Mucin genes: structure, expression and regulation.
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Amino Acid Sequence↗
Biomedical subjects
E A Davidson
Publications and source records attributed to E A Davidson.
Non-sequence-specific antimalarial activity of oligodeoxynucleotides.
The effects of exogenously applied oligodeoxynucleotides on Plasmodium falciparum proliferation was investigated. A fluorescence-activated cell sorter assay was employed to measure parasitemia after administration of either phosphodiester or phosphorothioate oligodeoxynucleotides. We report sequence-independent antimalarial activity preferentially with phosphorothioate congeners with IC50 values in the 1-2 microM range. Phosphorothioate oligodeoxynucleotides which were antisense, sense or nonsense to Plasmodium mRNA, as well as homopolymers (30-mers containing all A or T bases) were equally effective inhibitors of parasitemia. The antimalarial activity was dependent upon oligomer length, concentration, and time of addition to the cultures but was independent of the parasite strain tested. Four P. falciparum strains, including a multi-drug-resistant strain (MDR-K), a drug-sensitive strain (FCR-3), a erythrocyte membrane sialic acid-independent strain (7G8) and a strain isolated from a cerebral malaria patient (CM-87) were equally susceptible to treatment with a phosphorothioate oligomer. Inhibition of red cell invasion is primarily responsible for the observed decrease in proliferation as determined by a study of parasite maturation in the presence of a 30-mer nonsense phosphorothioate oligodeoxynucleotide.
Animals↗
Mucin synthesis in immortalized canine tracheal epithelial cells.
To study the regulation of mucin synthesis in canine tracheal epithelial cells, it is desirable to establish a cell line which synthesizes mucin continuously. We adopted the approach of immortalizing canine tracheal epithelial cells using a vector encoding the human papillomavirus (type 18) E6 and E7 genes. The E6 and E7 genes are essential and sufficient for the immortalization of human genital keratinocytes, as well as human tracheal epithelium. Primary epithelial cells from dog trachea were transfected with a vector containing HPV18 genes E6 and E7. The resultant cells (CT1) were cloned and maintained in selective medium supplemented with growth factors and hormones. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated the expression of the canine tracheal mucin (CTM) mRNA in these cells. The half-life of the CTM mRNA was found to be 45-60 min. Incorporation of labelled precursor (glucosamine) indicated that high-molecular-weight mucin glycoprotein was synthesized by these immortalized cells, which reacted with the antiserum to the native CTM. Equilibrium gradient centrifugation analysis showed that the buoyant density of the mucin synthesized in CT1 cells (1.486 g/ml) was similar to the reported value for native CTM (1.5 g/ml). Mucin which was isolated from immortalized cells was not a proteoglycan as chondroitinase treatment had no effect. These results suggest that CT1 cells synthesize a mucin glycoprotein which exhibits properties similar to native CTM. When characterized by immunostaining with a pool of monoclonal antibodies, these cells showed common epithelial antigens related to keratin expression. The CT1 cell line represents a unique resource for studying mucin biosynthesis and regulation.
Animals↗
New approach towards deglycosylation of sialoglycoproteins and mucins.
A modified procedure for chemical deglycosylation of glycoproteins containing sialylated and/or O-linked oligosaccharides, using anhydrous trifluoromethane sulfonic acid (TFMSA) is described. Although sialic acid residues are acid labile, it has been known that anhydrous TFMSA does not effectively remove carbohydrate side chains from glycoproteins if they are sialylated. In this procedure, sialic acid residues were removed by mild acid hydrolysis and the desialylated glycoprotein was treated with anhydrous TFMSA reagent under conditions which remove all the carbohydrate residues except the core D-GalNAc linked to serine/threonine. The core D-GalNAc residues were removed by reacting the glycoprotein with periodate followed by a second treatment with anhydrous TFMSA; this procedure gave a completely deglycosylated protein. The protein thus obtained was soluble in aqueous buffers and useful for biochemical and biophysical studies. The method was successfully employed to isolate polypeptides from alpha 1-acid glycoprotein (N-linked), fetuin, canine tracheal mucin and gastric mucin.
Amino Acids↗
Mucin coding sequences are remarkably conserved.
Mucins are complex glycoproteins expressed by glandular epithelia and the carcinoma which develop from these tissues. The core protein is aberrantly glycosylated in cancers, and some antibodies show specificity in their reactions with the cancer-associated mucins, which also contains epitopes recognized by T-cells from pancreatic and breast cancer patients. Based on the PCR amplification of the mucin coding sequences, hybridization analysis and determination of the sequence divergence we present the evidence that mucin coding sequences are conserved in a number of species. A broad series of organisms were examined for analogous sequences. Data show that mucin-type sequences are present in a variety of mammals, but less apparent in chicken and yeast. Divergence increased in the order human, monkey, rabbit/rat/cow, mouse; chicken and yeast exhibited minimal homology. Furthermore, nucleotide sequences not included in the tandem repeats, a common feature of mucin core structure, are more conserved than the flanking sequences which also suggests that the flanking sequences may be functionally significant while repeats are structurally important. The hybridization bands showed different restriction patterns (suggesting for the existence of the restriction fragment length polymorphism). Northern analysis indicates message polydispersity, commonly seen with this class of RNA. The major features of the protein appear broadly conserved in the different mammalian species examined. The evolutionary significance of the above studies has been discussed.
Animals↗
Canine U2 snRNA gene: nucleotide sequence, characterization and implications in RNA processing and cancer biology.
Abnormal RNA processing (splicing) may lead a cell to become cancerous. Transcription of a gene starts in the nucleus where genomic DNA is converted to precursor RNA by removing introns and joining exons. Splicing, mediated by small nuclear RNA (snRNA) and nuclear proteins, is tightly regulated during growth and development. U2 snRNAs are small, stable RNAs located in the nucleus of eukaryotic cells that recognize the branch point of the intron-exon junction. We describe here the organization of DNA sequences complementary to canine U2 snRNA. From a genomic library we isolated one recombinant containing the U2 gene. Southern analysis revealed that the canine species possesses only 3 to 5 U2 snRNA genes or very closely related sequences. The size of the U2 gene is 125 nt whereas in rat, Drosophila, trypanosome and yeast it is 189, 234, 141, and 192 nt respectively. The nucleotide sequence showed 82, 78, 72 and 95% homology with rat, Drosophila, yeast, and trypanosome U2 snRNA, respectively. The sensitivity of U2 snRNA towards alpha-amanitin suggests that it is transcribed by RNA polymerase II. The conserved nucleotide sequences which have been implicated in heterogeneous nuclear RNA splicing have been identified. The implications of the knowledge gained through above studies in cancer biology are discussed.
Animals↗
A monoclonal antibody capable of blocking the binding of Pf200 (MSA-1) to human erythrocytes and inhibiting the invasion of Plasmodium falciparum merozoites into human erythrocytes.
Glycophorin A is a major receptor on human erythrocytes for Plasmodium falciparum, the human malaria parasite. In this work, we have produced four glycophorin A-specific mAb: 2B10, 1E4, 3H12, and 3H2. 2B10 was mapped to the amino terminal region of glycophorin (amino acids 1-31), and its binding to erythrocytes was fully dependent on sialic acid residues. 3H2 bound to the region close to the cell membrane, and its binding to Wr (b-) erythrocytes was significantly decreased, compared with its binding to Wr (b+) erythrocytes. 1E4 and 3H12 recognized sites between those identified by 2B10 and 3H2. Pf200 (MSA-1) is a surface protein on the P. falciparum merozoite which has been shown to bind to erythrocytes. By reciprocal inhibition assays, 2B10 and MSA-1 could be shown to share the same determinant on erythrocytes. Using an in vitro assay, we have shown that 2B10 was the most efficient inhibitor of the invasion of human erythrocytes by P. falciparum merozoites. We conclude that the binding site for MSA-1 is primarily located on the amino terminal region, amino acids 1-31, of glycophorin A, and that 2B10 is valuable for additional study of the interactions between P. falciparum merozoites and human erythrocytes.
Animals↗
Molecular cloning and sequencing of a canine tracheobronchial mucin cDNA containing a cysteine-rich domain.
To date the complete sequence of only one mammalian mucin cDNA, MUC1, has been reported, although several mucin proteins have been partially characterized. Here we report the nucleotide sequence of a canine tracheal mucin cDNA containing two potential translation initiation codons, one translation termination codon and a poly(A) tail. A lambda gt11 cDNA library prepared from canine tracheal epithelial cells was screened with polyclonal anti-apo-canine tracheal mucin antibodies with the aim of obtaining the deduced amino acid sequence of the mucin core protein. Antibody-positive clones containing overlapping inserts of various lengths were purified and used for nucleotide sequencing. Based on the sequencing data, synthetic oligonucleotide primers were constructed and both ends (5' and 3') of the cDNA were determined. The complete sequence was 3.7 kb and included an open reading frame with coding capacity for 1118 aa, two translation initiation ATG codons in context with Kozak consensus sequences, one polyadenylylation site, and a poly(A) stretch. The protein was rich in Thr, Pro, Ser, Gly, and Ala and poor in Tyr, Phe, and Trp. Although tandem repeats of amino acids were absent in the deduced canine tracheal mucin sequence, motifs TPTPTP and TTTTPV appeared 13 and 19 times, respectively. The C-terminal region contained a Cys-rich domain (although a few Cys residues were also present in the middle of the protein) as has been reported for bovine submaxillary mucin, porcine submaxillary mucin, rat intestinal mucin, human intestinal mucin, and frog skin mucin. This suggested that a broad group of mucins contain such a Cys-rich domain whose functional significance is yet to be understood. Three potential N-glycosylation sites were present in canine tracheal mucin and the amino acid sequence showed homology with both human tracheal and intestinal mucins. The N-terminal domain showed more flexibility (probably due to a high number of Pro residues in this region) when analyzed by the University of Wisconsin Genetics Computer Group program package to determine the predicted secondary structure. Evaluation of the transcripts using the canine mucin cDNA as a probe indicated a polydisperse message with total RNA.
Amino Acid Sequence↗
Structural features of carbohydrate moieties in snake venom glycoproteins.
The structures of the carbohydrate moieties of glycoproteins in snake venoms are largely unknown. In the present study, we have analyzed venoms of several species of snakes as well as plasma and tissue glycoproteins from one species of cobra (Naja naja kaouthia) by lectin affinity staining of Western blots. The data demonstrate that glycoproteins in cobra venom invariably contain terminal alpha-galactosyl residues with negligible proportions of sialic acids. Interestingly, however, terminal alpha-galactosyl residues are present in significantly lower proportions in cobra tissues such as brain, liver, lung, kidney, spleen, muscle, and totally absent in cobra plasma glycoproteins. In sharp contrast to cobras, venom glycoproteins of other snakes do not contain terminal alpha-galactosyl residues but do contain terminal 2,3- and/or 2,6-linked sialic acids as well as beta-galactosyl residues. Cobra venom also contains high molecular weight heavily glycosylated proteins bearing poly-N-acetyllactosaminyl oligosaccharides, the majority of which appear to be linked to the protein core via O-glycosidic bonds.
Animals↗
Implications of inflammatory changes on cervical cytology.
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Cervix Uteri↗
Cloning and cDNA sequence of a bovine submaxillary gland mucin-like protein containing two distinct domains.
A lambda gt11 cDNA library prepared from bovine submaxillary gland mRNA was screened with polyclonal anti-apo-bovine submaxillary mucin antibodies with the aim of obtaining the deduced amino acid sequence of the mucin core protein. One of the positive clones had a 1.8 kilobase (kb) cDNA insert and coded for an incomplete protein. A 2.0-kb cDNA clone was isolated by rescreening the library with the 1.8-kb cDNA. Nucleotide sequencing of the full-length 2.0-kb cDNA revealed an open reading frame that coded for a 563-amino acid protein. A striking feature of the cloned protein is the skewed distribution of the amino acids, most notably that of the hydroxy amino acids and cysteine. The amino-terminal domain of 339 residues is very rich in threonine, serine, and glycine and poor in cysteine, aspartic acid, tyrosine, phenylalanine, and tryptophan. In contrast, the carboxyl-terminal domain of 224 residues is rich in cysteine, aspartic acid, tyrosine, lysine, and asparagine and relatively poor in threonine, serine, and glycine. A search of the protein data bank for homologies to the deduced amino acid sequence revealed statistically significant matches to several proteins, including the porcine submaxillary apomucin fragment. The cysteine-rich domain by itself was not statistically homologous with any of the registered polypeptide sequences. RNA blot analysis using DNA probes corresponding to the mucin-like and cysteine-rich regions detected a nearly identical pattern of transcripts, demonstrating that the characterized clones are not artifacts of cDNA library construction. The blots also showed the presence of polydisperse transcripts in bovine submaxillary gland but no detectable hybridization signals in liver or brain RNA.
Amino Acid Sequence↗
Modification of sialyl residues of glycoconjugates by reductive amination. Characterization of the modified sialic acids.
The sialic acid residues of alpha 1-acid glycoprotein and fetuin were modified by introduction of an amino residue, such as glycine and [3H]glycine. This modification involved (a) the selective periodate oxidation of the exocyclic carbon atoms of the sialic acid residue generating an aldehyde group at C-7, and (b) the reduction of the Schiff base formed with an amino compound by use of sodium cyanoborohydride. Thin layer chromatography, high pressure liquid chromatography, and amino acid composition data of the modified glycoprotein showed that the conversion was essentially quantitative. The glycine-modified sialic acids were isolated by mild acid hydrolysis and identified by g.l.c.-m.s. and n.m.r. spectroscopy, thus confirming that the quantitative modification produced a glycine-aminated C-7 sialic acid analog. Strong acid hydrolysis of the glycine-modified sialic acid yielded a fragment that had chromatographic characteristics similar to those of glycine.
Chemical Phenomena↗
Protein components of human tracheobronchial mucin: partial characterization of a closely associated 65-kilodalton protein.
A high-density mucin glycoprotein was isolated from human tracheobronchial secretions substantially free of contaminating protein, low-density glycoprotein, proteolytic enzymes, and lipid. A closely associated 65-kDa protein was discovered while investigating the effect of 2-mercaptoethanol treatment on the purified mucin glycoprotein. It has been established that the 65-kDa protein is neither alpha 1-antichymotrypsin nor human serum albumin, two proteins of similar molecular weight which are found in crude tracheobronchial secretions. This protein lacks cross-reactivity with antibodies directed against serum components and is presumably comparable to the 65-kDa protein similarly isolated from canine tracheal pouch secretions [Ringler et al. (1987) Biochemistry 26, 5322-5328]. Although both the presence of sulfhydryl groups and the ability to be reassociated with the mucin molecule have been established, it is not clear whether its association is due to direct disulfide bonding, hydrophobicity, or entrapment. It was found that 14C-methylated methemoglobin was an inappropriate substrate for measurement of proteolytic activity in mucin preparations due to inherent entrapment and clearance capabilities of mucin molecules.
Amino Acids↗
Purification and partial characterization of a malignancy-associated glycoprotein.
The isolation from cancer patient serum of a glycoprotein (Cc) associated with the presence of a variety of malignancies was previously reported. Although preliminary chemical and physical data indicated that Cc was different from identified circulating glycoproteins, subsequent immunological studies suggested that it was closely related to alpha 1-acid glycoprotein. Further analysis revealed the presence of two components in some Cc preparations and prompted a re-examination of the isolation and characterization data. In the present study, Cc was purified by a modified protocol which involved the use of pleural fluid obtained from individuals with cancer, and an alpha 1-acid glycoprotein antibody column to remove contaminating alpha 1-acid glycoprotein. Typically, the material not retained by the antibody column gave a single band with Mr 53,000 when subjected to sodium dodecyl sulfate-polyacrylamide electrophoresis. Amino terminal analysis revealed that the protein contained a blocked amino terminus, and carbohydrate analysis indicated that complex, asparagine-linked saccharide units were present. The product could be distinguished from alpha 1-acid glycoprotein and other previously described circulating glycoproteins by several criteria, including molecular weight, isoelectric point, and amino acid and carbohydrate composition. One of three preparations isolated had an apparent Mr of 59,000. Carbohydrate analysis as well as deglycosylation studies showed that the change in molecular weight was due to increased glycosylation.
Adsorption↗
Ultrastructural localization of erythrocyte cytoskeletal and integral membrane proteins in Plasmodium falciparum-infected erythrocytes.
The distributions of ankyrin, spectrin, band 3, and glycophorin A were examined in Plasmodium falciparum-infected erythrocytes by immunoelectron microscopy to determine whether movement of parasite proteins and membrane vesicles between the parasitophorous vacuole membrane and erythrocyte surface membrane involves internalization of host membrane skeleton proteins. Monospecific rabbit antisera to spectrin, band 3 and ankyrin and a mouse monoclonal antibody to glycophorin A reacted with these erythrocyte proteins in infected and uninfected human erythrocytes by immunoblotting. Cross-reacting malarial proteins were not detected. The rabbit sera also failed to immunoprecipitate [3H]isoleucine labeled malarial proteins from Triton X-100 and sodium dodecyl sulfate (SDS) extracts of infected erythrocytes. These three antibodies as well as the monoclonal antibody to glycophorin A bound to the membrane skeleton of infected and uninfected erythrocytes. The parasitophorous vacuole membrane was devoid of bound antibody, a result indicating that this membrane contains little, if any, of these host membrane proteins. With ring-, trophozoite- and schizont-infected erythrocytes, spectrin, band 3 and glycophorin A were absent from intracellular membranes including Maurer's clefts and other vesicles in the erythrocyte cytoplasm. In contrast, Maurer's clefts were specifically labeled by anti-ankyrin antibody. There was a slight, corresponding decrease in labeling of the membrane skeleton of infected erythrocytes. A second, morphologically distinct population of circular, vesicle-like membranes in the erythrocyte cytoplasm was not labeled with anti-ankyrin antibody. We conclude that membrane movement between the host erythrocyte surface membrane and parasitophorous vacuole membrane involves preferential sorting of ankyrin into a subpopulation of cytoplasmic membranes.
Animals↗
Deglycosylation studies on tracheal mucin glycoproteins.
Following several model experiments, conditions were developed for optimal deglycosylation of tracheal mucin glycoproteins. Exposure of rigorously dried material to trifluoromethanesulfonic acid at 0 degree C for up to 8 h results in cleavage of essentially all fucose, galactose, and N-acetylglucosamine, about 80% of the N-acetylneuraminic acid (NeuNAc), and a variable amount of N-acetylgalactosamine (GalNAc), the sugar involved in linkage to protein. Residual N-acetylneuraminic acid is sialidase susceptible and apparently in disaccharide units, presumably NeuNAc2----GalNAc. The remaining N-acetylgalactosamine is mostly present as monosaccharides, and a few Gal beta 1----3GalNAc alpha units are also present; both are cleaved by appropriate enzymatic treatment. The saccharide-free proteins obtained from either human or canine mucin glycoproteins have molecular weights of about 100,000 and require chaotropic agents or detergents for effective solubilization.
Amino Acids↗
Structure of canine tracheobronchial mucin glycoprotein.
Canine tracheal mucin glycoprotein was isolated from beagle dogs fitted with tracheal pouches. Following exclusion chromatography on Sepharose CL-4B, noncovalently associated proteins were further resolved by dissociative density gradient centrifugation in CsBr-guanidinium chloride, and the mucin was then extracted with chloroform-methanol. The delipidated high-density product obtained had a nominal molecular weight of about 10(6) and an overall composition characteristic for a mucin glycoprotein, viz., a high content of serine and threonine, about 80% carbohydrate by weight, the absence of mannose or uronic acid, measurable ester sulfate, and a Pronase-resistant domain of molecular weight (1.75-3.0) X 10(5) which contains essentially all of the saccharide residues. Noncovalently bound lipid amounted to 6-10% by weight and was primarily cholesterol and cholesteryl esters. Cleavage of disulfide bonds by performic acid oxidation resulted in the release of a protein (Mr 65,000) not otherwise resolved by sodium dodecyl sulfate gel electrophoresis or the purification scheme.
Amino Acids↗
Characteristics of two wheat germ agglutinin-resistant variants of B16 mouse melanoma cells with reduced tumorigenicity.
Two variants of B16 mouse melanoma cells, selected for their resistance to toxic levels of wheat germ agglutinin isolectin 1 (WGA-1) in serum-free medium, showed by chromosome analysis that they are still mouse cell-lines, continue to produce melanin, and are less tumorigenic in mice than the parent B16 cells. The variants showed a marked decrease in cell agglutination with the wheat germ lectin and a slight increase in cell agglutination with concanavalin A. The binding of 125I-labeled wheat germ agglutinin to the two variant lines was likewise decreased over a 10(3)-fold range of lectin concentrations. Terminal sialyl residues were critical in WGA-1 binding to the wild-type cells. The binding data indicated a decrease in high-affinity binding as well as a decrease in the total number of binding sites in the variants. Polyacrylamide gel electrophoresis, followed by affinity staining with 125I-wheat germ agglutinin, showed alterations in the wheat germ agglutinin-binding glycoproteins in the variants compared to those of the parent cell line. However, lactoperoxidase-catalyzed iodination revealed a similar cell-surface protein pattern among the three cell lines. Radioactive glycoproteins secreted or shed by the three cell lines grown in the presence of [3H]glucosamine in serum-free medium were fractionated on the basis of their interaction with WGA-Sepharose (2 mg/mL). The WGA-bound glycoproteins from the two variants had molecular weights of 92,000, 56,000, and 42,000. None of these components was detected in the parent cell-line. A major WGA-binding glycoprotein, which accounted for 37% of the total [3H]glucosamine incorporated, was isolated from the spent medium of the parent mouse melanoma cell-line. This glycoprotein was apparently absent in the WGA-1-resistant variants.
Agglutination Tests↗