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’VHE JOURNALOF BIOLOGICAL CHEMISTRY
Vol. ‘255. No. IO. Issue of May 25, pp. 45954598. 1980 In L1.S.A.
Prtnted
Oligosaccharides of Human Bronchial Glycoproteins NEUTRAL DI- AND TRISACCHARIDES ISOLATED FROM BRONCHITIS*
A PATIENT SUFFERING FROM CHRONIC
(Received for publication, September 4, 1979)
Genevieve Lamblin, Michel Lhermitte, Arnold Boersma, and Philippe Roussel From Unite des Proteines, Znserm No. 16, Place de Verdun, 59045 LilIe Cedex, France
Vernon Reinhold From Harvard Medical School, Boston, Massachusetts 02115
Human bronchial glycoproteins from a patient suffering from chronic bronchitis were subjected to alkaline borohydride degradation and aheterogeneous mixture of reduced oligosaccharides and glycopeptides was obtained. The shortest neutral oligosaccharides were subsequently fractionated by DAX4 anion exchange chromatography into 12 fractions. Five major oligosaccharides were purified by preparative paper chromatography. Their structures were determined by using periodic oxidation, methylation analysis, gas liquid chromatography-mass spectrometry, and enzymatic degradation. Two oligosaccharides and three trisaccharides were characterized as galactose (Gal) 81 + 3 N-acetylgalactosaminitol (GalNActitol), N-acetylglucosamine (GlcNAc) 81 + 3 GalNActitol, Gal 1 + 4 GlcNAc 1 + 3 GalNActitol, GlcNAc 1 + 3 Gal 1 + 3 GalNActitol andfucose a1 + 2 Gal 81 + 3 GalNActitol.
EXPERIMENTALPROCEDURES’ RESULTS’
DISCUSSION
Alkaline borohydride treatment of bronchial acidic glycoproteins leads to a heterogeneous population of reduced oligosaccharides and glycoproteins (2). The mixture, furthermore, wasfractionated onion exchange resinto give a neutral oligosaccharide Fraction IC,a neuraminic-rich oligosaccharide Fraction IIc, and two oligosaccharide Fractions IIIc and IVc having an elevated sulfate content. The neutral chains in Fraction IC were the shortest and they were subsequently fractionated by DAX4 anion exchange chromatography into 12 oligosaccharides. Oligosaccharides eluting in regions between peaks were not furtherpurified. The otherswere tested for purity by paper chromatography. Five major oligosaccharides were isolated,which accounted for 20% of the total included carbohydrate in Fraction IC. Such low yield may be Human acidicbronchialglycoproteinsisolated frompaa consequence of heterogeneity seen on paper chromatogratients suffering fromcysticfibrosis, chronicbronchitis,or phy and suggests the presence of many other minor typesof bronchial washings performedinmacroscopically healthy chains (as, for example, oligosaccharides(1,8,10))in amounts areas of the bronchialmucosa (which mayreflect normal too small to be isolated. Thus, a very restricted population secretion) had been shown to be very heterogeneous with has been fully characterized. These five oligosaccharides were regard to acidity and molecular size of their carbohydrate pure and sugar analysis showed the components to be in the chains (1-3). Such a wide variety of chain lengths has already proper molecular proportions. been demonstrated in pig submaxillary mucins (4, 5), in huFraction 4 from DAX4 could only be separated into two man ovarian cystblood group activeglycoproteins ( 6 ) ,and in different oligosaccharides by paper chromatography. human chronic bronchitis mucins (7). In human acidic bronPeriodicoxidation, methylation analysis, andgas liquid chial glycoproteins isolated from the sputum of patients suf- chromatography-mass spectrometry confvmed the presence fering from chronic bronchitis, we have previously shown that of GalNAc2 at thereducing end of all the carbohydrate chains neutral oligosaccharides, neuraminic-rich oligosaccharides, by detection of a terminal N-acetylgalactosaminitol, the exand two oligosaccharide fractions having an elevated sulfate pected product of alkaline borohydride elimination and subcontent might exist ( 2 ) . sequent reduction. This was found to be substituted at carbon Because of the difficulty in obtaining substantial amounts 3 in the five isolated oligosaccharides as in oligosaccharides of of normal bronchial glycoproteins, we attempted, in the present work, to isolate and purify five neutral oligosaccharides isolated from the sputumof a patient suffering from chronic ’ Portions of this paper (including “Experimental Procedure,” “Rebronchitis. Their structureswere determined in order tocom- sults,” Figs. 1 and 2, and Tables I to IV)are presented in miniprint a t pare them with oligosaccharides isolated in other bronchial the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from hypersecretions.
* This work was supported by contract Inserm 1980. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
the Journal of Biological Chemistry, 9650 Rockvdle Pike, Bethesda, Md. 20014. RequestDocument No. 79M-1803, citeauthor(s),and include a check or money order for $1.65 per set of photocopies. * Theabbreviations used are: GalNAc,N-acetylgalactosamine; GalNActitol, N-acetylgalactosaminitol; GlcNAc, N-acetylglucosamine; Fuc, fucose; Lac, lactose; Raf, raffinose; ‘GalNActitol, value of the oligosaccharide relative to that of N-acetylgalactosaminitol run in the samesolvent system and under thesame conditions.
4595
4596
Oligosaccharides ofBronchial Human Glycoproteins
hog submaxillary glycoprotein (4, 5) and rat intestinal glyco- setts Institute of Technology under the direction of Professor K. Biemann where all spectra were recorded. proteins (8). Two disaccharides have been isolated with the structure REFERENCES GlcNAc P l -+ 3 GalNActitol already characterizedin rat 1. Roussel, P., Lamblin, G., Degand, P., Walker-Nasir, E., and Jean(8) and Gal P l + 3 GalNActitol intestinalglycoproteins loz, R. W. (1975) J. Biol. Chem. 250,2114-2122 reported for many animal glycoproteins (9-12). The determi- 2. Lamblin, G., Humbert, P., Degand,P.,and Roussel, P. (1977) Clin. Chim. Acta 79,425-436 nation of configuration at the anomeric carbon atom of the 3. Lafitte, J. J., Lamblin, G., Lhermitte, M., Humbert, P., Degand, galactopyranosyl residue resulted in only partial cleavage with P., and Roussel, P. (1977) Carbohydr. Res. 56, 383-389 galactosidase from Escherichia coli. The resistance of the 4. Carlson, D. M. (1968) J . Biol. Chem. 243, 616-626 Gal -+ GalNActitol bond to cleavage by galactosidases has 5. Aminoff, D., Baig, M. M., and Gathmann, W. D. (1979) J. Biol. often been observed (13). Chem. 254,1788-1793 Gas chromatography-mass spectrometryof methylated al6. Rovis, L., Anderson, B., Kabat, E. A., Gruezd, F., and Liao, J . (1973) Biochemistry 12, 5340-5354 ditol acetates of oligosaccharide 12 as well as enzymatic diges7. Roberts, G . P. (1974) Eur. J . Biochem. 50,265-280 tion with a-fucosidase from emulsin (14) and /?-galactosidase 8. Carlsson, H. E., Sunblad, G., Hammarstrom, S., and Lonngren, J . from E . coli led to the following structure: Fuc a -+ 2 Gal (1978) Carbohydr. Res. 64, 181-184 pl -+ 3 GalNActitol. This component has been previously 9. Finne, J . (1975) Biochim. Biophys. Acta412, 317-325 characterized in oligosaccharides fromrat intestinalglycopro- 10. Margolis, R. K., and Margolis, R. U. (1973) Biochim. Biophys. teins (8) and recently in oligosaccharides of A' hog submaxActa 304,421-429 11. Vandenheede, J . R., Ahmed, A. I., and Feeney, R. E. (1972) J . illary glycoproteins (5). Biol. Chem. 247, 7885-7889 The two oligosaccharides isolated from the fourth peak of DAX4 are especially interesting. Methylation analysis and gas 12. Spiro, R. G., andBhoyroo, V. D. (1974) J . Biol. Chem.249,57045717 chromatography-mass spectrometry led to the two following 13. Codington, J. F., Yamasaki, T., van den Eijnden,D. H., Evans, N. structures: Gal 1 + 4 GlcNAc 1 3 GalNActitol and GlcNAc A., and Jeanloz, R. W. (1979) FEBS Lett. 99, 70-72 1-+ 3 Gal 1+ 3 GalNActitol. However, the determination of 14. Ogata-Arakawa, M., Muramatsu, T., and Kobata, A. (1977) Arch. Biochem. Biophys. 181,353-358 the anomeric configurations remains tobe confirmed because 15. Boersma, A., and Degand, P. (1976). C. R. Acad. Sci. 283, 397of lack of material. 399 These results show that theN-acetylgalactosamine residue 16. Reinhold, V. N., Wirtz-Peitz, F., and Biemann, K. (1974) Carboinvolved in the 0-glycosidic linkage may be substituted by hydr. Res. 37,203-221 two different carbohydrates corresponding to the effect of 17. Demaille, J., Dautrevaux, M., Havez, R., and Biserte, G. 11965) different glycosyltransferases giving rise to two types of carBull. SOC.Chem. Fr. 12,3506-3511 bohydrate chains. Whether these two different types of car- 18. Reissig, J . L., Strominger, J. L., and Leloir, L. F. (1955) J. Biol. Chem. 217,959-966 bohydrate chains are anchored on two different amino acids and Seakins, J. W. T. (1969) in Chromatographic (threonine or serine) or whether each is specifically responsi- 19. Menzies, I. S., and Electrophoretic Techniques (Smith, I., ed) 3rd Ed, Vol. 1, ble for subsequent sialylation or sulfation remains tobe dempp. 310-329, Interscience, New York onstrated. 20. Lee, Y. C., and Scocca, J. R.(1972) J . Biol. Chem.247,5753-5758 21. Hakomori, S . I. (1964) J . Biochem. (Tokyo) 55, 205-208 Achnoudedgments-We wish to thank MoniqueLuyckx, Marie- 22. Boersma, A,, and Degand, P. (1974) C. R. Acad. Sci. 278, 1903-+
Christine Tirlemont, and Daniele Vandeperre for technical assistance. We also acknowledge the Mass Spectrometry Facility at Massachu-
1906 23. Reinhold, V. N. (1972) Methods Enzymol.25, 244-249
OLigosaccharides ofBronchial Human Glycoproteins
4597
OLIUS3&CL'HARIDES OF W ERCNCHIAL GLYCOPROTEINS : NRltral dl- and tPLsacchaPLde5 1SOlated from patlent suffering from ChPOnlC bronchxtlS Genevieve Lamblln, Michel Lherrnltte, Arnold Boersma. Phllippe ROUSSel and Vernon Reinhold
* * EXPERIMENTAL
PROCEDURE
Material Detalls of thes o u ~ c eof Sugars, Sylon HTP and gas-llquld chromatographlc medla have beenp~ev1ous1y publlshed ( 2 ) . DAX4 10"s exchange ~ e s l nwas pul'chased from DURRUM. Eronchlal glycoproteins and pool Of neutral 0llgosacchaF.ldes were iaolated according to LAMELIN et (2). General methods Sugars were analysed as the>= trlmethylsllylated derlvatlves by gasllquld chromatography on a Perkln Elmer 900 instrument (flame-ionisatlon detector) with myo-Inositol as the Internal standard by the slighty modlfled method ( 1 ) of Reinhold (23). Separations were performed on stainless steel columns (180 x 0.3 cm) with ChrOmOSOrb W-AW-DMCS (1P-200 mesh) a5 support material carrylng 3 per 100 Of OV 1 7 . The partlally 0-methylated alditol acetates and 2-deoxy-P-(N-rnethyl) acetamldoalditol acetateS were ldentlfled using GC-MS. The instrumental analysis employed a Perkln-Elmer 990 gas chromatograph-Hltach1 RMU-6L mass Spectrometer combination Which 1s operated on llne vlth an IBM 1800 computer. Gas ChPOmatogLIaphy was performed Wlth a glass column ( 0 . 3 mrn 1.D. x 1 . 5 m ) packed Wlth 3 per 100 OV-17 OF 0 8 - 2 2 5 on 100/120 mesh gas chrom Q (Supelco. 1°C. Bellefante. Pennsylvania 1 6 8 2 3 ) . The column was heat programmed at a rate Of B o per mlnute ( 1 6 ) . The methylated sldltol acetates were ldentlfled by a cornbinatlon Of GC. Petention time. Selected ion recordlng and thelr mas6 Spectra. All Spectra were matched against a library Of known StandaPds for posltlve ldentlflcatlon. The effluent of 10"-exchange chromatography was monitored for hexose by the O F C ~ ~ O I - S U ~ ~ acld UI-IC method ( 1 7 ) . F r e e 2-acetamldo-Z-deoxy-D-glucose was measured by Ehrlich reagent (18) and free galactose and fucose by gas-liquld chromatography 88 thelr trlmethylsllylated derivatlves. Paper chromatography (analytical and preparative) Were performed on paper vlth 1-butanol-pyridine-water ( 6 : 4 : 3 ; v/v). Whatman N ' 3 " M Ollgosaccharldes references were located with periodate-benzidine (19) and ollg~saccharldes were eluted with0.1 5 acetic acid. Isolation Of O11RosacchaF.ldes
Acldlc glycoprotelnswere leolated from the Sputum Of a patlent suffePlng from chronlc bronchltls( 2 ) . They were furthermore degraded With alkaline borohydrlde and the degradation producte were fractionated by chromatography on D O W ~ XAG1 X 2 according to their acidity and by g e l filtratlon according to thelr molecular sire. Fractions referred to as IC are Small neutpal reduced carbohydratechains ( 2 ) . Seperatlon Of neutral
OI1gOSBCCharldeS
Neutral oligosaccharides I C ( 3 0 m g ) were subsequently fractlonatedaS thelr borate complexes by chromatography on anion-exchange LIeSln DAX4 ( 3 0 x 0.9 crn) eluted Wlth0.2 E sodium boratebuffer pH8.5. The eluate was collected ~n 2 rnl fractlons with a flow rate Of 20 m l p e r hour. Each fractlon was rnOnltoPed f o r hexose by the Orcinol SUlfUP1C method (17). A total of twelve peaks was found ~n the effluent(Figure 1). Fractlons w e ~ epooled as shown ~n Table I. The pooled fraCtlOnS2 , 5, 7 , 9 and 1 1 represented ~ ' e g l o n s between peaks and were not purified further. B o r l c acld was removed from ollgosaccharldes as its methyl eater by repeated evaporation wlth methanol. Composition and yield Of pools l , 3 , 4 . 6 , 8 , 10. 1 2 are shown in Table I.
TABLE I 1
FRACTION
NUMBER
Indicated by bars, w e ~ epooled and lyophlllzed.
640 463
0.70 0.43
1.2
0.8 0.8
1 1
Trlsaccharlde
0.55
1
1
1
431
6 : Dlsaccharlde 1 2 : Trlsaccharlde
0.65 0.60
1.2
1
1.2
I
1 266 401
3 : DlsacchaPlde 41 : Trlsaccharlde 42
'
1.2
4598
Oligosaccharides ofBronchial Human Glycoproteins
2.3.4 trl-o-me 2.3.4.6
0.9
Fuc
tetra-0-me
Gal
1.1 I
. , ~" . ~ and 2-deoxy-2 IN-m
