Developmental changes of ferret tracheal mucin composition and biosynthesis

We characterized the chemical composition of mucins secreted by ferret tracheal explants and the activities of key mucin glycosyltransferases in ferret tracheal epithelium during a period of rapid postnatal maturation of the mucin-secreting structures. Ferret tracheal explants secrete three major groups of high molecular weight glycoconjugates: (1) those susceptible to bovine testicular hyaluronidase; (2) those resistant to hyaluronidase and exhibiting high density (p greater than or equal to 1.60 g/mL); and (3) those resistant to hyaluronidase and exhibiting low density (1.45 less than or equal to p less than 1.60 g/mL). The hyaluronidase-resistant, low-density glycoconjugates have typical mucin properties and constitute 36% of total glycoconjugates released in newborns but only 8% in adult ferrets. Mucin secretory rate per unit surface area of trachea progressively decreases with age. Mucin amino acid and total carbohydrate contents do not vary; however, the sialic acid content increases, and fucose content as well as blood group A activity of the mucins decreases with age. Four glycosyltransferases involved in mucin biosynthesis [Gal beta 3GalNAc:(GlcNAc-GalNAc)beta 6 N-acetylglucosaminyl-, GalNAc:beta 3 galactosyl-, Gal:alpha 2 fucosyl-, and GalNAc alpha 2----6 neuraminyltransferase] are present in tracheal epithelium of ferrets at all ages. Activities of all but the neuraminyltransferase decrease with age. The relatively greater neuraminyltransferase activity is consistent with increased incorporation of sialic acid into secreted mucins over the same age span. Conversely, diminution of fucosyltransferase relative to galactosyltransferase activity may contribute to the lower fucose content and lower blood group A activity of mucins secreted by mature ferret tracheas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pseudomonas aeruginosa binds to neoglycoconjugates bearing mucin carbohydrate determinants and predominantly to sialyl-Lewis x conjugates.

Pseudomonas aeruginosa plays an important role in the colonization of the airways of patients suffering from cystic fibrosis. It binds to the carbohydrate part of respiratory and salivary mucins and its binding to cystic fibrosis mucins is even higher, suggesting that qualitative or/and quantitative modifications of the carbohydrate chains may be involved in this process. In order to find out the best carbohydrate receptors for P.aeruginosa, a flow cytometry technique using a panel of polyacrylamide based glycoconjugates labeled with fluorescein was developed. The neoglycoconjugates contained neutral, sialylated or sulfated chains analogous to carbohydrate determinants found at the periphery of respiratory mucins (Le(a), Le(y), Le(x), sialyl- and 3'-sulfo-Le(x), and blood group A determinants). We used also neoglycoconjugates containing Gal(alpha1-2)Galbeta and sialyl- N -acetyllactosamine determinants. The interaction of these glycoconjugates with the nonpiliated strain of P.aeruginosa, 1244-NP, was saturable except for the glycoconjugates containing blood group A or sialyl- N -acetyllactosamine epitopes. The measure of Kd indicated that strain 1244-NP had a higher affinity for the glycoconjugate bearing the sialyl-Le(x)determinant than for all the other glycoconjugates studied. The role of sialic acid was confirmed by competition assay using mainly sialylated mucin glycopeptides. In order to find out if this behavior was the same for pathological strains as for the 1244-NP mutant, four mucoid strains of P.aeruginosa isolated from cystic fibrosis patients were analyzed with the Le(x)neoglycoconjugate, its sialylated and its sulfated derivatives. Individual variations in the binding of these strains to the three glycoconjugates were observed. However, three strains out of four had a higher affinity for the sialyl-Le(x)than for the 3'-sulfo-Le(x)derivative.     

Mitochondrial DNA polymerase, deoxyribonuclease and ribonuclease H activities from brain of chick embryo

R-DNA polymerase, D-DNA polymerase, DNase and RNase H activities in mitochondria from chick embryonic brain were studied by ion-exchange chromatography. Two main fractions were separated according to their chromatographic behaviour: a fraction M Ib which is eluted with the washing buffer from two successive DEAE-cellulose columns and a fraction M IV which is eluted at 400 mM KC1 from a phosphocellulose column. Although the two fractions contain both the DNA polymerase and the degrading activities, all the specific activities are higher in fraction M IV than in fraction M Ib. Heat inactivation experiments have shown that R-DNA polymerase is inactivated in both fractions, whereas RNase H and DNase are not affected. Thus, degrading activities and R-DNA polymerase activity seem to be catalyzed by different molecular entities. However the fact that in most cases these activities co-chromatograph suggests that the corresponding molecules form rather stable complexes.     

Sialoglycopeptides isolated from bovine aorta.

Intima-media of bovine aorta was digested with pronase, after preliminary extraction of saline (1%)-soluble substances and fat. Crude glycopeptide fraction was then obtained from the resulting complex carbohydrate fraction by fractionation with CPC (cetylpyridinium chloride). Complete separation of sialoglycopeptides was achieved by chromatography on a DEAE-cellulose column at pH 7.2 followed by repeated chromatography on a DEAE-Sephadex A-25 column at pH 5.2. Nine sialoglycopeptides (SGP 1-SGP 9) thus obtained were homogeneous on high-voltage paper electrophoresis at pH 3.5 and pH 5.2. The analytical data showed great heterogeneity of the carbohydrate chains of these preparations, although they consisted of the same monosaccharides (galactose, glucose, mannose, glucosamine, galactosamine, fucose, and sialic acid), except that SGP 1 lacked galactosamine. Heterogeneity was also observed in their peptide chains. It was noticed, however, that the contents of hexose, hexosamine, and aspartic acid of the fractions (SGP 3, SGP 4, and SGP 5) which eluted from the DEAE-Sephadex A-25 column at lower molarity of the eluting salt were higher than those of the fractions (SGP 7, SGP 8, and SGP 9) which eluted at higher molarity, while the contents of sialic acid and hydroxyamino acids were in an opposite relationship. Representative fractions (SGP 7 and SGP 9) of the latter contained many more alkali-sensitive linkages than those (SGP 3 and SGP 5) of the former, indicating the presence of many more O-glycosidic linkages between hydroxyamino acid(s) and sugar(s) in the latter than in the former. The sialoglycopeptides contained significant amounts of sialic acid, ranging from 10% (sgp 1) to 32.4% (SGP 8). The highest contents were in SGP 8 and SGP 9, which contained equimolar amounts of sialic acid and hexosamine. Furthermore, infrared spectra indicated the presence of sulfate groups in most of the sialoglycopeptides.     

Isolation and partial characterization of two glycoproteins from human liver metastases of sigmoid colon carcinoma

1. Perchloric acid-soluble glycoprotein fraction (PASF) extracted from human liver metastases (LM) of sigmoid colon carcinoma was chromatographed on a DEAE-cellulose column. The main fraction (DEAE-nonadsorbed fraction) passed through the column was then subjected to Sephacryl S-200 superfine gel filtration and separated into 12 fractions. 2. Among 12 fractions, only both Fractions 3 and 4 were demonstrated to be chemically and immunologically homogeneous glycoproteins, respectively, by a combination of chemical composition analysis, SDS-PAGE and EITB assay using antisera against the DEAE-nonadsorbed fractions of PASFs from human LMs, normal liver (NL) and normal sigmoid colon (NSC). Each of Fractions 3 and 4 reacted with anti-LM serum to give one immuno complex on a nitrocellulose sheet in EITB assay, but did not react with anti-NL and -NSC sera. 3. Apparent molecular weights of 80,900 and 62,100, respectively, were found for Fractions 3 and 4. Both the fractions, respectively, had abnormal sugar compositions. Fraction 3 contained sialic acid, fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine, but lacked glucose and mannose, and Fraction 4 contained sialic acid, fucose, galactose and N-acetylglucosamine, but lacked glucose, mannose and N-acetylgalactosamine, as sugar components.     

Physicochemical properties of avian tracheal mucus.

Dual-radiolabelled avian tracheal secretions were obtained by giving Na235SO4 and D-[1-3H]glucosamine simultaneously into the lumen of the trachea in preparations in vitro. These secretions comprised fibrillar, gelatinous and soluble-phase mucins. These were eluted as single components in the non-retarded fractions from Bio-Gel A-15m. Although no evidence of the presence of subunit structure was found, chemical and radiolabelling analyses showed a high degree of internal inhomogeneity among the three types of mucins. The differences among these mucins could be attributed to the chemical nature of their constituent glycoproteins. Glycoprotein fractions separated by ion-exchange chromatography were found to contain sulphate and N-acetylneuraminic acid residues in differing amounts. The overall acidic properties appeared to be correlated with ester sulphate content. A close similarity in the carbohydrate composition and a reciprocal relationship between the total ester sulphate residue contents and 35S- and 3H-labelling suggested that, in addition to stepwise glycosylation and sulphation, some pre-existing sulphated oligosaccharides might have been utilized for the synthesis of acidic glycoproteins.     

Purification and characterization of a human pancreatic adenocarcinoma mucin.

Pancreatic mucins consist of core proteins that are decorated with carbohydrate structures. Previous studies have identified at least two physically distinct populations of mucins produced by a pancreatic adenocarcinoma cell line (HPAF); one is the MUC1 core protein, which includes an oligosaccharide structure identified by a monoclonal antibody (MAb) recognizing the DU-PAN-2 epitope. In this study, we purified and characterized a second mucin fraction, which also shows reactivity with the DU-PAN-2 antibody, but which has an amino acid composition that is not consistent with the MUC1 core protein. This new mucin was purified by ammonium sulfate precipitation, molecular sieve chromatography, and density gradient centrifugation. It eluted in the void volume of a Sepharose 4B column together with an associated low molecular weight protein, which could be further resolved. The mucin is highly polyanionic due to numerous sulfated and sialylated saccharide chains. Carbohydrate analyses of the purified mucin showed the presence of galactose, glucosamine, galactosamine, and sialic acid, but no mannose, glucose, or uronic acid. The purified and deglycosylated mucin shows no reactivity with anti-MUC1 apomucin antibody, but reacts with antiserum against deglycosylated tracheal mucins and antiserum against the MUC4 tandem repeat peptide. Analysis of mucin expression in HPAF cells revealed high levels of MUC1 and MUC4 mRNA, and moderate levels of MUC5AC and MUC5B mRNA. The amino acid composition of the purified mucin shows a high degree of similarity to the MUC4 core protein.     

Analysis of rat uterine estrogen receptors by high-pressure liquid chromatography methods

Cytosolic (ERc) and nuclear (ERn) estrogen receptors prepared from rat uteri were characterized by size-exclusion and ion-exchange HPLC. The oligomeric ERc eluted as a single, sharp peak near the exclusion volume of the gel column; ERn eluted as a broad peak. When salt-extracted ERn was partially purified sequentially by Sephadex G-200, DEAE-cellulose chromatography and polyacrylamide gel electrophoresis, the partially purified receptor moieties were not distinguishable by the sucrose gradient method, but showed characteristic retention times in the size-exclusion HPLC column. Further distinction in net surface charges was observed between ERc and ERn moieties by ion-exchange high-pressure liquid chromatography (HPLC). Molybdate-stabilized ERc was eluted as sharp peak at 0.27 M salt gradient. In contrast, fresh extracts of ERn emerged as a broad peak in the region of 0.1-0.2 M salt gradient. In the absence of molybdate, ERc dissociated into several 4-5 S molecules, which were well resolved in the DEAE column. This report, therefore, demonstrates the usefulness of size-exclusion and ion-exchange HPLC for steroid receptor analysis.     

Characterization of mono-, di-, and tri-O-acetylated sialic acids on human cells

The presence of mono-, di-, and tri-O-acetylated sialic acids on human cells was demonstrated by using radiochromatographic and chemical techniques. Human melanoma cells and fresh colon tissue were biosynthetically labeled with 6- (3H) glucosamine. Radiolabeled sialic acids were hydrolytically removed from cellular glycoconjugates, purified by ion-exchange chromatography, and separated by paper chromatography on the basis of the number of O-substitutions on each sialic molecule. This analytical technique characterized radiolabeled sialic acids that migrated with the same Rf as synthetic mono-, di-, and tri-O-acetylated 14C-labeled sialic acids. The mono-O-acetylated sialic acids were characterized by their sensitivity to sodium periodate oxidation and a crude mouse liver esterase preparation. The di- and tri-O-acetylated sialic acids were characterized by their resistance to sodium periodate oxidation and sensitivity to the action of crude mouse liver esterase. Chromatographically separated di- and tri-O-acetylated sialic acids from normal human colon tissue were characterized by their respective ion molecular weights by using fast-atom bombardment-mass spectrometry. Using these methods, we chemically characterized mono, di-, and tri-O-acetylated sialic acids expressed on human cells. Aberrant expression of O-acetylated sialic acids was associated with adenocarcinoma of the colon, leading to a nearly complete loss of di- and tri-O-acetylated sialic acids.     

Purification and characterization of two forms of soluble NADH cytochrome b5 reductases from human erythrocytes.

1. Two forms of soluble NADH cytochrome b5 reductase were purified from human erythrocytes. Two distinct fractions both having the NADH cytochrome b5 reductase activity eluted from the second DEAE-cellulose column were further purified by ultrafiltration and 5'-ADP-agarose affinity chromatography. 2. The final preparations were purified 9070- and 4808-fold, respectively, over hemolysate. Both reductases exhibited identical electrophoretic patterns when subjected to SDS-PAGE and apparent monomer Mr of each reductase was determined to be 32,000 +/- 1300. 3. Vmax values of reductase II for the various electron acceptors, namely, 2,6-dichlorophenolindophenol, ferricyanide and cytochrome c through cytochrome b5 were found to be 1.9, 1.8 and 2 times higher than those of reductase I. 4. Some differences were noted for reductase I and reductase II fractions. Their elution profiles from a second DEAE-cellulose column were quite different and that suggested that reductase II is more acidic than reductase I. Reductase II was found to be more sensitive to heat treatment than reductase I.     

Comparative rates of sialylation by recombinant trans-sialidase and inhibitor properties of synthetic oligosaccharides from Trypanosoma cruzi mucins-containing galactofuranose and galactopyranose

The mucin-like glycoproteins of Trypanosoma cruzi have novel O-linked oligosaccharides that are acceptors of sialic acid in the trans-sialidase (TcTS) reaction. The transference of sialic acid from host glycoconjugates to the mucins is involved in infection and pathogenesis. The O-linked chains may contain galactofuranose in addition to the acceptor galactopyranose units. Thus far, the galactofuranose form was found in the mucins of strains belonging to the less infective lineage. The acceptor properties of the chemically synthesized oligosaccharides were now studied in order to correlate their structure with the ability to act as substrates. Recombinant TcTS and sialyllactose as donor were used. The reactions were followed by HPAEC-PAD. The K(m) values were calculated for the free sugars, the sugar alditols and the benzyl glycosides. All the compounds showed to be good acceptors of sialic acid. Thus, the introduction of galactofuranose in the mucins of the strains of lineage 1 would not be responsible for the diminished virulence of the strains. The oligosaccharides and derivatives inhibited the transfer of sialic acid to the substrate N-acetyllactosamine with IC(50) values between 0.6 and 4 mM.     

Characterization of Fucosyl Oligosaccharides Associated with Synaptic Membrane and Synaptic Junctional Glycoproteins

Rats were administered [3H]fucose by intracranial injection and synaptic membranes (SMs) isolated 18 h later. Oligosaccharides associated with SM glycoproteins were prepared by hydrazinolysis and analyzed by a combination of affinity chromatography on concanavalin A (Con A)-agarose, ion-exchange chromatography on DEAE-cellulose, and gel permeation chromatography. Most (94%) of the [3H]fucose-labelled oligosaccharides were present in the fraction that did not bind to Con A. Of these 41% did not bind to DEAE-cellulose, indicating the absence of negatively charged groups and the remainder were resolved into four fractions of increasing acidity. Gel permeation chromatography of the fractions from the DEAE-cellulose column suggested that the major oligosaccharides corresponded to fucosylated triantennary structures containing varying amounts of sialic acid although more highly branched structures containing peripheral branches lacking one or more sugars may also have been present. Comparison of fucosyl oligosaccharides associated with SMs prepared from 10- and 28-day-old animals indicated that although the general oligosaccharide content was similar at both ages, membranes from younger animals were characterized by an increase in the proportion of highly acidic structures. Fucosylated glycans derived from synaptic junctional (SJ) glycoproteins were also characterized by a greater percentage of highly acidic structures than SMs. The results indicate that SMs and SJs are characterized by specific complements of fucosylated glycoprotein oligosaccharides.     

Histochemical alterations of mucin in normal colon, inflammatory bowel disease and colonic adenocarcinoma

Summary Loss of sialic acid o-acyl substitutions in colonic mucus was studied using specific histochemical techniques in individuals with a variety of large-bowel diseases and in a control population. Changes found included a focal or field (diffuse) loss of side-chain substitutions which were qualitatively similar in all groups studied. The results were tested statistically using a variety of assumptions that field and/or focal loss of o-acyl substitution may be either abnormal or a normal variant. No statistically significant differences in the prevalence of substitutions were detected between normal males and females or between normal individuals aged 0–29 years and 30–80 years. Significant differences were found between ascending and descending colon in both normal individuals and in the non-neoplastic mucosa of patients with cancer. There were also significant differences between the normal descending colon and cases with cancer of the descending colon. These differences seem unlikely to be due to non-specific factors, since for most assumptions there were also differences between colons containing cancer and those from patients with inflammatory bowel disease. In agreement with the work of other investigators, it seems likely that focal loss of o-acetylation results from an acquired gene mutation. It is not clear whether or not this plays a role in carcinogenesis. Deceased 21 May, 1994.     

A Simple Device for Assuring High Reproducibility in Fluorometric Measurement of Deoxyribonucleic Acid in Yeast Cells

The heterogeneity and chemical composition were investigated in κ-casein from colostrum. The acid casein was obtained from four different Holstein cow colostra. The yield of acid casein from colostrum was higher than that from normal milk. κ-Casein from colostrum was prepared by the gel filtration method of Yaguchi et al. The gel filtration profiles differed among the four colostrum acid caseins.

Colostrum κ-casein was fractionated on a DEAE-cellulose column into one nonadsorbed and six adsorbed fractions with increasing salt concentration. Six adsorbed fractions had the same molecular weight and stabilizing ability for α_s1-casein in the presence of calcium ion. The amino acid composition and the phosphorus content of the adsorbed fractions were identical, but fractions eluted with high salt concentrations had more carbohydrates (galactose, sialic acid, glucosamine, galactosamine). Colostrum κ-casein was characterized by a higher content of carbohydrate moiety in comparison with normal κ-casein. Also glucosamine which has not been found in normal κ-casein was detected in colostrum κ-casein. The κ-casein component from colostrum contained at least one molecule of carbohydrate, though the carbo hydrate-free component was detected in normal κ-casein.     

Glycopeptides in pus of acute pleurisy patients

Glycopeptides were isolated from tryptic digests of pus from acute pleurisy patients. The hexose, hexosamine, and sialic acid contents rose over the first 2-4 days after admission to the hospital, continued at high levels for 5-8 hospital days, and fell to low levels after 9 hospital days. The course of duration after admission to the hospital was divided into three stages: 1-4 days after admission to the hospital, 5-8 hospital days, and 9-21 hospital days. Materials corresponding to these three stages were then collected for fractionation by DEAE-cellulose column chromatography. Fractionation of glycopeptides by DEAE-cellulose column chromatography yielded three glycopeptide fractions at 0.05 to 0.2 M NaCl. Column chromatography of crude material on DEAE-cellulose showed an increase in the 0.2 M NaCl fraction from the concentrate over a period of 4-8 hospital days due to a large increase in sialic acid-rich glycopeptide fraction.     

Fractionation of sialyl oligosaccharides of human milk by ion-exchange chromatography

The sialyl oligosaccharides of human milk are partially separated by ion-exchange chromatography on DEAE-cellulose in acetic acid-pyridine buffers, pH 5.4. One neutral and seven sialyl oligosaccharide fractions are obtained in essentially quantitative yields. The method permits the large-scale preparation of sialyl oligosaccharide fractions from which individual sialyl oligosaccharides may be purified without the necessity of preparative high-voltage paper electrophoresis.     

Purification and chemical characterization of human hexosaminidases A and B.

N-Acetyl-beta-hexosaminidases A and B were purified to homogeneity from human placenta. In the initial step of purification, the enzymes were adsorbed on concanavalin A-Sepharose 4B and eluted from the column with alpha-methyl D-mannosides. Subsequent purification steps included DEAE-cellulose column chromatography, QAE-Sephadex [diethyl-(2-hydroxypropyl)aminoethyl-Sephadex] column chromatography, Sephadex G-200 gel filtration and preparative disc polyacrylamide-gel electrophoresis, followed by another QAE-Sephadex chromatography for the hexosaminidase A preparation, and DEAE-cellulose column chromatography, calcium phosphate gel chromatography, Sephadex G-200 gel filtration, QAE-Sephadex chromatography and CM-cellulose chromatography for the hexosaminidase B preparation. The purified preparations, particularly hexosaminidase A, had significantly higher specific enzyme activities than previously reported. The preparations moved on polyacrylamide-gel electrophoresis as single protein bands, which also stained for enzyme activity. Sedimentation-equilibrium centrifugation indicated homogenous dispersion of the enzymes, and the molecular weight was estimated as about 110000 for both enzymes. Complete amino acid and carbohydrate compositions of the two isoenzymes were determined, and, in contrast with previous suggestions, no sialic acid was found in the enzymes.     

Synthesis of lactosaminoglycan-containing glycoproteins by vascular endothelial cells

Human vascular endothelial cells synthesize lactosaminoglycan-type glycoproteins which are found both associated with cells and secreted into the culture medium. Pronase-derived glycopeptides prepared from [³H]glucosamine-labeled glycoproteins were found to contain about 10% of the labeled products as a large size (M_r > 5000) ³H-labeled glycopeptide. Digestion of these ³H-labeled glycopeptides with endo-β-galactosidase resulted in the release of smaller size saccharides, which were characterized as having the structure sialic acid → Gal → GlcNAc → Gal. Treatment of [³H]glucosamine-labeled cells with melittin caused ³H-labeled glycoconjugates to be released from the cells. Separation of released glycoproteins from proteoglycans by DEAE-cellulose chromatography indicated that melittin had released 25% of the total ³H-labeled glycoproteins from the cell and 3% of the ³H-labeled proteoglycans. The ³H-labeled glycoproteins were digested with Pronase and the resulting ³H-labeled glycopeptides were fractionated on Sephadex G-50. The large size fraction (M_r > 5000) now comprised about 30% of these released ³H-labeled glycopeptides. These high molecular weight ³H-labeled glycopeptides were degraded with endo-β-galactosidase but not with testicular hyaluronidase. Analysis of the released ³H-labeled glycoproteins indicated a preferential release of glycoproteins of 70–90 kDa enriched in lactosaminoglycan-type oligosaccharides.     

Multiple forms of calpastatin in pig brain

Pig brain was found to contain two calpain-specific, heat-stable inhibitory fractions which could be separated by DEAE-cellulose chromatography. CS-0.1, which was eluted from the column at 0.1 M NaCl, was identified as an ordinary, well-known calpastatin. CS-0.2, eluted at 0.2 M NaCl, was different from CS-0.1 in that it inhibited calpain 1 more strongly than calpain II and that it did not cross-react with anti-calpastatin antibodies. Partial purification indicated that CS-0.2 contained inhibitor proteins smaller than ordinary calpastatin, but whether they are the products derived from CS-0.1 or entirely different genetic products has not yet been determined.