Purification and structures of oligosaccharide chains in swine trachea and Cowper's gland mucin glycoproteins

Mucin glycoproteins were purified from extracts of swine trachea mucosa and Cowper's gland. The gelatinous extracts were solubilized by reduction and carboxymethylation and then purified by chromatography on Sepharose CL-6B and DEAE-Sepharose. The structure of some of the carbohydrate units in these glycoproteins were determined and compared. Alkaline borohydride treatment indicated that more than 85% of the carbohydrate chains in these glycoproteins were linked to serine or threonine residues in the polypeptide chain through O-glycosidic bonds with N-acetylgalactosamine. Reduced oligosaccharides released by treatment with alkaline borohydride were isolated by gel filtration on Bio-Gel P-6 and chromatography on DEAE-cellulose and paper. The structures of the oligosaccharides were established by methylation analysis, gas chromatography, and sequential hydrolysis with specific exoglycosidases. The major oligosaccharides in Cowper's gland mucin glycoproteins were sialylated short chains: NeuAc alpha 2,6GalNAcol and NeuAc alpha 2,3Gal beta 1,3(NeuAc alpha 2,6)GalNAcol. In marked contrast, branched chains containing a Gal beta 1,3(GlcNAc beta 1,6)GalNAc core unit were the major components of trachea mucin glycoprotein. Ten of these chains had the following structures: (Formula: see text).     

Structural and compositional differences between intracellular and secreted mucin of rat small intestine.

An investigation was undertaken to discover whether mucin purified from secretions in the lumen of rat small intestine differed in structure or composition from intracellular mucin purified from rat intestinal tissue. To do this, ligated loops were constructed in situ from previously washed intestinal segments and mucin purified separately from tissue homogenates or loop fluid. Secreted mucin (SM) differed from intracellular mucin (IM) by having a higher proportion of 'minor' mucin amino acids (aspartic acid, glutamic acid, glycine and alanine) and a lower proportion of 'major' amino acids (serine, proline and threonine). SM also contained less N-acetylgalactosamine and a small, but measureable, amount of mannose. Gel electrophoresis showed that SM penetrated the gel more readily and, unlike IM, gave a rather prominent, but diffuse, band having a midpoint position of Mr 200,000. After reduction both IM and SM gave rise to the putative 'link' component of Mr 118,000 and the 200,000-Mr band of SM disappeared. SM was included to a greater extent than IM on Sepharose CL-2B chromatography, suggesting a smaller size. With the use of CsCl-density-gradient ultracentrifugation of SM, a lighter species [buoyant density (rho) = 1.38 g/ml] enriched in the 200,000-Mr component, was separated from a heavier, more glycosylated, species (rho = 1.50 g/ml). Purified 200,000-Mr component had a composition identical with that of the 118,000-Mr 'link' component of IM, reacted in Western blots with an antibody specific for the 118,000-Mr 'link' component, and after reduction gave rise to a 118,000-Mr component on gel electrophoresis. Thus secreted mucin contains a 200,000-Mr component which appears to represent a disulphide-linked dimer of the previously described 118,000-Mr 'link' component of intracellular mucin.     

Regulation of the synthesis of mucin glycoproteins in swine trachea explants

Swine tracheal epithelium has been cultured as explants in a chemically defined medium for periods of up to 2 wk. The viability of the explants was shown by the preservation of the ultrastructural features of cells in the epithelial layer and by the active incorporation of radioactive glucosamine and sulfate into secreted mucin glycoproteins. The rate of secretion of mucin glycoprotein was about 0.035 mg per cm2 per d. After initial 24 h lag period was shown to be due to the equilibration of intracellular mucin glycoprotein pools with radioactive precursors. The rate of secretion of glycoprotein showed a linear dependence on the area of the explant, and maximal incorporation was observed at 200 microM glucosamine. A higher concentration of 35SO4, 1000 microM, was required for maximal incorporation of the precursor. Insulin at 0.1 to 1 microgram/ml increased the rate of secretion twofold, whereas 0.1 to 100 micrograms/ml of hydrocortisone and 0.1 to 100 micrograms/ml of epinephrine significantly decreased the rate of secretion. Vitamin A had little or no effect of normal trachea explants at low concentrations, and, at higher concentrations, 10(-5) M, it decreased the secretion of mucin glycoproteins. Vitamin A, at a concentration of 10(-9) M, increased the rate of synthesis of glycoprotein at least fourfold in trachea explants from vitamin A-deficient rats. Mucus secretions collected from the surface of swine trachea and from the culture medium of trachea explants were purified. The mucus was solubilized by reduction and carboxymethylation, and the high molecular weight mucin glycoproteins were purified by chromatography on Sepharose CL-6B columns under dissociating conditions in 2 M guanidine HCl. The mucin glycoproteins purified from swine trachea and from the culture medium of trachea explants were virtually indistinguishable. They showed the same properties when examined by gel electrophoresis and immunoprecipitation. The purified glycoproteins contained about 25% protein, and serine, threonine, and proline were the principal amino acids present. More than 80% of the carbohydride chains in both samples were released by treatment with alkaline borohydride. Nearly the same molar ratio of N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose, sulfate, and sialic acid was found in both preparations.     

Isolation from human urines of a mucin with blood group Sda activity

A mucin with Sd^a blood group activity was isolated from human group 0 urines by a multistep procedure including an affinity chromatography on $\text{Helix pomatia}$ - Ultrogel. About 8 mg of active material was obtained from 100 litres of urines. The purified substance of apparent molecular weight 340,000 dalton is not stained by Coomassie blue but gave a single periodic acid-Schiff positive band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analytical composition indicated the absence of mannose, a high content of N-acetylgalactosamine and a molar ratio galactose: N-acetylgalactosamine: N-acetylglucosamine: sialic acid of 2:2:1:2. Amino acid composition is typical of a mucin substance with high values of serine, threonine, proline and alanine. The urinary mucin inhibited human anti-Sd^a antibodies as strongly as the Tamm-Horsfall glycoprotein isolated from Sd(a+) urines. However, the two substances clearly have different composition and properties. It is suggested that oligosaccharide chains with Sd^a blood group activity might be carried by different glycoconjugates of human arines and tissues.     

A galactose-specific lectin from the hemolymph of the pearl oyster, Pinctada fucata martensii

1. A lectin in the serum of Pinctada fucata martensii was purified by a combination of affinity chromatography on Sepharose 4B coupled with bovine submaxillary gland mucine, anion exchange chromatography on Mono Q and gel filtration on Superose 6. 2. The purified lectin was indicated to be homogeneous by polyacrylamide electrophoresis and rechromatography on Mono Q. 3. The purified lectin was approximately 440,000 in molecular weight and was composed of identical subunits with a molecular weight of approximately 20,000. 4. D-galactose and N-acetylgalactosamine gave a 50% inhibition of agglutination of horse erythrocytes by the lectin at 0.3 and 1.2 mM, respectively. 5. The antibody obtained from rabbit immunized with the purified lectin was monospecific to the lectin judged from the hemagglutination blocking test, immunoelectrophoresis and immunoblotting.     

Isolation and partial characterization of a soluble mucin in human pancreatic juice.

Morphological and histochemical abnormalities in pancreatic mucin occur in many pancreatic disorders. However, the composition of pancreatic mucin is poorly understood. Purified mucin was isolated from pure pancreatic juice by sequential chromatography on Sepharose CL-2B and CL-4B followed by CsCl density gradient ultracentrifugation. The mucin preparation consists of 24% protein and 73% carbohydrate. Reduction of the macromolecule (greater than 2 x 10(6)) by mercaptoethanol resulted in the formation of subunits of molecular weight 500,000 and released several small molecular weight proteins, including a glycoprotein of an average molecular weight of 116,000. Cellulose acetate electrophoresis separated the mucin into three species of different staining properties for periodic acid-Schiff reagent and Alcian blue, suggesting the presence of microheterogeneity with respect to sulphation and sialation. Threonine, serine, and proline composed 48% of the total amino acids, while the oligosaccharide moiety contained N-acetylglucosamine, N-acetylgalactosamine, fucose, galactose, sialic acid, and sulphate. We also detected the presence of C16:0 and C18:0 fatty acids which were probably noncovalently bound to the pancreatic mucin.     

Synthesis and macromolecular organization of gastrointestinal mucin.

Mucus glycoproteins (mucins), the principal determinants of mucus protective qualities and mucosal defense, are studied extensively to define pathological aberrations in the relation to gastrointestinal disease and to develop the mucous barrier strengthening agents. Recent work from our laboratory provided evidence as to the initial stages of the gastrointestinal mucin synthesis, molecular size of the apomucin, its macromolecular organization and interaction with other elements of gastrointestinal mucus. Using monoclonal antibodies against apomucin (clone 1H7), O-glycosylated with N-acetylgalactosamine apomucin (clone 2B4), and that against carboxyl terminal of the apomucin (clone 3G12), the mucin synthesizing polysomes were isolated and glycosylated peptides ranging in size from 6-60 kDa identified. The in vitro synthesis in the cell-free system also afforded 60-64 kDa products recognized by 1H7 and 3G12 antimucin MAbs. The obtained results provided evidence that the mucin core consists of 60 kDa peptide which at cotranslational stage is O-glycosylated with N-acetylgalactosamine. Studies on mucin polymer assembly revealed that mucin preparations prepared by equilibrium density gradient centrifugation and Sepharose 2B chromatography (Mantle, M., Mantle, D., and Allen, A. (1981) Biochem. J. 195, 277-285) are not completely purified and contain DNA and extraneous proteins. The evidence was obtained that so called mucin "link protein", 118 kDa glycopeptide, is a N-glycosylated fragment of fibronectin, whereas the supposedly native undegraded mucin isolated by Carlstedt et al. (Biochem. J. (1983) 211, 13-22) was found to contain mucin-fibronectin-DNA complexes. The general picture that emerged from the studies is that the pure mucin consists of 60 kDa glycosylated peptides only. The carboxyl terminal (8-12 kDa fragment) of these peptides is not glycosylated (naked) and is responsible for mucin interaction with fibronectin and other fibronectin-like extracellular matrix proteins. While the formation of the mucosal coat depends on many other factors and extracellular components, our findings on mucin structure and interaction with the extracellular matrix proteins provide explanation as to the possible mechanism of mucin adherence to the epithelial surfaces.     

A fibronectin-binding glycoprotein from human platelet membranes.

Fibronectin ('cold-insoluble globulin') has been suggested as a possible mediator of platelet adhesion. A fibronectin-binding protein as partially purified from washed solubilized human platelet membranes by affinity chromatography on fibronectin-Sepharose. The isolated protein migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an Mr (relative molecular mass) of approx. 125 000 under reducing conditions. The protein migrated as a dimer in non-reduced gels. The purified protein did not react with immunoglobulins against fibrinogen or fibronectin when tested in crossed immunoelectrophoresis or electroimmunoassay. The protein and purified fibronectin formed a complex that had a significantly faster mobility in crossed immunoelectrophoresis than did native fibronectin. The presence of heparin in the binding-protein-fibronectin mixture resulted in an even faster mobility of the complex, whereas the mobility of native fibronectin was unaffected. Crossed affinoimmunoelectrophoresis of the complex using different lectins suggested that the binding protein is a glycoprotein containing N-acetylglucosamine residues. The complex, but not purified fibronectin, bound to phenyl-Sepharose on crossed hydrophobic-interaction immunoelectrophoresis. The results strongly suggest the presence of a fibronectin-binding glycoprotein in the platelet membrane.     

Comparison of physicochemical properties of purified mucus glycoproteins isolated from respiratory secretions of cystic fibrosis and asthmatic patients

The major nonreduced mucus glycoproteins (mucins) from sputa of cystic fibrosis (CF) and asthmatic patients have been purified to electrophoretic homogeneity and subjected to physical and chemical characterization. The sputum specimens were solubilized in buffer containing 0.22 M KSCN and fractionated on Bio-Gel A-5m, followed by digestion with DNase, rechromatography on the same column, and chromatography on hydroxylapatite. Sodium dodecyl sulfate gel electrophoresis of purified mucins gave a single band. Carbohydrate analyses of the purified mucins showed no significant differences in the sugar components from the two mucins. However, the CF mucin contained substantially higher (11%) sulfate content than that observed for the asthmatic mucin (5.9%). Amino acid analyses indicated that the CF mucin had higher levels of serine plus threonine (35%) as compared to the asthmatic mucin (29%). In contrast, CF mucin contained a lower content of aspartate, glutamate, and glycine than that observed for the asthmatic mucin. Molecular weights of 3.8 X 10(6) and 3.5 X 10(6) were obtained for CF and asthmatic mucins, respectively, from light-scattering studies of mucins in the presence of 6 M guanidine hydrochloride. Reduction of the disulfide bonds of the two mucins did not alter their molecular weights. Liquid chromatographic studies on Sepharose CL2B showed that CF mucin forms aggregates sufficiently large to be excluded from the gel. As compared to the CF mucin, the asthmatic mucin formed fewer of these large aggregates under identical experimental conditions. Reduction and alkylation of the mucins resulted in their inability to form aggregates. The higher state of aggregation of CF mucin may influence the viscoelastic properties of the CF lung's mucus secretions.     

Galactosyl transferases of baby hamster kidney (BHK) cells. Characterization of two oligosaccharide products synthesised using bovine asialo submaxillary-gland mucin as acceptor

Extracts of BHK (baby hamster kidney) cells catalyse incorporation of galactose from UDP-galactose into asialo bovine submaxillary gland mucin. The galactosylated oligosaccharide products were released by alkaline-borohydride treatment and purified by Bio-Gel P2 chromatography and high-performance liquid chromatography. The structures of the oligosaccharide sequences synthesised have been identified unequivocally by high resolution 500 MHz 1H-NMR as galactosyl-(beta 1----3) N-acetylgalactosamine and galactosyl (beta 1----4) N-acetylglucosaminyl (beta 1----3)-N-acetylgalactosamine. Characterization of the latter sequence shows the presence in bovine mucin of the type III core sequence N-acetylglucosamine-(beta 1----3) N-acetylgalactosamine. Fractionation of BHK cell extracts on alpha-lactalbumin-Agarose has shown that the (beta 1----4)-galactosyl transferase responsible for synthesis of the trisaccharide binds to alpha-lactalbumin, a modulator of the (beta 1----4)-galactosyl transferase involved in N-glycan assembly. The evidence that the same transferase activity may be responsible for galactose transfer to both O-glycans and N-glycans is discussed.     

Purification and characterization of UDP-GalNAc:polypeptide N-acetylgalactosamine transferase from an ascites hepatoma, AH 66

The membrane-bound UDP-GalNAc:polypeptide N-acetylgalactosamine transferase from an ascites hepatoma, AH 66, has been purified 48,100-fold, mainly by affinity chromatography in aqueous Triton X-100 on apomucin (deglycosylated bovine submaxillary mucin) coupled to Sepharose. The purified preparation behaved homogeneously on gel filtration on Sephadex G-150 in aqueous Triton X-100 and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of about 55,000. The enzyme requires Mn2+, and only UDP-GalNAc served as a sugar donor. Apomucin, A1 protein, kappa-casein, apofetuin, and apoantifreeze glycoproteins served as acceptors, but the rate and amount of the transfer varied considerably from one acceptor to another. The transfer reaction terminated at the level of glycosylation of from only a few to at most about 40% of the serine plus threonine residues from which mucin-type oligosaccharides had been removed. This indicates that the transferase requires a certain conformation surrounding the acceptor site, but suggests also that a special mechanism may be functioning in vivo for frequent glycosylation of the abundant serine plus threonine residues of mucins. Lacto-N-fucopentaose I, ceramide di- and trihexosides, and globoside were not acceptors.     

Highly purified, functionally active human fibronectin preparation

Fibronectin has been purified by gelatin-Sepharose affinity chromatography from fresh frozen human plasma. The bound fibronectin was eluted with 3 M urea. The purity of the fibronectin obtained has been checked on (immunoelectrophoresis, polyacrylamide gel electrophoresis, FPLC). Biological activity of the purified molecule has been monitored by means of three assays: quantitation of the gelatin-binding activity by ELISA, quantitation of the fibronectin-mediated attachment of fibroblasts on plastic and evaluation of the opsonic activity (uptake of gelatin latex particles by a murine macrophage line). When deep-frozen, fibronectin retains all of its properties. This highly purified and functional fibronectin fulfills the basic requirements for a standard reagent. It will allow to investigate physicochemical and functional alterations of various fibronectins.     

Purification and characterization of an agglutinin from mucus of the snail Achatina fulica

The mucus of the snail Achatina fulica shows the presence of an agglutinin that nonspecifically agglutinates human erythrocytes. The agglutinin has been purified by affinity chromatography using Sepharose 4B-hog gastric mucin as the affinity matrix. Homogeneity was checked by polyacrylamide gel electrophoresis, immunodiffusion, immunoelectrophoresis, and gel filtration. The agglutinin is a glycoprotein of native molecular weight 70,000. The isoelectric point of the protein was found to be 8.0. The predominant amino acids are aspartic acid and glutamic acid (or amides) and serine, which account for 32% of the total amino acid residues. The agglutinin has 10% carbohydrate (wt/wt) and the most abundant sugar is N-acetylglucosamine. The cd spectra of the agglutinin show the presence of random coil conformation. The inhibition of hemagglutination data indicates that the agglutinin is specific for beta glycosides of D-Gal and D-GalNAc.     

Ca2+-binding protein from human kidney. Purification and properties.

A Ca2+-binding protein (CaBP) from human kidney was purified by two different procedures. The first involved heat-precipitation of a kidney cytosol fraction followed by gel filtration and chromatofocusing. This resulted in a 200-fold increase in the specific Ca2+-binding activity with a yield of 10%. A specific antibody was raised against the purified CaBP, as demonstrated by one precipitate in crossed immunoelectrophoresis of a kidney cytosol fraction. The antibody was coupled to Sepharose 4B and CaBP was then purified by immunoadsorbent chromatography. Applying this technique, a 500-fold purification of CaBP with a yield of 50% was obtained. Both preparations appeared homogeneous in crossed immunoelectrophoresis against a polyvalent antiserum and migrated as a single band corresponding to a mol.wt. of 26000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In gel filtration under non-denaturing conditions CaBP was eluted corresponding to a mol.wt. of 28000. The association constant for the high-affinity Ca2+-binding sites of CaBP was estimated by gel filtration to be 0.1 X 10(6)M-1, and the protein displayed Ca2+-dependent electrophoretic mobility, with more rapid anodic migration in the presence of EDTA. The protein eluted at a position corresponding to a pI of 4.5 in chromatofocusing. Immunochemical experiments with the specific antibody showed no cross-reaction between renal and intestinal CaBP.     

Inhibition of urokinase by complex formation with human alpha1-antitrypsin.

Human alpha1-antitrypsin was prepared from fresh human plasma by (NH4)-SO4-precipitation, gel filtration, affinity chromatography on concanavalin A, ion exchange chromatography and isotachophoresis. Human urokinase (EC 3.4.99.26) (plasminogen activator from urine) with M, 46 000 and 36 000 was further purified from Urokinase Leo reagent preparation by gel filtration on Sephadex G-100 Superfine. The hydrolytic activity of urokinase on acetyl-glycyl-L-lysine methyl ester acetate (Ac-Gly-Lys-OMeAc) was inhibited in a strong time-dependent manner by alpha1-antitrypsin. Complex formation between enzyme and inhibitor could be demonstrated in crossed immunoelectrophoresis against anti-alpha1-antitrypsin and anti-urokinase serum as well as by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The latter method revealed the formation of 1:1 and 2:1 molar enzyme-inhibitor complexes.     

Further characterization of the cold agglutinin from the snail Achatina fulica.

The cold agglutinin from the albumin gland of the snail Achatina fulica was purified to homogeneity by using sheep gastric mucin-Sepharose 4B as affinity column followed by gel filtration on Bio-Gel P-300. The homogeneity was checked by alkaline gel electrophoresis, immunodiffusion and immunoelectrophoresis. The purified cold agglutinin is a glycoprotein of native M2 220,000 consisting of three non-covalently bound subunits of Mr 84,000, 74,000 and 62,000 and having a pI value of 4.5. The predominant amino acids are aspartic acid and glutamic acid (or amides) and serine, which account for 39% of the residues. About 3% of the residues are half-cystine. The lectin is a glycoprotein with about 30.7% carbohydrate, the most abundant sugars being galactose, N-acetylgalactosamine and N-acetylglucosamine. Mannose, xylose and fucose are also present. The inhibition of agglutination of human umbilical-cord erythrocytes by the cold agglutinin is specific for methyl beta-D-galactoside and also for glycolipids present on cord erythrocytes. The c.d. data show only negative ellipticity values in the far-u.v. region for the protein at various concentrations and temperatures and also in the presence of the hapten lactose (at different concentrations), indicating the presence of a random-coil conformation in the agglutinin that varies according to temperature.     

Rapid, simplified method for production and purification of tetanus toxin

A rapid, simplified method for production and purification of tetanus toxin from bacterial extracts was described. The extracts were prepared by stirring young cells (ca. 45-h culture) of Clostridium tetani in 1 M NaCl-0.1 M sodium citrate, pH 7.5, overnight at 0 to 4 degrees C. The toxin was purified by a combination of (i) ammonium sulfate fractionation (0 to 40% saturation), (ii) ultracentrifugation for removal of particulate materials, and (iii) gel filtration by high-pressure liquid chromatography on a TSK G3000 SW-type column. This method required 6 days as follows: (i) overnight incubation of the seed culture, (ii) 2 days for growing the bacteria for toxin production, (iii) overnight extraction of the toxin from the bacteria, (iv) overnight precipitation of the toxin with ammonium sulfate, (v) 2 h for ultracentrifugation of the ammonium sulfate concentrate of the bacterial extract, and (vi) 1 h for high-pressure liquid chromatography. The minimum lethal dose of the purified toxin preparations for mice was 1.4 X 10(7) to 1.5 X 10(7) per mg of protein and they showed 360 to 390 Lf (flocculating activity) per mg protein and a 280/260 nm absorbance ratio of 2.0 to 2.1. The final recovery of the toxin from bacterial extracts was 90 to 93%. The purified preparations gave a single band of toxin protein with a molecular weight of 150,000 +/- 5,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On crossed immunoelectrophoresis, the purified toxin preparations gave a single precipitation arc against anti-crude toxin serum.     

Rapid, simplified method for production and purification of tetanus toxin.

A rapid, simplified method for production and purification of tetanus toxin from bacterial extracts was described. The extracts were prepared by stirring young cells (ca. 45-h culture) of Clostridium tetani in 1 M NaCl-0.1 M sodium citrate, pH 7.5, overnight at 0 to 4 degrees C. The toxin was purified by a combination of (i) ammonium sulfate fractionation (0 to 40% saturation), (ii) ultracentrifugation for removal of particulate materials, and (iii) gel filtration by high-pressure liquid chromatography on a TSK G3000 SW-type column. This method required 6 days as follows: (i) overnight incubation of the seed culture, (ii) 2 days for growing the bacteria for toxin production, (iii) overnight extraction of the toxin from the bacteria, (iv) overnight precipitation of the toxin with ammonium sulfate, (v) 2 h for ultracentrifugation of the ammonium sulfate concentrate of the bacterial extract, and (vi) 1 h for high-pressure liquid chromatography. The minimum lethal dose of the purified toxin preparations for mice was 1.4 X 10(7) to 1.5 X 10(7) per mg of protein and they showed 360 to 390 Lf (flocculating activity) per mg protein and a 280/260 nm absorbance ratio of 2.0 to 2.1. The final recovery of the toxin from bacterial extracts was 90 to 93%. The purified preparations gave a single band of toxin protein with a molecular weight of 150,000 +/- 5,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On crossed immunoelectrophoresis, the purified toxin preparations gave a single precipitation arc against anti-crude toxin serum.     

Altered O-glycosylation and sulfation of airway mucins associated with cystic fibrosis

Cystic fibrosis (CF) is the most lethal genetic disorder in Caucasians and is characterized by the production of excessive amounts of viscous mucus secretions in the airways of patients, leading to airway obstruction, chronic bacterial infections, and respiratory failure. Previous studies indicate that CF-derived airway mucins are glycosylated and sulfated differently compared with mucins from nondiseased (ND) individuals. To address unresolved questions about mucin glycosylation and sulfation, we examined O-glycan structures in mucins purified from mucus secretions of two CF donors versus two ND donors. All mucins contained galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), fucose (Fuc), and sialic acid (Neu5Ac). However, CF mucins had higher sugar content and more O-glycans compared with ND mucins. Both ND and CF mucins contained GlcNAc-6-sulfate (GlcNAc-6-Sul), Gal-6-Sul, and Gal-3-Sul, but CF mucins had higher amounts of the 6-sulfated species. O-glycans were released from CF and ND mucins and derivatized with 2-aminobenzamide (2-AB), separated by ion exchange chromatography, and quantified by fluorescence. There was nearly a two-fold increase in sulfation and sialylation in CF compared with ND mucin. High performance liquid chromatography (HPLC) profiles of glycans showed differences between the two CF samples compared with the two ND samples. Glycan compositions were defined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Unexpectedly, 260 compositional types of O-glycans were identified, and CF mucins contained a higher proportion of sialylated and sulfated O-glycans compared with ND mucins. These profound structural differences in mucin glycosylation in CF patients may contribute to inflammatory responses and increased pathogenesis by Pseudomonas aeruginosa.     

Ox spleen ferritin: an isoelectrofocusing and crossed immunoelectrofocusing study

Ox spleen ferritin was purified and its purity checked by two-dimensional immunoelectrophoresis and polyacrylamide plate electrophoresis. Microheterogeneity was shown with a preparation of purified ferritin by isoelectric focusing. The protein was separated into at least 6 fractions; two large fractions in the 4.50-4.55 pH range and another 4 in the 4.65-4.80 interval. Microheterogeneity was confirmed in purified preparations by crossed immunoelectrofocusing. Seven fractions were observed, the most acid ones (4.50-4.55) also being the most abundant. In the crossed IEF procedure, exactness in the isoelectrophoretic separation time is important in that excessive time may impair the resolution potential.