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Oligosaccharides of human milk
ARCHIVES
OF
BIOCHEMISTRY
AND
160, 273-281 (1972)
BIOPHYSICS
Oligosaccharides IV. Isolation
and Characterization
of Human
of a New Hexasaccharide,
A. KOBATA’
Institutes of Health,
Received
January
Lacto-N-neohexaose
V. GINSBURG
AND
National
A new hexasaccharide,
Milk
Bethesda, Maryland
13, 1972; accepted
“la&o-N-neohexaose,”
February
with
20014 24, 1972
the probable
structure
Galpl4GlcNacpl 6 has been isolated from human milk. In addition some fucosyl and sialyl derivatives of la&o-N-neohexaose and a previously described hexaose, lacto-N-hexaose, have been partially characterized.
A hexasaccharide, the structure2
lacto-N-hexaose,
with
EXPERIMENTAL
Galpl-4GlcNAcpl I 6 Gal~1-3GlcNAc~l-3Galpl4Glc
was recently isolated from human milk (1). The present paper describes the characterization of another hexasaccharide “lacto-Nneohexaose” which differs from Iacto-Nhexaose only in that both of its terminal galactose residues are linked fi1-4 as follows:
NANA~2-3Ga1~1-3GlcNAcpl-3Galpl-4Glc 6 I
Galpl-4GlcNAcpl I
6
NAN Aor;
Gal~l-4GlcNAc~1-3Gal~l4Glc.
(disialyllacto-N-tetraose)
In addition, the structures of some fucosyl and sialyl derivatives of both Iacto-N-hexaose and lacto-N-neohexaose have been investigated.
while NANAa26Gal was prepared by acetolysis of NANAor2-GGalfll-4Glc (6’-sialyllactose) (7). The disaccharides were purified from other acetolysis products by paper chromatography using solvent I (the relative mobility of the three disaccharides in this solvent are shown in Fig. 5). GlcNAc$14sorbitol and GlcNAcpl-6Ga@l4sorbitol were prepared flom lacto-N-hexaitol by partial hydrolysis (0.1 N HCl at 100°C for 40 min) and purified by paper chromatography using solvent II (1).
i Present address: Department of Biochemistry, Kobe University School of Medicine, Kusunoki-cho 7 Chrome, Ikuta-Ku, Kobe, Japan. 2 All monosaccharides mentioned in this paper have a D configuration except for fucose which has an L configuration. 273 Copyright
@I 1972 by Academic Presls, Inc.
PROCEDURE
Paper chromatography. Descending paper chromatography wm performed with the following solvents: I, ethyl acetate-pyridine-acetic acidwater (5:5:1:3); and II, ethyl acetate-pyridinewat,er (12:5:4). Sugars were located with AgNOs reagent (2) or with periodate-benzidine reagent (3). Thiobarbituric acid reagent was used to detect oligosaccharides that contain sialic acid (4). Standard oligosaccharides. Known oligosaccharides were isolated from human milk by methods described previously (1, 5, 6). NANAol23Gal and NANA~Y~-GGIcNAc were prepared by acetolysis of
274
KOBATA
AND
Labeling oligosaccharides with 3H. Oligosaccharides were labeled at their reducing ends by reduction with PHIsodium borohydride a follows: One micromole of oligosaccharide was dissolved in with a 0.5 ml of 0.01 M [3H] sodium borohydride specific activity of 100 &i/pmole (New England Nuclear Co., Boston, MA). The reaction mixture was kept at room temperature for 1 hr and then cooled to 0°C. A drop of glacial acetic acid was added to destroy excess reagent and the solution was passed through a column (0.5 X 3 cm) of Amberlite AG 50(H+) in the cold. The column was washed with 3 ml of HZ0 and the effluent and washing combined and evaporated to dryness under vacuum. Boric acid was removed by the repeated addition and removal under vacuum of methanol (live times). The oligosaccharide was then purified by chromatography as a 3-cm band on Whatman No. 1 paper developed with solvent I. Development time varied with the size of the oligosaccharide: 3 days for tetra- and pentasaccharides and 10 days for hexa-, hepta-, and octasaccharides. The reduced oligosaccharides, which move slightly slower than the corresponding unreduced oligosaccharides, were located by scanning the chromatogram for radioactivity and eluted from the paper with water. Glycosidases. fl-Galactosidase and ,9-N-acetyiglucosaminidaae from Jack bean (8) were the same preparations reported previously (1). p-Galactosidase from almond emulsin was purified by the method of Schwartz et al. (9). *H-Labeled oligosaccharides were treated with the glycosidases aa follows : The 3H-labeled oligosaccharide (about 5 nmoles containing 1.0 X lo5 cpm) was incubated with 10 ~1 of the glycosidase solution and 2 ~1 of toluene for 20 hr at 37°C in a sealed tube. The glycosidase solution w&s prepared by diluting the purified preparations with buffer so that 10 pl of the diluted enzyme cleaved about 20 nmoles of the appropriate p-nitrophenyl p-glycoside per hour at 37°C. The p-galactosidase from almond emulsin and Jack bean was diluted with 0.1 M phosphate-citrate buffer, pH 6.0; the p-N-acetyglucosaminidase from Jack bean was diluted with 0.1 M phosphate-citrate buffer, pH 5.4. Under the above conditions of incubation the fl-galactosidase from Jack bean can be used to distinguish Galpl3GlcNAc and Galpl4GlcNAc bonds: the enzyme cleaves the Ga@l4GlcNAc bond of la&o-N-neotetraose but not the Galpl3GlcNAc bond of lacto-N-tetraose.3 The p-galactosidase from almond emulsin, in contrast, cleaves both tetraoses equally well and at about 10% the rate of p-pnitrophenylgalactoside. Hapten-inhibition studies. Hapten-inhibition 8 A. Kobat)a,
unpublished
data.
GINSBURG studies were studied with quantitative precipitin reactions between human Lea, Leb, and H substances and goat anti-Lea and anti-Leb sera and Eulex europeaus lectin, respectively (10). RESULTS
AND
DISCUSSION
Isolaticn
of lack-N-neoh.e.xaose. Lacto-Nneohexaose is best isolated from the milk of donors with a Lewis negative blood type as described for lacto-N-hexaose (1). During gel filtration with Sephadex G-25 and preliminary paper chromatography the two hexaoses do not separate; they are separated by paper chromatography for 10 days using solvent II as lacto-N-neohexaose migrates 5 % faster than lacto-N-hexaose (1). About 5 mg of lacto-N-neohexaose is obtained from 1 liter of milk. of la&o-N-neohexaose. Characterization Lacto-N-neohexaose, like lacto-N-hexaose contains three galactose residues and two N-acetylglucosamine residues for each glucose residue (Table I). Glucose is at the reducing end of the chain as all the glucose disappears and sorbitol is produced upon reduction of the intact oligosaccharide with NaBH4 as evidenced by paper chromatography after acid hydrolysis. Thus, the sugar is a hexaose and isomeric with lacto-N-hexaose. Enzymatic and chromatographic evidence presented below suggests that it differs from lacto-N-hexaose only in the linkage of one of its two terminal galactose residues. Products of the action of &galactosidases and P-N-acetylglucosaminidase on lacto-Nhexaitol and lacto-N-neohexaitol are shown in Fig. 1. One of the two terminal galactose residues of lacto-N-hexaitol is hydrolyzed by Jack bean /3-galactosidase to yield the pentasaccharide GlcNAcfil I 6 Galpl-3GlcNAcpl3Gal@l-4Sorbitol
as the Jack bean enzyme cleaves Gal@l4GlcNAc linkages faster than Galpl-3GlcNAc linkages (see Experimental Procedures). The chromatographic mobility of lacto-N-hexaitol and the pentasaccharide derived from it are shown in Fig. 1A and lB, respectively. In contrast, treatment of lactoN-neohexaitol with Jack bean P-galactosid-
OLIGOSACCHARIDES OF HUMAN MILK
275
charide GlcNAcBl I
6 GlcNAc,V1-3Gal~1-4sorbitol
which is formed from lacto-N-hexaose on treatment with almond emulsin p-galactosidase (Fig. 1C). Almond emulsin p-galactosidase, unlike the Jack bean enzyme, does not distinguish between Galbl-3GlcNAc and Gal/314GlcNAc linkages (see Experimental Procedure) and releases both terminal galactose residues from lacto-N-hexaose at the same rate. The tetrasaccharides derived from la&o-N-neohexaose and lacto-N-hexaose were both hydrolyzed to lactitol on incubation with /3-N-acetylglucosaminidase (not shown in Fig. 1). When lacto-N-neohexaitol was briefly treated with Jack bean /3-galactosidase a cWFS ORIGIN km: X’CPCE pentaitol intermediate was detected (Fig. lF, peak 2) in addition to the final tetraitol Fro. 1. Action of glycosidases on [aH] lecto-Nproduct (Fig. lF, peak 1) and residual lactohexaitol and [SH] lacto-N-neohexaitol. The condiN-neohexaitol (Fig. lF, peak 3). The pentations of incubation are given in Experimental Procedure. The radioactive sugars were chromatitol intermediate had the same chromatoographed for 4 days using solvent II and the graphic mobility as the pentssaccharide resulting chromatograms scanned for radioactivderived from lacto-N-hexaitol (Fig. 1B). ity. A, la&o-N-hexaitol; B, lacto-N-hexaitol However, upon incubation with P-N-acetyltreated with Jack bean o-galactosidase; C, lactoglucosaminidase it gave rise to two tetraitols N-hexaitol treated with almond emulsin @-galacin approximately equal amounts (Fig. 2G, tosidase; D, la&o-N-neohexaitol; E, lacto-Npeaks 4 and 5). The chromatographic mobilneohexaitol treated with Jack bean p-galactosiity of one tetraitol (peak 4) was the same as daae; F, lacto-N-neohexaitol treated with Jack while the mobility bean p-galactosidase for a shorter time than in E standard lacto-N-tetraitol of the other (peak 5) was slower. Incubation (1 hr instead of 20 hr); G, peak 2 (from F) treated with almond emulsin @-N-acetylglucosaminidase; of both tetraitols with Jack bean ,8-galactoH, peak 4 (from G) treated with Jack bean ,%galac- sidase resulted in the formation of triitols: tosidaae; I, peak 5 (from G) treated with Jack the triitol derived from peak 4 had the bean @-galactosidase; and J, standard sugars as chromatographic mobility of standard Glcfollows: T-3 is GleNa@3Galfll4sorbito1, T-6 is NAcfl13Gal@l-4sorbitol (Fig. 1H) while GlcNAcpl-6Gal@4sorbitol, LNnT-OH is lactothe triitol derived from peak 5 had the N-neotetraitol, and LNnH-OH is la&o-N-neo chromatographic mobility of standard Glchexaitoi. NAcfil-C,Galfil-4sorbitol (Fig. 11). The most probably structure of lacto-Nase under the same conditions releases two neohexaitol based on these studies and by galactose residues instead of one resulting in analogy to the structure of other milk oligothe formation of a tetrasaccharide. The saccharides is shown in Fig. 2 along with a chromatographic mobilities of lacto-N-neosummary of the action of the glycosidases. hexaitol and the tetrasaccharide derived La&o-N-neohexaose would be the correfrom it are shown in Fig. 1D and lE, respec- sponding unreduced sugar. tively. The chromatographic mobility of the Isolation of higher oligosaccharide fractions N-d, N-S, S-5, and S-6. Higher oligosactetrasaccharide is identical to the tetrasac-
276
KOBATA
AND GINSBURG
GalOl-4GlcNAcDl I 6 GalPl-4GlcNAc~l-3Gal~1-4Sorbitol lacto-&neohexaitol (Fig. 1D)
GlcNAcO
1
k
+
I 6 GalPt-4GlcNAcL31-3Galpl-4Sorbitol (Fig. lF, peirk 2)
~~dfli--dGdN~~flj
GlcNAcPl-3GalSl-4Sorbitol
I [ P-GlcNAcare GlcNacPl
I Gal~l-4GlcNAc~l-3Galpl-46orbitol lacto-N-neotetraitol (Fig. 1G. peak 4)
1 p-G&S
GlcNAcPl-3Galpl-4Sorbitol (Fig. 1H)
I 6 GldUAcfll-3GalPl-4Sorbitol (Fig. 1E)
I
Gal~1-4GlcNAcpl-6Gal~l-4Sorbitol (Fig. 1G. peak 5)
fl-GlcNAcare
GalpldSorbitol lactitol
GlcNAcpl-GGalpl-46orbitol (Fig. II)
FIG. 2. Probable structure of la&o-N-neohexaitol and the products of glycosidase action (see text and Fig. 1). fl-Galase is 8-galactosidase; &GlcNAcase is &N-acetylglucosaminidase.
charide fractions N-2, N-3, S-5, and S-6 were 0.79) and N-3 (RLNDeI = 0.50) were eluted separately with HzO, passed through a obtained as follows: the oligosaccharides from 1 liter of milk were fractionated by gel mixed-bed column (0.5 X 3 cm) containing Amberlite AG5O(H+) and AG3(OH-) and filtration with Sephadex G-25 aa previously lyophilized. From the paper containing the described (1, 5, 6). The fractions containing hexaoses and higher oligosaccharides [for acidic fraction, S-5 (RDLN = 0.77) and S-6 example, fractions l-23 in Fig. 1 of Ref (l)] (RDLN= 0.61) were eluted separately with were combined and lyophilized. The white water and lyophilized directly. The yield residue was dissolved in HzO, applied as a of each fraction varied with different samples streak on Whatman No. 3MM paper (2 mg of milk but about 30 mg of N-2, 35 mg of of sugar per cm), and subjected to electroN-3,20 mg of S-5, and 20 mg of S-6 are obtained . phoresis at 73 V/cm for 1 hr using HzOpyridine-glacial acetic acid (3870 :30 : 11.5) Fraction N-2 isolated from the milk of as a buffer (pH 5.4). Four acidic sugar bands “secretors” (but not from the milk of “nonand one neutral sugar band were detected by secretors”) can be further resolved by paper staining guide strips with AgNOs reagent. chromatography for 14 days using solvent one into two fractions (N-2-l and N-2-2) ; The neutral fraction and the slowest moving acidic fraction were eluted from the paper the Rf of N-2-l is 5 % higher than the RI of with water and applied separately as streaks N-2-2. Milk samples from nonsecretors conon Whatman No. 3 MM paper (1 mg of sugar tain only N-2-2; N-2-l is missing. Fractions per cm). The papers were then developed N-3, S-5, and S-6 are not further resolved by with solvent I for 10 days; lacto-hr-difucothe same chromatographic treatment. hexaose I (LND-I) was used as a standard Characterization of the higher oligosacdmride fractions. The monosaccharide composition for the neutral fraction and disialyllacto-l\rtetraose (DLN) was used as a standard for of oligosaccharide fractions N-2-1, N-2-2, the acidic fraction. From the paper con- N-3, S-5, and S-6 are given in Table I. Their composition differs from lacto-N-neohexaose taining the neutral fraction, N-2 (RLNDmI =
OLIGOSACCHARIDES
OF HUMAN
TABLE
MILK
277
I
MONOSACCHARIDE COMPOSITION 0~ LAcTo-N-neoHEx.4osq LACTO-N-HEXAOSE, HIGHER OLIGOSACCHARIDE FRACTIONS OF MILKY
AND SOME
Monosaccharide ratio Oligosaccharide Lacto-N-neohexaose Lacto-N-hexaose N-2-l N-2-2 N-3 s-5 S-6
Glucose
Galactose
N-Acetylglucosamine
Fucose
Sialic acid
1.00 1.00 1.00 1.00 1.00 1.00 1.00
2.73 2.86 2.95 2.88 2.95 2.84 2.75
1.79 1.83 2.01 1.91 2.11 1.96 1.81
0.92 1.11 1.88 0.82
1.11 1.08
= The sugar composition of the oligosaccharides was determined by gas chromatography of the reduced and acetylated derivatives of acetolysis (11). Lacto-N-tetraose and lacto-N-fucopentaose I were used as standards. Sialic acid waz measured calorimetrically after hydrolysis of the oligosaccharide in 0.1 N HzS04 at 80°C for 1 hr (12). N-2-1, N-2-2, N-3, S-5, and S-6 refer to higher oligosaccharide fractions described in the text.
and lacto-N-hexaose in that they contain fucose or siliac acid or both. The results of acid hydrolysis shown in Fig. 3 suggest that these oligosaccharides include fucosyl and sialyl derivatives of these hexaoses. Under conditions which liberate most of the fucose, N-2 and N-3 give rise to oligosaccharides with the chromatographic mobility of the hexaoses (lacto-N-hexaose and lacto-hi-neohexaose are not distinguished under the chromatographic conditions of Fig. 3) (lanes 7 and 9 in Fig. 3). Milder acid hydrolysis of N-3 yields, in addition, some N-2 (lane 10 in Fig. 3). Hydrolysis conditions, which liberate sialic acid but not fucose, converts S-6 into N-2 and S-5 into a hexaose (lanes 3 and 5, respectively, in Fig. 3). Longer hydrolysis of the N-2 derived from S-6 results in the formation of hexaose (not shown). Lacto-Nhexaose and lacto-N-neohexaose can easily be distinguished by paper chromatography after reduction by [3H]sodium borohydride. The oligosaccharide fractions N-2-1, N-2-2, N-3, S-5, and S-6 were labeled with 3H by reduction (see Experimental Procedure) and the reduced derivatives subjected to mild acid hydrolysis to liberate fucose and sialic acid. In all fractions except S-6, two hexaitols mereformed whosechromatographic mobility corresponded to lacto-N-hexaitol and lactoN-neohexaitol. A typical result is shown in Fig. 4A. The relative amounts of the two
hexaitols varied but generally there was more lacto-N-hexaitol than lacto-N-neohexaitol (oligosaccharide fractions obtained from five different samples of milk were tested). Surprisingly, hydrolysis of samples of S-6 obtained from five different donors gave rise to only one hexaitol, corresponding in chromatographic mobility to lacto-N-neohexaitol as shown in Fig. 4B. These results indicate that fractions N-2-l and N-2-2 contain monofucosyl derivatives of lacto-N-hexaose and lacto-N-neohexaose; N-3 contains difucosyl derivatives of lactoN-hexaose and lacto-N-neohexaose; S-5 contains monosialyl derivatives of lacto-N-hexaose and lacto-N-neohexaose; and S-6 contains monofucosyl monosialyl derivatives of only la&o-N-neohexaose. Fucose occurs in the oligosaccharides of human milk in at least 4 different linkages: al-2 to gala&se, al-3 to N-acetylglucosamine, (~14 to N-acetylglucosamine, and al-3 to glucose; sialic acid occurs in at least 3 different linkages: ~~2-3 to galactose, ~~2-6 to galactose, and ~~2-6 to N-acetylglucosamine [for structures see (5, S)]. Using only these linkages, there are theoretically possible 6 isomeric monofucosyl derivatives of lacto-N-hexaose and 6 of lacto-N-neohexaose in N-2, 15 isomeric difucosyl derivatives of each hexaose in N-3, 6 isomeric monosialyl derivatives of each hexaose in S-5, and 36
278
KOBATA
AND GINSBURG
FIG. 4. Pr0duct.s of partial acid hydrolysis of some higher oligosaccharides of human milk labeled by reduction with [*HI sodium borohydride (see Experimental Procedure). The products of hydrolysis were chromatographed for 9 days using solvent I. A, hydrolysis products of reduced fraction S-5 (0.01 N HCl for 10 min at 1OO’C); B, hydrolysis products of reduced fraction S-6 (0.01 N HCl for 60 min at 100°C); C, standard sugars as follows: LNnH-OH is lacto-2\i-neohexaitol, LNHOH is lacto-iV-hexaitol, N-2-2-OH, N3-OH, S-5-OH, and S-6-OH are reduced fractions N-2-2, N-3, S5, and S-6, respectively. FIG. 3. Paper chromatography of the products of partial acid hydrolysis of the oligosaccharide fractions N-2, N-3, S-5, and S-6. The chromatogram was developed for 6 days with solvent I and the sugars visualized with AgNOa reagent (2). The sugars circled with dotted lines also reacted with thiobarbituric acid reagent (4) aft.er hydrolysis (5). Lane 1 and 8, N-2: lane 2, S-6; lane 4, S-5; lane 6, lacto-iV-hexaose; lane 11, N-3; lanes 3 and 5, hydrolysis products of S-6 and S-5, respectively (0.01 N KC1 at 100°C for 8 min); lanes 7 and 9, hydrolysis products of N-2 and N-3, respectively (0.01 N HCl at 100°C for 60 min); and lane 10, hydrolysis products of N-3 (0.01 N KC1 at 100°C for 40 min).
isomeric monofucosyl monosialyl derivatives of lacto-N-neohexaose in S-6. As isolated, N-2-1, N-2-2, N-3, S-5, and S-6 are probably mixtures of isomers with fucosyl and sialyl residues attached at different positions. As mentioned above, fraction N-2-l occurs only in milk from secretors and is absent from the milk of nonsecretors while fraction N-2-2 occurs in both types of milk. As the
biochemical basis for secretor status is the presence or absence of the fucosyltransferase responsible for forming Fuccrl-2Gal linkages (13), the fucose residues in N-2-l are probably linked (~1-2 to galactose while in N-2-2 the fucose residues are linked to other positions (e.g., al-3 and ~~1-4 to N-acetylglucosamine or al4 to glucose). This supposition was supported by testing N-2-l and N-2-2 for their ability to accept N-acetylgalactosamine in the reaction catalyzed by the human N-acetylgalactosaminyltransferase responsible for the formation of the determinants of blood group A in man (14). This enzyme, which forms GalNAcal3Gal linkages, requires the acceptor galactose to be substituted on the two position with fucose. Under standard assay conditions (14), N-2-l was as active as lacto-N-fucopentaose I in functioning as an acceptor for the A gene enzyme; N-2-2 was completely inactive. However, even with the assignment of fucose in an al-2 linkage to a terminal galactose in N-2-1, four monofucosyl derivatives are
OLIGOSACCHARIDES
possible, two of lacto-N-hexaose and two of lacto-N-neohexaose. Fraction N-3 also appears to be a complex mixture of isomers as its serologic properties depend on the blood type of the donor from whose milk it is isolated. N-3 isolated from a donor belonging to blood group Le(a-b+) is a haptenic inhibitor of anti-Leb, anti-Lea, and anti-H reagents; N-3 isolated from a nonsecretor belonging to the blood group Le(a+b-) inhibits only the anti-Lea reagent; and N-3 isolated from a secretor belonging to the blood group Le(a-b-) inhibits only the anti-H reagent. These results are consistent with the known distribution of other fucose-containing oligosaccharides and fucosyltransferases in these individuals (5, 6, 13, 15, 16) and with the structural determinants of Leb, Lea, and H specificities (17): the Leb determinant is FuccJ-2Galpl-3GlcNAc
OF HUMAN
MILK
279
was removed from the CHCL extract by the addition of anhydrous NazSOd, the extract was filtered to remove NazSOd, and the filtrate was evaporated to dryness. Methanol saturated with NH+ 3 ml, was added to the residue and the reaction mixture left 3 days at room temperature with intermittent shaking under anhydrous conditions. The solution was evaporated to dryness and residual NH3 was removed by the repeated addition and evaporation (three times) of a mixture containing equal volumes of methanol, ethyl acetate, and toluene. The residue was dissolved in 0.5 ml of Hz0 and passed
...,
i’ Fuccvl
the Lea determinant
is
Galpl-3GlcNAc.
,
7 Fucorl
and the H determinant is either Fuccul2Gal@l-3GlcNAc.. . or Fuccrl-2Gal/314GlcNAG . . . . Acetolysis of oligosaccharides that contain sialic acid is a useful method for structural studies as sialyl linkages are far more resistant to acetolysis than hexosyl or N-acetylhexosaminyl linkages and fragments containing sialic acid can be obtained (7). Fractions S-5 and S-6 were acetolyzed as follows: 5 mg of oligosaccharide were dissolved in 0.25 ml of a mixture of acetic anhydride, glacial acetic acid, and cone HzS04 (10: 10: 1 by volume) and kept at room temperature for 3 days under anhydrous conditions. The reaction mixture was added to 5 g of ice and neutralized to pH 4.0 by the addition of powdered sodium carbonate. After standing for 1 hr at 0°C the acetylated sugars were extracted three times with 5-ml portions of CHCI,. The combined extract was backwashed once with 15 ml of HzO. Water
FIG. 5. Chromatographic analysis of the sialic acid-containing oligosaccharides obtained by acetolysis of fractions S-5 and S-6. The chromatogram was developed for 3 days with solvent I. Lanes 1 and 6, standard NANAa2-GGal; lanes 2 and 7, standard NANAa2-3Gal; lanes 3 and 8, standard NANAor2-6GlcNAc; lanes 4 and 5, acetolysis products of oligosaccharide fractions S-5 and S-6, respectively. The black arrows indicate disaccharide products while the white arrows indicate tetrasaccharide products; lane 9, standard 6’-sialylactose; lane 10, standard 3’-sialyllactose; and lane 11, standard NANAorZ-GGal@l4GlcNAc@l-3Galpl-4Glc (LST-c).
280
KOBATA
AND
through a column (0.5 cm diam) containing 3 cm of Amberlite AG 5O(H+) in a lower layer and 3 cm of Dowex 1 (acetate) in an upper layer. The column was washed with water, and the sialic acid-containing sugars eluted wit,h 4 ml of 0.1 M sodium acetate. The eluate was lyophilized and a portion of the residue was analyzed by paper chromatography. As shown in Fig. 5, lanes 4 and 5, the acetolysis products from S-5 and S-6 were the same: only one disaccharide containing sialic acid was obtained from each (indicated I
I
I
!I? I 5 5 IO
I
GlcN e
I
I
. 1
, NANA*it,
s
,
z,
I
2
3
4
,::~i'":'r"
5
s
Fra. 6. Chromatographic analysis of the hydrolysis products of the sialic acid-containing oligosaccharides obtained by acetolysis of fractions S-5 and S-6. The chromatogram was developed for 24 hr with solvent II. The black spots represent sugars visualized with AgN08 reagent (2) while the spots circled with dotted lines represent sugars that also reacted with thiobarbituric acid reagent for sialic acid (4). Lane 1, the disaccharide formed by acetolysis of either S-5 or S-6; lane 2, products arising from the hydrolysis of either disaccharide in 0.01 N HCl for 10 min at 100°C; lane 3, the tetraaaccharide formed by acetolysis of either S-5 or S-6; lane 4, products arising from the hydrolysis of either tetraaaccharide in 0.01 N HCl for 10 min at 100°C; lane 5, products arising from the hydrolysis in 1 N HCl for 1 hr at 100°C of the neutral trisaccharide indicated by the arrow in lane 4; and S, standard sugars.
GINSBURG
by the black arrows). Both disaccharides corresponded in chromatographic mobility to NANAcr2-6Gal and on hydrolysis gave rise to galactose and sialic acid (Fig. 6, lane 2). The conditions of acetolysis also gave rise to sialic acid-containing tetraoses from S-5 and S-6 (indicated by the white arrows in Fig. 4, lanes 4 and 5). Hydrolysis of both tetraoses gave rise to sialic acid and triaoses (indicated by the black arrow in Fig. 6, lane 3). The triaoses had the chromatographic mobility of Galpl-3GlcNAcpl-3Gal but are clearly not Gal/?l-3GlcNAcpl3Gal as the unknown triaoses were cleaved by Jack bean /3-galactosidase under conditions in which standard Gal813GlcNAc/314Glc was resistant (see “Glycosidases” in Experimental Procedure). Both triaoses gave rise to galactose and glucosamine after complete hydrolysis (Fig. 6, lane 5). As the triaoses are chromatographically distinct from Galpl4GlcNAcfilSGal (see Fig. 5) their probable structure is GalPl4GlcNAc/31-6Gal. From the foregoing data it is likely that oligosa,ccharide fraction S-5 consists predominantly of the monosialyl derivative of lactoN-hexaose, NANArr2-GGalpl4GlcNAcpl I 6 Galfll-3GlcNAc~1-3Gal@l4Glc
and the monosialyl derivative of lacto-N-neohexaose, NANAor2-GGal@l-4GlcNAcfll I 6 Galpl-4GlcNAc@1-3Gal@1-4Glc.
Oligosaccharide fraction S-6 consists predominantly of monofucosyl derivatives of the sialyllacto-N-neohexaose isomer shown above. The absence of lacto-N-hexaose from S-6 may be due to enzyme specificity. For example, if sialic acid is added last in the synthesis of S-6 it is conceivable that the sialyltransferase responsible for the addition of sialic acid to monofucosyl derivatives of la&o-N-neohexaose cannot use monofucosyl derivatives of lacto-N-hexaose as substrates. The linkage of sialic acid to galactose in S-5 and S-6 is consistent with its linkage to galactose in the other sialic acid-containing
OLIGOSACCHARIDES
oligosaccharides of human milk, NANAa23Gal/3l-/3-3GlcNAc~l-3Galpl-4Glc (LSTa) (18)) NANAa2-6Gal/314GlcNAc$3-3Gal/31-4Glc (LST-c) (IS), and NANA~2-3Gal~l-3GIcNAc~l-3Gal~14Glc 7 NANAa2 (disialyllacto-N-tetraose)
(19) :
when the galactose is linked /314 to Nacetylglucosamine (as it is in S-5, S-6, and LST-c) the sialic acid is linked a2-6 to galactose; when the galactose is linked pl-3 to N-acetylglucosamine (as it is in LST-a and in disialyllacto-N-tetraose) the sialic acid is linked a2-3 to galact.ose. Again, this phenomenon could be explained by the specificity of the sialyltransferases that form the a2-6 and a23 linkages. ACKNOWLEDGMENT We thank Dr. Donald Marcus the hapten-inhibition studies.
for carrying
out
REFERENCES 1. KOBATA, A., AND GINSBURG, V., (1972) J. Biol. Chem. %47,1525. 2. ANET, E. F. L. J., AND REYNOLDS, T. M. (1956) Nature (London) 174, 930.
OF HUMAN
MILK
281
3. GORDON, H. T., THORNBURG, W., AND WERUM, L. N. (1966) Anal. Chem. 28,849. 4. WARREN, L. (1969) Nature (London) 186, 237. 5. KOBATA, A., TSUDA, M., AND GINSBURG, V. (1969) Arch. Biochem. Biophys. 130, 599. 6. KOBATA, A., AND GINSBURG, V. (1969) J. BioL Chem. 244, 5496. 7. KUHN, R., AND WIEGANDT, H. (1963) Chem. Ber. 96, 866. 8. LI, Y. T., AND LI, S. C. (1968) J. Biol. Chem. 243, 3994. 9. SCHWARTZ, J., SLOAN, J., AND LEE, Y. C. (1970) Arch. Biochem. Biophys. 137. 122. 10. MARCUS, D. M., AND GROLLMAN, A. P. (1966) J. Immunol. 97. 867. 11. YOUNG, II., AND HAKOMORI, S. (1971) J. Biol. Chem. !Z46, 1192. 12. WARREN, L. (1959) J. Biol. Chem. 234, 1971. 13. SHEN, L., GROLLMAN, E. F., AND GINSBURG, V. (1968) Proc. Nat. Acad. Sci. U.S.A. 69, 224. 14. KOBATA, A., AND GINSBURG, V. (1970) J. Biol. Chem. 246, 1484. 15. GROLLMAN, E. F., AND GINSBURG, V. (1967) Biochem. Biophys. Res. Commun. 28. 59. 16. GROLLMAN, E. F., KOBATA, A., AND GINSBURG, V. (1969) J. Clin. Invest. 48, 1489. 17. WATKINS, W. M. (1966) in Glycoproteins (Gottschalk, A., ed.), p. 462. Elsevier, London. 18. KUHN, R., AND GAUHE, A. (1965) Chem. Ber. 98, 395. 19. GRIMMONPREZ, L., AND MONTREUIL, J. (1968) Bull. Sot. Chim. Biol. 60, 843.
OF
BIOCHEMISTRY
AND
160, 273-281 (1972)
BIOPHYSICS
Oligosaccharides IV. Isolation
and Characterization
of Human
of a New Hexasaccharide,
A. KOBATA’
Institutes of Health,
Received
January
Lacto-N-neohexaose
V. GINSBURG
AND
National
A new hexasaccharide,
Milk
Bethesda, Maryland
13, 1972; accepted
“la&o-N-neohexaose,”
February
with
20014 24, 1972
the probable
structure
Galpl4GlcNacpl 6 has been isolated from human milk. In addition some fucosyl and sialyl derivatives of la&o-N-neohexaose and a previously described hexaose, lacto-N-hexaose, have been partially characterized.
A hexasaccharide, the structure2
lacto-N-hexaose,
with
EXPERIMENTAL
Galpl-4GlcNAcpl I 6 Gal~1-3GlcNAc~l-3Galpl4Glc
was recently isolated from human milk (1). The present paper describes the characterization of another hexasaccharide “lacto-Nneohexaose” which differs from Iacto-Nhexaose only in that both of its terminal galactose residues are linked fi1-4 as follows:
NANA~2-3Ga1~1-3GlcNAcpl-3Galpl-4Glc 6 I
Galpl-4GlcNAcpl I
6
NAN Aor;
Gal~l-4GlcNAc~1-3Gal~l4Glc.
(disialyllacto-N-tetraose)
In addition, the structures of some fucosyl and sialyl derivatives of both Iacto-N-hexaose and lacto-N-neohexaose have been investigated.
while NANAa26Gal was prepared by acetolysis of NANAor2-GGalfll-4Glc (6’-sialyllactose) (7). The disaccharides were purified from other acetolysis products by paper chromatography using solvent I (the relative mobility of the three disaccharides in this solvent are shown in Fig. 5). GlcNAc$14sorbitol and GlcNAcpl-6Ga@l4sorbitol were prepared flom lacto-N-hexaitol by partial hydrolysis (0.1 N HCl at 100°C for 40 min) and purified by paper chromatography using solvent II (1).
i Present address: Department of Biochemistry, Kobe University School of Medicine, Kusunoki-cho 7 Chrome, Ikuta-Ku, Kobe, Japan. 2 All monosaccharides mentioned in this paper have a D configuration except for fucose which has an L configuration. 273 Copyright
@I 1972 by Academic Presls, Inc.
PROCEDURE
Paper chromatography. Descending paper chromatography wm performed with the following solvents: I, ethyl acetate-pyridine-acetic acidwater (5:5:1:3); and II, ethyl acetate-pyridinewat,er (12:5:4). Sugars were located with AgNOs reagent (2) or with periodate-benzidine reagent (3). Thiobarbituric acid reagent was used to detect oligosaccharides that contain sialic acid (4). Standard oligosaccharides. Known oligosaccharides were isolated from human milk by methods described previously (1, 5, 6). NANAol23Gal and NANA~Y~-GGIcNAc were prepared by acetolysis of
274
KOBATA
AND
Labeling oligosaccharides with 3H. Oligosaccharides were labeled at their reducing ends by reduction with PHIsodium borohydride a follows: One micromole of oligosaccharide was dissolved in with a 0.5 ml of 0.01 M [3H] sodium borohydride specific activity of 100 &i/pmole (New England Nuclear Co., Boston, MA). The reaction mixture was kept at room temperature for 1 hr and then cooled to 0°C. A drop of glacial acetic acid was added to destroy excess reagent and the solution was passed through a column (0.5 X 3 cm) of Amberlite AG 50(H+) in the cold. The column was washed with 3 ml of HZ0 and the effluent and washing combined and evaporated to dryness under vacuum. Boric acid was removed by the repeated addition and removal under vacuum of methanol (live times). The oligosaccharide was then purified by chromatography as a 3-cm band on Whatman No. 1 paper developed with solvent I. Development time varied with the size of the oligosaccharide: 3 days for tetra- and pentasaccharides and 10 days for hexa-, hepta-, and octasaccharides. The reduced oligosaccharides, which move slightly slower than the corresponding unreduced oligosaccharides, were located by scanning the chromatogram for radioactivity and eluted from the paper with water. Glycosidases. fl-Galactosidase and ,9-N-acetyiglucosaminidaae from Jack bean (8) were the same preparations reported previously (1). p-Galactosidase from almond emulsin was purified by the method of Schwartz et al. (9). *H-Labeled oligosaccharides were treated with the glycosidases aa follows : The 3H-labeled oligosaccharide (about 5 nmoles containing 1.0 X lo5 cpm) was incubated with 10 ~1 of the glycosidase solution and 2 ~1 of toluene for 20 hr at 37°C in a sealed tube. The glycosidase solution w&s prepared by diluting the purified preparations with buffer so that 10 pl of the diluted enzyme cleaved about 20 nmoles of the appropriate p-nitrophenyl p-glycoside per hour at 37°C. The p-galactosidase from almond emulsin and Jack bean was diluted with 0.1 M phosphate-citrate buffer, pH 6.0; the p-N-acetyglucosaminidase from Jack bean was diluted with 0.1 M phosphate-citrate buffer, pH 5.4. Under the above conditions of incubation the fl-galactosidase from Jack bean can be used to distinguish Galpl3GlcNAc and Galpl4GlcNAc bonds: the enzyme cleaves the Ga@l4GlcNAc bond of la&o-N-neotetraose but not the Galpl3GlcNAc bond of lacto-N-tetraose.3 The p-galactosidase from almond emulsin, in contrast, cleaves both tetraoses equally well and at about 10% the rate of p-pnitrophenylgalactoside. Hapten-inhibition studies. Hapten-inhibition 8 A. Kobat)a,
unpublished
data.
GINSBURG studies were studied with quantitative precipitin reactions between human Lea, Leb, and H substances and goat anti-Lea and anti-Leb sera and Eulex europeaus lectin, respectively (10). RESULTS
AND
DISCUSSION
Isolaticn
of lack-N-neoh.e.xaose. Lacto-Nneohexaose is best isolated from the milk of donors with a Lewis negative blood type as described for lacto-N-hexaose (1). During gel filtration with Sephadex G-25 and preliminary paper chromatography the two hexaoses do not separate; they are separated by paper chromatography for 10 days using solvent II as lacto-N-neohexaose migrates 5 % faster than lacto-N-hexaose (1). About 5 mg of lacto-N-neohexaose is obtained from 1 liter of milk. of la&o-N-neohexaose. Characterization Lacto-N-neohexaose, like lacto-N-hexaose contains three galactose residues and two N-acetylglucosamine residues for each glucose residue (Table I). Glucose is at the reducing end of the chain as all the glucose disappears and sorbitol is produced upon reduction of the intact oligosaccharide with NaBH4 as evidenced by paper chromatography after acid hydrolysis. Thus, the sugar is a hexaose and isomeric with lacto-N-hexaose. Enzymatic and chromatographic evidence presented below suggests that it differs from lacto-N-hexaose only in the linkage of one of its two terminal galactose residues. Products of the action of &galactosidases and P-N-acetylglucosaminidase on lacto-Nhexaitol and lacto-N-neohexaitol are shown in Fig. 1. One of the two terminal galactose residues of lacto-N-hexaitol is hydrolyzed by Jack bean /3-galactosidase to yield the pentasaccharide GlcNAcfil I 6 Galpl-3GlcNAcpl3Gal@l-4Sorbitol
as the Jack bean enzyme cleaves Gal@l4GlcNAc linkages faster than Galpl-3GlcNAc linkages (see Experimental Procedures). The chromatographic mobility of lacto-N-hexaitol and the pentasaccharide derived from it are shown in Fig. 1A and lB, respectively. In contrast, treatment of lactoN-neohexaitol with Jack bean P-galactosid-
OLIGOSACCHARIDES OF HUMAN MILK
275
charide GlcNAcBl I
6 GlcNAc,V1-3Gal~1-4sorbitol
which is formed from lacto-N-hexaose on treatment with almond emulsin p-galactosidase (Fig. 1C). Almond emulsin p-galactosidase, unlike the Jack bean enzyme, does not distinguish between Galbl-3GlcNAc and Gal/314GlcNAc linkages (see Experimental Procedure) and releases both terminal galactose residues from lacto-N-hexaose at the same rate. The tetrasaccharides derived from la&o-N-neohexaose and lacto-N-hexaose were both hydrolyzed to lactitol on incubation with /3-N-acetylglucosaminidase (not shown in Fig. 1). When lacto-N-neohexaitol was briefly treated with Jack bean /3-galactosidase a cWFS ORIGIN km: X’CPCE pentaitol intermediate was detected (Fig. lF, peak 2) in addition to the final tetraitol Fro. 1. Action of glycosidases on [aH] lecto-Nproduct (Fig. lF, peak 1) and residual lactohexaitol and [SH] lacto-N-neohexaitol. The condiN-neohexaitol (Fig. lF, peak 3). The pentations of incubation are given in Experimental Procedure. The radioactive sugars were chromatitol intermediate had the same chromatoographed for 4 days using solvent II and the graphic mobility as the pentssaccharide resulting chromatograms scanned for radioactivderived from lacto-N-hexaitol (Fig. 1B). ity. A, la&o-N-hexaitol; B, lacto-N-hexaitol However, upon incubation with P-N-acetyltreated with Jack bean o-galactosidase; C, lactoglucosaminidase it gave rise to two tetraitols N-hexaitol treated with almond emulsin @-galacin approximately equal amounts (Fig. 2G, tosidase; D, la&o-N-neohexaitol; E, lacto-Npeaks 4 and 5). The chromatographic mobilneohexaitol treated with Jack bean p-galactosiity of one tetraitol (peak 4) was the same as daae; F, lacto-N-neohexaitol treated with Jack while the mobility bean p-galactosidase for a shorter time than in E standard lacto-N-tetraitol of the other (peak 5) was slower. Incubation (1 hr instead of 20 hr); G, peak 2 (from F) treated with almond emulsin @-N-acetylglucosaminidase; of both tetraitols with Jack bean ,8-galactoH, peak 4 (from G) treated with Jack bean ,%galac- sidase resulted in the formation of triitols: tosidaae; I, peak 5 (from G) treated with Jack the triitol derived from peak 4 had the bean @-galactosidase; and J, standard sugars as chromatographic mobility of standard Glcfollows: T-3 is GleNa@3Galfll4sorbito1, T-6 is NAcfl13Gal@l-4sorbitol (Fig. 1H) while GlcNAcpl-6Gal@4sorbitol, LNnT-OH is lactothe triitol derived from peak 5 had the N-neotetraitol, and LNnH-OH is la&o-N-neo chromatographic mobility of standard Glchexaitoi. NAcfil-C,Galfil-4sorbitol (Fig. 11). The most probably structure of lacto-Nase under the same conditions releases two neohexaitol based on these studies and by galactose residues instead of one resulting in analogy to the structure of other milk oligothe formation of a tetrasaccharide. The saccharides is shown in Fig. 2 along with a chromatographic mobilities of lacto-N-neosummary of the action of the glycosidases. hexaitol and the tetrasaccharide derived La&o-N-neohexaose would be the correfrom it are shown in Fig. 1D and lE, respec- sponding unreduced sugar. tively. The chromatographic mobility of the Isolation of higher oligosaccharide fractions N-d, N-S, S-5, and S-6. Higher oligosactetrasaccharide is identical to the tetrasac-
276
KOBATA
AND GINSBURG
GalOl-4GlcNAcDl I 6 GalPl-4GlcNAc~l-3Gal~1-4Sorbitol lacto-&neohexaitol (Fig. 1D)
GlcNAcO
1
k
+
I 6 GalPt-4GlcNAcL31-3Galpl-4Sorbitol (Fig. lF, peirk 2)
~~dfli--dGdN~~flj
GlcNAcPl-3GalSl-4Sorbitol
I [ P-GlcNAcare GlcNacPl
I Gal~l-4GlcNAc~l-3Galpl-46orbitol lacto-N-neotetraitol (Fig. 1G. peak 4)
1 p-G&S
GlcNAcPl-3Galpl-4Sorbitol (Fig. 1H)
I 6 GldUAcfll-3GalPl-4Sorbitol (Fig. 1E)
I
Gal~1-4GlcNAcpl-6Gal~l-4Sorbitol (Fig. 1G. peak 5)
fl-GlcNAcare
GalpldSorbitol lactitol
GlcNAcpl-GGalpl-46orbitol (Fig. II)
FIG. 2. Probable structure of la&o-N-neohexaitol and the products of glycosidase action (see text and Fig. 1). fl-Galase is 8-galactosidase; &GlcNAcase is &N-acetylglucosaminidase.
charide fractions N-2, N-3, S-5, and S-6 were 0.79) and N-3 (RLNDeI = 0.50) were eluted separately with HzO, passed through a obtained as follows: the oligosaccharides from 1 liter of milk were fractionated by gel mixed-bed column (0.5 X 3 cm) containing Amberlite AG5O(H+) and AG3(OH-) and filtration with Sephadex G-25 aa previously lyophilized. From the paper containing the described (1, 5, 6). The fractions containing hexaoses and higher oligosaccharides [for acidic fraction, S-5 (RDLN = 0.77) and S-6 example, fractions l-23 in Fig. 1 of Ref (l)] (RDLN= 0.61) were eluted separately with were combined and lyophilized. The white water and lyophilized directly. The yield residue was dissolved in HzO, applied as a of each fraction varied with different samples streak on Whatman No. 3MM paper (2 mg of milk but about 30 mg of N-2, 35 mg of of sugar per cm), and subjected to electroN-3,20 mg of S-5, and 20 mg of S-6 are obtained . phoresis at 73 V/cm for 1 hr using HzOpyridine-glacial acetic acid (3870 :30 : 11.5) Fraction N-2 isolated from the milk of as a buffer (pH 5.4). Four acidic sugar bands “secretors” (but not from the milk of “nonand one neutral sugar band were detected by secretors”) can be further resolved by paper staining guide strips with AgNOs reagent. chromatography for 14 days using solvent one into two fractions (N-2-l and N-2-2) ; The neutral fraction and the slowest moving acidic fraction were eluted from the paper the Rf of N-2-l is 5 % higher than the RI of with water and applied separately as streaks N-2-2. Milk samples from nonsecretors conon Whatman No. 3 MM paper (1 mg of sugar tain only N-2-2; N-2-l is missing. Fractions per cm). The papers were then developed N-3, S-5, and S-6 are not further resolved by with solvent I for 10 days; lacto-hr-difucothe same chromatographic treatment. hexaose I (LND-I) was used as a standard Characterization of the higher oligosacdmride fractions. The monosaccharide composition for the neutral fraction and disialyllacto-l\rtetraose (DLN) was used as a standard for of oligosaccharide fractions N-2-1, N-2-2, the acidic fraction. From the paper con- N-3, S-5, and S-6 are given in Table I. Their composition differs from lacto-N-neohexaose taining the neutral fraction, N-2 (RLNDmI =
OLIGOSACCHARIDES
OF HUMAN
TABLE
MILK
277
I
MONOSACCHARIDE COMPOSITION 0~ LAcTo-N-neoHEx.4osq LACTO-N-HEXAOSE, HIGHER OLIGOSACCHARIDE FRACTIONS OF MILKY
AND SOME
Monosaccharide ratio Oligosaccharide Lacto-N-neohexaose Lacto-N-hexaose N-2-l N-2-2 N-3 s-5 S-6
Glucose
Galactose
N-Acetylglucosamine
Fucose
Sialic acid
1.00 1.00 1.00 1.00 1.00 1.00 1.00
2.73 2.86 2.95 2.88 2.95 2.84 2.75
1.79 1.83 2.01 1.91 2.11 1.96 1.81
0.92 1.11 1.88 0.82
1.11 1.08
= The sugar composition of the oligosaccharides was determined by gas chromatography of the reduced and acetylated derivatives of acetolysis (11). Lacto-N-tetraose and lacto-N-fucopentaose I were used as standards. Sialic acid waz measured calorimetrically after hydrolysis of the oligosaccharide in 0.1 N HzS04 at 80°C for 1 hr (12). N-2-1, N-2-2, N-3, S-5, and S-6 refer to higher oligosaccharide fractions described in the text.
and lacto-N-hexaose in that they contain fucose or siliac acid or both. The results of acid hydrolysis shown in Fig. 3 suggest that these oligosaccharides include fucosyl and sialyl derivatives of these hexaoses. Under conditions which liberate most of the fucose, N-2 and N-3 give rise to oligosaccharides with the chromatographic mobility of the hexaoses (lacto-N-hexaose and lacto-hi-neohexaose are not distinguished under the chromatographic conditions of Fig. 3) (lanes 7 and 9 in Fig. 3). Milder acid hydrolysis of N-3 yields, in addition, some N-2 (lane 10 in Fig. 3). Hydrolysis conditions, which liberate sialic acid but not fucose, converts S-6 into N-2 and S-5 into a hexaose (lanes 3 and 5, respectively, in Fig. 3). Longer hydrolysis of the N-2 derived from S-6 results in the formation of hexaose (not shown). Lacto-Nhexaose and lacto-N-neohexaose can easily be distinguished by paper chromatography after reduction by [3H]sodium borohydride. The oligosaccharide fractions N-2-1, N-2-2, N-3, S-5, and S-6 were labeled with 3H by reduction (see Experimental Procedure) and the reduced derivatives subjected to mild acid hydrolysis to liberate fucose and sialic acid. In all fractions except S-6, two hexaitols mereformed whosechromatographic mobility corresponded to lacto-N-hexaitol and lactoN-neohexaitol. A typical result is shown in Fig. 4A. The relative amounts of the two
hexaitols varied but generally there was more lacto-N-hexaitol than lacto-N-neohexaitol (oligosaccharide fractions obtained from five different samples of milk were tested). Surprisingly, hydrolysis of samples of S-6 obtained from five different donors gave rise to only one hexaitol, corresponding in chromatographic mobility to lacto-N-neohexaitol as shown in Fig. 4B. These results indicate that fractions N-2-l and N-2-2 contain monofucosyl derivatives of lacto-N-hexaose and lacto-N-neohexaose; N-3 contains difucosyl derivatives of lactoN-hexaose and lacto-N-neohexaose; S-5 contains monosialyl derivatives of lacto-N-hexaose and lacto-N-neohexaose; and S-6 contains monofucosyl monosialyl derivatives of only la&o-N-neohexaose. Fucose occurs in the oligosaccharides of human milk in at least 4 different linkages: al-2 to gala&se, al-3 to N-acetylglucosamine, (~14 to N-acetylglucosamine, and al-3 to glucose; sialic acid occurs in at least 3 different linkages: ~~2-3 to galactose, ~~2-6 to galactose, and ~~2-6 to N-acetylglucosamine [for structures see (5, S)]. Using only these linkages, there are theoretically possible 6 isomeric monofucosyl derivatives of lacto-N-hexaose and 6 of lacto-N-neohexaose in N-2, 15 isomeric difucosyl derivatives of each hexaose in N-3, 6 isomeric monosialyl derivatives of each hexaose in S-5, and 36
278
KOBATA
AND GINSBURG
FIG. 4. Pr0duct.s of partial acid hydrolysis of some higher oligosaccharides of human milk labeled by reduction with [*HI sodium borohydride (see Experimental Procedure). The products of hydrolysis were chromatographed for 9 days using solvent I. A, hydrolysis products of reduced fraction S-5 (0.01 N HCl for 10 min at 1OO’C); B, hydrolysis products of reduced fraction S-6 (0.01 N HCl for 60 min at 100°C); C, standard sugars as follows: LNnH-OH is lacto-2\i-neohexaitol, LNHOH is lacto-iV-hexaitol, N-2-2-OH, N3-OH, S-5-OH, and S-6-OH are reduced fractions N-2-2, N-3, S5, and S-6, respectively. FIG. 3. Paper chromatography of the products of partial acid hydrolysis of the oligosaccharide fractions N-2, N-3, S-5, and S-6. The chromatogram was developed for 6 days with solvent I and the sugars visualized with AgNOa reagent (2). The sugars circled with dotted lines also reacted with thiobarbituric acid reagent (4) aft.er hydrolysis (5). Lane 1 and 8, N-2: lane 2, S-6; lane 4, S-5; lane 6, lacto-iV-hexaose; lane 11, N-3; lanes 3 and 5, hydrolysis products of S-6 and S-5, respectively (0.01 N KC1 at 100°C for 8 min); lanes 7 and 9, hydrolysis products of N-2 and N-3, respectively (0.01 N HCl at 100°C for 60 min); and lane 10, hydrolysis products of N-3 (0.01 N KC1 at 100°C for 40 min).
isomeric monofucosyl monosialyl derivatives of lacto-N-neohexaose in S-6. As isolated, N-2-1, N-2-2, N-3, S-5, and S-6 are probably mixtures of isomers with fucosyl and sialyl residues attached at different positions. As mentioned above, fraction N-2-l occurs only in milk from secretors and is absent from the milk of nonsecretors while fraction N-2-2 occurs in both types of milk. As the
biochemical basis for secretor status is the presence or absence of the fucosyltransferase responsible for forming Fuccrl-2Gal linkages (13), the fucose residues in N-2-l are probably linked (~1-2 to galactose while in N-2-2 the fucose residues are linked to other positions (e.g., al-3 and ~~1-4 to N-acetylglucosamine or al4 to glucose). This supposition was supported by testing N-2-l and N-2-2 for their ability to accept N-acetylgalactosamine in the reaction catalyzed by the human N-acetylgalactosaminyltransferase responsible for the formation of the determinants of blood group A in man (14). This enzyme, which forms GalNAcal3Gal linkages, requires the acceptor galactose to be substituted on the two position with fucose. Under standard assay conditions (14), N-2-l was as active as lacto-N-fucopentaose I in functioning as an acceptor for the A gene enzyme; N-2-2 was completely inactive. However, even with the assignment of fucose in an al-2 linkage to a terminal galactose in N-2-1, four monofucosyl derivatives are
OLIGOSACCHARIDES
possible, two of lacto-N-hexaose and two of lacto-N-neohexaose. Fraction N-3 also appears to be a complex mixture of isomers as its serologic properties depend on the blood type of the donor from whose milk it is isolated. N-3 isolated from a donor belonging to blood group Le(a-b+) is a haptenic inhibitor of anti-Leb, anti-Lea, and anti-H reagents; N-3 isolated from a nonsecretor belonging to the blood group Le(a+b-) inhibits only the anti-Lea reagent; and N-3 isolated from a secretor belonging to the blood group Le(a-b-) inhibits only the anti-H reagent. These results are consistent with the known distribution of other fucose-containing oligosaccharides and fucosyltransferases in these individuals (5, 6, 13, 15, 16) and with the structural determinants of Leb, Lea, and H specificities (17): the Leb determinant is FuccJ-2Galpl-3GlcNAc
OF HUMAN
MILK
279
was removed from the CHCL extract by the addition of anhydrous NazSOd, the extract was filtered to remove NazSOd, and the filtrate was evaporated to dryness. Methanol saturated with NH+ 3 ml, was added to the residue and the reaction mixture left 3 days at room temperature with intermittent shaking under anhydrous conditions. The solution was evaporated to dryness and residual NH3 was removed by the repeated addition and evaporation (three times) of a mixture containing equal volumes of methanol, ethyl acetate, and toluene. The residue was dissolved in 0.5 ml of Hz0 and passed
...,
i’ Fuccvl
the Lea determinant
is
Galpl-3GlcNAc.
,
7 Fucorl
and the H determinant is either Fuccul2Gal@l-3GlcNAc.. . or Fuccrl-2Gal/314GlcNAG . . . . Acetolysis of oligosaccharides that contain sialic acid is a useful method for structural studies as sialyl linkages are far more resistant to acetolysis than hexosyl or N-acetylhexosaminyl linkages and fragments containing sialic acid can be obtained (7). Fractions S-5 and S-6 were acetolyzed as follows: 5 mg of oligosaccharide were dissolved in 0.25 ml of a mixture of acetic anhydride, glacial acetic acid, and cone HzS04 (10: 10: 1 by volume) and kept at room temperature for 3 days under anhydrous conditions. The reaction mixture was added to 5 g of ice and neutralized to pH 4.0 by the addition of powdered sodium carbonate. After standing for 1 hr at 0°C the acetylated sugars were extracted three times with 5-ml portions of CHCI,. The combined extract was backwashed once with 15 ml of HzO. Water
FIG. 5. Chromatographic analysis of the sialic acid-containing oligosaccharides obtained by acetolysis of fractions S-5 and S-6. The chromatogram was developed for 3 days with solvent I. Lanes 1 and 6, standard NANAa2-GGal; lanes 2 and 7, standard NANAa2-3Gal; lanes 3 and 8, standard NANAor2-6GlcNAc; lanes 4 and 5, acetolysis products of oligosaccharide fractions S-5 and S-6, respectively. The black arrows indicate disaccharide products while the white arrows indicate tetrasaccharide products; lane 9, standard 6’-sialylactose; lane 10, standard 3’-sialyllactose; and lane 11, standard NANAorZ-GGal@l4GlcNAc@l-3Galpl-4Glc (LST-c).
280
KOBATA
AND
through a column (0.5 cm diam) containing 3 cm of Amberlite AG 5O(H+) in a lower layer and 3 cm of Dowex 1 (acetate) in an upper layer. The column was washed with water, and the sialic acid-containing sugars eluted wit,h 4 ml of 0.1 M sodium acetate. The eluate was lyophilized and a portion of the residue was analyzed by paper chromatography. As shown in Fig. 5, lanes 4 and 5, the acetolysis products from S-5 and S-6 were the same: only one disaccharide containing sialic acid was obtained from each (indicated I
I
I
!I? I 5 5 IO
I
GlcN e
I
I
. 1
, NANA*it,
s
,
z,
I
2
3
4
,::~i'":'r"
5
s
Fra. 6. Chromatographic analysis of the hydrolysis products of the sialic acid-containing oligosaccharides obtained by acetolysis of fractions S-5 and S-6. The chromatogram was developed for 24 hr with solvent II. The black spots represent sugars visualized with AgN08 reagent (2) while the spots circled with dotted lines represent sugars that also reacted with thiobarbituric acid reagent for sialic acid (4). Lane 1, the disaccharide formed by acetolysis of either S-5 or S-6; lane 2, products arising from the hydrolysis of either disaccharide in 0.01 N HCl for 10 min at 100°C; lane 3, the tetraaaccharide formed by acetolysis of either S-5 or S-6; lane 4, products arising from the hydrolysis of either tetraaaccharide in 0.01 N HCl for 10 min at 100°C; lane 5, products arising from the hydrolysis in 1 N HCl for 1 hr at 100°C of the neutral trisaccharide indicated by the arrow in lane 4; and S, standard sugars.
GINSBURG
by the black arrows). Both disaccharides corresponded in chromatographic mobility to NANAcr2-6Gal and on hydrolysis gave rise to galactose and sialic acid (Fig. 6, lane 2). The conditions of acetolysis also gave rise to sialic acid-containing tetraoses from S-5 and S-6 (indicated by the white arrows in Fig. 4, lanes 4 and 5). Hydrolysis of both tetraoses gave rise to sialic acid and triaoses (indicated by the black arrow in Fig. 6, lane 3). The triaoses had the chromatographic mobility of Galpl-3GlcNAcpl-3Gal but are clearly not Gal/?l-3GlcNAcpl3Gal as the unknown triaoses were cleaved by Jack bean /3-galactosidase under conditions in which standard Gal813GlcNAc/314Glc was resistant (see “Glycosidases” in Experimental Procedure). Both triaoses gave rise to galactose and glucosamine after complete hydrolysis (Fig. 6, lane 5). As the triaoses are chromatographically distinct from Galpl4GlcNAcfilSGal (see Fig. 5) their probable structure is GalPl4GlcNAc/31-6Gal. From the foregoing data it is likely that oligosa,ccharide fraction S-5 consists predominantly of the monosialyl derivative of lactoN-hexaose, NANArr2-GGalpl4GlcNAcpl I 6 Galfll-3GlcNAc~1-3Gal@l4Glc
and the monosialyl derivative of lacto-N-neohexaose, NANAor2-GGal@l-4GlcNAcfll I 6 Galpl-4GlcNAc@1-3Gal@1-4Glc.
Oligosaccharide fraction S-6 consists predominantly of monofucosyl derivatives of the sialyllacto-N-neohexaose isomer shown above. The absence of lacto-N-hexaose from S-6 may be due to enzyme specificity. For example, if sialic acid is added last in the synthesis of S-6 it is conceivable that the sialyltransferase responsible for the addition of sialic acid to monofucosyl derivatives of la&o-N-neohexaose cannot use monofucosyl derivatives of lacto-N-hexaose as substrates. The linkage of sialic acid to galactose in S-5 and S-6 is consistent with its linkage to galactose in the other sialic acid-containing
OLIGOSACCHARIDES
oligosaccharides of human milk, NANAa23Gal/3l-/3-3GlcNAc~l-3Galpl-4Glc (LSTa) (18)) NANAa2-6Gal/314GlcNAc$3-3Gal/31-4Glc (LST-c) (IS), and NANA~2-3Gal~l-3GIcNAc~l-3Gal~14Glc 7 NANAa2 (disialyllacto-N-tetraose)
(19) :
when the galactose is linked /314 to Nacetylglucosamine (as it is in S-5, S-6, and LST-c) the sialic acid is linked a2-6 to galactose; when the galactose is linked pl-3 to N-acetylglucosamine (as it is in LST-a and in disialyllacto-N-tetraose) the sialic acid is linked a2-3 to galact.ose. Again, this phenomenon could be explained by the specificity of the sialyltransferases that form the a2-6 and a23 linkages. ACKNOWLEDGMENT We thank Dr. Donald Marcus the hapten-inhibition studies.
for carrying
out
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MILK
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