP)基因cDNA电子序列, 经RT-PCR生物验证正确, 登录GenBank (No. DQ311195)。MLP基因cDNA长2 327 bp, ORF全长1 485 bp, 编码产生494个氨基酸。该MLP基因组DNA含有11个外显子, 10个内含子, 所有内含子/外显子边界都符合典型的GT/AG剪切模式。MLP基因编码的蛋白富含Gly (14.4%), 分子量约为53.03 kDa, 等电点(PI)为8.29。通过BLAST分析发现该基因编码的家蚕肌肉LIM蛋白, 含有5个保守的LIM结构域, 家蚕的另一种LIM蛋白(AAR23823)含一个LIM结构域, 两者可能是通过可变剪切产生; 后者可能通过竞争作用调节前者在肌细胞中的功能。MLP的克隆为进一步研究其体内功能奠定了基础。     

MLP参与心肌肥大发生过程与Calcineurin/NFAT信号通路有关

心肌肥大是心肌细胞面对多种病理刺激时的共同反应,以心肌细胞体积增大和胚胎期基因的重新表达为标志.心肌发育调控基因肌肉LIM蛋白(muscle LIM protein,MLP)的表达异常与心肌肥大有关.为研究MLP参与心肌肥大发生的分子机制,采用去氧肾上腺素（phenylephrine, PE）刺激大鼠原代培养心肌细胞,建立心肌细胞肥大模型,采用RNAi技术敲减MLP的表达,分析MLP与肥大信号通路钙调神经磷酸酶（calcineurin）/活化T细胞核因子（nuclear factor of activated T-cells, NFAT）的关系.结果显示, 原代培养的心肌细胞经一定浓度的PE刺激后细胞表面积增加,肥大标志蛋白ANP、BNP表达增高,并伴有MLP表达上调. RNAi方法敲减MLP的表达则明显抑制PE诱导的心肌细胞表面积增加和BNP表达增高,并且直接 影响NFAT的转录激活活性,提示MLP与心肌肥大的发生密切相关,并且可能是通过calcineurin/NFAT信号通路而参与心肌肥大的发生.     

Molecular cloning of rat intestinal mucin. Lack of conservation between mammalian species

We have prepared antisera to deglycosylated rat intestinal mucin and used it to obtain immunoreactive clones from a rat jejunum cDNA library. Four of these clones were sequenced, and all were found to be partial cDNAs that contained 18-base pair tandem repeats characteristic of a mucin. These cDNAs encoded a repetitive peptide with a consensus sequence of TTTPDV. Thus, they bear little resemblance to either of the two human intestinal mucin cDNAs isolated previously (Gum, J. R., Byrd, J. C., Hicks, J. W., Toribara, N. W., Lamport, D. T. A., and Kim, Y. S. (1989) J. Biol. Chem. 264, 6480-6487 and Gum, J. R., Hicks, J. W., Swallow, D. M., Lagace, R. E., Byrd, J. C., Lamport, D. T. A., Siddiki, B., and Kim, Y. S. (1990) Biochem. Biophys. Res. Commun. 171, 407-415). One of these rat mucin clones, designated RMUC 176, was chosen for further analysis. This clone recognized a band of approximately 9 kilobases when used to probe RNA blots. A strong hybridization band was present using rat small intestine and colon RNA but was not detectable when RNA isolated from heart, liver, or kidney was tested. The RMUC 176 clone and the two previously isolated human intestinal mucin cDNA clones were used to probe blots prepared from BamHI-digested DNA of various species. Here, the human probes detected fragments present only in human and chimpanzee DNA, whereas the RMUC 176 clone recognized fragments only in rat and mouse DNA. Thus, the repetitive portions of intestinal mucin genes are apparently not well conserved between phylogenetically distant species.     

cDNA cloning and chromosomal assignment of the endothelin 2 gene: vasoactive intestinal contractor peptide is rat endothelin 2.

Four members of the endothelin family of vasoactive and mitogenic peptides have been identified: human endothelins 1, 2, and 3 (ET1, ET2, and ET3, respectively) and mouse vasoactive intestinal contractor (VIC). To characterize the mRNA encoding ET2, a 192-bp fragment of the ET2 gene, amplified by the polymerase chain reaction from human genomic DNA, was used to screen cell lines and tissues for ET2 gene expression. ET2 mRNA was detected in a cell line (HTB119) derived from a human lung small cell carcinoma, and an ET2 cDNA was cloned from a cDNA library prepared from HTB119 mRNA. DNA prepared from human-mouse somatic hybrid cell lines was used to assign the gene encoding ET2 (EDN2) to the 1p21----1pter region of chromosome 1, demonstrating that EDN2 is not linked to genes encoding ET1 (EDN1; chromosome 6) and ET3 (EDN3; chromosome 20). Southern blot hybridization revealed a single gene in human and rat genomes that hybridized with the ET2 gene fragment, and the rat gene was cloned. The endothelin peptide encoded by the rat gene differed from ET2 at 1 of 21 residues and was identical to mouse VIC. We conclude that VIC is the mouse and rat analogue of the human ET2 gene.     

肌肉LIM蛋白增强生肌素对AChRγ启动子的反式激活作用

采用RT PCR技术克隆了大鼠肌肉LIM蛋白 (MLP) 6 40bp的全长cDNA序列 .以此cDNA为探针进行的Northern印迹表明 ,MLP于C2C12细胞在分化的第 3d至第 5d表达 .将MLPcDNA亚克隆至pcDNA3,构建真核表达质粒pcDNA3 MLP ,同时构建AChRγ启动子序列 (96 0bp)调控的荧光素酶报告基因真核表达质粒pGL3 γ .C2C12细胞转染及荧光素酶活性分析表明 ,复合转染pcD NA3 MLP和pGL3 γ的分化肌细胞表达的荧光素酶活性约为对照的 4倍 ;而在 3T3或未分化肌细胞复合转染pcDNA3 MLP和pGL3 γ均未检出报告基因表达 ,说明MLP可促进生肌素对AChRγ亚基基因启动子的反式激活作用 .     

The DHE cell line as a model for studying rat gastro-intestinal mucin expression: effects of dexamethasone

The expression of mucin genes was evaluated in rat intestinal cell lines in order to establish an in vitro model for investigating the regulation of intestinal mucin expression in this species. Two rat intestinal cancer cell lines (DHE, LGA) and three nontumoral rat intestinal cell lines (IEC6, IEC17, IEC18) were screened. The mRNA expression of rMuc1, rMuc2, rMuc3, rMuc4, and rMuc5AC mucin genes was studied by semiquantitative RT-PCR, real-time RT-PCR and Northern-blot analysis. Results were correlated with immunohistochemical expression of rat gastric and intestinal mucin proteins, and secretion of glycoconjugates was examined by enzyme-linked lectin assay. We showed that mRNA of rMucl and rMuc2 were constitutively expressed in all IEC cell populations but periodic acid Schiff staining of these cells did not reveal the presence of glycoproteins. DHE cells expressed rMuc1-5AC mRNA and LGA expressed the same mucins but the level of rMuc4 was much lower. Mucin mRNA expression also differed in relation with the length of cultivation. Immunocytochemical studies revealed the presence of gastric and intestinal mucins in the two tumoral cell lines. Functional experiments showed that bethanechol, A23187 and PMA stimulated release of glycoconjugates in DHE but not in LGA cells. Treatment of DHE cells with dexamethasone (10(-7) mol/l) enhanced rMuc2 mRNA but decreased rMuc1 and rMuc5AC mRNA. Real-time RT-PCR showed that the expression of rMuc1 and rMuc5AC genes was reduced by more than tenfold after 24 h. The increased expression of rMuc2 gene was confirmed by Northern blot analysis. In conclusion, DHE cells provide a valuable cellular model for research on rat mucin secretion and expression.     

Characterization and localization of the putative ''link'' component in rat small-intestinal mucin.

Rat intestinal mucin is polymerized by a putative 'link' component of Mr 118,000 that can be released from the native mucin by thiol reduction [Fahim, Forstner & Forstner (1983) Biochem. J. 209, 117-124]. To confirm that this component is an integral part of the mucin and independent of the mucin purification technique, rat mucin was purified in the present study by three independent techniques. In all cases, the 118,000-Mr component was released after reduction. The 118 kDa band was electroeluted from SDS/polyacrylamide gels and its composition shown to resemble closely that of the link component of human intestinal mucin [Mantle, Forstner & Forstner (1984) Biochem. J. 224, 345-354]. Carbohydrates were present, including significant (10 mol/100 mol) amounts of mannose, suggesting the presence of N-linked oligosaccharides. Monospecific antibodies prepared against the rat 118,000-Mr component established its tissue localization in intestinal goblet cells. Mucins subjected to SDS/polyacrylamide-gel electrophoresis and Western blots using the same antibody, established that the link components of rat and human intestinal mucin are similar antigenically. Brief exposure (10 min) of native rat mucin to trypsin or Pronase (enzyme/mucin protein, 1:500, w/w) also released a 118,000-Mr component that reacted with the monospecific antibody. Thus the 118,000-Mr component is an integral part of the mucin and, although linked to large glycopeptides by disulphide bonds, this component also has proteinase-sensitive peptide bonds, presumably at terminal locations such that brief treatment with proteinases releases the molecule in a reasonably intact form. Under physiological conditions, therefore, one might expect that, after mucin is secreted into the intestinal lumen, luminal proteinases would rapidly remove the link component, thereby causing the mucin to depolymerize.     

CDX-2, MUC-2 and B-catenin as intestinal markers in pure mucinous carcinoma of the breast

Background

Pure mucinous adenocarcinoma of the breast is a rare entity characterized by the production of variable amounts of mucin comprising 1% to 6% of breast carcinomas. Some mucinous adenocarcinomas have shown expression of intestinal differentiation markers such as MUC-2. This study examines the expression of intestinal differentiation markers in this type of breast carcinoma.

Results

Twenty-two cases of pure mucinous adenocarcinoma of the breast were assessed. Immunochemistry was performed for beta-catenin, CDX-2 and MUC-2. All cases were positive for B-catenin. MUC-2 positivity was observed in all cases; 63. 6% were 3 plus positive. All cases were negative for CDX-2.

Conclusions

These results suggest that mucinous breast carcinomas express some markers of intestinal differentiation, such as MUC-2 and beta-catenin; however, future studies with a larger series of cases and using molecular techniques that help affirm these results are needed.     

Binding of Shigella to rat and human intestinal mucin

Invasion of epithelial cells by Shigella is an early step in their pathogenesis. Adherence is generally presumed to be a prerequisite for invasion. This study examined the possibility of intestinal mucins serving as initial binding sites for clinical isolates of S. boydii and S. sonnei. The interactions of Shigella with rat and human small intestinal and colonic mucin were investigated. In solid phase binding assays, [³⁵S] labelled Shigella did not show any preferential binding to rat/human small intestinal mucin or to rat colonic mucin. On the other hand, Shigella bound specifically to human colonic mucin in a concentration-dependent manner. This specific binding to human colonic mucin was not by weak hydrophobic interactions and could not be attributed to the presence of contaminating glycolipids in the mucin preparation. The human colonic mucin receptor was sensitive to periodate treatment suggesting the involvement of the carbohydrate portion of the mucin. Reduction and alkylation of mucin enhanced adherence probably by exposing buried binding sites. The monosaccharides present in mucins were ineffective as hapten inhibitors as was the lectin wheat germ agglutinin suggesting that the mucin receptor is a more complex one. This study identifies, for the first time, the presence of a specific Shigella-binding site on the carbohydrate portion of human colonic mucin, which is not present in rat colonic mucin or in rat/human small intestinal mucin.     

The Mucin Degrader Akkermansia muciniphila Is an Abundant Resident of the Human Intestinal Tract

A 16S rRNA-targeted probe, MUC-1437, was designed and validated in order to determine the presence and numbers of cells of Akkermansia muciniphila, a mucin degrader, in the human intestinal tract. As determined by fluorescent in situ hybridization, A. muciniphila accounted more than 1% of the total fecal cells and was shown to be a common bacterial component of the human intestinal tract.     

MUC-6 mucin is a major component of ‘blood group substance’ from human ovarian cyst fluid

Ovarian cyst fluid has been a valuable source of the mucins (traditionally termed ‘blood group substances’) that were used for the elucidation of the structures of the ABO Lewis blood group determinants, but the identity of the mucin peptide core(s) carrying these carbohydrate specificities is not known. An ovarian cyst fluid mucin was purified, deglycosylated with HF and digested with trypsin or chymotrypsin to yield a number of peptides. Amino acid sequencing of these peptides yielded five different sequences which showed complete or partial homology to the MUC-6 apomucin deduced from DNA sequencing. As no other sequences were identified, it is concluded that MUC-6 is the major mucin core structure of ovarian cyst fluid mucin.     

MUC-6 mucin is a major component of "blood group substance" from human ovarian cyst fluid

Ovarian cyst fluid has been a valuable source of the mucins (traditionally termed "blood group substances") that were used for the elucidation of the structures of the ABO Lewis blood group determinants, but the identity of the mucin peptide core(s) carrying these carbohydrate specificities is not known. An ovarian cyst fluid mucin was purified, deglycosylated with HF and digested with trypsin or chymotrypsin to yield a number of peptides. Amino acid sequencing of these peptides yielded five different sequences which showed complete or partial homology to the MUC-6 apomucin deduced from DNA sequencing. As no other sequences were identified, it is concluded that MUC-6 is the major mucin core structure of ovarian cyst fluid mucin.     

Human scFv antibody fragments specific for the epithelial tumour marker MUC-1, selected by phage display on living cells

New anti-cancer agents are being developed that specifically recognise tumour cells. Recognition is dependent upon the enhanced expression of antigenic determinants on the surface of tumour cells. The tumour exposure and the extracellular accessibility of the mucin MUC-1 make this marker a suitable target for tumour diagnosis and therapy. We isolated and characterised six human scFv antibody fragments that bound to the MUC-1 core protein, by selecting a large naive human phage display library directly on a MUC-1-expressing breast carcinoma cell line. Their binding characteristics have been studied by ELISA, FACS and indirect immunofluorescence. The human scFv antibody fragments were specific for the tandem repeat region of MUC-1 and their binding is inhibited by soluble antigen. Four human scFv antibody fragments (M2, M3, M8, M12) recognised the hydrophilic PDTRP region of the MUC-1 core protein, which is thought to be an immunodominant region. The human scFv antibody fragments were stable in human serum at 37 °C and retained their binding specificity. For imaging or targeting to tumours over-expressing MUC-1, it might be feasible to use these human scFv, or multivalent derivatives, as vehicles to deliver anti-cancer agents. Received: 2 November 2000 / Accepted: 11 January 2001