Goverdhan P. Sachdev

Versatile fluorescent derivatization of glycans for glycomic analysis.

The new field of functional glycomics encompasses information about both glycan structure and recognition by carbohydrate-binding proteins (CBPs) and is now being explored through glycan array technology. Glycan array construction, however, is limited by the complexity of efficiently generating derivatives of free, reducing glycans with primary amines for conjugation. Here we describe a straightforward method to derivatize glycans with 2,6-diaminopyridine (DAP) to generate fluorescently labeled glycans (glycan-DAP conjugates or GDAPs) that contain a primary amine for further conjugation. We converted a wide variety of glycans, including milk sugars, N-glycans, glycosaminoglycans and chitin-derived glycans, to GDAPs, as verified by HPLC and mass spectrometry. We covalently conjugated GDAPs to N-hydroxysuccinimide (NHS)-activated glass slides, maleimide-activated protein, carboxylated microspheres and NHS-biotin to provide quantifiable fluorescent derivatives. All types of conjugated glycans were well-recognized by appropriate CBPs. Thus, GDAP derivatives provide versatile new tools for biologists to quantify and covalently capture minute quantities of glycans for exploring their structures and functions and generating new glycan arrays from naturally occurring glycans.

Glycan reductive isotope labeling for quantitative glycomics

Many diseases and disorders are characterized by quantitative and/or qualitative changes in complex carbohydrates. Mass spectrometry methods show promise in monitoring and detecting these important biological changes. Here we report a new glycomics method, termed glycan reductive isotope labeling (GRIL), where free glycans are derivatized by reductive amination with the differentially coded stable isotope tags [(12)C(6)]aniline and [(13)C(6)]aniline. These dual-labeled aniline-tagged glycans can be recovered by reverse-phase chromatography and can be quantified based on ultraviolet (UV) absorbance and relative ion abundances. Unlike previously reported isotopically coded reagents for glycans, GRIL does not contain deuterium, which can be chromatographically resolved. Our method shows no chromatographic resolution of differentially labeled glycans. Mixtures of differentially tagged glycans can be directly compared and quantified using mass spectrometric techniques. We demonstrate the use of GRIL to determine relative differences in glycan amount and composition. We analyze free glycans and glycans enzymatically or chemically released from a variety of standard glycoproteins, as well as human and mouse serum glycoproteins, using this method. This technique allows linear relative quantitation of glycans over a 10-fold concentration range and can accurately quantify sub-picomole levels of released glycans, providing a needed advancement in the field of glycomics.

Respiratory mucous secretions in patients with cystic fibrosis: relationship between levels of highly sulfated mucin component and severity of the disease.

The tracheobronchial secretions from cystic fibrosis patients contained higher levels of protein, DNA and sialic acid than the tracheobronchial secretions from healthy donors. In contrast, the neutral hexose content in CF secretions was strikingly lower than in secretions from normal subjects. The levels of neutral hexose and sialic acid in the CF secretions were found to increase with increasing severity of the disease. The alterations in the levels of these chemical parameters in the secretions of patients with increased disease severity are as a result of increased levels of the mucin content of the secretions, especially of the highly sulfated mucin component. Since mucins are considered, to a large extent, responsible for the viscoelastic properties of the secretions, the enhanced levels of the highly sulfated mucin component in the secretions of the patients with increased disease severity, may contribute to altered rheological properties and hence decreased mucociliary transport of the secretions.

IL-4 induced MUC4 enhancement in respiratory epithelial cells in vitro is mediated through JAK-3 selective signaling

BackgroundRecent studies have identified MUC4 mucin as a ligand for activation of ErbB2, a receptor tyrosine kinase that modulates epithelial cell proliferation following epithelial damage in airways of asthmatics. In this study, we investigated the potential role of IL-4, one of the Th2 inflammatory cytokines persistent in asthmatic airways, in regulating MUC4 expression using a cell line NCI-H650.MethodsReal time PCR analysis was performed to determine concentration and time dependent effects of IL-4 upon MUC4 expression. Nuclear run on experiments were carried out to explore potential transcriptional modulation. Western blotting experiments using a monoclonal antibody specific to ASGP-2 domain of MUC4 were performed to analyze MUC4 glycoprotein levels in plasma membrane fractions. To analyze potential signal transduction cascades, IL-4 treated confluent cultures were co-incubated, separately with a pan-JAK inhibitor, a JAK-3 selective inhibitor or a MEK-1, 2 (MAPK) inhibitor at various concentrations before MUC4 transcript analysis. Corresponding transcription factor activation was tested by western blotting using a monoclonal p-STAT-6 antibody.ResultsMUC4 levels increased in a concentration and time specific fashion reaching peak expression at 2.5 ng/ml and 8 h. Nuclear run on experiments revealed transcriptional enhancement. Corresponding increases in MUC4 glycoprotein levels were observed in plasma membrane fractions. Pan-JAK inhibitor revealed marked reduction in IL-4 stimulated MUC4 levels and JAK3 selective inhibitor down-regulated MUC4 mRNA expression in a concentration-dependent fashion. In accordance with the above observations, STAT-6 activation was detected within 5 minutes of IL-4 stimulus. No effect in MUC4 levels was observed on using a MAPK inhibitor.ConclusionThese observations signify a potential role for IL-4 in MUC4 up-regulation in airway epithelia.

Isolation, chemical composition, and properties of the major mucin component of normal human tracheobronchial secretions

Abstract The major mucin-type glycoprotein component of normal human tracheobronchial secretions was isolated in electrophoretically homogeneous form, and subjected to physical and chemical characterization. The respiratory mucus, collected from healthy young male volunteers by fiberoptic bronchoscopy, was subjected to initial fractionation on a column of Sephadex G-200. The excluded fraction (mucins) was then chromatographed on a column of Bio-Gel A-15m. Gel electrophoretic examination of the material from the included peak of the latter column indicated the presence of a single mucin component, whereas the pattern of the excluded peak showed two bands, a minor component with the mobility of the included mucin and a major band of slower-migrating, large-molecular-weight mucin. Chemical analysis of the electrophoretically homogeneous mucin present in the included peak showed 73% carbohydrate (galactose, fucose, glucosamine, galactosamine, and sialic acid), 4% sulfate monoester, and 23% protein. The amino acid composition showed a high content (29%) of serine plus threonine, and the presence of carbohydrate chains linked to these hydroxyamino acids was demonstrated by reductive β-elimination. A molecular weight of 69,400 daltons was estimated by sedimentation equilibrium.

Differential expression of MUC genes in endometrial and cervical tissues and tumors

BackgroundMucin glycoproteins are major components of mucus and are considered an important class of tumor associated antigens. The objective of this study was to investigate the expression of human MUC genes (MUC1, MUC2, MUC5B, MUC5AC and MUC8) in human endometrium and cervix, and to compare and quantitate the expression of MUC genes in normal and cancerous tissues.MethodsSlot blot techniques were used to study the MUC gene expression and quantitation.ResultsOf the five-mucin genes studied, MUC1, MUC5B and MUC8 showed high expression levels in the normal and cancerous endometrial and cervical tissues, MUC2 and MUC5AC showed considerably lower expression. Statistically, higher levels of MUC1, MUC5B and MUC8 were observed in endometrial adenocarcinomas compared to normal tissues. In contrast, only MUC1 levels increased with no significant changes in expression of MUC5B and MUC8 in cervical tumors over normal cervical tissues.ConclusionEndometrial tumors showed increased expression of MUC1, MUC5B and MUC8 over normal tissues. Only MUC1 appears to be increase, in cervical tumors. All the studied tissues showed high and consistent expression of MUC8 mRNA. Low to neglible levels of MUC2 and MUC5AC were observed in all studied endometrial and cervical tissues.

Physical Properties of Purified Human Respiratory Mucus Glycoproteins: Effects of Sodium Chloride Concentration on the Aggregation Properties and Shape

The biophysical properties of purified native (nonreduced) mucus glycoproteins (mucins) isolated from lung mucus secretions of cystic fibrosis (CF) patients and subjects with normal lungs were studied using the technique of light scattering. The effects of different NaCl concentrations and 6 M guanidine hydrochloride on the molecular size of mucins, their ability to form aggregates, and their shape were investigated. Under the concentration range studied (0.05-3.5 mg/ml), in buffered 0.03 and 0.01 M NaCl, the CF mucins had higher molecular weights (12.2 x 10(6) to 17.1 x 10(6) and 9.5 x 10(6) to 10.4 x 10(6), respectively) than those observed in buffered 0.15 M NaCl (4.3 x 10(6) to 6.6 x 10(6]. These results were interpreted in terms of CF mucins self-aggregating in buffered 0.03 and 0.01 M NaCl. In contrast, in the both buffered 0.3 and 0.15 M NaCl, the normal respiratory mucins had molecular weights of 6.3 x 10(6) to 8.6 x 10(6), thus suggesting the absence of normal mucin aggregation in buffered 0.03 M NaCl. In the presence of 6 M guanidine HCl both CF and normal mucins had molecular weights of about 5 x 10(6) and showed more extended structure (i.e., larger radius of gyration) than in the presence of 0.03 or 0.15 M NaCl. Studies of the relationship of the light scattering intensity with scattering angle showed that, under the above experimental conditions studied, both CF and normal respiratory mucins were polydisperse flexible coil-shaped molecules. The increased aggregation of CF mucins observed at lower salt concentrations may alter the viscoelastic properties of CF lung mucus secretions.

Hydrophobic interaction of fluorescent probes with fetuin, ovine submaxillary mucin, and canine tracheal mucins

The presence of hydrophobic sites in fetuin, ovine submaxillary mucin and two homogeneous canine tracheal mucins was established by fluorescence probe techniques. The interaction between the above-mentioned glycoproteins and two hydrophobic fluorescent compounds, sodium mansate and mansylphenylalanine, was accompanied by an enhancement in fluorescence and a shift of the fluorescence maxima to shorter wavelengths. The introduction of a phenylalanine residue to the mansyl group enhanced the binding affinity of the probe for the hydrophobic sites of these glycoproteins as evidenced by lower values for the dissociation constants. The high molecular weight (581 600) tracheal mucin, which had the highest carbohydrate content (80%) of all the glycoproteins investigated, exhibited the highest fluorescence enhancement and the largest number of binding sites for these fluorescent probes.

Pseudomonas aeruginosa mucoid strain 8830 binds glycans containing the sialyl-Lewis x epitope

Pseudomonas aeruginosa infection of patients with cystic fibrosis (CF) is a leading cause of their morbidity and mortality. Pathogenesis is initiated in part by molecular interactions of P. aeruginosa with carbohydrate residues in airway mucins that accumulate in the lungs of patients with this disease. To explore the nature of the glycans recognized by a stable, mucoid, alginate-producing strain P. aeruginosa 8830 we generated a genetically modified Pa8830 expressing green fluorescent protein (Pa3380-GFP). We tested its binding to a panel of glycolipids and neoglycolipids in which selected glycans were covalently attached to dipalmitoyl phosphatidylethanolamine and analyzed on silica gel surfaces. Among all glycans tested, Pa8830-GFP bound best to sialyl-Lex-containing glycan NeuAc(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc-R and bound weakly to H-type blood group Fucα1-2Galβ1-4GlcNAc-R, sialyl-lactose, and Lex, and exhibited little binding toward non-fucosylated derivatives. Interestingly, while Pa8830-GFP bound to the glycosphingolipid asialoGM1, it did not appear to bind to a wide variety of other glycosphingolipids including GM1, GM2, asialoGM2, and sulfatide. These results indicate that P. aeruginosa 8830 has preferential binding to sialyl-Lex-containing glycans and has weak recognition of related fucose- and sialic acid-containing glycans. The finding that Pa8830 binds sialyl-Lex-containing glycans, which occur at increased levels in mucins from CF patients, is consistent with studies of other strains of P. aeruginosa and further suggests that such glycans on CF mucins contribute to disease pathogenesis.

Evidence for secretion of high molecular weight mucins by canine tracheal epithelial cells in primary culture: Effects of select secretagogues on mucin secretion

SummaryThe purpose of this investigation was to provide evidence for the secretion of high molecular weight mucins, CTM-A and CTM-B, in primary culture of canine tracheal epithelial (CTE) cells. The cells were isolated from tracheas of mongrel dogs by pronase treatment. Primary cultures of the epithelial cells were established using ICN cellagen inserts in Dulbecco’s modified Eagle’s/F12 medium supplemented with growth factors and could be maintained for up to 23 days. The evidence for the mucin secretion in culture medium and their localization in the cells was established by a) positive immunocytochemical staining using specific antibodies developed against purified native as well as deglycosylated CTM-A and CTM-B; b) incorporation of labeled amino acids, followed by electrophoresis and autoradiography detection of glycoconjugates purified from the culture medium; c) comparison of the amino acid compositions of mucin purified from canine tracheal pouch secretions and that purified from the culture medium; and d) Western blot analyses using specific polyclonal antibodies directed against deglycosylated CTM-A and CTM-B. Immunoaffinity purified secreted labeled glycoconjugates were resistant to hyaluronidase treatment. The effects of cyclic AMP (1 × 10−5M), dibutyryl cyclic AMP (1 × 10−5M), 8-bromocyclic AMP (1 × 10−5M), and prostaglandin E1 (1 × 10−6M) on mucin secretion by CTE cells were also investigated. Secretion of mucins by CTE cells in culture was considerably more enhanced by 8-bromocyclic AMP than that observed for other secretagogues used in this study.