Mar 1, 1982 - School of Medicine for the degree requirement of Doctor of Medical ...... works as the determinant of stage-specific embryonic antigen ... REFERENCES. 1. ... Fournet, B., Montreuil, J., Strecker, G., Dorland, L., Haverkamp,.
THE.JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 16, Issue of August 25, pp. 9612-9621, 1982 Printed in U S.A.
Structures of the Carbohydrate Moieties of Secretory Component Purified from Human Milk* (Received for publication, March 1, 1982)
Akira Mizoguchi,Tsuguo Mizuochi, and Akira Kobata From the Department of Biochemistry, Kobe University School of Medicine, Chuo-ky Kobe, Japan
tween SC and J chain (7). The mechanism of SC molecule to SecretorJ component purified from human milk contains four asparagine-linked sugar chains in one moletransport macromolecules through epithelial layer is of parcule. The sugar chains were released from the poly- ticular interest. In the light of recent knowledge that the sugar peptide portion of secretory component by hydrazino- chains of many glycoproteins are playing roles as signals of lysis as oligosaccharidesandfractionatedbypaper several cellular recognition phenomena (S), the structure of electrophoresis and paper chromatography. The sugar the carbohydrate which comes up to more than 20% of SC chains of secretory component show an extraordinarily molecule might be important. Purkayastha et al.(9) reported high multiplicity. By sequential exoglycosidase diges- that SC purified from human milk contains an asparaginetion in combination with methylation analysis, more linked sugar chain, asfollows, than 40 structurally different sugar chains were found. They were allof biantennary complex type with either Fuccul Manal + G(Mana1 -+ 3)Manfil .+ 4GlcNAcfil -+ 1 6 4GlcNAc or Manal + G(Mana1 -+ 3)Manfil ”+ Sia2 + GGal01- 4GlcNAc?l- 2Man?l 4GlcNAcfil -+ 4(Fucal + 6)GlcNAc as their core porI tions and the multiplicityis produced by varietyof the 6 Gal/31-+ 4GlcNAc/31 Man?l “-f 4GlcNAc two outer chain moieties. The structures of outer chains 3 3 I foundareGlcNAcPl + 2,kNeuAca2 + 6Galfi1 ”+ 4 7 t 4GlcNAcfil-+ 2, Galfil -+ 4(Fucal-+ 3)GlcNAcfil3 2, Manal Fucal + 3Fucal GalP1 + 4GlcNAcP1 -+ 3Galfi1 -+ 4(+Fucal ”+ 2 7 3)GlcNAcfil- 2, NeuAca2 -+ GGalDl-+ 4GlcNAcfi1-+ Gal/3l-+4GlcNAcBl 3 Galfil + 4GlcNAcPl + 2, Galfil + 4(Fucal -+ 3)GlcNAcfil+ 3Gal/31”+4(Fucal+ 3)GlcNAcfi13 2, andNeuAca2 6Galfi14GlcNAcfil + 3Galfi1 -+ as a major component. This sugar chain contains many unu4(Fucal+ 3)GlcNAcfl1+ 2. sual structural groups, such as Galpl- 4(Fuccul-+ 6)GlcNAc and F u c d 3 3Fuc, which havenot been found in other glycoproteins. However, ourstudy of thesugarchains of Secretory component, which has also been called as secre- human SC, which will be described in detail in this paper, tory piece, is a glycoprotein synthesizedin mucosal epithelium gave very different results. (1).Since all IgA in secretions areof dimeric formto which an EXPERIMENTAL PROCEDURES’ SC] molecule is linked by two disulfide bonds, SC is considered RESULTS to play a role as a specific receptor to transport IgA dimer Fractionation of Oligosaccharides Liberatedfrom Human through the epithelial layer (1-3). This hypothesis was subof the stantiated by the finding of a secretory componentdeficiency SC by Paper Etectrophoresis-Paper electrophoresis who had normal serum IgA levels without IgA in his secretions tritium-labeled oligosaccharide fraction obtained by hydrazinolysis of human SC gave one neutral (N) and two acidic (AI (4). Actually, SC purified from human milk binds with dimeric and AII) fractions. The molar ratio of N, AI, and AI1 calcuIgA but not with IgA monomer and IgG in vitro (5). Since SC lated on thebasis of their radioactivities was 19:57:24. When a part (1 X IO4 cpm) of N, AI, and AI1 were hydroalso binds with IgM (5), theJ chain is believed to play a role in the binding. This interaction,however, may arise from the lyzed in 4 N HCI at 100 “C for 2 h and the hydrolysates were conformational change imposed on dimeric IgA and IgM analyzed by paper electrophoresis (borate buffer) after N detected as a molecules (6) because no covalent interaction is found be- acetylation, only N-acetylglucosaminitol was radioactive component in all cases. Therefore, the reducing * This study was supported in part by research grants from the termini of all oligosaccharides were N-acetylglucosamine, as Yamanouchi Foundation for Research on Metabolic Disorders, and expected from hydrazinolysis reaction on asparagine-linked the Ministry of Education, Science and Culture of Japan. This paper sugar chains.
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is a part of the dissertation submitted by A. M. to Kobe University School of Medicine for the degree requirement of Doctor of Medical Sciences. 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. 1 The abbreviations used are: SC, secretory component; Fuc, fucose; GlcNAc, N-acetylglucosamine; Sia, sialic acid NeuAc, N-acetylneuraminic acid; N, AI, and AII, neutral and acidic oligosaccaride fractions, respectively, of human SC.
* Portions of this paper (including “Experimental Procedures,” Figs. 2-5 and 7 and Tables I, 11, and V) 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. 812M-507, cite the authors, and include a check or money order for $6.00 per set of photocopies. Full size copies are also included in the microfilm edition of the Journal that is available from Waverly Press.
9612
Sugar Chainsof Human Secretory Component
9613
Structures of Nl a n d N2-In order to determine the anoWhen incubated exhaustively with sialidase, both AI and meric configuration and sequence of each monosaccharide, AI1 were completely converted to neutral components (data not shown). These results indicated that theacidic nature of radioactive N1 and N2 were subjected to sequential exoglywere analyzed by AI and AI1 are due to their sialic acid residues. The sialic cosidase digestion and the reaction products acids released from intact SC by sialidase digestion were all Bio-Gel P-4 column. Both N1 and N2 released a galactose residue by incubation with jack bean ,&galactosidase (Fig. N-acetylneuraminic acid and no N-glycolylneuraminic acid was detected (data not shown). Bymild acid hydrolysis (0.01 2 s ) . T h etwo radioactive products then released an N-acetylN HC1 at 100 “C for 2.5 min), whereby a part of the original glucosamineresidue by incubation with P-N-acetylhexosaacidic oligosaccharidesstill remained, AI gave onlythe neutral minidase (Fig. 2C). The products derived from N1 and N2 at this stage showed the same mobilities as authentic Man.,. component upon paper electrophoresis (pyridine-acetate GlcNAc GlcNAcoT and Mancj. GlcNAc Fuc. GlcNAcoT, rebuffer), while AI1 gave another acidic component together spectively.” That these two components have the structures with a neutral fraction in addition to their originalacidic and components. These results indicated that AI and AI1 contain (Manal”-$)pManfll ”+ GlcNAcPl + GICNAC~T 1 and 2 mol of N-acetylneuraminic acid residues in one mol- (Manal-+)2ManPl -+ GlcNAcPl ”-$ (Fucal-+)GlcNAcoT was confirmed by sequential digestion with a-mannosidase (Fig. ecule, respectively. Fractions N, AI, and AI1 were recovered from the electro- 20), /3-mannosidase (Fig. 2E),&N-acetylhexosaminidase (Fig. a-fucosidase(Fig.2G),respecphoresis paper by elution with water. Deuterium-labeledoli- 2F), and Charonia lampas gosaccharide fraction was also fractionated in the same man- tively. The series of sequential exoglycosidase digestion described above indicated that the structure of N1 canbe written ner into N,AI, and AII. P-4 Fractionation of as GalPl + GlcNAcPl + Manal + (Manal+)ManPl + Paper Chromatographic and Bio-Gel GlcNAcPl -+ GlcNAcor and thatof N2 as GalPl-+ GlcNAcPl the Neutral Oligosaccharides-When the radioactive fraction N was subjected to paper chromatography, a fractionation --f Manal -+ (Manal+)Man/31 -+ GlcNAcPl -+ pattern, as shown in Fig. lA, was obtained. Components in (Fucal+)GlcNAcor. Methylation analysis was performed on deuterium-labeled the area indicated by an open bar were recovered and subjected to a longer paper chromatography. As shown in Fig. N1 and N2 and the data are summarizedin Table I. MethyllB, they were separated into at least seven fractions. The ation data of the trimannosyl core portions of N1 and N2 eleven fractions (Nl-N11) in total, indicatedby black bars in (corresponding to the peaks of Fig. 2C) are also summarized Fig. 1, A and B, were recovered from paper by elution with in Table 11. These results indicated that the structure of N1 water, and N3-N8 were further purified by rechromatography should be GalPl -+ 4GlcNAcPl ”-$ 2Manal ”-$ 6 or 3(Manal of the neighboring com- --$ 3 or 6)Manpl- 4GlcNAcPl- 4GlcNAco~ and thatof N2 to remove the mutual contamination ponents. The per cent molar ratio of N1-N11 in the neutral should be Gal/3l+ 4GlcNAcPl ”-$ 2Manal -+ 6 or d(Mana1 oligosaccharide fraction N as calculated by their radioactivi- -+ 3 or G)ManPl--$ 4GlcNAcP1 ”-$ 4(Fucal”+ 6)GlcNAco~. ties was 0.8, 2.4, 2.7, 8.0, 9.9, 30.1, 6.8, 20.2, 5.5, 7.3, and 6.3, Both radioactive N1 and N2 were resistant to a-mannosirespectively. For further confirmation of the purity of the dase digestion (data not shown). Since theenzyme releases a eleven fractions, they were subjected to Bio-Gel P-4 column mannose residue from R --f Manal ”-$ 6(Manal”-$ 3)ManBl chromatography. Nl-N8 behaved as single components with -+ 4GlcNAc - - - group but not from Manal + 6(R --f Manal mobilities of 10.4, 11.5, 12.4,13.4,13.5, 14.5, 14.2, and 15.1, -+ 3)ManBl- 4GlcNAc - - - group (lo), the structuresof N1 respectively, while N9 wasseparated intotwo peaks (Fig. 4 A ) and N2 should be as shown in Table 111. and N10 and N11 eluted as a broad peak as shown in Fig. 5A Structures of N3 and N4”When the radioactive N3 and and in Fig. 4C, respectively. Deuterium-labeled N was also N4 were incubated with jack beanP-galactosidase, one galacfractionated in the same manner intoeleven fractions. tose was removed from both oligosaccharides. Two N-acetylglucosamineresidues were released fromboth radioactive products by ,8-N-acetylhexosaminidase digestion. That the products of N3and N4 at this stage have the structures Manal”-$ G(Mana1 ”-$ S)Man/31+ 4GlcNAc/31”-$4GlcNAc~”-$ 6(Manal ”-$ 3)ManPl + 4GlcNAcPl -+ andManal 4(Fucal -+ 6)GlcNAco~,respectively, was confirmed by sequential exoglycosidase digestion and methylation study, as in the case of two components in Fig. 2C. t Methylation analysis of the deuterium-labeled N3 and N4 > F -’ (Table I) indicated that their structures should be Gal/31 -+ 4GlcNAcPl”-$ 2Mannl”-$ 6 or 3(GlcNAcPl + 2Manal- 3 or6)ManPl ”-$ 4GlcNAcP1 -+ 4GlcNAc~randGalPl -+ 4GlcNAc/31”-$ 2Manal”-$ 6 or 3(GlcNAcPl+ 2Manal-+ 3 or 6)Manpl ”-$ 4GlcNAcPl + 4(Fucal ”-$ 6)GlcNAco.,, respectively. When radioactive N3 andN4 were incubated with P-N-acetylhexosaminidase, one N-acetylglucosamine residue was released from both oligosaccharides. These two products 0 10 20 30 40 were completely resistant to a-mannosidasedigestion. ThereDISTANCE FROM ORIGIN (cm 1 fore GalPl ”-$ 4GlcNAc group in both N3 and N4 should be FIG. 1. Paper chromatograms of neutral oligosaccharides. located on Manal”-$ 3 side as shown in Table 111. A, fraction N obtained by paper electrophoresis (see text) was applied Structures of N 5 a n dN6-Radioactive N5 and N6 showed to a sheet of Whatman No. 3MM paper and developed for 6 days thesame mobilities as authenticGal2. GlcNAc,,.Man:j with ethylacetate/pyridine/acetic acid/water (5:5:1:3);B , oligosacchaGlcNAc -GlcNAcoT and Gal2. GlcNAc2. Man:, .GlcNAc. Fuc. rides in the area indicated by an open bur in A were recovered from paper by elution with water, and subjected to the same paper chromatography for 19 days. Arrows indicate theposition where authentic Gal,. GlcNAcz. Man.3. GlcNAc. Fuc. GlcNAcc~~ migrated.
‘‘I Subscript OT is used in this paper to indicate NaB[’H],-reduced oligosaccharides. All sugars mentioned in thispaper have the 0 configuration except for fucose, which has the I, configuration.
Sugar Chainsof Human Secretory Component
9614
TABLE111 Structures of neutral oligosaccharides obtained from human SC
-R=GlcNAcOT
&=Fucal+6GlcNAcOT
Manal N2 ‘$Man61+4GlcNAc61+4R N1 Gal~1+4BlcNAc61+2Manalfl
Gl~NAc61+2Manal\~ ManB1+4GlcNAC61+4R
N3
N4
Gal61+4Gl~NAc61+2Manal’~
Gal~1+4GlcNAc61+2Manal >ManBt+4GlcNAc61+4R N5 Gal61+4GlcNAc61+2Manal
N6
Fucal 4 3
GalB1+4GlcNAcB1+2Manal~ /jMan61+4GlcNAc61+4R Gal~1+4GlcNAcB1+2Manal NB Gal61+4GlcNAcB1+2Mana ~Man61+4GlcNAct31+4R Gal61+4GlcNAcgl+2Manal
N7
3
t
Fucal Fucal
v
4
3 Ga1@1+4GlcNAc81+2Manal ~Manfi1+4GlcNAc61+4R
N9
Gal,31+4GlcNAcB1+2Manalf3 3
t Fucal
N10-1
Fucal J.
3
Fucal t
N10-3
+ Fucal Fucal c 3
Fucal 4 3
N10-4
N11
Chains Sugar
of Secretory Human Component
9615
GlcNAco~ in Bio-Gel P-4 column chromatography, respec- sequential exoglycosidase digestion and methylation study. tively. Sequential digestion with jack bean P-galactosidase The product whichmoved at 14.5 glucose units was also and P-N-acetylhexosaminidase released two galactoses and identical with N6 in its structural analyses. Therefore, N9 two N-acetylglucosamines from both oligosaccharides. That should be a mixture of difucosyl derivatives of N5 and N6. Detection of 2mol of 6-mono-0-methyl 2-N-methylacetathe radioactive products N5 and N6 a t this stage have the mido-2-deoxyglucitols in the methylation data of N9 (Table I) respective structuresManal G(Mana1 3)ManPl 4GlcNAcPl -+ 4GlcNAco~andManal -+ G(Mana1 --* indicated that the two a-fucosyl residues were linked at the 3 ) M a n f l l d 4GlcNAcP1-+ 4(Fucal-+ 6)GlcNAco~was con- C-3 position of N-acetylglucosamine residues of two GalPl f m e d by sequential exoglycosidase digestion and methylation "-f 4GlcNAcPl- outer chains of N5 and N6. Therefore, N9 study as in the case of the two radioative components inFig. should be amixture of two oligosaccharides, as shown in Table 111. 2c. Structure of NlO"N10 gave a broad peak both in paper These results, together with the methylation studiesof N5 chromatography (Fig. 1B) and in Bio-Gel P-4 column chroand N6, confirmed their structures, as shown in Table 111. Structures of N 7 and N8-Radioactive N8 migrated at the matography (Fig. 5A), indicating its heterogeneous nature. When radioactive N10 was incubated with jack bean P-galacsame position as authentic Galp-Fuc.GlcNAcz.Mana. GlcNAc. Fuc. GlcNAco~ inBio-Gel P-4 column chromatog- tosidase, two radioactivepeaks were detected in approxiraphy (Fig. 3A). When N7 and N8 were incubated with jack mately a 4 to 3 ratio (Fig. 5B, dotted line). Thesizes of these bean &galactosidase, one galactose was removed from both two peaks (17.5 and 16.2 glucose units) indicated thatone and oligosaccharides (Fig. 3B). P-N-Acetylhexosaminidase treat- two galactose residueswere removed fromoriginal N10 by the enzymatic digestion, respectively. By P-N-acetylhexosaminimentliberatedan N-acetylglucosamineresidue from both products (Fig. 3C). The products of Fig. 3C were completely dase digestion, the two peaks were converted to two pairs of mobilities resistant to further digestion with P-galactosidase and P-N- components as shown in Fig. 5B (solid lines). The acetylhexosaminidase, but released a fucose residue by al- of each major peaks indicated that one (component 2) and monda-fucosidase treatment (Fig. 30). A fucose was not two (component 1) N-acetylglucosamines were removed from released when they were incubated with Bacillus a-fucosi- the dotted line peaks 2 and 1, respectively. That the dotted dase. When the products in Fig. 3 0 were incubated sequen- linepeak 1 was converted to component1 and the dotted line tially with jack bean P-galactosidase and P-N-acetylhexosa- peak 2 tocomponent 2 by the P-N-acetylhexosaminidase minidase, another galactose and N-acetylglucosamine were digestion was confirmed by separate experiments (data not released (Fig. 3, E and F, respectively). The products from shown). The major andminor solid linepeaks infraction1 of P-galactosidase and N7 and N8 at this stageshowed the samemobilities as authen- Fig. 5B were totally resistant to jack bean P-N-acetylhexosaminidasedigestion (data not shown). Howtic Manj GlcNAc G~cNAc~T and Mans. GlcNAc Fuc GlcNAco~,respectively. Their respective structures were con- ever, they released one fucose residue by incubation with almond a-fucosidase (Fig. 5C). Sequential digestion of the two f i e d as Manal -+ 6(Manal-+ 3)bIanPl-+ 4GlcNAcPl4GlcNAcoT and Manal -+ G(Mana1 -+ 3)ManPl + radioactive peaks with either jack beanor diplococcal P4GlcNAcPl-+ 4(Fucal-+ 6)GlcNAco~by sequential exogly- galactosidase and then with P-N-acetylhexosaminidaserecosidase digestion and by methylation studies, as in the case leased 1mol each of galactose and N-acetylglucosamine resiof two components as Fig. 2C (data not shown). Almond a- dues from both peaks, respectively (Fig. 5, D and E ) . That the fucosidase digestion of N7 and N8 released a fucose residue structures of major and minor oligosaccharides a t this stage G(Mana1 -+ 3)ManPl + 4GlcNAcPl -+ from both oligosaccharides (Fig. 3G) and theradioactive prod- were Manal ucts showed the same results of sequential exoglycosidase 4(Fucal-+ 6)GlcNAco~ and Manal 6(Manal-+ 3)ManPl digestion as N5 and N6, respectively (data notshown). These -+ 4GlcNAcPl- ~G~cNAcoT, respectively, was confirmed by results indicated thatN7 and N8 havea fucose on one of the sequential exoglycosidase digestion and methylation analysis two GalPl -+ 4GlcNAcPl -+ outer chains of N5 and N6, (datanotshown).Theseresultsindicatedthatthe initial oligosaccharides in N10 which were led to fraction 1 and Fig. respectively. Methylation analysis of N7 and N8 before and after almond 5B (which will be called as N10-1 in the following part of this a-fucosidasedigestion are summarized in Tables I and 11, paper)havestructures: GalBl -+ GlcNAcPl -+ GalPl -+ respectively. The data indicated that the additional fucose 4(Fucal +)GlcNAcPl-+ Manal-+ 6 or 3(Gal/31-+ GlcNAcPl residue in both oligosaccharides is linked at the C-3 position -+ Manal -+ 3 or 6)ManPl + 4GlcNAcPl -+ 4(+-Fucal -+ of N-acetylglucosamine residue of an GalPl -+ 4GlcNAcP1 6)GlcNAco~. Methylation analysis of N10 (Table I) gave only -+ outer chain. 3,6-di-0-methyl-2-N-methylacetamido-2-deoxyglucitol as diIn order to determine on which outer chain the fucose 0-methyl glucosamine derivativeand only 6-mono-0-methylresidue is located, the two radioactive peaks in Fig. 3C were 2-N-methylacetamido-2-deoxyglucitol as mono-0-methylgluincubated with a-mannosidase and the reaction mixtures were cosamine derivative. These results indicated that the GalPl analyzed by Bio-Gel P-4 column. As shown in Fig. 3H, about -+ GlcNAc and GalPl-+ 4(Fucal -+)GlcNAc groups in NIO70% of both radioactive oligosaccharides released a mannose 1 should occur as G a v l -+ 4GlcNAc and GalPl -+ 4(Fucal residue, and 30%remained unchanged. The resistantfractions -+ 3)GlcNAc groups, respectively. Detection of only 2,4,6-triof both samples were not hydrolyzed a t all by the second a- 0-methylgalactitol as tri-0-methylgalactose derivative and of mannosidase treatment (data notshown). Therefore, N7 and only 3,4,6-tri-0-methylmannitol as tri-0-methylmannose deN8 should be mixtures of two isomeric oligosaccharides, as rivative indicated that the GlcNAcPl --* Gal group occurs as shown in Table 111. GlcNAcPl-+3Gal and the two outer chains should be linked Structure of N9-Upon Bio-Gel P-4 column chromatogra- at c-2position of the two a-mannosyl residues. phy, N9 wasseparated intotwo radioactive components (Fig. In order to confirm the location of GalPl -+ 4GlcNAcPl 44).These components were totally resistant to jackbean p- -+ 3Galfll-+ 4(Fucal-+ 3)GlcNAcPl--* outer chain, the two galactosidase and P-N-acetylhexosaminidasedigestion (data peaks in solid line fraction 1 of Fig. 5B, in which the anot shown). However, they released two fucose residues by mannose residue originally substituted by GalPl -+ almond a-fucosidase digestion (Fig. 4B). The product with a 4GlcNAcPl -+ group hadbecome a nonreducing terminal, was mobility of 13.5 glucose units gave the same results asN5 by incubated separatelywith a-mannosidase. Approximately 70% -+
e
-
-
-+
-
-
-
-
Chains Sugar
9616
of Human Secretory Component
of both peaks released a mannose residue and the remaining 6)GlcNAco~. Interpretation of the methylation data of N10 as 30% remainedunchanged (data not shown). These results in the case of N10-1 conf'iied the structures of N10-3 and indicated that N10-1 was a mixture of the four oligosaccha- NlO-4, as shown in Table 111. So far, we have notbeen able to rides, as shown in Table 111. assign the location of the two outer chains in N10-3 and N10When fraction 2 (shown as a solid line in Fig. 5B) was 4. incubated with jack bean P-galactosidase, a complicatedpatStructure of Nll-The elution pattern of N11 in Bio-Gel tern (shown by a dotted line in Fig. 5F) was obtained. This P-4 column chromatography indicated that it contained sevresult indicated that a galactose residue was removed from eral oligosaccharides (Fig. 4C). The elution pattern of N11 approximately 50% of the oligosaccharides in fraction 2. Fur- eluted from paper with water as indicatedby bars in Fig. 6A ther digestion of the productswith /3-N-acetylhexosaminidase was not changed prominently by incubation with jack bean or gave more clear cut results (Fig, 5F, solid line), indicating diplococcal P-galactosidase, P-N-acetylhexosaminidase or althat the oligosaccharides, which were susceptible to P-galac- mond a-fucosidase alone (data not shown).However, incubatosidase digestion, further released an N-acetylglucosamine tion of N11 with a mixture of jack bean /3-galactosidase and residue by the enzymatic digestion. P-N-acetylhexosaminidaseresulted in pairs of a major and a The major and the minorpeahs in fraction 3 in Fig. 5 F gave minor peak shown in Fig. 4 0 . The mobilities of major peaks the same results as those solid in Line fraction 1 in Fig. 5B in were 12,16, 19,22,and 25 glucose units. By repeatingdigestion sequential exoglycosidase digestion andmethylationstudy with almond a-fucosidase and witha mixture of jack bean P(data not shown). These results indicated that the oligosac- galactosidase and P-N-acetylhexosaminidase, all major peaks charides in N10 which were led to fraction 3 in Fig. 5 F (which were converted to Mana.GlcNAc Fuc GlcNAcoT and all miwill be called N10-2 in the following part of this paper) have nor peaksto Man3.GlcNAc .GlcNAcoT (Fig. 4E). The number structures: GalD1 -+ 4(Fucal -+)GlcNAcPl -+ Manal -+ 6 of cycles of the sequential enzymatic digestion needed to and 3 ( G a l P l d GlcNAcPl -+ GalDl-+ GlcNAcPl -+ Manal convert the peaks 1, 2, 3, 4, 5, and 6 to the trimannosyl core -+ 3 and 6)ManPl- 4GlcNAcP1-+ 4(fFucal-+ 6)GlcNAco~. were 1, 1 and 2, 2, 2, 3, and 3, respectively. The possibility of the structures: Gal@l-+ GlcNAcPl -+ GalPl These results, together with the methylation data of N11 -+ GlcNAcPl Galj3l -+ 4(Fucal "+)GlcNAcPl -+ Manal (Table I), indicated that the oligosaccharides in N11 have --* 6 or 3(Manal-+ 3 or 6)Manpl --f 4GlcNAc,f?l+ 4 ( f F u c a l either Manal -+ 6(Manal -+ 3)Manbl -+ 4GlcNAcPl -+ -+ 6)GlcNAco~ was denied by the complete absence of 2,3,4,6- 4(Fucal -+ 6)GlcNAcoT or Manal -+ G(Mana1 -+ 3)ManBl tetra-0-met,hylmannitol in the methylation dataof N10 (Ta- "+ 4GlcNAcPl -+ 4GlcNAco~as their core and GalP1 -+ ble I). Interpretation of the methylation dataof N10 as in the 4(fFucal-+ 3)GlcNAcP14 3 repeating groups as their outer case of NlO-1 confirmed the complete structures of oligosac- chains. Since almost all radioactive oligosaccharides in N11 charides in N10-2 as shown in Table 111. were bound to a ConA-Sepharose column and eluted by 0.1 When the fraction 4 (shown as solid line in Fig. 5 F ) , which M a-methyl mannopyranoside,alloligosaccharides in N11 remained unhydrolyzed during thesecond cycle of sequential should have two outer chainslinked at theC-2 position of two digestion with ,L?-galactosidaseand P-N-acetylhexosaminidase, a-mannosyl residues of their cores. Therefore, N11 should be was incubated with almond a-fucosidase, a complex pattern, a mixture of oligosaccharides, as shown in Table 111. No as shown in Fig. 5G, was obtained. Thiscomplex pattern was further structural study was performed on N11 because of the interpreted that a fucose residue was released from parts of limited amount of sample and of its complex nature. Structural Studies ofMonosialy1 Oligosaccharides-When the oligosaccharides and two fucoses from the remaining parts of oligosaccharides allowing for the following results of exo- fraction AI was subjected to thesecond paper electrophoresis at least ten acidic glycosidase digestion. The four peaks in Fig. 5G were con- using 1.2-m long paper, it was separated into verted into two pairs of a major and a minor peak by incuba- components(Fig. 6A). Thesecomponents were separately tion with either jack bean ordiplococcal P-galactosidase (Fig. and named AI-1-AI-10, respectively. Thetencomponents were purified further by paper electrophoresis under the same 5H, dotted line), releasing one and two galactose residues. The pair of peaks in fraction 5 of the dotted line in Fig. 5 H conditions, and converted to neutral oligosaccharides by sialreleased two N-acetylglucosamines by P-N-acetylhexosamin- idase digestion. By sequential exoglycosidase digestion and idase digestion (Fig. 5H, solid line fraction5 ) . while those in methylation analysis (data not shown), the neutral oligosacfraction 6 of the dotted line in Fig. 5H releasedone N- charide portions of AI-1-AI-10 were confirmed as summarized acetylglucosamine by the enzymatic digestion (Fig. 5H, solid in Table IV. Methylation data of AI-1-AI-10 are summarized in Table line fraction 6 ) .The two components in the solid line fraction 5 were confirmed as Manal -+ 6(Manal -+ 3)Manbl -+ V. By comparing each data with the corresponding neutral 4GlcNAc/?l-+ 4(fFuctul+ 6)GlcNAcor by sequential exogly- Oligosaccharides in Table I, it was concluded that 1 mol of cosidase digestion and by methylation study (data not shown).sialic acid residue in all ten acidic fractions is linked at theCTherefore, the original oligosaccharides in N10 which were 6 position of a galactose residue of their neutral oligosacchaled to thisfraction (whichwill be called N10-3 in thefollowing ride portions. Since N1, N2, N3, and N4 contain only one galactose, the structures of AI-1, Al-2, AI-3, and AI-4 should -+ part of thispaper) should havethestructures:GalPl GlcNAcPl-+ GalPl-+ 4(Fucal -+)GlcNAcPl--+ Manal+ 6 be as shown in Table VI. When radioactive AI-5 and AI-6 was incubated fist with a and/or 3[GalP1-+ 4(Fucal -+)GlcNAcPl-+ Manal-+ 3 and/ mixture of jack bean P-galactosidase and P-N-acetylhexosaor 61 ManPl-+ 4GlcNAcPl- 4(-+Fucal-+ 6)GlcNAco~. The two radioactive components in the solid line fraction minidase, and then with sialidase, radioactive products with 6 in Fig. 5 H gave the same results as those in solid Line mobilities of 10.4 and 11.4 glucose units, respectively, in Biofraction 1 in Fig. 5B in sequential exoglycosidase digestion Gel P-4 column chromatography were obtained (Fig. 7A). (data not shown). Therefore, theoriginal oligosaccharides in These results indicated that oneof the Galpl-+ 4GlcNAcBl N10 which were led to thisfraction (which will be called N10- -+ groups of AI-5 and AI-6 which was not substituted witha 4 in the following part of this paper) should have the struc- sialic acid was removed by the fist enzymatic digestion. When tures: GalP1 -+ 4(Fucal -+)GlcNAcPl -+ GalP1 -+ 4(Fucal these radioactive products were incubated with a-mannosi-+)GlcNAcPl -+ Manal -+ 6 or 3(GalP1 -+ GlcNAcPl -+ dase, approximately70% of both oligosaccharides releasedone mannose residue and 30% of both oligosaccharides remained Manal -+ 3 or6)Manfll -+ 4GlcNAcPl --+ 4 ( f F u c a l -+
-
-+
Sugar Chains ofSecretory Human Component
9617
isomeric oligosaccharides, as shown in Table VI. When radioactive AI-7 and AI-8 were incubated first with a mixture of jack bean /3-galactosidase and P-N-acetylhexosaminidase and then withsialidase, N7 andN8, respectively, were obtained asradioactive products (data not shown). These results indicated that the sialic acid residue in both acidic oligosaccharides is linked only to the Galpl + 4GlcNAcPl + group of N7 andN8andprotecteditsremovalfrom digestion with P-galactosidase and P-N-acetylhexosaminidase. c 0 Therefore, AI-7 and AI-8 should be mixturesof two isomeric oligosaccharides, as shown in Table VI. 4 When radioactive AI-9 was incubated with a mixture of jack beanP-galactosidase and P-N-acetylhexosaminidase and the reaction mixture wassubject,ed to paper electrophoresis, approximately 50% of AI-9 was converted to a pair of smaller acidicoligosaccharides and 50% remained unchanged(Fig. 6 B ) . Fractions “I” and “11” in Fig. 6B were recovered from paper and incubated with siahdase. Elution patterns of the neutral oligosaccharides thus obtainedin Bio-Gel P-4 column chromatography are shown in Fig. 7C. As expected, the neutral oligosaccharides obtained from fractionI1 were eluted at the N10 area (solid line), and the eluting positions of those DISTANCE FROM ORIGlN t cm) FIG. 6. Paper electrophoresis of acidic oligosaccharides lib- from fraction I (15.2 and 14.2 glucose units) indicated that erated from human SC and their fragments.Open arrows indi- they are one Gal/3l + 4GlcNAc group smaller than N10. cate the migrating positions of authentic oligosaccharides: a, NeuAc. When incubated sequentially with jack bean,B-galactosidase Ga12.Fuc.GlcNAcp.Mans.GlcNAc~Fuc~GlcNAc~~; b, NeuAc-Gal?. and P-N-acetylhexosaminidase, both products from fractions GlcNAcs. Man3 .GlcNAc .Fuc .GlcNAcol; c, NeuAcz .Gal,. GlcNAc;.. I and I1 decreased their sizes by 3.0 glucose units, indicating ManafGlcNAc .Fuc .GlcNAcoT. A, the radioactive AI fraction (see text) was subjected to paper electrophoresis using pyridine-acetate that one GalD1- 4GlcNAc group was removed by the enzya Galpl buffer, pH 5.4, and 120-cm-long paper at 80 V/cm for 6 h; B , AI-9 matic digestion (Fig.7 0 ) . These results indicated that + 4GlcNAc group was newly exposed by the desialylation. incubated with a mixture of jack bean P-galactosidase and P-A“ acetylhexosaminidase was subjected to paper electrophoresis as in A ; Based on the results, the oligosaccharides in AI-9 fraction, C, the radioactive AI1 fraction (see text) was subjected to the paper which were converted to the fractionI in Fig. 6B should have electrophoresis using 120-cm-long paper for 3 h. the structures shown as AI-91 in Table VI. That the major peak of fraction I in Fig. 6B showed the same mobility as TABLEIV authentic NeuAc. Galz. Fuc. GlcNAc:! Man:r-GlcNAc Fuc. Per cent molar ratio of acidic oligosaccharides and their neutral GlcNAcoT also supports this conclusion. Whether the sialic portions acid residue is linked to GalPl + 4GlcNAcPl + 3GalP1 -+ Per cent 4(Fuca 3)GlcNAcPl -+ outerchainorto GalP1 -+ molar ratio in Neutral oligosacchaOligosaccharide fraction 4GlcNAc/31+ outer chain or both was not confirmed in this ride portion acidic study. The results described above also indicated that the fraction oligosaccharides inAI-9, which were converted to the fraction Monosialyl oligosaccharides I1 in Fig. 6B, should have the structures shown in AI-911 in N1 AI-1 0.6 Table VI,inwhich bothouterchainsareprotectedfrom AI-2 2.0 N2 degradation with P-galactosidase and P-N-acetylhexosaminiAI-3 2.8 N3 AI-4 6.6 N4 dase mixture by a sialic acid and a fucose residue. AI-5 6.3 N5 Because of the limited amount of sample, the structuresof AI-6 18.7 N6 oligosaccharides in AI-10 were not studiedin detail. However, AI-7 6.1 N7 based on theevidence that its neutral portionwas N11 and on AI-8 17.9 N8 the data of methylation analysis (Table V), the structures of N10-1, N10-2 AI-9 6.1 oligosaccharides in AI-10 were estimated as shown in Table N10-3, N10-4 AI-10 3.3 N11“ VI. Disialyl oligosaccharides Structural Studiesof Disialyl Oligosaccharides-Fraction AII-1 5.8 N5 AI1 was also separated into five components by longer paper AII-2 16.3 N6 electrophoresis (Fig.6C). The five components were recovered AII-3 1.5 N10-1 (-Fuc*) from paper, as indicated by bars in Fig. 6C, and namedAII-1AII-4 4.1 N10-1 AII-5. The five components were purified further by paper AII-5 1.9 N11 “Identification was made only on the basis of elution pattern in electrophoresis under the same condition and converted to neutral oligosaccharides by sialidase treatment. By sequential Bio-Gel P-4 column chromatography. The missing fucose was confirmed to be that in the core portion exoglycosidase digestion and methylation analysis (data not and not that in the outer chain moiety of N10-1. shown), the neutral oligosaccharides liberated from AII-1AII-5 were confirmed as summarized in Table IV. unchanged (Fig. 7B). These results indicated that a sialic acid Methylation data of AII-1-AII-4 are summarized in Table residue in 70% of both AI-5 and AI-6 is linked to thegalactose V. By comparing each data with the corresponding neutral residue on the Manal * 6 side, and in 30% to that on the oligosaccharides, it was concluded that sialic acid residues in 3 side, protectingthe Gal/3l+ 4GlcNAc/31- group all five disialyl oligosaccharide fractions arelinked at theC-6 Manal frQm digestion by /?-galactosidase and /3-N-acetylhex- position of two terminal galactoses. Therefore, the structures osaminidase. Therefore, AI-5 and AI-6 are mixtures of two of the five acidic fractions were proposed as shown in Table
51B
a a
-
i
-
-
96 18
Sugar Chainscf Human Secretory Component TABLEVI Structures of acidic oligosaccharides obtained from human SC m + n > 2. Mean value of fucose content was 3.6. R=GlcNACOT Manalb _13ManB1+4GlcNAcBl+R NeuAca2+6GalBl+4GlcNAcB1’2Manal
&=Fucal+6GlcNAc
AI-1
AI-
AI-2
3
AI-4
AI- 5
AI-6
AI-7
AI-8
Fucal
+
3 Gal61+4GlcNAc61+2Manal~ and
and 6ManBl+4GlcNAcB1+R NeuAca2+6GalB1+4GlcNAcBl+2ManalP Fucal
+
v
3
AI-91
and 3ManB1+4GlcNAcB rR GalB1+4GlcNAc61+2Manal/a and
NeuAca2+6
Fuca 1 &
.R
3
Fucal
Fucal
f
f
3 G a l B l + 4 G l c N A c ~ 1 + 3 G a l B l + 4 G l c N A c ~ l + 2 M a n aand ~
and 6Man61+4GlcNAcB1+R NeuAcu2+6GalB1+4GlcNAc~1+2ManalP Fucal t
3
4
Fucal tFucal
tFucal
+
3 NeuAca2*6
+
3 (GalB1+4GlcNAcgl+3)m.Gal~l*4GlcNAcB1+2Mana~~ pMan61+4GlcNAct31+R (Gal51+4Gl~NAc~1+3)~~Gal61+4GlcNAc61+2Manal 3 t
4
tFucal
tFucal
AI-1 0
AII-I
AII-2
Fucal f
AII-4
AII-3
* Fuca 1
3 (NeuAca2+6)2
tFucal t t 3 ~Ga~~1+4GlcNAcEl+3)m.Ga161+4GlcNAc61+2Manal~ f13Mant31+4GlcNAcBl+R (Gal61~4GlcNA~61+3)~.GalB1+4GlcNAcBl+2Manal 3 t f
Fuca 1
tFucal
AII-5
OT
Sugar Chains of Human Secretory Component
9619
VI. Because of the limited amount of sample, the structures 3GlcNAc/?1+ repeating structure. In the case of human aiacid glycoprotein, the trisaccharide group is included not in of oligosaccharides in AII-5 fraction were rather arbitrary. biantennary but in tri-and tetraantennary sugar chains (15, DISCUSSION 23). These structural evidences indicate the complex nature that forms the GalP1 -+ As pointed out by several previousreports (9,20,21), human of the biosyntheticmachinery 4(Fucal + 3)GlcNAcfll + group and suggests the possibile SC contains only asparagine-linked sugar chains. This paper confimed that the sugar chains show an extraordinarilyhigh presence of multiple fucosyl transferases responsible for the structural multiplicity, and more than 40 structurally different formation of the trisaccharide group in the outer chainmoiesugar chains were found. Since an SC molecule contains four ties of asparagine-linked sugar chains. In view of the recent finding that the trisaccharide group asparagine-linked sugar chains, it must be heterogeneous in works as the determinantof stage-specific embryonic antigen its carbohydrate moiety. It must be pointed out here that an interesting sugar chain reported by Purkayastha et al. (9) is (24, 25), the multiple occurrence of the trisaccharide group is elucidate the enzymatic not included among the oligosaccharides in TablesI11 and VI. of particular interest. In order to The reason for this discrepancy is hard todiscuss because no background of the occurrence of the stage-specific antigen, detailed description about the purityof the glycopeptide and the multiplicity of the fucosyl transferase must be kept in the oligosaccharide samples obtained from SC was given in mind. their paper. It might well be that the sample theyused was a Acknowledgment-Wethank I. Ueda for herskillful secretarial mixture containing most of the sugar chainslisted in Tables assistance. I11 and IV in thispaper. Structural analysis of such a mixture might have led them to a false conclusion. REFERENCES The sugar chains of SC reported here all contain either 1. Brandtzaeg, P. (1974) J . Immunol. 112, 1553-1559 Manal + G(Mana1 + 3)Manpl + 4GlcNAcPl -+ 4(Fuccul 2. Poger, M. E., and Lamm, M. E. (1974) J. Exp. Med. 139,629-642 -+ 6)GlcNAco~ or Manal + G(Mancu1 -+ 3)ManPl + 3. Crago, S. S., Kulhavy, R., Prince, S . J., and Mestecky, J. (1978) J . Exp. Med. 147,1832-1837 4GlcNAcPl + 4GlcNAco~as their core portion, indicating 4. Strober, W., Krakauer, R., Klaeveman, H. L., Reynolds, H. Y., that they are formed by the processing pathway (22) proposed and Nelson, D. L. (1976) N. Engl. J . Med. 294, 351-356 for the biosynthesis of many complex-type asparagine-linked 5. March, J.-P. (1970) Nature 228, 1278-1282 sugar chainsof glycoproteins. Several sugar chains of SC, such 6. Brandtzaeg, P. (1976) Scand. J. Immunol. 5, 411-419 as N1, N2, AI-1, and AI-2, contain only one outer chain.That 7. Mestecky, J., Kulhavy, R., Wright, G. P., and Tomana, M. (1974) these outer chains areexclusively located on the Manal -+ 3 J. Immunol. 113,404-412 side of the core accords with the suggestion that the p-N8. Neufeld, E. F., and Ashwell, G . (1980) inThe Biochemistry of Glycoproteins and Proteoglycans (Lennarz,W. J., eds) pp. 241acetylglucosamine residue onManal-+ 3 sideis added during 266, Plenum Press, New York the processing pathway (22). 9. Purkayastha, S., Rao, C. V. N., and Lamm, M. E. (1979) J . Biol. The sugarchain pattern of human SC summarized in Tables Chem. 254,6583-6587 I11 and VI shows some similarity to that of lactoferrin (11) 10. Yamashita, K., Tachibana, Y., Nakayama, T., Kitamura, M., which occurs also in human milk: Galfll -+ 4(Fuccul + Endo, Y., and Kobata, A. (1980) J . Biol. Chem. 255,5635-5642 3)GlcNAcPl+ group and Galpl-+ 4GlcNAcBl+ 3 repeating 11. Matsumoto, A,, Yoshima, H., Takasaki, S., and Kobata, A. (1982) J. Biochem. (Tokyo) 91, 143-155 group are included in the outer chain moieties of both glycoproteins. However, several prominent structural differences 12. Takasaki, S., Yamashita, K., Suzuki, K., and Kobata, A. (1980) J. Biochem. (Tokyo) 88, 1587-1594 are also found among the sugar chains of the two glycopro13. Takasaki, S., and Kobata, A. (1974) J. Biochem. (Tokyo) 76,783teins. Abasic difference resides in the core structure. The core 789 portion of lactoferrin is Manal + G(Mana1 + 3)Manpl + 14. Yamashita, K., Mizuochi, T., and Kobata, A. (1982) Methods 4GlcNAcPl- 4(Fucal+ 6)GlcNAc only in contrast to that Enzymol. 83, 105-126 of SC which is a mixture of Manal -+ G(Mancu1 -+ 3)Manpl 15. Yoshima, H., Matsumoto, A., Mizuochi, T., Kawasaki, T., and Kobata, A. (1981) J. Biol. Chem. 256, 8476-8484 + 4GlcNAcPl+ 4 ( + F u c a l - + 6)GlcNAc. In addition, many 16. Ohkura, T., Yamashita, K., and Kobata, A. (1981) J . Biol. Chem. structural differences were found also in theouterchain 256,8485-8490 moiety. Galpl + 4GlcNAcfiI + 3Galp1 -+ 4(Fuca1 + 17. Takasaki, S., and Kobata, A. (1978) Methods Enzymol. 50, 50-54 3)GlcNAcP1-+ group and Galpl+ 4(Fucal+ 3)GlcNAcPl 18. Bjork, I., and Lindh, E. (1974) Eur. J . Biochem. 45, 135-145 + repeating structures were found in S C but notin lactoferrin, 19. Takasaki, S., Mizuochi, T., and Kobata, A. (1982) Methods Enwhile Galpl -+ 4(Fucal + 3)GlcNAcPl + 3Galpl + zymol. 83, 263-268 4GlcNAcpl + group was found in lactoferrin but not in SC. 20. Kobayashi, K. (1971) Immunochemistry 8, 785-800 Furthermore, the Galpl+ 4(Fucal -+ 3)GlcNAcPl- groups 21. Van Munster, P. J . J., Stoelinga, G. B. A,, Clamp, J. R., Gerding, J. J. T., Reijnen, J. C. M., and Voss, M. (1972) Immunology 24, in the sugar chains of lactofenin were always found in the 249-256 outer chain linked to the Manal+ 6 side, while those of SC 22. Kornfeld, S., Li, E., and Tabas, I. (1978) J. Biol. Chem. 253, 7771were in both outer chainmoieties. 7778 Galpl- 4(Fucal+ 3)GlcNAc/31+ group was also found 23. Fournet, B., Montreuil, J., Strecker, G., Dorland, L., Haverkamp, J., Vliegenthart, J. F. G., Binette, J. P.,and Schmidt, K. (1978) in the outer chain moieties of human parotid a-amylase (10) Biochemistry 17, 5206-5214 and human al-acid glycoprotein (15, 23). In a-amylase, the trisaccharide group was found in both outer chainmoieties of 24. Hakomori, S., Nudelman, E., Levery, S., Solter, D., and Knowles, B. B. (1981) Biochem. Biophys. Res. Commun. 100, 1578-1586 biantennary structure, asin the case of human SC. However, 25. Hounsell, E. F., Gooi, H. C., and Feizi, T. (1981) FEBS Lett 131, the outer chain of a-amylase does not include any GalPl + 279-282
Chains Sugar
9620
o f Human Secretory Component
H ,.
/I
',
'\,
400 300 ELUTION VOLUME (ml) Secretary Cornpunent-€Free s e c r e t o r yc o m p n e n t IsCL was p u r l f l e d from 2.1 l o f human m l l k c o l l e c t e d a n d p o o l e d f r o m € o o u r l n d l v l d u a l s by modifying t h e method r e p o r t e d by HSdrk andLlndh I 1 81. 'The m o d l f l e d procedures are as f o l l o w s . i n c a ~ ruf CM-cellulose column c h r o m a t o g r a p h y , elution Of S C was p e r f o r m e d by l r n e a r g r a d l e n to f sodium a c e t a t e b u i f e r , pH 5 . 0 from 5 to 500 mM l n s t e a d o f sodlum a c e t a t e b u f f e r , p H . 5 . 2 from 20 t o 200 mM I" t h e orlglndl methodbecause W E found t h a t most Sc d l d n o t b m d to t h e column e q u l l l b r a t e d n t h 20 &4 acetatc b u f f e r , pH 5.2. Another Sephaden G-200 column I5 x 90 c m l chromatography was performed a t t h e f l n a l S t e p to remove a mlnor p r o t e l n peak t h a t f o l l o w e d the malor SC p e a k . The p u r l f l e d Sc (266 m g l , t h u so b t a m e dm w r a t e d as a slngle band stainable by Coomassir blueandPAS-reagent Ln SDS polyacrylamide gel e l e c t r o p h a r a s l s ~n the presence and absence of 8 - m e r c a p t o e t h a n o l . I n Ouchterlony t e s t , the Sc Preparation gave a f u s l n g p r e c l p l t l n l m e W i t hr a b b l ta n t i - h u m a n sc m t l s e r u ma n dr a b b l ta n t l - h u m a n mllk whey a n t i s e r u m . No p r e c l p l t l n l m e was formed agilnst r a b b l t antl-human I q G , antz-human IgA a n da n t l - h u m a nl a c t o f e r r l n a n t l s t x d and goat antl-normal human serum antiserum. The r a b b l ta n t l - h u m a n IgG were p u r c h a s e i antl-human IgA, antl-human SC and a n t i - h u m a n l a c t o f e r r l n a n t i s e r a from Behrlngwerkc AG, Marburg-Lahn. M a ta n t l - n o r m a l human serum antiserum was p u r c h a s e df r o mI g a k u - S e l b u t s u g a k u Kenkyusho Nagoya. Rabhlt anti-human mllk whey ilntlserum was prepared ~n our l a b o r a t o h y .T h e m l a r CiEltLO O f monosacchaI r d e s oi t h e Sc sample as d e t e r m r n e d by t h er a d l o e l e c t r o p h o r e t l cm e t h o do f T a k a s a k l andKobata 1171 was 4 . 4 mol N - a c e t y l g l u c o s a m m e 2.0mol f u c o s e , 2 . 5 mol g d l a c t o s e a n d 0.9 m o l N - a c e t y l n e u r a m i n l c acld per 3 k l mannose. valneb m o s t l ya c c o r dw l t h those r e p o r t e d by Kobayashl (20) and MunSterTehE al. 1211. Slnce no N - a c c t y l g a l a c t o s a m l n e was d e t e c t e d , S C s h o u l dn o tc o n t a i na n y mucln-type sugar c h a m .
D
E Details o fo t h e rr n a t e r l a l s
have b e e n d e s c r l b r d
and a n a l y t r c a l methodsused
~n our recent p u b l l c a t r o n s (11, 14-16].
IB
i n 1- h e p r e s e n t s t u d y
- - . ~
>-
400 300 ELUTIONVOLUME
(ml)
Chains Sugar
400 300 ELUTIONVOLUME
of Human Secretory Component
962 1
(ml)
Table V.
Molar ratio of aldrtol acetates obtarned from the hydrolysates of permerhylated neutral oligosaccharldea. tablewere calculated by making the values of 2 . l - d i - O - n e t h ~ 1 , " a n n o t ~ ~1.0. ~~
Nmbers i n the
Methylated sugar
111-1 AI-2 AI-3AI-4
M - 5 AI-6 AI-7 111-8 A I - 9 11-10 RII-1
AII-2 AII-3 X I - 4
FYCltOl
2.3.4-Trl-0-methyi 11,5-d1-o-&eeyl>
0
1.0
0
0
0
0
0
0.9
0.9
0
1.2
1.2
2.1 2.3 3.5
0
0
0
0
1.1
1.1
1.0
1.1
0
0
0
0
1.0
0.9
1.1
1.0
1.2
1.1
1.1
1.0
1.0
1.2
0
0
1.0
1.1 1.9 2.0
0
0
1.0
1.0
1.0
0
0
1.1
1.1
0
0
0
0
0.9 2.7
0
0
0.9
1.0
2.0
1.9
1.9
2.1
0.9
2.1
Galactitol
2.3.4.6-Tetra-0-methyl 11.5-dl-~-acet7ll 2.4.6-Trl-g-methyl 11,3,5-trl-~-acetyll 2.3.4-Tri-0-methyl 11,5,6-tri~~-acetyl)
1.0
1.1
Ma""it01
2.3.4.6-Terra-0-nethyl I1 ,5-di-Q-acetyl) 3.4,6-Tri-~-methyl
U
t
Numbers ~n the
Methylated s u q a r
...
n
; '
1
2,l-Dl-g-methyl
I
",
.,
tablewere calculated by rnaklng the values of 2.4dr72-methyl m a n n i t o l N2
0
0
2.1
1.9
1.9 2.1
1.0
1.0
1.0
1.0
1.0
1.1
0
0.9
0
0.9
0
0
1.1
0
1.1
0
1.0
0.9
0.8
0.9
0
0
0
0
0
0
0
0
0
0
0
0
2.2
2.0
2.1
2.0
2.2
1.0
1.0
1.0
1.0
1.0
0.3 0 . 3
0.9
0
1.0
0
0
1.1
0
1.1
N5
N4
N8
N6
N7
0
1.0
1.1
2.8 2.0
2.1
2.1 0
0
0.9
0
1.1
1.0
1.1
1.2
2.0
1.9
0
0
0
0
0
0
1.1
1.0
l1,1.5,6-t~tra-~-acetyl~
2-pKethylacetan~do -2-dwxyglucitol 1.3.5.6-retra-o-methyl 14-mono-2-acetyl) 1.3.5-Tri-0-methyl 14.6-dl-2-hcetyl) 3.4.6-Tri-g-methyl 11.5-d~-O-acetyl)
300 400 ELUTION VOLUME (mi)
N1 N3
0
2.0
11,2,5-tri-l)-acetyl)
0
a6 1.0.
N10
Nll
2.2
3.6
2.0
2.0
2.0
0
1.0
2.8
N9
1.1
7.1
0
0
0
0
0
0
0
0
0
0.9
1.1
2.0
2.1
2.1
2.0
2.0
2.1
1.9
2.0
2.1
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
0
1.0
0
0.3
0
1.7
0
0.2
0.3
0.3
0
1.1
0
1.1
0
0.9
0
1.1
0.7
0.8
0.9
0
0
1.1
1.0
0
0
0
0
0
0
0
2.2
2.0
2.1
2.2
3.0
9.i
2.1
2.0
1.1
2.1
3.5
0
0
0
0
0
0
1.0
1.1
2.0
7.3
2.7
1.1
0
1.0
0
0
0.9
0
0
0
3.1
2.9
3.0
0
1.1
1.1
