Structure determination of five sulfated oligosaccharides derived from tracheobronchial mucus glycoproteins.

The structure of five sulfated oligosaccharide units of highly anionic tracheobronchial mucous glycoproteins, isolated from a cystic fibrosis patient's sputum, were established. Reduced oligosaccharides (84%) were released under alkaline borohydride conditions, and the acidic oligosaccharides (63%) were isolated by Dowex 1-X2 chromatography. Following the removal of acidic oligosaccharides possessing N-acetylneuraminic acid and L-fucose by lectin affinity chromatography a heterogeneous mixture of sulfated oligosaccharides was obtained. From this fraction, five short chain monosulfated oligosaccharides (S-I to S-V) were purified by sequential separation by SynChroprep AX300 anion exchange high pressure liquid chromatography, gel filtration on Bio-Gel P-2, and high voltage paper electrophoresis. Based on the results of carbohydrate composition, sequential exoglycosidase degradation, permethylation analysis, lectin affinity chromatography, and periodate oxidation, the following structures (where GalNAcol is N-acetylgalactosaminitol) were proposed for these oligosaccharides. (formula; see text)


S-V SO,(6)G@(1~4)Gl~NA~fl(1+3)Galfl(14) GlcNAcf3(1+3)GalNAeol
TracheQbronchial mucous glycoproteins are a class of large molecular weight components that are the primary macromolecular constituents of a mucous viscoelastic gel among whose functions may include protection of the pulmonary airways from inhaled particulate matter, irritants, microorganisms, and rapid drying by evaporation. In the absence of pulmonary disease, tracheobronchial mucous glycoproteins are secreted in low quantities and have been shown to possess both an anionic nature and side-chain oligosaccharides of small length (1). Notably, the acidic characteristic of these tracheobronchial mucous glycoproteins is primarily due to the presence of N-acetylneuraminic acid and sulfate esters.
A major feature observed in many chronic obstructive pulmonary diseases is the hypersecretion of tracheobronchial mucous glycoproteins (2). Several reports indicate that in * This research was supported by Grant HL-32026 from the National Heart, Lung, and Blood Institute, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. certain pathological states, such as chronic bronchitis and cystic fibrosis, these hypersecreted tracheobronchial mucous glycoproteins reflect an increased anionic characteristic due to their increased content of N-acetylneuraminic acid and sulfate esters (3). These same investigations have suggested that the content of sulfate esters was greatest in the larger oligosaccharides isolated from these glycoproteins and that the N-acetylneuraminic content was greatest in the smaller oligosaccharides (3, 4). Although structures of several oligosaccharides containing N-acetylneuraminic acid isolated from these glycoproteins have been reported (5), there have been no similar reports on the primary structures of oligosaccharides containing sulfate esters.
Since normal human tracheobronchial secretions are attainable only in small quantities and possess relatively low percentages of sulfate esters (6), we chose to investigate the sulfated oligosaccharides isolated from tracheobronchial mucous glycoproteins found in the sputum of patients suffering from cystic fibrosis. From these studies we hope to accrue structural data that will provide better understanding of the roles of the sulfate ester and sulfated oligosaccharides in health and disease. In the present investigation, we report on the separation and complete structural determination of five sulfated oligosaccharides consisting of a disaccharide, two trisaccharides, a tetrasaccharide, and a pentasaccharide, purified from the tracheobronchial mucous glycoproteins isolated from the sputum of a patient suffering from cystic fibrosis. Though the general acceptance, based upon compositional analyses, is that the sulfate ester is predominant on long-chain oligosaccharides the current study indicates that they can be present on oligosaccharide structures as short as a disaccharide. Sputum from a single patient was employed in this investigation rather than a pool from several individuals in an effort to decrease the introduction of any heterogeneity due to individual variation.

DISCUSSION
Tracheobronchial glycoproteins are readily purified from contaminating proteins, cell debris, and DNA typically found in purulent sputum obtained from patients suffering from cystic fibrosis following thiol reduction, treatment with deox-Portions of this paper (including "Experimental Procedures," "Results," Tables I-XIV, and Figs. 1-7) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-2063, cite the authors, and include a check or money order for $14.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

Sulfated Oligosaccharides of
yribonuclease, and Bio-Gel A-5m gel chromatography (20). The highly anionic glycoproteins can then be separated by anion exchange chromatography (20). Alkaline borohydride treatment of these acidic tracheobronchial mucous glycoproteins has been shown to produce a heterogenous population of P-eliminated and reduced oligosaccharides (4, 39).
In the present study, separation of monosulfated oligosaccharides from this mixture was accomplished by anion exchange chromatography (Fig. 3), by lectin affinity chromatography (Fig. 4) to remove oligosaccharides possessing either N-acetylneuraminic acid or L-fucose residues, and by high pressure liquid chromatography (Fig. 5). Further separation by Bio-Gel P-2 (Fig. 6) chromatography produced four welldefined low molecular weight oligosaccharide fractions with elution patterns corresponding to standard disaccharides, trisaccharides, tetrasaccharides, and pentasaccharides. Though oligosaccharides of higher degrees of polymerization were present, no distinct separation could be achieved on this column. By subsequent high voltage paper electrophoresis (Fig. 7) of these four fractions, five sulfated oligosaccharides of high purity were attained.
Determination of the primary carbohydrate structure of each of the five sulfated oligosaccharides was performed by analyzing them and their respective desulfated analogs by classical carbohydrate chemistry employing total carbohydrate analysis, specific glycosidase degradation, methylation analysis, and periodate oxidation and analysis. Determination that the sulfate ester on each oligosaccharide resided on the C6 primary alcohol of the nonreducing terminus galactose residue was based upon several analytical results. These included methylation and periodate oxidation analysis of the intact sulfated and desulfated oligosaccharides, periodate oxidation analysis of the sulfated galactose residue that was liberated from each oligosaccharide by 8-galactosidase, the generation of 3,6-anhydrogalactose following alkaline hydrolysis, and, last, the time required to acid hydrolyze the sulfate ester.
To our knowledge, this is the first report presenting the complete structural analysis of sulfated oligosaccharides isolated from human tracheobronchial mucous glycoproteins. It should be noted, though, that the neutral carbohydrate cores of each sulfated oligosaccharide presented have been reported. The neutral disaccharide Gal/3(1+3)GalNA~ol,~ which is the neutral carbohydrate core of the sulfated oligosaccharide S-I, has been isolated from sputum from a patient suffering from chronic bronchitis (40) and also from a patient suffering from cystic fibrosis (41). It also has been isolated from hog submaxillary gland mucin glycoproteins (42) and from human salivary mucin oligosaccharides from normal patients and patients with cystic fibrosis (43). The trisaccharides GalP(1-3)GlcNAcP( 1+3)GalNAcol and GalP(1-4)GlcNAc/3(1-3)GalNAcol, which form the neutral carbohydrate cores of S-I1 and S-111, respectively, have both been identified from bronchial glycoproteins from a patient suffering with cystic fibrosis (41). S-I11 has also been isolated from human colonic mucin (44, 45) and from bronchial glycoproteins from a patient with chronic bronchitis (40). The tetrasaccharide Galp(l+I)GlcNAcp( 1-3)Galj3(1+3)GalNAcol, the neutral carbohydrate core of S-IV, has been identified from sputum glycoproteins from a patient with cystic fibrosis (41). Last, the pentasaccharide Gal@( 1-4)GlcNAcP( 1+3)GalP( 1-4)GlcNAcP( 1-3)GalNAcol that forms the neutral core of the sulfated oligosaccharide S-V has been reported from human colonic mucins (44,45).
In a report on the primary-structure determination of 14 The abbreviation used is: GalNAcol, N-acetylgalactosaminitol.
neutral oligosaccharides isolated from human bronchial glycoproteins utilizing 500-MHz 'H NMR spectroscopy, it was suggested that bronchial glycoprotein oligosaccharides could be grouped into two classes based upon the type of core they possessed (41). These two cores were, specifically, galactose or N-acetylglucosamine linked P( 1-3) to N-acetylgalactosaminitol. Both of these cores were found to be present in this study. S-I and S-IV were found to have Galp(1-3)GalNAcol at their original reducing termini, whereas S-11, S-111, and S-V were found to possess GlcNAc~(1~3)GalNAcol. It should also be noted that with regard to the four common types of 0-glycosidic glycoprotein linkages found in nature, as described and categorized by Schachter and Williams (46), S-I and S-IV possess type 1 cores, whereas S-11, S-111, and S-IV are type 3 core structures.
Last, it has been generally accepted within recent years that N-acetylneuraminic acid is the primary anionic moiety on short chain oligosaccharides and that sulfate is predominant on longer chains. This view has been based primarily on compositional analysis of column chromatographic fractions possessing oligosaccharides of different molecular weight ranges (3,4). The present study clearly shows that, though present in small quantities in the present study, oligosaccharides as small as a disaccharide can possess a sulfate ester.
The overall goal of our research is to elucidate the structures of the sulfated oligosaccharides that occur on tracheobronchial mucous glycoproteins in man and to determine what changes occur from the normal to the disease state. This information is fundamental to our understanding of the role(s) that mucinous glycoproteins play in the defense mechanisms that are triggered by chronic lung disease.