Daniel Tetaert

O-Glycosylation potential of lepidopteran insect cell lines

The enzyme activities involved in O-glycosylation have been studied in three insect cell lines, Spodoptera frugiperda (Sf-9), Mamestra brassicae (Mb) and Trichoplusia ni (Tn) cultured in two different serum-free media. The structural features of O-glycoproteins in these insect cells were investigated using a panel of lectins and the glycosyltransferase activities involved in O-glycan biosynthesis of insect cells were measured (i.e., UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, UDP-Gal:core-1 beta1, 3-galactosyltransferase, CMP-NeuAc:Galbeta1-3GalNAc alpha2, 3-sialyltransferase, and UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase activities). First, we show that O-glycosylation potential depends on cell type. All three lepidopteran cell lines express GalNAcalpha-O-Ser/Thr antigen, which is recognized by soy bean agglutinin and reflects high UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity. Capillary electrophoresis and mass spectrometry studies revealed the presence of at least two different UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases in these insect cells. Only some O-linked GalNAc residues are further processed by the addition of beta1,3-linked Gal residues to form T-antigen, as shown by the binding of peanut agglutinin. This reflects relative low levels of UDP-Gal:core-1 beta1,3-galactosyltransferase in insect cells, as compared to those observed in mammalian control cells. In addition, we detected strong binding of Bandeiraea simplicifolia lectin-I isolectin B4 to Mamestra brassicae endogenous glycoproteins, which suggests a high activity of a UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase. This explains the absence of PNA binding to Mamestra brassicae glycoproteins. Furthermore, our results substantiated that there is no sialyltransferase activity and, therefore, no terminal sialic acid production by these cell lines. Finally, we found that the culture medium influences the O-glycosylation potential of each cell line.

Architecture of the large membrane-bound mucins.

Epithelial membrane-bound mucins are high molecular mass glycoproteins that may be also secreted or released into the extracellular environment. The genomic and multi-domain organizations of human large epithelial membrane-bound mucins are reviewed here with the purpose to clarify the literature on the subject with the help of mouse sequences. This family of complex molecules contains at least MUC3A, MUC12, MUC17, all organized in a cluster of genes, MUC4 and likely MUC16. In addition, we discuss the splicing events reported for these mucins with an emphasis on the human mucin MUC4.

Gonadotropic dysfunction produced by Trypanosoma brucei brucei in the rat

Hormonal disorders have been frequently observed in humans and animals infected with tsetse-transmitted (African) trypanosomes. We studied the pituitary gonadal axis (plasma concentrations of testosterone, luteinizing hormone (LH), follicle stimulating hormone (FSH) and the pituitary gonadotropin (LH, FSH) concentrations) in rats as an experimental model infected with an acute stock of Trypanosoma brucei brucei (AnTat 1.1A). The same investigations in vivo were carried out with rats inoculated by trypanosomal preparations: surface coat components slowly released at pH 5.5 and the parasitic cellular pellet. The releasing procedure as firstly described by Baltz et al. (1976) was performed in the presence or the absence of protease inhibitors. We noted a testicular hypogonadism produced by the acute infection with the decrease of the testosterone level and an increase of the pituitary LH concentration, although the other circulating FSH and LH hormone levels were stable. The injection of the trypanosomal pellet, obtained in the presence of antiproteases, generated a similar clinical hormonal picture: decrease of testosterone level; increase in pituitary LH, FSH content; absence of significant variation of circulating FSH and LH rates. When the trypanosomal pellet was prepared in absence of antiproteases the circulating gonadostimuline levels were significantly decreased. In the same conditions (absence of antiproteases) the trypanosomal supernatant pH 5.5 induced the decrease of the testosterone and plasma LH levels. These results suggested that component(s) from trypanosomes generated hormonal perturbations.

Dietary n-3 fatty acids have suppressive effects on mucin upregulation in mice infected with Pseudomonas aeruginosa.

BackgroundMucin hypersecretion and mucus plugging in the airways are characteristic features of chronic respiratory diseases like cystic fibrosis (CF) and contribute to morbidity and mortality. In CF, Pseudomonas aeruginosa superinfections in the lung exacerbate inflammation and alter mucus properties. There is increasing evidence that n-3 polyunsaturated fatty acids (PUFAs) exhibit anti-inflammatory properties in many inflammatory diseases while n-6 PUFA arachidonic acid (AA) favors inflammatory mediators such as eicosanoids prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) that may enhance inflammatory reactions. This suggests that n-3 PUFAs may have a protective effect against mucus over-production in airway diseases. Therefore, we hypothesized that n-3 PUFAs may downregulate mucins expression.MethodsWe designed an absolute real-time PCR assay to assess the effect of a 5-week diet enriched either with n-3 or n-6 PUFAs on the expression of large mucins in the lungs of mice infected by P. aeruginosa.ResultsDietary fatty acids did not influence mucin gene expression in healthy mice. Lung infection induced an increase of the secreted gel-forming mucin Muc5b and a decrease of the membrane bound mucin Muc4. These deregulations are modulated by dietary fatty acids with a suppressive effect of n-3 PUFAs on mucin (increase of Muc5b from 19-fold up to 3.6 × 105-fold for the n-3 PUFAs treated group and the control groups, respectively, 4 days post-infection and decrease of Muc4 from 15-fold up to 3.2 × 104-fold for the control and the n-3 PUFAs treated groups, respectively, 4 days post-infection).ConclusionOur data suggest that n-3 PUFAs enriched diet represents an inexpensive strategy to prevent or treat mucin overproduction in pulmonary bacterial colonization.

The primary sequence of badger myoglobin.

Abstract The badger apomyoglobin was first submitted to tryptic digestion. The tryptic hydrolysate of whole protein was fractionated by resin chromatography and each tryptic peptide was sequenced. The apomyoglobin was also cleaved by cyanogen bromide and the resulting fragments were fractionated by gel filtration; three segments were obtained; the N-terminal segment (55 residues), the C-terminal segment (22 residues) and the median segment (76 residues). Each segment was submitted to a variety of enzymatic digestions and the complete amino acid sequence of resulting peptides was established. In addition, the whole protein and the median segment were analyzed by using a sequenator (Edman); the first 31 N-terminal residues of the intact protein and the first 28 N-terminal residues of the median segment of CNBr resulting peptide were confirmed. The complete sequence of the C-terminal segment (22 residues) was also established and confirmed by manual Edmans degradation. Between badger and harbor seal myoglobins, 19 differences out of 153 residues were found. Of these amino acid replacements, one corresponds to the exchange of two bases and 18 to the exchange of one base in the coding triplets.

Influence of the amino acid sequence on the MUC5AC motif peptide O-Glycosylation by Human gastric UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase(s)

The present work was carried out to study the role of the peptide moiety in the addition of O-linked N-acetylgalactosamine to human apomucin using human crude microsomal homogenates from gastric mucosa (as enzyme source) and a series of peptide acceptors representative of tandem repeat domains deduced from the MUC5AC mucin gene (expressed in the gastric mucosa). Being rich in threonine and serine placed in clusters, these peptides provided several potential sites for O-glycosylation. The glycosylated products were analysed by a combination of electrospray mass spectrometry and capillary electrophoresis in order to isolate the glycopeptides and to determine their sequence by Edman degradation. The O-glycosylation of our MUC5AC motif peptides gave information on the specificity and activity of the gastric microsomal UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase(s). The proline residues and the induced-conformations are of great importance for the recognition of MUC5AC peptides but they are not the only factors for the choice of the O-glycosylation sites. Moreover, for the di-glycosylated peptides, the flanking regions of the proline residues strongly influence the site of the second O-glycosylation.

Characterization of different proteolytic activities in Trypanosoma brucei brucei.

The variant surface glycoprotein of African trypanosomes is released after overnight incubation of parasites at 4 degrees C in pH 5.5 phosphate glucose buffer and may be purified by Concanavalin A Sepharose affinity chromatography. The addition of proteinase inhibitors during the parasite incubation is necessary to prevent the proteolysis of the variant surface glycoprotein by the trypanosomal released proteinases. Using this procedure without the addition of proteinase inhibitors, the proteolytic activities, released from the bloodstream forms Trypanosoma brucei brucei variant AnTat 1.1, were separated by Concanavalin-A Sepharose affinity chromatography. The unretained material (F1) shows hydrolytic activity against the two synthetic substrates Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, which is stimulated by dithiothreitol, but not inhibited by E-64, and characterized by an alkaline pH optimum and an estimated molecular mass of 80-100 kDa. The Michaelis constant for the substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC was, respectively, 2.8 and 6.7 microM. The retained material eluted by addition of 1% methyl-alpha-D-mannopyranoside (F2) shows hydrolytic activity against the synthetic substrate Z-Phe-Arg-AMC, which is stimulated by dithiothreitol, inhibited by E-64, active between pH 6.0 and 8.0, and could be separated into two peaks of activity by HPLC, one peak of high molecular mass (greater than 70 kDa) and the other peak of lower molecular mass (30-70 kDa). By electrophoresis in gels containing gelatin as substrate, this fraction contains several proteins with gelatinolytic activity, whereas the unretained fraction F1 did not have any gelatinolytic activity.

The characterization of the first anti-mouse Muc6 antibody shows an increased expression of the mucin in pancreatic tissue of Cftr-knockout mice.

Gel-forming mucins are large high-molecular weight secreted O-glycoproteins responsible for the gel-properties of the mucus blanket. Five orthologous gel-forming mucins have been cloned in human and mouse. Among them, the mucin MUC6 has been less studied, particularly in rodents and no anti rodent-Muc6 antibody has been reported yet. In order to further study Muc6 in mice, our aims were to obtain a specific Muc6 antibody, to validate it and to test it in Cftr deficient mice. A polyclonal serum named CP4 was isolated from a rabbit immunized by a mouse Muc6 peptide. In Western blot experiments, the antibody detected a high-molecular weight molecule secreted by the gastric tissue. Using immunohistochemistry, we showed that the antibody reacted strongly with deep glands of duodenum and ileum and mucous neck cells of gastric body. CP4 also recognized Muc6 protein secreted at the surface of the stomach and renal collecting tubules. The centroacinar cells of pancreatic tissue also reacted with the antibody. Cftr−/− mice showed a higher expression of Muc6 at both protein and RNA levels compared with their control Cftr+/+ littermates suggesting that as in the human disease, Muc6 may contribute to the formation of materials that block pancreatic acini and ducts in mouse models of cystic fibrosis. The rabbit anti-mouse Muc6 polyclonal antibody seems highly specific to the mouse mucin and will be useful to study pancreatic pathology in cystic fibrosis.

Physical and immunological analysis of the two domains isolated from a variant surface glycoprotein of Trypanosoma brucei.

A specific surface glycoprotein of a variant of Trypanosoma brucei was cleaved with trypsin and the two major domains of the molecule have been purified. We have studied the chemical composition of each domain and compared the data to published results of the specific cDNA sequence. Circular dichroism measurements show that the amino-terminal domain includes preferentially alpha-helical or beta-sheet structure. The physicochemical analyses are supplemented by a prediction of secondary structure and a statistical pattern of hydrophilicity-hydrophobicity. The results are discussed in light of the internal limits that were described in the process of partial gene conversion occurring between the variant gene sequence and related members of the same gene family. Immunoblots with homologous antiserum indicate that the amino-terminal domain is implicated in antigenicity. In addition, immunoblotting with heterologous antiserum on native antigen, tryptic hydrolysates, or purified domains suggests a site of interaction supported by the two domains.

Studies of acceptor site specificities for three members of UDP-GalNAc:N-acetylgalactosaminyltransferases by using a synthetic peptide mimicking the tandem repeat of MUC5AC.

The acceptor specificity of three major isoforms of UDP-GalNAc:polypeptide N-acetylgalactosaminyltranferases (murine recombinant proteins GaNTase-T1, -T2 and -T3) was investigated using the synthetic peptide (GTTPSPVPTTSTTSAP) containing clusters of threonine residues mimicking the mucin tandem repeat unit of MUC5AC. The O-glycosylated products obtained after in vitro reactions were fractionated by capillary electrophoresis and the purified glycopeptides were characterized by MALDI mass spectrometry (number of O-GalNAc residues) and by Edman degradation (site location). A maximum of three GalNAc residues was transferred into the MUC5AC motif peptide and the preferential order of incorporation for each GaNTase isoform was determined. Our results suggest that clusters of threonine appear to be essential for site recognition of peptide backbone by the ubiquitous GaNTases and also support the notion that the different GaNTase isoforms with varying substrate specificities are involved in a hierarchical order of O-glycosylation processing of the mucin-type O-glycoproteins.