Primary structure determination of five sialylated oligosaccharides derived from bronchial mucus glycoproteins of patients suffering from cystic fibrosis. The occurrence of the NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)] GlcNAc beta(1----.) structural element revealed by 500-MHz 1H NMR sp

The structure of sialylated carbohydrate units of bronchial mucins obtained from cystic fibrosis patients was investigated by 500-MHz 1H NMR spectroscopy in conjunction with sugar analysis. After subjecting the mucins to alkaline borohydride degradation, sialylated oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by high performance liquid chromatography. Five compounds could be obtained in a rather pure state; their structures were established as the following: A-1, NeuAc alpha(2----3)Gal beta(1----4) [Fuc alpha(1----3)]GlcNAc beta(1----3)Gal-NAc-ol; A-2, NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)-[GlcNAc beta (1----3)]GalNAc-o1; A-3, NeuAc alpha(2----3)Gal beta-(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----3)Gal beta(1----3) GalNAc-o1; A-4, NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)]Glc-NAc NAc beta(1----6)[GlcNAc beta(1----3)]GalNAc-o1; A-6,NeuAc alpha-(2----3) Gal beta(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----6)[Gal beta-(1----4) GlcNAc beta(1----3)]GalNAc-o1. The simultaneous presence of sialic acid in alpha(2----3)-linkage to Gal and fucose in alpha(1----3)-linkage to GlcNAc of the same N-acetyllactosamine unit could be adequately proved by high resolution 1H NMR spectroscopy. This sequence constitutes a novel structural element for mucins.

(14)GlcNAc~(l+3)]GalNAc-ol. The simultaneous presence of sialic acid in a(2-+3)-linkage to Gal and fucose in a(ld3)-linkage to GlcNAc of the same Nacetyllactosamine unit could be adequately proved by high resolution 'H NMR spectroscopy. This sequence constitutes a novel structural element for mucins.

A c a ( 2 4 3 ) G a ( 1 + 4 ) [ F u~( 1+3)]GlcNAcB( 1+3)Gal-
Human bronchial mucus glycoproteins (or "mucins") that are prepared from lavages of macroscopically healthy areas of bronchial mucosa, have been shown to be of acidic, predominantly sialylated nature, while their average carbohydrate chain length is relatively small (1). Nevertheless, no detailed structural characterization of these chains could be achieved as yet, because of the difficulty in obtaining sufficient amounts of normal bronchial mucins. Therefore, most studies related to human bronchial mucins have concerned material * This investigation was supported by the Association Francaise de Lutte contre la Mucoviscidose, the Netherlands Foundation for Chemical Research, and Grant UUKC 83-13 from the Netherlands Foundation for Cancer Research. A preliminary account of this study was presented at the 7th International Symposium on Glycoconjugates, held in Ronneby, Sweden, July 17-23, 1983. 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. isolated from sputum of patients suffering from bronchial hypersecretion ( 2, 3).
For the structure determination of such sialylated carbohydrate chains, substantial amounts of these oligosaccharides have been isolated by us from the sputum of six patients with blood group 0, who suffered from cystic fibrosis (4). Here, we report the fractionation and the purification by hplcl of these sialylated oligosaccharides, followed by their primary structure determination by combination of sugar analysis and 500-MHz 'H NMR spectroscopy. This approach has proved to be powerful in characterizing mucin-type oligosaccharide-alditols in amounts down to about 20 nmol, even if present in complex mixtures of closely related structures (3,5,6).

DISCUSSION
Acidic mucus glycoproteins can be purified after thiol reduction of the bronchial mucus gel of patients suffering from cystic fibrosis or from chronic bronchitis (4, 7). Comparison of their structural features is relevant with respect to possible alteration of their carbohydrate metabolism; the results may provide a clue to the study of mucus glycoprotein biosynthesis by the bronchial mucosa under various pathological conditions.
Alkaline borohydride treatment of such purified glycoproteins leads to heterogeneous populations of glycopeptides and reduced oligosaccharides; the smallest size oligosaccharides of each population can be obtained in a neutral fraction IC, a sialic acid-rich fraction IIc, and two fractions having an elevated sulfate content (4, 7). High pressure liquid chromatography is progressively unveiling the remarkable heteroge-' The abbreviations used are: hplc, high performance liquid chromatography; glc, gas-liquid chromatography; NMR, nuclear magnetic resonance; GalNAc-01, N-acetylgalactosaminitol.
'Portions of this paper (including "Experimental Procedures," "Results," Tables I and 11, Schemes 1 and 2, Figs. 1-6, and Footnote 3) 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.
83M-3606, cite the authors, and include a check or money order for $8.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.
Sicslylateed Ol$oswcharides from Bronchial Mucins neity of each of these fractions (8), the significance of which remains to be explained.
The structures of 15 neutral oligosaccharides present in fraction IC obtained from purified bronchial acidic glycoproteins secreted by six patients suffering from cystic fibrosis have already been reported (3). The corresponding sialic acidrich fraction IIc represents 7% (by weight) of the various fractions recovered after alkaline borohydride treatment (fraction IC represented 16.8%). It was subfractionated into two main peaks (see Fig. I).
Structural investigation by 500-MHz 'H NMR spectroscopy in combination with sugar analysis of hplc-purified oligosaccharide fractions obtained from the first peak ( Fig. 1) afforded the primary structures of five sialylated mucin-type oligosaccharide-alditols (see Scheme 2). NMR spectroscopy proved to be an adequate approach for complete structural analysis; it enabled to define the core type of the various compounds, that is, the substitution pattern of GalNAc-01, as well as the types of linkage and the locations in the chains of the terminal NeuAc and Fuc residues, even when they were present in the NeuAca( 2-3) GalP( 1 4 ) [ Fuca ( 1 4 ) ] GlcNAcP ( 14. ) sequence which had not been observed by 'H NMR before. The 'H NMR features of this element have now been established.
Even more unusual is the finding that four of five identifiable oligosaccharide-alditols described herein contain N-acetyllactosamine units that bear NeuAc in a(2*3)-linkage to Gal, as well as Fuc in a( 143)-linkage to GlcNAc. In contrast to the mutual exclusiveness of a( 1+3)-fucosyltransferase and a(2-&)-sialyltransferase in acting upon the same N-acetyllactosamine unit (23), apparently such an antagonist relationship does not exist for a(2-+3)-sialyltransferase and a(1-3)fucosyltransferase. The aforementioned structural element has been reported rarely (24, 25). Here it is described for the first time to occur in mucins.
Human bronchial mucins are secreted by goblet cells of the bronchial epithelium and also by mucous cells of the submucosal glands (26, 27). Goblet cells have been shown to secrete both mucin with a high content of sialic acid as well as mucin rich in sulfate. Cells from mucous glands, however, have been shown to secrete neutral mucin and sialic acid-containing mucin, but no sulfated mucin (27). Preliminary studies using labeled lectins have pointed out that lectins could discriminate between the different bronchial cell types (28-30). For example, Limulus polyphemus agglutinin, which has an affinity H. van  for sialic acid 4 2 4 ) (or a(2-3))-linked to GalNAc but not for sialic acid linked cr(2+3) (or 4 2 4 ) ) to galactose (31) has been shown to label goblet and serous cells but it does not label mucous cells (30). These data suggest that there are structural differences between the sialylated mucins synthesized by goblet cells and those made by mucous cells, probably with respect to the linkage of NeuAc. The sialylated oligosaccharides described herein might stem from mucins produced by the mucous cells.
(6.9-14). FxaCLlOns A-1 to A-11 contain NeuAic ol2-3l-linked to a GalB(I*4>valved. the '4-NMR spectra of fractiOnS A-5. and A-7 tO a-11, Could not be interpreted I n terms of prlmary structural assignments of COnStltUtinq 01190hydrate material that made ImpOSalble the undisturbed ObServatian Of some saccharides. This was a t h e r due to contamination of the sample by non-carbosrrucrural-reporter-4roUP regions of the spectra (as with A-5 and A-01, 01 to apparent mlxturer of aldrtols, prohibited a detailed interpretation for A-7, the low amount of material available whlch, together with the complexity of the and A-9 to A-11. However. the spectra of A-1 to A-4, which are depicted I n Figs. 3 to 6, and that of A-6 could be unraveled, a6 will be Shorn below. The molar carbohydrate Compositions of these fractions are Suarmariled in Table I.

Apart frcm the recognition of the type of linkage 2" which NevAc is in-
The chemical shifts of the structural-reporter groups for these fractions have been compxled ~n Table 11. Four Out Of the Six identifiable oligosaccharidefound to be present I" the neutral fractron (IC) from cystic fibrosis Bputtm 131.
-alditols appeared to be sialylatad analogs Of compounds which *ere p~eviously The neutral analog of A-1 (denoted N -I ) , which had not been encountered before, was prepared by trea-nt   Table 11). The st~~Ct ( Table 11). It should be mentioned that N-1 is the NHR features Of Which were described previously ( Table 111   In conjunction rzth Its carbohydrate composltlon (Table I1 reveals