Analysis of hexosaminitol-containing disaccharide alditols from rat brain glycoproteins and gangliosides as O-trimethylsilyl derivatives by gas chromatography mass spectrometry

The O-trimethylsilyl derivatives of five reference disaccharide alditols composed of a N-acetylhexosaminitol substituted at C-3, C-4 or C-6 with a hexose moiety were studied by gas chromatography mass spectrometry. The data were used in the determination of the linkage in disaccharide alditols derived from rat brain glycoproteins and gangliosides. The O-glycosidically linked carbohydrate units of brain glycoproteins contained two oligosaccharides, alpha-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol and beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol, whereas only the latter was obtained from brain gangliosides after partial acid hydrolysis and reduction.     

Electrophoretic studies of the cystic fibrosis ciliary inhibitor and its interaction with immunoglobulin G

The cystic fibrosis ciliary inhibitor (CFCI) has been partially purified from serum and plasma of cystic fibrosis (CF) homozygotes and heterozygotes, and from media of cultured fibroblasts derived from cystic fibrosis genotypes. Characterization and comparison of fractions containing the CFCI were carried out by polyacrylamide gel electrophoresis. Gel electrophoresis confirmed previous molecular weight estimations of 4,500 to 11,000 for the CFCI and provided an estimate of the number of proteins present in the fractions. Low molecular weight proteins from serum and media were combined with IgG preparations. No specific binding to IgG by the media fraction containing the CFCI could be demonstrated by the techniques employed. There was decreased binding of the low molecular weight serum fraction containing CFCI to native IgG molecules from cystic fibrosis patients as compared to IgG from normal individuals. However, IgG from CF individuals demonstrated increased binding of the cfci-containing low molecular weight serum fraction after gel filtration in the presence of guanidinium chloride. This suggests: 1) that very low concentrations of CFCI are present in media fractions; and 2) that native CF IgG cannot bind the low molecular weight CFCI fractions to the same degree as native IgG from normals or CF IgG that has been dissociated from non-covalently bound components.     

Heterogeneity of carbohydrate chains of acidic bronchial mucin isolated from the spatum of two subjects with chronic bronchitis

Acidic glycoproteins having blood group H activity were isolated from the sputum of two patients suffering from chronic bronchitis by reduction of the fibrillar mucus, chromatography on ECTEOLA-cellulose and gel filtration on Sepharose 4-B. These glycoproteins were degraded with alkaline borohydride and the degradation products were fractionated by chromatography on ionexchange resins and by gel filtration. The carbohydrate chains have a wide heterogeneity with regard to acidity and molecular size. Therefore carbohydrate chain heterogeneity which was already observed for bronchial glycoproteins isolated from a patient suffering from cystic fibrosis is not specific for cystic fibrosis.     

Ovine submaxillary mucin. Primary structure and peptide substrates of UDP-N-acetylgalactosamine:mucin transferase

Tryptic digests of ovine submaxillary apomucin were fractionated by gel filtration and ion exchange chromatography to give 14 peptide fractions. Three purified tryptic peptides, representing 106 of the 650 residues in apomucin, were submitted to automated sequence analysis. The NH2-terminal 50 of the 74 residues in one peptide and the entire sequence of the other two hexadecapeptides were established. These studies suggest that purified ovine submaxillary, mucin is chemically homogeneous, containing a unique primary structure without substantial repeating sequences in its polypeptide chain. The sequences adjacent to 28 known O-glycosidically substituted seryl and threonyl residues were compared. No homologies were apparent around the glycosylated seryl and threonyl residues which might define the specificity of the UDP-N-acetylgalactosaminyl:mucin polypeptide transferase that incorporates N-acetylgalactosamine into O-glycosidic linkage in glycoproteins. However, there appears to be a minimum size requirement for glycosylation, because the transferase catalyzes glycosylation of tryptic peptides efficiently, while chymotryptic and thermolytic peptides were much poorer substrates for the transferase.     

Deglycosylation by gaseous hydrogen fluoride of mucus glycoproteins immobilized on nylon membranes and in microtiter wells

Strongly reacting antibodies specific for defined mucin gene products are often directed against the mucin protein backbone of the heavily glycosylated serine/threonine rich regions. A prerequisite for the use of such antibodies is often the complete removal of the oligosaccharides from the protein. This paper describes an efficient one-step deglycosylation method using gaseous hydrogen fluoride on nylon blotting membranes and microtiter wells.     

Sulfated lewis X determinants as a major structural motif in glycans from LS174T-HM7 human colon carcinoma mucin

This article describes oligosaccharide structures of mucin isolated from nude mouse xenograft tumors produced by LS174T-HM7 cells, a subline of the human colon carcinoma LS174T with higher metastatic tendency and higher mucin production. A striking feature of the oligosaccharides of the LS174T-HM7 xenograft tumor mucin was a predominance of sulfated Lewis X determinants: HSO3-Galbeta1-4(Fucalpha1-3)GlcNAc. In addition to one previously known saccharide with one sulfated Lewis X determinant, the HM7 xenograft tumor mucin contained multiple novel structures containing one, two, or three sulfated Lewis X determinants. This determinant, known to act as a selectin ligand, has been found previously in minor saccharide components of human milk as well as mucins, but never before as a predominant structure in one mucin source.     

Real-time analysis of immunogen complex reaction kinetics using surface plasmon resonance

The role of the N-terminal sequence of myeloperoxidase in the intracellular targeting was examined by using glycosylated lysozyme as a reporter. A fusion protein was constructed in which the presequence residues-18 through -6 of the lysozyme moiety had been replaced by residues 1-158 of prepromyeloperoxidase. Expression of the fusion protein in Chinese hamster ovary cells demonstrated its partial secretion and partial intracellular retention. The latter was accompanied by trimming the myeloperoxidase prosequence off the lysozyme moiety. The rate of the retention of the lysozyme fusion protein was higher than that of glycosylated lysozyme that had been expressed in cells transfected with cDNA of glycosylated lysozyme. The retention was insensitive to NH4Cl. In the secreted protein, lysozyme contained predominantly complex oligosaccharides as demonstrated by a proteolytic fragmentation in vitro and resistance to endo-beta-N-acetylglucosaminidase H. In contrast, when targeted to lysosomes, the lysozyme moiety of the fusion protein contained predominantly mannose-rich oligosaccharides. In baby hamster kidney cells, the trimming of the oligosaccharides in the lysozyme fragment was less vigorous, and a selective targeting of molecules bearing mannose-rich oligosaccharides to lysosomes was more apparent than in Chinese hamster ovary cells. In the presence of monensin, the formation of complex oligosaccharides in the fusion protein and its secretion were strongly inhibited, whereas the intracellular fragmentation was not. We suggest that the prosequence of myeloperoxidase participates in the intracellular routing of the precursor and that this routing operates on precursors bearing mannose-rich rather than terminally glycosylated oligosaccharides and diverts them from the secretory pathway at a site proximal to the monensin-sensitive compartment of the Golgi apparatus.     

Mucin genes and the proteins they encode: structure, diversity, and regulation

Mucins are the structural components of the mucus gels that protect the respiratory, gastrointestinal, and reproductive tracts. These polydisperse glycoproteins (250,000 to 20,000,000 D) are approximately 80% carbohydrate on a mass basis and have a high intrinsic viscosity due to their large size and extreme hydrophilicity. Mucin oligosaccharides, the structures responsible for this hydrophilicity, are heterogeneous in size and structure but are chiefly O-linked, i.e., they initiate from N-acetylgalactosamine residues attached to threonine and serine residues of the polypeptide backbone. Our understanding of the structure of mucins has advanced rapidly in the last few years with the isolation and sequencing of cDNA clones that encode mucin polypeptide backbones. All currently well-characterized mucins have been found to contain extended arrays of tandemly repeated peptides rich in potential O-glycosylation sites. Less is known about the unique sequences that flank the tandem repeat arrays of secretory mucins, but currently available information indicates that these flanking regions contain cysteine-rich stretches that participate in mucin oligomer formation. Thus, secretory mucins appear to consist of oligomers containing heavily glycosylated domains flanked by unique sequences required for polymerization. Progress has also been made in characterizing the genes that encode mucins. At least four human mucin genes are known at present, although many others may remain to be discovered. Moreover, much work remains before we gain an understanding of the mechanisms involved in the expression of mucin genes and their tissue-specific regulation.     

An N-linked high-mannose type oligosaccharide, expressed at the major outer membrane protein of Chlamydia trachomatis, mediates attachment and infectivity of the microorganism to HeLa cells

The structure of the carbohydrate of the 40-kD major outer membrane component of Chlamydia trachomatis and its role in defining infectivity of the organism were investigated. The oligosaccharides were released from the glycoprotein by N-glycanase digestion, coupled to a 2-aminopyridyl residue, and subjected to two-dimensional sugar mapping technique. The major fractions consisted of "high-mannose type" oligosaccharides containing 8-9 mannose residues. Bi- and tri-antennary "complex type" oligosaccharides having terminal galactose were detected as minor components. These oligosaccharides were N-linked and contained no sialic acid. This structural profile is consistent with our previous characterization based on lectin-binding and glycosidase digestion. Functional specificity of identified chlamydial oligosaccharides was analyzed using glycopeptides fractionated from ovalbumin and structurally defined oligosaccharides from other sources. The glycopeptide fraction having high-mannose type oligosaccharide, as compared to those having complex or hybrid-type, showed a stronger inhibitory effect on attachment and infectivity of chlamydial organisms to HeLa cells. Among high-mannose type oligosaccharides, the strongest inhibition was observed with mannose 8 as compared with mannose 6, 7, or 9. These results indicate that a specific high-mannose type oligosaccharide linked to the major outer membrane protein of C. trachomatis mediates attachment and infectivity of the organism to HeLa cells.     

Porcine submaxillary mucin forms disulfide-linked multimers through its amino-terminal D-domains

COS-7 cells expressing 1,360 residues from the amino terminus of porcine submaxillary mucin were used to determine whether this region, containing the D1, D2, and D3 domains, is involved in forming mucin multimers. Analysis of the proteins immunoprecipitated from the medium of transfected cells by reducing SDS-gel electrophoresis showed a single N-glycosylated protein with no indication of proteolytically processed forms. Without prior reduction, only two proteins, corresponding to monomeric and disulfide-linked trimeric species, were observed. The expressed protein devoid of N-linked oligosaccharides also formed trimers, but was secreted from cells in significantly less amounts than glycosylated trimers. Pulse-chase studies showed that the disulfide-linked trimers were assembled inside the cells no earlier than 30 min after protein synthesis commenced and after the intracellular precursors were N-glycosylated. Trimer formation was inhibited in cells treated with brefeldin A, monensin, chloroquine, or bafilomycin A1, although only brefeldin A prevented the secretion of the protein. These results suggest that trimerization takes place in compartments of the Golgi complex in which the vacuolar H+-ATPase maintains an acidic pH. Coexpression in the same cells of the amino-terminal region and the disulfide-rich carboxyl-terminal domain of the mucin showed that these structures were not disulfide-linked with one another. Cells expressing a DNA construct encoding a fusion protein between the amino- and carboxyl-terminal regions of the mucin secreted disulfide-linked dimeric and high molecular weight multimeric species of the recombinant mucin. The presence of monensin in the medium was without effect on dimerization, but inhibited the formation of disulfide-linked multimers. These studies suggest that disulfide-linked dimers of mucin are subsequently assembled into disulfide-linked multimers by the amino-terminal regions. They also suggest that the porcine mucin forms branched disulfide-linked multimers. This ability of the amino-terminal region of mucin to aid in the assembly of multimers is consistent with its amino acid identities to the amino-terminal region of human von Willebrand factor, which also serves to form disulfide-linked multimers of this protein.     

Structural studies of the sugar chains of hen ovomucoid. Evidence indicating that they are formed mainly by the alternate biosynthetic pathway of asparagine-linked sugar chains

The carbohydrate moieties of hen ovomucoid were released as oligosaccharides by hydrazinolysis. The neutral oligosaccharide fraction which comprised about 85% of the total sugar was fractionated into eight oligosaccharide fractions by Bio-Gel P-4 column chromatography. Occurrence of novel penta-antennary oligosaccharides in the larger three fractions was reported in the preceding paper (Yamashita, K., Kamerling, J.P., and Kobata, A. (1982) J. Biol. Chem. 257, 12809-12814). Structural studies of the remaining smaller oligosaccharides indicated that they all have Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc as their common core. The alpha-mannosyl residues occur either free or as one of the following five groups: GlcNAc beta 1 leads to 2Man, GlcNAc beta 1 leads to 4Man, GlcNAc beta 1 leads to 4(GlcNAc beta 1 leads to 2)Man, GlcNAc beta 1 leads to 6(GlcNAc beta 1 leads to 2)Man, and GlcNAc beta 1 leads to 6(GlcNAc beta 1 leads to 4)(GlcNAc beta 1 leads to 2) Man. In most oligosaccharides, a beta-N-acetylglucosamine residue is linked at the C-4 position of the beta-mannosyl residue of the core. The structural characteristic of the sugar chains of hen ovomucoid indicated that they are not formed by the ordinary processing pathway of the asparagine-linked sugar chains.     

Chemotactic factors in bronchial secretions of cystic fibrosis patients

To understand chronic neutrophil attraction into cystic fibrosis airways, both global chemotactic activity and individual chemotactic factors were studied in bronchial secretions. Bronchial secretions of 8 cystic fibrosis patients, collected on the first day of admission for antibiotic treatment, showed a high chemotactic index (19.4 +/- 5.7, n = 8). Fractionation by gel filtration of bronchial secretions resulted in three chemotactic fractions. The first factor corresponded to interleukin-8, and the second activated neutrophils via the FMLP receptor. The third factor, which was of lower molecular weight, did not activate FMLP or leukotriene B4 receptors, and its nature is still under investigation. Treating patients with antibiotics reduced global chemotactic activity, mainly by reducing the activity due to stimulation of the FMLP receptor.     

Retinoic acid-regulated cellular differentiation and mucin gene expression in isolated rabbit tracheal-epithelial cells in culture

Rabbit tracheal epithelial cells were cultured in a serum-free and hormone-supplemented medium with and without retinoic acid. The cells showed time-dependent mucin gene expression when cultured in the medium with retinoic acid. In the absence of retinoic acid, however, mucin mRNA was barely detectable in the cells. When retinoic acid was added back to the medium, the mucin message was prominent again. Actinomycin D and cycloheximide did not inhibit mucin gene expression. The mucin message was slightly elevated by cAMP agonists. A mucin antisense oligomer inhibited the retinoic acid-induced mucin mRNA expression and secretion, thus offering an alternate approach in the management of mucus hypersecretion in upper airway respiratory diseases such as chronic bronchitis, asthma, and cystic fibrosis.     

How do cystic fibrosis transmembrane conductance regulator mutations produce lung disease?

In recent years, several functions of the cystic fibrosis transmembrane conductance regulator have been discovered, yet the pathophysiology of the pulmonary disease in cystic fibrosis remains unclear. At the cellular level, functions of this protein include regulation of chloride and sodium transport at the cell membrane and in intracellular organelles, regulation of protein trafficking, and posttranslational processing of glycoconjugates. Elucidation of these functions has led to several hypotheses to account for how defects in the cystic fibrosis transmembrane conductance regulator produce pulmonary disease, but a clear understanding of the pathophysiologic links between the cellular functions of the cystic fibrosis transmembrane conductance regulator and organ dysfunction has been hampered by the lack of ideal model systems. Current evidence suggests that defects in the cystic fibrosis transmembrane conductance regulator lead to alterations in periciliary fluid homeostasis, mucus hydration, mucin secretion, and apical membrane protein structure. In turn, these alterations impair mucociliary clearance and promote bacterial infection, which then leads to chronic airway inflammation and the development of bronchiectasis.     

Neutral core oligosaccharides of bovine submaxillary mucin--use of lead tetraacetate in the cold for establishing branch positions

Oligosaccharide alditols were released from bovine submaxillary mucin by alkaline borohydride treatment. The fractions containing smaller neutral oligosaccharides were separated by HPLC, to give, in the mono-, di- and trisaccharide-alditol sizes, 19 different structures, in addition to three fucosylated tetrasaccharide alditols. Molecules were identified by NMR spectroscopy, electrospray-mass-spectrometry-mass-spectrometry, permethylation analyses, and highly selective Pb(OAc)4 oxidation at -30 degrees C, followed by borohydride reduction. Pb(OAc)4 oxidation was found to be generally applicable in identifying the branch position(s) of substitutions for all core structures. Among the isolated oligosaccharide alditols were structures not previously reported, including 12 structures not reported from bovine submaxillary mucin, and four structures (three core structures) not found in the Carbbank database.     

Identification of Man alpha1-3Man alpha1-2Man and Man-linked phosphate on O-mannosylated recombinant leech-derived tryptase inhibitor produced by Saccharomyces cerevisiae and determination of the solution conformation of the mannosylated polypeptide

The production of recombinant leech-derived tryptase inhibitor (rLDTI) by two different strains of Saccharomyces cerevisiae resulted in the secretion of non-glycosylated and glycosylated rLTDI. Monosaccharide analysis and a-mannosidase treatment demonstrated that glycosylated rLDTI was exclusively alpha-mannosylated. A trypsin digest of reduced and S-carboxymethylated glycosylated rLDTI was separated on a reverse-phase HPLC column. Glycopeptides identified by a combination of matrix-assisted laser desorption mass spectrometry, amino acid sequence analysis, and monosaccharide analysis revealed the presence of different glycoforms. It was found that Ser24, Ser33 and Ser36 were partially glycosylated with a single mannose residue, whereas Thr42 in glycosylated rLDTI from both strains was fully occupied with manno-oligosaccharides with a degree of polymerization ranging over 1-3 and 1-13 depending on the yeast strain. In phosphorylated rLDTI a single phosphate group was predominantly located at the innermost Man residue of units of mannobiose, mannotriose, mannotetraose and mannopentaose at Thr42. Oligosaccharides released by alkaline treatment were reduced by sodium borohydride and separated by high-pH anion-exchange chromatography on a CarboPac MA1 column, and analyzed by one- and two-dimensional 1H-NMR spectroscopy. Besides the major oligosaccharide Man alpha1-2Man-ol, the (for yeast protein O-glycosylation) unusual Man alpha1-3Man alpha1-2Man-ol was determined. The solution conformation of glycosylated rLDTI was investigated by two-dimensional NMR spectroscopy. Structure calculations by means of distance geometry showed that glycosylated rLDTI is compactly folded and contained small secondary structure elements. Analysis of the chemical shifts showed that amino acids Val32-Ser33, Ser36-Ser39 and Thr42 were affected by the O-mannosylation. In addition, changes in chemical shift were observed within the beta-hairpin peptide regions Val13-Ser16 and Gly18-Tyr21 attributed to direct interactions of the mannose residue at Ser36. Furthermore, the protein-linked oligosaccharides were spatially grouped in a position opposite of the canonical binding loop.     

The number of oligosaccharides borne by porcine thyroglobulin is variable

Purified porcine thyroglobulin (Tg) was fractionated on a concanavalin A-Sepharose 4B column by a step-wise elution with increasing concentrations of methyl alpha-mannoside (fraction A, 50 mM; B, 100 mM; C, 200 mM; D, 500 mM, and E, 1 M), and its fractional ratio was 12.8:28.6:26.4:19.7:12.4. These five fractions showed the same profile in polyacrylamide gel electrophoresis. The subfractions were analyzed for their relative contents in oligosaccharides of each structure type and for their monosaccharide contents. In fractions B, C, D, and E the former varied between 15-22% for triantennary complex-type, 47-60% for biantennary complex-type, and 22-30% for high mannose-type oligosaccharide. Fraction A showed a higher percentage of triantennary complex-type structures (36%) and a lower percentage of biantennary complex-type structures (17%). The monosaccharide numbers increased from fraction A to E: 85 to 135 mannose residues, 60 to 82 galactose residues, 84 to 115 N-acetyl glucosamine residues, and 22 to 28 sialic acid residues. After analysis of the number of mannose residues contained in the high mannose-type structures, it was possible to calculate the number of oligosaccharides borne by each Tg subfraction. This number was approximately the same for fractions A and B (22.4 and 21.7), then it increased from B to E (21.8 to 32.9). These results account for the separation obtained on the concanavalin A-Sepharose 4B column. Separation of the two first subfractions bearing the same number of oligosaccharides is certainly due to the higher number of high mannose-type structures in B. In conclusion, the studies reported here show that porcine Tg is heterogeneous, and mainly so in terms of total number of N-glycan structures.     

Cytologic diagnosis of pancreatic cystic lesions. A prospective study of 28 percutaneous aspirates

PURPOSE: To prospectively evaluate fine needle aspiration biopsy (FNAB) of pancreatic cystic lesions. STUDY DESIGN: We performed a blind, prospective study on percutaneous aspirates from 28 radiographically identified cysts, including 6 inflammatory cysts (5 pseudocysts and 1 abscess), 4 serous cystadenomas, 1 cystic islet cell tumor, 5 mucinous cystic neoplasms, 6 mucinous cystadenocarcinomas and 6 nonpancreatic cysts. RESULTS: Four of six (67%) cystadenocarcinomas were identified as malignant, and the other two, which lacked sufficient morphologic criteria for malignancy, as consistent with mucinous cystic neoplasm. Two of five mucinous cystic neoplasms were correctly classified. One, which contained atypical cells, did not appear to be mucinous on the ThinPrep, and one, which lacked an epithelial component, was suggested because of the presence of mucin in the background. The fifth one contained inflammatory cells only. One of four serous cystadenomas produced a diagnostic specimen. FNAB of the cystic islet cell tumor was nondiagnostic. Five of six inflammatory cysts (83%) were correctly diagnosed, whereas one case produced an acellular, nondiagnostic specimen. Six of 28 (23%) cases were nonpancreatic cysts, aspirated under the presumption that they were pancreatic cysts based on radiologic studies: only one case, a papillary cystadenocarcinoma of the stomach, was correctly diagnosed; the other five cases were nondiagnostic, and in two of these the assumption that the cysts were pancreatic in origin precluded an accurate classification. CONCLUSION: FNAB of pancreatic cystic lesions can differentiate mucinous from nonmucinous pancreatic cysts and provide definitive evidence of malignancy. In some cases, serous cystadenomas can be diagnosed. Pseudocysts can be suspected on the basis of an inflammatory smear lacking both epithelial cells and background mucin, but this finding is not specific. Nonpancreatic lesions constitute a significant percentage of cases aspirated as pancreatic cysts and present a major pitfall in cytologic interpretation.