Heterogeneity in the protein cores of mucins isolated from human middle ear effusions: evidence for expression of different mucin gene products

High molecular weight mucins were isolated and purified from human middle ear effusions of children with Otitis Media with Effusion (OME) classified into three groups, (1) thick and (2) thin from anatomically normal children and (3) effusions from cleft palate patients. Amino acid analyses of the purified mucins from the three pools were similar but not identical with characteristic contents of serine threonine and proline (32%, 28%, and 38% for pools (1) (2) and (3) respectively). Proteinase resistant glycopeptide fragments corresponding to the tandem repeat domains of cloned mucin genes showed marked differences both between the three mucin pools and with the composition of the tandem repeat sequences of the cloned mucin genes expressed in the airways. Studies on the antigenic identity of middle ear mucins found an epitope likely to be present on MUC5AC, but only accounting for a maximum of 15% by weight and no reactivity was found with antibodies to MUC2 or MUC1. A polyclonal antibody raised to thick effusion mucins reacted strongly with human salivary mucin suggesting the presence of MUC5B epitopes. These studies suggest that more than one mucin gene product is secreted by the human middle ear mucosa and that there may be further mucin genes expressed by the middle ear that have yet to be cloned.     

Membrane-bound mucin modular domains: From structure to function

Mucins belong to a heterogeneous family of large O-glycoproteins composed of a long peptidic chain called apomucin on which are linked hundreds of oligosaccharidic chains. Among mucins, membrane-bound mucins are modular proteins and have a structural organization usually containing Pro/Thr/Ser-rich O-glycosylated domains (PTS), EGF-like and SEA domains. Via these modular domains, the membrane-bound mucins participate in cell signalling and cell interaction with their environment in normal and pathological conditions. Moreover, the recent knowledge of these domains and their biological activities led to the development of new therapeutic approaches involving mucins. In this review, we show 3D structures of EGF and SEA domains. We also describe the functional features of the evolutionary conserved domains of membrane-bound mucins and discuss consequences of splice events.     

The antimicrobial peptide cathelicidin interacts with airway mucus

Antimicrobial peptides (AMPs) and mucins are components of airway secretions and both contribute to the innate host defense system. At neutral pH, AMPs are positively charged, mucins negatively. It was the aim of the study to test whether these opposite charges result in interactions between AMPs and mucins. We measured binding of mucins isolated from porcine gastric mucosa to the cathelicidin LL-37 coated to multiwell plates and found that LL-37 electrostatically interacts with mucins. Circular dichroism spectra of the peptide revealed the induction of -helical conformation by mucins. Addition of mucins to solutions of LL-37 significantly decreased the antimicrobial activity of the peptide against Pseudomonas aeruginosa and Streptococcus pneumoniae. We then tested whether LL-37 is bound to mucins in airway secretions from human subjects and found that a significant proportion of the peptide and its propeptide are bound to high molecular weight components. Together these data show that cationic AMPs interact with anionic mucins in airway secretions. Functions of AMPs are modulated by this interaction.     

The membrane-bound mucins: From cell signalling to transcriptional regulation and expression in epithelial cancers

The membrane-bound mucins belong to an ever-increasing family of O-glycoproteins. Based on their structure and localization at the cell surface they are thought to play important biological roles in cell–cell and cell–matrix interactions, in cell signalling and in modulating biological properties of cancer cells. Among them, MUC1 and MUC4 mucins are best characterized. Their altered expression in cancer (overexpression in the respiratory, gastro-intestinal, urogenital and hepato-biliary tracts) indicates an important role for these membrane-bound mucins in tumour progression, metastasis, cancer cell resistance to chemotherapeutics drugs and as specific markers of epithelial cancer cells. Some mechanisms responsible for MUC1 and MUC4 role in tumour cell properties have been deciphered recently. However, much remains to be done in order to understand the molecular mechanisms and signalling pathways that control the expression of membrane-bound mucins during the different steps of tumour progression toward adenocarcinoma and evaluate their potential as prognostic/diagnostic markers and as therapeutic tools. In this review we focus on the molecular mechanisms and signalling pathways known to control the expression of membrane-bound mucins in cancer. We will discuss the mechanisms of regulation at the promoter level (including genetic and epigenetic modifications) that may be responsible for the mucin altered pattern of expression in epithelial cancers.     

MUC genes: a superfamily of genes? Towards a functional classification of human apomucins

The MUC genes encode epithelial mucins. Eight different human genes have been well characterized, and two others identified more recently. Among them, a family of four genes, expressed in the respiratory and digestive tracts, is clustered to chromosome 11p15.5; and these genes encode gel-forming mucins which are structurally related to the superfamily of cystine-knot growth factors. A second group is composed of three independent genes encoding various isoforms of mucins including membrane-bound mucins associated to carcinomas. In this second group, MUC3 and MUC4 encode large apomucins containing EGF-like domains.     

The contribution of tandem repeat number to the O-glycosylation of mucins

The serine- and threonine-rich tandem repeat (TR) units that make up the characteristic feature of mucin glycoproteins are often polymorphic with substantial genetic variation in TR number. The precise effect of TR number on O-glycosylation is not fully understood, although the TR number of several mucins may be associated with apparent susceptibility to certain human diseases. To evaluate the contribution of TR number to O-glycosylation, we generated a series of chimeric mucins carrying increasing numbers of TR units from the MUC5B mucin in the context of an epitope-tagged MUC1 mucin backbone. These mucins were expressed in Caco2 colon carcinoma cell clones and purified by immunoprecipitation. O-Glycosylation was investigated by western blotting with antibodies to known carbohydrate structures and by fast atom bombardment-mass spectrometry. Additional carbohydrate epitopes were detected with antibodies on chimeric mucins with a higher TR number in comparison to those with fewer TRs. Using mass spectrometry, higher-molecular-weight glycans were detected more frequently on the mucins with extended TRs compared to those with fewer TRs. However no novel carbohydrate structures were seen, suggesting that TR number does not affect the specificity of O-glycosylation.     

LPS up-regulates mucin and cytokine mRNA expression and stimulates mucin and cytokine secretion in goblet cells

Bacterial inflammation in mucosa is accompanied by morphological and proliferative changes in goblet cells and mucin hypersecretion. Main stimulators of bacterial inflammation are bacterial lipopolysaccharides (LPS). In vitro investigation of the LPS effect on the molecular processes in goblet cells, using the human mucin-secreting goblet cell line HT29-MTX, showed the following results. LPS up-regulated mucin and cytokine mRNA expression and secretion in goblet cells in a concentration and time-dependent manner, with a maximum output at an LPS concentration of 100 ng/ml. LPS (100 ng/ml) increased mRNA expression of MUC5AC (2.4x), MUC5B (2.1x), and IL-8 (2.3x) and stimulated secretion of mucins (MUC5AC up to 39%, MUC5B up to 31%) and the inflammatory cytokine IL-8 (up to 10x). A significant correlation was found between the LPS-induced IL-8 secretion and secretion of mucins. These results suggest: (1) goblet cells, responding to the direct stimulation of bacterial LPS by two inflammatory-related processes such as production and secretion of the gel-forming mucins and the inflammatory cytokine IL-8, can be considered as an important part of mucosal immunity and (2) LPS- induced goblet cell mucin secretion can occur partly via IL-8-dependent pathway.     

Nucleotide sequence and organization of full length human U4 RNA pseudogenes

Two loci encoding human U4 RNA, designated U4/7 and U4/14, have been isolated and sequenced. Both are pseudogenes in that their sequences do not match any identified human U4 RNA species perfectly. The U4/7 locus harbours a full-length pseudogene of 144 bp with eight base substitutions in the structural region. This pseudogene might be derived from a hitherto unidentified human U4 RNA gene. The second locus, U4/14, has a complex structure; the structural sequence of a U4 gene has apparently been integrated into an Alu sequence.     

Mouse fatty acid transport protein 4 (FATP4): characterization of the gene and functional assessment as a very long chain acyl-CoA synthetase

FATP4 (SLC27A4) is a member of the fatty acid transport protein (FATP) family, a group of evolutionarily conserved proteins that are involved in cellular uptake and metabolism of long and very long chain fatty acids. We cloned and characterized the murine FATP4 gene and its cDNA. From database analysis we identified the human FATP4 genomic sequence. The FATP4 gene was assigned to mouse chromosome 2 band B, syntenic to the region 9q34 encompassing the human gene. The open reading frame was determined to be 1929 bp in length, encoding a polypeptide of 643 amino acids. Within the coding region, the exon-intron structures of the murine FATP4 gene and its human counterpart are identical, revealing a high similarity to the FATP1 gene. The overall amino acid identity between the deduced murine and human FATP4 polypeptides is 92.2%, and between the murine FATP1 and FATP4 polypeptides is 60.3%. Northern analysis showed that FATP4 mRNA was expressed most abundantly in small intestine, brain, kidney, liver, skin and heart. Transfection of FATP4 cDNA into COS1 cells resulted in a 2-fold increase in palmitoyl-CoA synthetase (C16:0) and a 5-fold increase in lignoceroyl-CoA synthetase (C24:0) activity from membrane extracts, indicating that the FATP4 gene encodes an acyl-CoA synthetase with substrate specificity biased towards very long chain fatty acids.     

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

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 toNHERF1 (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 coprecipitates with the scaffold protein PDZK1 as identified byMS. 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 brushborder 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.     

On the mechanism of biosynthesis of leukotrienes and related compounds

[10D-³H; 3-¹⁴C]- and [10L-³H; 3-¹⁴C]arachidonic acids were incubated with human polymorphonuclear leukocytes and with human platelets. Leukotriene B₄ and 5(S),12(S)-dihydroxy-6trans,8cis,10trans,14-cis-eicosatetraenoic acid (5,12-DHETE) were isolated and the ³H/¹⁴C ratios determined. It could be concluded that the 10D (pro-R)-hydrogen is eliminated in the conversion of 5(S)-hydroperoxy-6trans,8cis,11cis,14cis-eicosatetraenoic acid into leukotriene A₄ whereas in the conversion of arachidonic acid into 5,12-DHETE the 10L (pro-S)-hydrogen is lost. Incubation of the doubly labeled arachidonic acids with human platelets confirmed and extended previous data on the stereochemistry of the hydrogen removal from C-10 during the conversion into 12(S)-hydroperoxy-5cis,8cis,10trans,14cis-eicosatetraenoic acid, i.e., the 10L (pro-S)-hydrogen is eliminated and the 10D (pro-R)-hydrogen retained.     

Bambi is coexpressed with Bmp-4 during mouse embryogenesis

Signaling of TGF-β superfamily members is tightly controlled by an elaborate network of regulators (for recent review see Trends Genet. 15 (1999) 3; Genes Dev. 14 (2000) 627). Recently, the transmembrane protein BAMBI (BMP and activin membrane-bound inhibitor) has been shown to interfere with Bmp and activin-like signaling by inhibiting Tgf-β type I receptor activation (Nature 401 (1999) 480). In striking contrast to other Bmp antagonists like noggin (Cell 86 (1996) 599) or chordin (Cell 86 (1996) 589), BAMBI is strictly coexpressed with Bmp-4 during early Xenopus embryogenesis. The grouping of genes according to their shared complex spatial expression pattern and their involvement in the same biological signaling pathway has been referred to as synexpression group. This concept facilitates prognoses about the roles of a group member with unknown function. Apparently, only a minority of genes is organized in synexpression groups and up to now they have mainly been described in yeast and Xenopus (for review see Nature 402 (1999) 483). In the frog, BAMBI is a member of the Bmp-4 synexpression group (Nature 401 (1999) 480). We identified two murine homologues of BAMBI one of which, named Bambi-ψ, is a pseudogene. We show that the spatiotemporal expression pattern of Bambi closely matches that of Bmp-4 during mouse embryonic development. Moreover, we show that Bambi expression is induced in mouse embryonic fibroblasts by Bmp-4. Hence, we provide first evidence for the existence of an evolutionarily conserved Bmp-4 synexpression group in mammals.     

Comparative analysis of structure, expression and PSD95-binding capacity of Lrfn, a novel family of neuronal transmembrane proteins

Leucine-rich repeat and fibronectin III domain-containing (Lrfn) has five members in mouse and human (Lrfn1, Lrfn2, Lrfn3, Lrfn4, Lrfn5), and homologues in other vertebrates. Lrfn proteins share leucine-rich repeat (LRR)–immunoglobulin-like (Ig)–fibronectin type III (Fn)–transmembrane domain structure, which is also found in LRR–Ig–Fn superfamily proteins. Mouse Lrfn genes were expressed at adult stage predominantly in the brain. In the course of development, expression of Lrfn1, Lrfn3, and Lrfn4 started from immature neural cells, whereas that of Lrfn2 and Lrfn5 was limited to mature ones. Lrfn1–5 commonly encode glycoproteins spanning the plasma membrane, with their N-terminus located on the extracellular side. C-termini of Lrfn1, Lrfn2 and Lrfn4 were bound by PDZ domains of postsynaptic protein PSD95, re-distributing PSD95 to cell periphery where the Lrfn proteins were detected. These results suggest that Lrfn proteins are neuronal components with a role in the developing or mature vertebrate nervous system.     

Characterization and tissue distribution of multiple agouti-family genes in pufferfish, Takifugu rubripes

Four types of agouti-family genes (AGRP1, AGRP2, ASIP1 and ASIP2) were obtained from torafugu, Takifugu rubripes. Their characterization and structure were analyzed to elucidate the relationship among the torafugu agouti-family genes. Both AGRP1 and AGRP2 showed genomic synteny with the human AGRP gene. Phylogenetic tree analysis showed that AGRP1 formed a cluster with human AGRP. We inferred that torafugu AGRP1 and AGRP2 are orthologs of human AGRP and that they are paralogous genes derived from genome duplication occurred in the teleost phylogeny. Torafugu ASIP1 showed genomic synteny with the human ASIP, but ASIP2 did not. The ASIP1 expression level was about five times higher in the white ventral skin than in the black dorsal skin. Therefore, we concluded that torafugu ASIP1 is an ortholog of human ASIP, nevertheless, we are unable to determine if torafugu ASIP2 is a paralog of ASIP1 or not.     

Enzymatic large-scale synthesis of MUC6-Tn glycoconjugates for antitumor vaccination

In cancer, mucins are aberrantly O-glycosylated, and consequently, they express tumor-associated antigens such as the Tn determinant (alpha-GalNAc-O-Ser/Thr). As compared with normal tissues, they also exhibit a different pattern of expression. In particular, MUC6, which is normally expressed only in gastric tissues, has been detected in intestinal, pulmonary, colorectal, and breast carcinomas. Recently, we have shown that the MCF7 breast cancer cell line expresses MUC6-Tn glycoproteins in vivo. Cancer-associated mucins show antigenic differences from normal mucins, and as such, they may be used as potential targets for immunotherapy. To develop anticancer vaccines based on the Tn antigen, we prepared several MUC6-Tn glycoconjugates. To this end, we performed the GalNAc enzymatic transfer to two recombinant MUC6 proteins expressed in Escherichia coli, using UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), which catalyze in vivo the Tn antigen synthesis. We used either a mixture of ppGalNAc-Ts from MCF7 breast cancer cell extracts or a recombinant ppGalNAc-T1. In both cases, we achieved the synthesis of MUC6-Tn glycoconjugates at a semi-preparative scale (mg amounts). These glycoproteins displayed a high level of Tn antigens, although the overall density depends on both enzyme source and protein acceptor. These MUC6-Tn glycoconjugates were recognized by two anti-Tn monoclonal antibodies that are specific to human cancer cells. Moreover, the MUC6-Tn glycoconjugate glycosylated using MCF7 extracts as the ppGalNAc-T source was able to induce immunoglobulin G (IgG) antibodies that recognized a human tumor cell line. In conclusion, the large-scaled production of MUC6 with tumor-relevant glycoforms holds considerable promise for developing effective anticancer vaccines, and further studies of their immunological properties are warranted.     

Wide intra-genomic G+C heterogeneity in human and chicken is mainly due to strand-symmetric directional mutation pressures: dGTP-oxidation and symmetric cytosine-deamination hypotheses

The intra-strand Parity Rule 2 of DNA (PR2) states that A=T and G=C within each strands. Useful corollaries of PR2 are G/(G+C)=A/(A+T)=0.5, G/(G+A)=C/(C+T)=G+C, G/(G+T)=C/(C+A)=G+C. Here. A, T, G, and C represent relative contents of the four nucleotide residues in a specific strand of DNA, so that A+T+G+C=1. Thus, deviations from the PR2 is a sign of strand-specific (or asymmetric) mutation and/or selection pressures. The present study delineates the symmetric and asymmetric effects of mutations on the intra-genomic heterogeneity of the G+C content in the human genome. The results of this study on the human genome are: (1) When both two- and four-codon amino acids were combined, only slight departures from the PR2 were observed in the total ranges of G+C content of the third-codon position. Thus, the G+C heterogeneity is likely to be caused by symmetric mutagenesis between the two strands. (2) The above result makes the deamination of cytosine due to double-strand breathing of DNA [Mol. Biol. Evol. 17 (2000) 1371] and/or incorporation of the oxidized guanine (8-oxo-guanine) opposite adenine during DNA replication (dGTP-oxidation hypothesis) as the most likely candidates for the major cause of the diversities of the G+C content. (3) Patterns of amino acid-specific PR2-biases detected by plotting PR2 corollaries against the G+C content of third codon position revealed that eight four-codon amino acids can be divided into three types by the second codon letter: (a) C₂-type (Ala, Pro, Ser4, and Thr), (b) G₂-type (Arg4 and Gly), and (c) T₂-type (Leu4 and Val). (4) Most of the asymmetric plot patterns of the above three classes in PR2 biases can be explained by C₂→T₂ deamination of C₂pG₃ of C₂-type to T₂pG₃ (T₂-type) in both human and chicken. This explains the existence of some preferred codons in human and chicken. However, these biases (asymmetric) hardly contribute to the overall G+C content diversity of the third codon position.     

MUC5AC,but not MUC2, is a prominent mucin in respiratory secretions

Airway mucus was collected from healthy and chronic bronchitic subjects. The chronic bronchitic sputum was separated into gel and sol phase by centrifugation and mucins were isolated using isopycnic density-gradient centrfugation in CsCl. The presence of the MUC5AC and MUC2 mucins was investigated with antisera raised against synthetic peptides with sequences from the respective apoproteins. The gel and sol phase of chronic bronchitic sputum as well as healthy respiratory secretions were shown to contain MUC5AC whereas the MUC2 mucin could not be detected. Rate-zonal centrifugation showed that the MUC5AC mucin was large, polydisperse in size and that reduction yielded subunits. Ion-exchange HPLC revealed the presence of two subunit populations in all secretions, the MUC5AC subunits always being the more acidic. MUC5AC is thus the first large, subunit-based, gel-forming respiratory mucin identified and this glycoprotein is biochemically distinct from at least one other population of large, gel-forming mucins also composed of subunits but lacking a genetic identity.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate - CF cystic fibrosis - DFP diisopropylphosphofluoridate - DTT dithiothreitol - EDTA ethylenedinitrilotetraacetic acid - NEM N-ethylmaleimide - PAS periodic acid/Schiffs - PMSF phenylmethylsulphonyl fluoride - Tris Tris(hydroxymethyl)aminomethane - VNTR variable number of tandem repeats