Wheat g e m agglutinin chromatography of GlcNacBl-3(GlcNAc#ld)Gal and GlcNAc~l-3(GlcNAc@l~6)Ga1@1-4GlcNAc, obtained by in vitro synthesis and by

partial cleavage of teratocarcinoma poly-~-acetyl~actosaminoglycansl

ANTTI SEPPO,' LEENA PENT TI LA,^ ANNE MAKKONEN, ANNE LEPPANEN,' RITVA NIEMELA,~ JUSSI JANTTI, JARI HELIN,' AM) OSSI RENKONEN~ '~

Department of Biochemistry, University of Helsinki, Unioninkatu 3 5, SF-001 70 Hefsinki, Finland Received April 25, 1989

SEPPO, A., PENTTILA, L., MAKKONEN, A., LEPPANEN, A,, NIBMELA, R., J A ~ I , J., HELIN, J., and RENKONEN, 0. 1996. Wheat germ agglutinin chromatography of GlcNAcO 1 -3(GlcNAcb 1 -6)Gal and GlcNAcP I -3(GlcMAc@ 14)- Gal@l4GlcNAc, obtained by in vitro synthesis and by partial cleavage of teratocarcinoma poly-N-acetyllactos- aminoglycans. Biochem. Cell Biol. 68: 44-53.

G ~ C N A C ~ 1 -~(G~cNAc/~ 1 - 6 ) [ 1 4 ~ ( ~ ) ] ~ a l and G ~ C N A C ~ 1 - ~ ( G ~ C N A C ~ 1-6) [14c(U)] Gala 1 - 4 ~ l c ~ ~ c were prepared by in vitro synthesis. They were characterized by enzymatic sequencing, by partial acid Bydrotysis, and by peridate oxidation experiments. The two saccharides were isolated also from partial acid hydrolysates of metabolically labeled poly-N- acetyllactosaminogly~s of murine embryonal carcinoma cells (line PC 13). The tetrasaccharide was retarded in a column of agarose-linked wheat germ agglutinin; the trisaccharide was strongly bound. Chromatography in this column separated the trisaccharide into two distinct peaks, which represented interconvertible molecules. Together with our previous data on linear teratocarcinoma saccharides, these findings show that affinity chromatography with immobilized wheat germ agglutinin can be advantageously used in fractionating radiolabeled oligo-N-acetyllactosaminoglycans and saccharides related to them. Key words: GlcNAcb 1-3(GlcNAcj3 1 -6)Qal, GlcNAcp 1 -3(GlcNAq3 1 -6)Galfll-4GlcNAc, wheat germ agglutinin -

agarose chromatography, in vitro biosynthesis, teratocarcinoma cell.

SEPPO, A., PENTTILA, L., MAKKONEN, A., L E P P ~ ~ N , A., NIEMELA, R., JANTTI, J., HELIN, J., et RENKONEN, 0. 1990. Wheat germ agglutinin chromatography of GlcNAc~l-3(GlcNAc~l-6)Gal and GIcMAcP 1 -3(GlcNAca 1-6)- Galpl-4GlcNAc, obtained by in vitro synthesis and by partial cleavage of teratocarcinoma poly-N-acetyllactos- aminoglycans. Biochem. Cell Biol. 68 : 44-53.

Le G I C N A C @ ~ - ~ ( G ~ ~ N A C ~ ~ 1 -6)[l4~(U)]Gal et le G~CNACP 1 -3(~lcNAcfll-6)[ 14c(v)] alp 1 - 4 ~ l c c sont prCpares par synthbe in v i t r ~ . NOUS les avons carzectQisCs par des expkriences de skquen~age enzymatique, d'hydrolyse acide par- tielle et d'oxydation par le periodate. Les deux saccharides sont Cgalement isolCs des Rydrolysats acides partiels des poly-N-acCtyllactosaminoglycanes mCtaboliquement marquts des cellules (lignde PC 13) d'un carcinome embryonnaire murin. Le tbtrasacchtuide est retard6 dans une colonne d'agglutinine de germe de blb liCe B lkguose; le trisaccharide

. est fortement lib. Dans cette colonne, Ia chromatographie sCpare le trisaccharide en deux pics distincts qui reprtsentent des mol6cules interchangeables. Ces resuftats,.avec nos donnCes antCrieures s u r les saccharides d'un tCratocarcinome, montrent que la chromdographie d"affinit6 avec l'agglutinine de germe de blC immobiliste peut Stre une mCthode avan- tageuse pour fractionner les oligo-N-acbtyllactosaminoglycanes radiomarquCs et les saccharides qui y sont relies.

Mots elks : GlcNAcfl l-3(GicNAc@ 1 -6)Gal, GlcNAcfl 1 -3(GlcNAcp 1 -6)Galfll ACilcNAc, chromatograp hie avec l'agglutinine de blC liCe B l'agarose, biosynthbe in vitro, cellule d'un tdtratocarcinorne.

[Traduit par la revue]

Introduction The present report describes two branched "backbone" We have recently obtained two oligosaecharides,

GlcNAcP 1 -6Gd and GlcNAcP 1-6GdD 1 -4GlcNAc, from partid acid hydrolysates of radiolabeled poly-N-acetyuactos- aminoglycans from embryonal carcinoma cells (Renkonen et sf. 1988). Their chromatography in a column of agarose- linked WGA gave interesting results: the trisaccharide was moderately retarded by the column, while the disaccharide was very tightly bound. In contrast, a number of other sac- charides were not bound to the column or were bound only weakly.

ABBWVIATIQNS: GlcNAc, N-acetylglucosan%ine; Gal, 8-galac- tose; WGA, wheat germ agglutinin; fl1,3-GlcNAc-transferase, N-acetyllactosaminide 0-l,3-N-acetylglucosabminyltransferase; HPLC, high performance Liquid chromatography; OMe, methoxy ; LactNAc, N-acetyllactosamine.

h his paper is dedicated to Dr. Morris Kates in honour of his valuable contributions to biochemistry in Canada.

'present address: Institute of Biotechnology, University of Helsinki, Valimotie 7, SF-00380 Helsinki, Finland.

'~u thor to whom all correspondence should be addressed.

oligosacch~ides, GlcNAcP 1 -3(GlcNAcfll-6)Gal and GlcNAcP 1-3(GlcNAc@ 1-6)GdP 1 -4GlcNAc, from teratocar- cinoma poly-N-acetyllactosaminoglycms. Their behavior in WGA chromatography was as refnarkable as that of their linear companions: the tetrasaccharide was retarded by the lectin column, while the trisaccharide was very strongly bound. The new findings were confirmed by synthesizing the branched saccharides with an enzymatic procedure pre- viously studied by Piller et al. (1 9841.~

Materials and methods Sakharides

D-LYxoS~, 8-threose, 8-galactose, N-acetylgalactosamine, N - a ~ e t y l g l u c o ~ e ~ lacme, N-acetyl lactde, Galpl-3GlcNAc, Gal0 1 -6GlcNAc, GlcNAcBl -Gal , maltotriose, rnaltotetraose, dtopentaose, maltoheptaose, UDP-Gal, and UDP-GlcNAc were purchased from Sigma, St. Louis, MO. UDP-[ '~C(U)JG~~ (155 mCi/mmol; 1 Ci = 37 GBQ) was from hersham, England. Radioactive, metabolically labeled Galpl-QGlcNAc,

'A preliminary report of the present data was previously pre- sented by Segpo et a/. (1988).

htd in Canada / Imprim6 ou Cafada

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GlcNAcj9 1 -3Gal, GlcNAcP 1 -6Ga1, GlcNAcP 1 -3-Gal@ 1 -4GlcNAc, and GlcNAcPl-6Gal@l-4GlcNAc were obtained as described (Renkonen et al. 1988).

[ 1 4 ~ ] ~ a l f l 1 - 4 G l c ~ ~ c was obtained by incubating N-acetyl- glucosamine with UDP-['~C(LJ)]G~~ and N-acetyllactosamine synthase (EC 2.4.1.90) from bovine milk (Sigma), essentially as described by Brew et al. (1968). The resulting disaccharide migrated in paper chromatography with solvents A and B like authentic Galp 1 -4GlcNAc, but unlike GaW1-3GlcNAc a d GalB 1 -6GlcNAc (data not shown).

G ~ C N A C ~ 1-3 [ 1 4 ~ ] ~ a l l 1 -4GlcNAc was obtained by incubating UDP-GlcNAc and [' CIGalBl-4GlcNAc with human serum, which acted as a fll,3-GlcNAc-transferase @C 2.4.1.149) (Yates and Watkins 1983; Biller and Cartron 1983). The product (15% of the label) and the starting disaccharide (85% of the label) were separated by paper chromatography in solvent A; the product migrated like authentic GlcNAcP 1 -3Gallp1-4GlcNAc marker (data not shown), and it was cleaved by 0-N-acetylhexosaminidase that released labeled Galal-4GlcNAc (data not shown). G ~ c N A c ~ ~ - ~ [ ' ~ c ( u ) ] G ~ ~ was obtained by cleaving GlcNAc@l-3 [ 1 4 ~ ( ~ ) ] ~ a l f 1 1 - 4 ~ l c ~ ~ c with endo-P-galactosidase from Escherichiafreundii. The disaccharide (93Vo of the label) and the starting trisaccharide (7% of the label) were separated by paper chromatography in solvent B. The disaccharide migrated like authentic Glcsrl~c@l -3[14c]Gal (data not shown) and it was cleaved by 0-N-acetylhexosaminidase that released labeled galactose (data not shown).

/3l,Q-GlcNAc-transferase reactions Hog gastric mucosal microsomes were used as /31,6-GlcNAc-

tramsferase (EC 2.4.1.148) (Piller et al. 1984). The microsomes were prepared as described @rockhausen et al. 1983). The reaction mix- ture contained in a volume of 100 pL, 50-706 pmol radioactive acceptor saccharide, 4.5 pmol UDP-GIcNAc, 5 pmol sodium cacodylate @H 7.01, 0.2 pmol EDTA, 0.2 pmol ATP, 6.8 pmol NaN,, 3 pmol GlcNAc, 20 pmol sucrose, and mucosal microsomes corresponding to 2.5 mg protein. The mixture was incubated in 37°C for 16 h and the reaction was terminated by heating at 100°C for 3 min. The reaction mixture was desalted and deproteinized by a passage through Dowex AG SOW x 8 (H9) and Dowex AG 1 x 8 (AcO-); sucrose was eliminated by Bio- Gel P-2 chromatography (see below).

Partial acid hydrolysis Radiolabeled saccharides were dissolved in 200 pL of 0.1 M

trifluoroamtic acid in capped 6.0-mL tubes and incubated at 100°C for 40 min. Hydrolysis was terminated by adding 3.0 mL ice-cold water, after which the mixture was evaporated to dryness with a rotary evaporator.

Peridate oxidation Oligosaccharides that contained a reducing end [ '4~(~)]galac-

tose unit were oxidized with periodate as described (Rmkonen et al. 1989). Briefly, the oligosaccharides were cleaved with 15 mM NaIO, in Hough's sodium acetate buffer (pH 3.6) (Nough 1965) for 16 h at room temperature. The oxidation product was desalted, then cleaved with 1 M WC1 (lOO°C, 4 h), and finally deacidified by ion exchange.

Exo- and endo-hydrolase digestions P-N-Acetylhexosaminidase from jack bean (EC 3,2.1.52) was

purchased from Seikagaku, Tokyo, Japan. Reaction mixtures (40 pL) containing 2.5 U / d of the enzyme, 0.05 M sdiurn citrate (pH 4.0), and 30 mM of y-gdactonolactone were incubated at 37°C for 6 h. The reactions were terminated by heating at 100°C for 3 min. In control tests the enzyme cleaved synthetic [ ' 4 ~ ~ l c ~ ~ c - @ 1 -3Gal@ 1 -4GlcNAc completely, liberating [ B 4 ~ ] ~ l c ~ ~ c . In con- trast, synthetic [ 1 4 ~ ] ~ a l ~ l - 4 ~ l c ~ ~ c was recovered intact after control incubations with the enzyme.

63-Galactssidase from jack bean (BC 3.2.1.23) (Sigma) was also Used at a concentration of 2.5 U/mL in 0.05 M sodium citrate

(pH 4.0). The reactions were carried out in a volume of 46 pL at 37°C for 6 h and they were terminated by heating at 100°C for 3 min. Synthetic [ 1 4 ~ ] ~ l c ~ ~ c f i l -3GalP 1-4GlcIV~c resisted the enzyme in control tests, but 90% or more of synthetic [I4c] Gal0 1 -4GlcN~c was cleaved into [14c] Gal.

Endo-@-galactosidase (EC 3.2.1.103) from E. freundii (Seikagaku, Tokyo, Japan) was used in a concentration of 125 mU/mL as described (Rasilo and Renkonen 1982~). In con- trol experiments the enzyme cleaved 85% or more of s nthetic G ~ C N A C ~ ~ ~ -3 [14~]~alf11 ~ G ~ C N A C , liberating GlcNAcPl-3 [ Y'ClGal.

WGA -agarose chromatography Chromatography on immobilized WGA was performed as

described (Renkonen et al. 1988). Briefly, the elution was carried out with a sugar-free buffer, followed by the same buffer containing 0.2 M N-acetylglucosamine. Recovery of radioactivity was 75% or more in all experiments. HPLC experiments revealed that trace amounts s f unlabeled N-amtylglucosamine often contaminated the radioactive oligosaccharides, even when they emerged from the WGA-agarose column with the "sugar-free" buffer. This con- tamination resulted from imperfections in washing the column between consecutive runs. For reliable results, the contamination, obviously, must be carefully controlled.

Auxiliary procedures Descending paper chromatography was carried out using

Whatman 3 paper and the upper phase of n-butanol - acetic acid - water (4: 1 :5, by volume) (solvent A), n-butanol-ethanol-water (10:1:2, by volume) (solvent B), or ethyl acetate - pyridine - water (2:1:2, by volume) (solvent C). N-Acetylhexosamines were separated using borate impregnated Whatman 1 paper and ethyl acetate - isopropanol - pyridine - water (7:3:2:2, by volume) according to (Rasilo and Renkonen 1982b).

HPLC was carried out using the system 1 of Blanken et al. (Blanken et al. 1985).

Gel filtration was performed in a 1.1 x 145 cm column of Bio- Gel P-2 (200-400 mesh) (Bio-Rad, Richmond, CA) equilibrated with water. Samples were applied to the column in 580 fiL of water. Elution was carried out with water. Fractions of 1.74 mL were collected.

Results Etqyrnatic synthmii of G~CNA~SI J(GICNAC@ 1 -6)[14C.(~)]~al

The trisaccharide was synthesized by incubating UDP- GlcNAc and G~CNACB 1 -3 [14c(u)] Gal with hog gastric mucosal microsomes (Piller et al. 1984). The product migrated like a maitotriose marker in paper chromatogra- phy with solvent A; its retardation in comparison with the acceptor disaccharide suggested that only one GlcNAc unit was introduced (see Table 1, glycan A). The trisaccharide product was cleaved by P-M-acetylhexosaminidase into [14~(~)]galactose that was identified by paper chromatog- raphy (data not shown), indicating that the newly introduced GlcNAc group was @-linked.

Periodate oxidation and subsequent acid hydrolysis (Hough 1965) of the synthetic GlcNAcal-3(GlcNAcj3 1-6) [ 1 4 ~ ~ ) ] ~ a l gave ["C]lyxose (26% yield based on the start- ing trisaccharide); no (14~]threose or ["k]galactose was observed (F'ig. 1). The formation of labeled lyxose was com- patible with a trisaccharide structure, where the reducing end galactose unit was monosubstituted at position 3 or disubstituted at positions 3,4 or 3,6. Moreover, the forma- tion of lyxose was incompatible with d l other structures. The absence of labeled threose among the oxidation prod- ucts excluded the presence of a galactose disubstituted at positions 4 and 6.

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48 BIWHBM. CELL BIOL. VOL. 68, 1998

Lac * Gal 9

Thre 9

0 10 20 30 40

Fractions

FIG. 1. Analysis of the reaction product obtained by periodate oxidation and subsequent hydrolysis of synthetic GlcNAc/31-3- (G~cNAC/~~-~)['~C(U)~G~~. The reaction product was chromatographed on paper with solvent A for 28 h. The markers were lactose (Lac), galactose (Gal), Iyxose (Lyx), and threose (Thre).

Partial acid hydrolysis of the synthetic trisaccharide gave four major fractions (Fig. 2A) (see Table 1, glycans J and K, for migration rates in paper chromatography). Peak 1 was the original trisaccharide, eak 2 was G~CNAC~!~ 1 - 6 [ 1 4 ~ ] ~ a l , P peak 3 was G~cNAc/?I-~[' CIGal, and peak 4 was [14~]Gal. The formation of two GlcNAcPGd species confirmed that the synthetic trisaccharide was branched,

GlcNAcfl 1-3 [l4C(LJ)]Gal from the partial acid hydrolysate was converted into [ 14~]lyxose (29% yield), but not to ( 14C] threose, by periodate oxidation and hydrolysis (data not shown). This indicated the presence of a 3-substituted galactose, and the absence s f a 4-substituted galactose. The same treatment of the G~CNACB 1 -6 [14C.]Gal gave no ['4~jlyxose or ["Clthreose. In WGA-agarose chrornritography the GlcNAc@ 1-61 14C]Gal behaved Like the labeled GlcNAcBl -6Gal marker from teratocarcinoma cells (data not shown). In HPLC this disaccharide was eluted several minutes later than authentic G ~ c N A c @ ~ - ~ ~ - ' ~ c ] G ~ (data not shown), indicating that it was not GlcNAc@l-4Gal, which is known to run faster than GlcNAcb 1 -3Gal in the system used (Bianken et al. 1985). Our inability to detect GlcNAc/?l4GaI excluded the presence s f a contaminating G ~ C N A C ~ 1 -3(GlcNAcfl 1 -4)[ l4c(u)]Gd trisaccharide.

Put together, our data established the structure of the syn- thetic trisaccharide as GlcNAcP 1 -3(GlcNAc@ 1-6)- [ 1 4 ~ ~ ) ] ~ a l .

Enzymatic synthesis of ~ l c ~ ~ c ~ 1 - 3 ( ~ l d ~ ~ c 0 1 - 6 ) { ~ ~ ~ ( u)] Gal6 I -4GlNAc

The tetrasaccharide was synthesized from UDP-GlcNAc and GlcNAcfi 1-3 [l4~(~)]Gztl/?l -4GlcNAc using the hog gastric mucosal microsomes. The resulting tetrasaccharide migrated a little slower than maltotriose marker in paper chromatography with solvent A (see Table 1, glycan B), sug- gesting that only one GlcNAc-unit had been transferred to the acceptor trisaccharide. 6-N-Acetylhexosaminidase digestion of the synthetic

tetrasaccharide gave [ 1 4 ~ ( U ) ] ~ a l ~ 1-4GlcN~c (see Table 1, glycan L). This showed that even the newly introduced GlcNAc unit was 6-linked.

Partial acid hydrolysis of the synthetic tetrasaccharide was expected to give six major oligosacchai-ide fractions, as

890 1 MT Lac Gal 9 9 9

A I

MT Lac Gal GlcNAc 60 p

I + + + +

Fractions FIG. 2. Paper chromatographic analysis of partial acid

hydrolyzates of radiolabeled GlcNAc~l-3(GlcNAc/3l-6)Gal. The saccharides were treated with 0.1 M trifluoroacetic acid at 100°C for 40 min and the digests were chromatographed on paper with solvent A for 48-64 h. The saccharides digested were (A) synthetic ~ lcNAc/3 1 -3(GlcN~c/3l - 6 ) [ ' 4 ~ ( ~ ) ] ~ a l , (B) teratocarcinoma trisaccharide [ 3 ~ ] ~ l c ~ ~ c f l 1 - ~ ( L ~ H J G ~ C N A C / ~ ~ - ~ ) G ~ ~ , and (C) teratocarcinoma trisaccharide G~CNAC$ 1 -3(GlcNAc/3 1 -6)['4C]~al. The separated peaks were (1) intact trisaccharide, (2) GlcNAcfl 1 -6Ga1, (3) GlcNacfl 1 -3Gal, and (4) galactose in panels A and C, and N-acetylglucosamine in panel B. The markers were M-acetylglucosamine (GlcNAc), galactose {Gal), lactose (Lac), and maltotriose (MT).

shown in Fig. 3. The actual reaction mixture was frac- tionated by paper chromatography and, indeed, the six cleavage products were visible (Fig. 4A). Peak 1 represented the intact tetrasaccharide, but it contained also GlcNA@l-3- (GlcNAc@l -6)[ 1 4 C ( ~ G a l ; peak 2 was GlcNAc01-6- [ 14~ (U) ]~a lg 1-4Glc~Ac; peak 3 was G~CNAC/? 1 -3[14C(U)]- Gal6 1 -4GlcNAc; component 4 was Glc~Ac@ 1 -6 [14C&J)] - Gal; peak 5 was G ~ C N A C ~ ~ - ~ [ ' ~ C ( U ) ] G ~ ; and peak 6 was I l4~(U)]~a.l/3 1 -4Glc~Ac. All these components were even- tually obtained in pure form (see Table 1, glycans C-H for the migration rates) and characterized as shown below. The material running ahead of peak 6 was not identified.

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1 ET AL. 49

Partial acid hydrolysis

FIG. 3. Labeled oligosaccharide products expected to be pres- ent in partid acid hydrolysates of GlcNAcD 1 -3(GlcNAc@ 1-6) [ 1 4 ~ ( ~ ) ] ~ a l / 3 1 - 4 ~ l c ~ ~ c . Gal* denotes [14c(U)]C?al.

a 100 1 M T ~ MT Lac a + + +

Peak 1, in a long run with solvent B, gave two peaks that migrated like GlcN~cfl 1 -3 (G~CNAC@ 1 -6) [ i 4 ~ & J ) ] Gal@ 1 -4- GlcNAc (90% of the label) and GlcNAcB 1 -3(GlcNA@ 1 -6)- [ 1 4 ~ ( ~ ) ] G a l (10% of the label). The latter was chromatographed once more in a long run with solvent B to obtain a pure sample (Table 1, glycan C; Fig. 5A). Periodate oxidation and hydrolysis converted it into [14C] lyxose (1 9% yield) and some intact [14~]galactose, but not to labeled threose (data not shown). The formation of labeled lyxose in a good yield showed that most of the galac- tose in the trisaccharide was disubstituted at positions 3,6 or 3,4. The formation of labeled galactose in the periodate experiment may indicate that the oxidation was incomplete; alternatively a minor component of the trisaccharide con- tained galactose units that were substituted at positions 2,3 or 2,4.

Peak 2 of the acid hydrolysate migrated like authentic GlcNAc@l-6@al/31-4GlcNAc from teratocarcinoma cells during paper chromatography with solvent B (see Table 1, glycan D). It was cleaved by 6-N-acetylhexosaminidase into [ ' 4 C ] ~ & 3 1 - 4 ~ l c ~ ~ c , which was identified by paper chro- matography with solvent B (data not shown).

Peak 3 saccharide was cleaved by endo-P-galactosidase from E. freundii to give G ~ C N A C ~ ~ I -3 [ 1 4 ~ ~ ) ] ~ a l (90% of the label), which was identified by paper chromatography with solvent B (data not shown). The disaccharide gave lyxose but no labeled thrmse in periodate oxidation and sub- sequent hydrolysis (data not shown). This excluded the pres- ence of radiolabeled GlcNAcp 1 -4Galb 1 -4GlcNAc in peak 3 saccharide.

The saccaride of component 4 was separated from peak 3 in auxiliary long runs with solvent B. In pure form, it migrated in paper chromatography like authentic GlcNAc- p1 -6@al @g .5B) (see Table 1, glyean F, for the exact migra- tion rate). It was cleaved by 0-N-acetylheiosaminidase into [14~&J)]galactose, which was identified by paper chroma- tography in solvent A (data not shown). In HPEC the com- ponent 4 saccharide migrated several minutes behind the authentic GlcNAcP1-3Gd; this shows that it could not be

0 10 20 30 40

Fractions FIG. 4. Paper chromatographic analysis of partial acid

hydrolysates of radiolabeled GlcNAcB 1 -3(GlcNAc@l-@Gal- @14GlcNAc. The saccharides were treated with 0.1 M trifluoroacetic acid at 100°C for 40 min, and the digests were chromatographed on paper with solvent B for 11 days. The saccharides were (A) synthetic G ~ c N A c / ~ ~ - ~ ( G ~ c N A ~ ~ ~ - ~ ) [14c(LJ)]~alfl 1 - 4 G l c ~ ~ c and (£3) teratocarcinoma tetrasaccharide GlcNAcfl 1 -3(GlcNAcfl l-6)- [ ' 4 ~ ] ~ a l / 3 1 4 ~ l c ~ ~ c . The separated labeled components were (1) a mixture of GlcNAcpl -3(GlcNA@ 1 -6)GaqS 1 -4GlcNAc and GlcNAcfll -3(GicNAcfl 1 -6)Gals (2) GlcNAcfl 1 GlcNAcfl l -3GdO 1 -4GlcNAc, (4) GlcNAcD 1 -6Ga.1, (5) GlcNAc- 01-3Gd, and (6) Galpl-4GlcNAc. The markers were lactose (Lac), maltotriose (MT), and rnaltotetraose (MTe).

GlcNAcfl 1-4Gd (Blanken et al. 1985). In WGA-agarose chromatography the disaccharide of component 4 behaved characteristically like authentic G ~ c N ~ l - 6 G a l (data not shown).

Peak 5 glycm migrated like GlcNAcPl-3Gd in paper chromatography (see Table 1, glycan G). Its periodate oxida- tion and subsequent hydrolysis gave labeled lyxose (30% yield), but labeled threose was not detected (data not shown). These findings were compatible with the presence of a 3-substituted galactose.

Peak 6 glycan migrated in paper chromatography like Gdal-4GlcNAc. It was completely cleaved by fl- galactosidase into [14~]galactose (data not shown). It resisted the action of @-N-acetylhexosaminidase (data not shown). These findings excluded the presence of a radiolabeled GlcNAcPGal species in peak 6.

Preparation of metabolically labeled teratocarcinorna saccharides

Murine embryonal carcinoma cells (line PC 13) were labeled metabolically with [ ' 4~(~) ]ga lac tose or 2-amiin0-2-deoxy[6-~~lglucose, and peptide-linked poly-N- acetyllactosaminoglycans were isolated from the pronase digests by gel filtration and ion-exchange chromatography

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Fractions FIG. 5. Isolation of key saccharides by prolonged paper chro-

matography with solvent B. (A) GlcNAcfll-3(GlcNAc@1-6)- ["C(U)]G~~ from peak 1 in Fig. 4A was rechromatographed with solvent B for 25 days. The calculated position of radiolabeled GlcNAcfll-3(GlcNAc@l-6)Gal synthesized in the present experiments coincided with the peak in fraction 10. (B) G ~ c N A c ~ ~ ~ - ~ [ ' ~ c ( u ) ] G ~ ~ from fractions 13-1 5 in Fig. 4A (component 4) was rechromatographed for 20 days with solvent B, The calcuIated position of radiorabeled CilcNAcfll-6G.l marker coincided with the peak in fraction 24. (C) Mixed GlcN~cjl1 -3(GlcN~c/31 -6)[I4c]@d and GlsNAcfll3(GlcNAc- ~ 1 - 6 ) [ 1 4 ~ ] ~ a l @ l - 4 ~ l c ~ ~ c from metabolically labeled teratocarcinoma cells were chromatographed on paper with solvent B for 20 days. T h e p a k in fractions 7-9 proved to be GICNAC~S~ - 3 ( ~ l c ~ ~ @ 1 - 6 ) [ ' CjGd, while that in fractions 4-6 was @@ICNAC@ 1 - 3 ( G l c ~ f ? 1-6) [ " c ] G ~ ~ 1 -4Glc~Ac. The markers were lactose (Lac), maitotriose (MIF), an8 rnaltopentaose (MP).

(Renkonen 1983). Partial acid hydrolysis of the labeled polysaccharide, followed by a series of chromatographic procedures, gave a fraction containing neutral penta- to octa-saccharides. This fraction was digested with @- galactosidase, whereafter a product migrating like mdtotriose was isolated by paper chromatography using sol- vent A. This material was then reaolved into two peaks in

Fractions

FIG. 6. Paper chromatographic identification of N-acetyl- hexosamines. The saccharide from peak 4, Fi. 2B, was chroma- tographed on borate-containing paper as described in Materials and methods. The markers were N-aeetylgalactosamine (GdNAc) and N-acetylgluco~mine (GlcNAc).

long runs with solvent B pig. 5C; see Table 1, glycans N and R, for the migration rates). The faster migrating peak proved to be labeled GlcNAcfll-3(GlcNAc@ 1 -6)Gaf, while the slower peak was labeled GlcNAcBl-3(GlcNAcPl-6)- Gal@ 1 -4GlcNAc.

Characterization of GleNAcfll-3(GlcNAcfi i-6)Gal from teratocarcinorns cells

The [ 3 ~ ] ~ l c ~ ~ c - and [14~]~al-labeled versions of the teratocarcinoma saccharide migrated like the synthetic GlcNAcfl 1-3(GlcNAcfl l - 6 ) [ 1 4 ~ ] ~ a l in paper chromatogra- phy with solvents A and B (see Table 1, glycans M and N). Also in HPLC the galactose-labeled teratocarcinoma trisac- charide migrated like the synthetic GlcNAcPl-3(GlcNAc- @la)Gal, emerging from the column 3.5 Hnin after maltotriose marker. Both versions of the trisaccharide migrated also in WGA-agarsse chromatography like the synthetic GlcNAc- pl -3 (Glc~Ac@ 1 -6) [ 1 4 ~ ( ~ ) ] ~ d (see below); this behavior is very characteristic.

0-N-Acetylhexosaininidase converted the ['4~]galactose- labeled teratocarcinoma trisaccharide completely into labeled gafactose, which was identified by paper chroma- tography with solvent A (data not shown).

Partial acid hydrolysis of the ['H]G~CNAC-labeled teratocarcinoma trisaccharide gave four major components (Fig. 2B). Peak 1 migrated like the original trisaccharide in paper chromatography with solvent A, peak 2 migrated like GlcNAcP 1 -6Gal, peak 3 migrated like GlcNAcP 1 -3Ga1, and peak 4 migrated like N-acetylglucosamine and N- acetylgalactosamine (see Table 1, glycans O and P). Peak 2 glycan migrated like authentic GlcNAc@l4Gal also in sol- vent B (see Table 1, glycan P) and solvent C (data not shown). Moreover, peak 2 was cleaved completely by P-N- acetylhexosaminidase, liberating ['HIO~CNAC which was identified by b ~ r a t e paper chromatography (data not shown). Peak 3 migrated like GlcNAcfil-3Gal in solvent C (data not shown). Peak 4 was identified as GlcNAc by borate paper chromatography (Fig. 6).

The [14~]gdactose-labeled teratocarcinoma trisaccharide gave also four major components after partial acid hydrolysis (Fig. 2C). The " finger-printing" pattern was very similar to that obtained from the synthetic trisaccharide G~CNACP 1-3(~lcNAc~1-6)[ 146(u) ]Gal (see above). The

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Gal t

Fractions

FIG. 7. WGA-agarose chromatography of synthetic GlcNAcj3 1-3 (GlcNAcpl-6) [ 1 4 ~ ( ~ ) ] ~ a l . The saccharide (6 pmol) was chromatographed in a (0.7 x 12 cm) column of WGA- agarose (1.65 ~zlg WGMmL of 4% beaded agarose). Fractions 1-40 were collected with 10 m M sodium phosphate buffer (pH 7.1) containing 0.9% NaCl and 0.02% NaN3. Fractions 41-70 were collected with the same buffer containing 0.2 M GlcNAc. Fraction size was 0.55 mL throughout. The yield of radioactivity was 86%. The markers were galactose (Gal) and reduced N1,N", NW- triacetylchitotriose (CT,,) .

GlcNAcfll - 6 [ 1 4 ~ ] ~ a l from the finger-printing experiment (peak 2 in Fig. 2C) migrated more slowly than GlcNAc- /31-3Gal in HPLC (data not shown), indicating that it was not GlcNAcB1-4Gal (Blanken et al. 1985). In WGA-agarose chromatography most of the label of this saccharide behaved like radiolabeled GlcNAcpl-Gal marker (data not shown). Peak 3 glycan from the finger-printing experiment of Fi . 2C was cleaved by 0-N-acetylhexosaminidase into H: [' Clgalactose to the extent of 85% (data not shown).

Put tagether, these findings showed that the branched teratocarcinoma trisaccharide was GlcNAcpl-3(GlcNAc- pl -6)Gal.

Characterizstian sf the teratocarcinoms tetrasaccharide G~~NAC~~-~(G~~NAC@~-~)GU~@~-~G~CNAC

The ['HI G~CNAC- and ["c]Gal-labeled versions of the teratocarcinoma tetrasaccharide migrated in paper chroma- tography (Table 1, glycans Q and R) and in HPLC (data not shown) like the synthetic GlcNAcpl -3(GlcNAc/3 1-6)- [ 1 4 ~ ~ ) ] ~ a l ~ 1 - 4 G l c ~ ~ c . Also in WGA-agarose chroma- tography the [14~]galactose-labeled teratocarcinoma tetrasaccharide migrated like the synthetic GlcNAc- 6 1 - 3 ( G l c ~ ~ c @ 1 -6)-[14c(u)]~al@1 -4GlcN~c (see below); this behavior was very characteristic.

The [14~]galactose-labled teratocarcinoma tetrasac- charide was cleaved completely by 6-N-acetylhexosaminidase into [ ' 4 ~ ] ~ a l @ 1 - 4 - ~ l c ~ ~ c , which was identified by paper chromatography with solvents A and B (see Table 1, glycan S). The disaccharide was cleaved further by P-gdactosidase, releasing [14~]galactose that was identified by paper chro- matography with solvent A (data not shown).

Partial acid hydrolysis of the galactose-labeled title tetrasaccharide gave six components that could be resolved by paper chromatography (Fig. 4B, Table -1, glycans U-W) . This finger-printing pattern of cleavage products was prac- tically identical with the pattern obtained with synthetic G ~ C N A C ~ 1 - 3 ( G l c ~ ~ c @ 1-61 [ 1 4 ~ ( ~ ) ] ~ a l f l - 4 G l c c (see

- above).

Gal

1, Fractions

FIG. 8 . WGA-agarose chromatography of synthetic GlcNAc/31-3(GlcNAcj3 1-6) [U- 14c] alp 1 - 4 ~ l c N ~ c . A 3 pmol sample of the saccharide was chromatographed as described in Fig. 7. The yield of radioactivity was 77%. Markers were as in Fig. 7.

The trisaccharides of the partial acid digest were characterized further by the following experiments. Peak 1 from Fig. 4B was resolved in a long run with solvent B into two peaks that migrated like the synthetic GlcNAc- p l -3(GlcNAcpl-6)[ 1 4 ~ ( ~ ) ] ~ a l ~ 1 -4GlcNAc and GlcNAc- @ 1 - 3 ( G l c ~ ~ c f l l - 6 ) [ ' 4 ~ & J ) ] ~ a l , respectively (see Table 1, glycan U). The trisaccharide migrated in WGA-agarose chromatography like synthetic GlcNAcB 1 -3(GlcNAc- f i l - 6 ) [ 1 4 ~ ( ~ ) ] ~ a l (data not shown). Most of the label of G~cNAc@~-~[~~c]G~~~~~-~G~cNAc of peak 2 from Fig. 4B migrated like the GlcNAcp 1 -6GalPl-4GlcNAc marker in WGA-agarose chromatography (data not shown). Peak 3 from Fig. 4B was cleaved almost completely by endo-6- galactosidase into G ~ c N A c ~ ~ ~ - ~ [ ' ~ c ] G ~ , which was iden- tified by paper chromatography with solvent B (see Table 1, glycm X).

These data established the most likely structure of the branched teratocarcinoma tetrasaccharide as GlcNAc- p 1 -3(GkNAc@ 1 -6)calfl-4GlcNAc.

Chromatography of the oligosaccharik on immobilized WGA

The synthetic trisaccharide GlcNAcP 1 -3(GlcNAcp 1-6)- [ 1 4 ~ ( ~ ] ~ a l was strongly bound to WGA-agarose column (Fig. 7). The elution profile revealed two major peaks; in addition, 10Vo of the label was located between the peaks. The first peak (32Vo of total radioactivity) was retarded, but could be eluted from the column with a sugar-free buffer; the second peak (58Vo of total radioactivity) was eluted with the same buffer that contained additionally 0.2 M N- acetylglucosamine.

The saccharide in the first peak, in fractions 20-32 in Fig. 7, was pooled, desalted, and then rechromatographed in the WGA-agarose column (data not shown). A major part of radioactivity was shifted to fractions 49-52. The distribution of label between fractions 28-32 (29%) and frac- tions 49-52 (59Vo) resembled that of the original chromatogram. The "sharp-peak" saccharide in fractions 49-52 in Fig. 7 was also desalted and separated from N- acetylglucosamine by Bio-Gel P-2 chromatography. Upon rechromatography in the WGA-agarose column it revealed again the characteristic profile of two peaks (data not shown). These findings suggested that GlcNAc-

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/3 1 -3(GlcNAc(3 1-6)Gal existed in two interconvertible forms, which were bound to different degrees to immobilized WGA. The interconversion of the different forms of the trisaccharide may be caused by mutarotation.

The teratocarcinoma trisaccharide GlcNAcp 1-3(GlcNAc- f11-6)[14~]~al behaved like the synthetic trisaccharide in WGA-agarose chromatography. Also, the elution profile of the ['HIG~CNAC-labeled teratocarcinoma trisaccharide was identical with that of the synthetic trisaccharide (data not shown).

The synthetic tetrasaccharide GlcNAcfll-3(GlcNAc- pl -6)[ 14c(LJ)]~al/3 1 - 4 ~ l c ~ ~ c was chromatographed in the WGA-agarose column, together with [ '~l~alactose as an internal marker. The data (see Fig. 8) showed that the branched tetrasaccharide was eluted three to four fractions after galactose. The retardation was equivalent to about 0.4 column volumes. The branched teratocarcinoma tetrasac- charide migrated in WGA-agarose column identically with the synthetic tetrasaccharide (data not shown).

Discussion The present report describes in vifrs, synthesis of two

branched saccharides, GlcNAc@l-3(GlcNAcp 1 -6)Gal and GlcNAcP 1 -3(GlcNAcP 1 -6)GalP 1 -4GlcNAc, in radiolabeled form. Their synthesis was undertaken to help characteriza- tion and identification of cleavage products obtained from poly-N-acetyllactosaminoglycans of embryonal carcinoma cells. The two glycans were synthesized from known linear acceptor saccharides, using a Pl ,d-GlcNAc-transferase present in epithelial microsomes of hog stomach. This enzyme preparation had been used previously by Piller et ul. (1984) to transfer GlcNAc units from UBP-GlcNAc, in P- linkage, to position 6 of the gdactose residues in GlcNAcP 1 -3Gal/31-4Glc@OMe. In view of the findings of Brockhausen ef ul. (1 983) showing that Gal@l4GIcNAc and lactose do not always react with porcine gastric GlcNAc- transferases in the same way, we subjected the synthetic products to a careful study. The characterization was carried out largely by partial acid hydrolysis, followed by chromatographic separation and identification of the oligosaccharide products (Figs. 2 and 4). This approach was selected, instead of radiochernial methylation analysis (Stoffyn et al. lwl ) , because no IiqGd chromatographic method was available for the separation and firm identifica- tion of the six radiolabeled di-0-methylgalactose species.

The characterization data suggested that the synthetic G~CNACS~ - 3 ( ~ l c ~ ~ c p l - 6 ) [ 1 4 ~ ~ ] ~ a l was rather pure; the only minor contaminant that was not definitely excluded was GlcNAcfll-2(GlcNAcP 1-6)[ 14c]~a l .

The synthetic tetrasaccharide gave two major species of GlcNAcPLadNAc in partial acid hydrolysis, which sug- gested that it, too, was branched at the galactose residue. The tetrasaccharide was cleaved by (3-N-acetyl- hexosaminidase into labeled GalPl4GlcNAc, showing that both nonreducing GlcMAc units were P-linked. The partial acid hydrolysate of the tetrasaccharide contained GlcNAc- 01 -3Gal and GlcNAcPl -6Gd as the two major GlcNAcW species, indicating that there was a branching galactose unit that was 3,6-disubstitutd. This conclusion was confirmed by the identity of the two major GlcNAcPLactNAc species of the acid hydro1 ysate as GlcNAcP 1 -6Galfll-4GlcNAc and GlcNAcpl -3GalP 1 -4GlcNAc. Periodate oxidation experi-

ments, in turn, excluded the presence of 2,3-, 2,4-, 2,6-, and 4,6-disubstituted galactose units in the tetrasaccharide.

Put together, our data showed that the hog gastric epithelial /31,6-GlcNAc-transferase acted in the same man- ner with G ~ c N A c @ ~ - ~ [ ~ ~ c ( u ) ] G ~ I and GlcNAcPl-3- [14c(U)]~alfl 1 - ~ G ~ C N A C , as it did with GlcNAcO 1-3- Gal01 -4Glc@OMe in the experiments of Pifler et sl. (1 984). It catalyzed the transfer of a GlcNAc unit from UBP- GlcNAc to the 6-position of the branching galactose of all three acceptors; the new GlcNAc unit was introduced in P-linkage in all three reactions. These findings are very helpful for the in vifro synthesis of branched oligo-N- acetyllactosaminoglycans.

The present report further showed that the teratowcinoma poly-N-acetyllactosaminoglycan from PC 13 EC cells con- tained a branched sequence very similar to GlcNAcfl1-3- (GlcNAcP 1 -6)Galpl-4GlcNAc. Unfortunately, structures containing GlcNAcPl-2Gal unit could not be excluded on the basis of the present data; this unit had been found in a branched hexasaccha-ide that was strongly bound to WGA (Wran and Tuppy 1978). Our data confirmed previous methylation studies that suggested the presence of 3,6-disubstituted galactose units in the polysaccharide of PC 13 EC cells (Renkonen 1983) and also in the closely related F9 cells (Muramatsu e f sl. 1983); the presence of other disubstituted galactose structures was not excluded in these early studies.

Radiolabeled GlcNAcP 1 -3(GlcNAc@ 1 -6)Gal was strongly bound to immobilized WGA, resembling the disaccharide GlcMAcfl l -6Gal (Wenkonen et sl. 1988). Furthermore, the branched trisaccharide was eluted from the WGA-agarose column as two distinct peaks, which seemed to represent two interconvertible molecules. This behavior has been observed also with radiolabeled GlcNAc/31-&3al menkonen 1988). To our knowledge previous 0bservatio.n~ of this kind have not been reported in chromatography of oligosaccharides on immobilized lectins or antibodies. In reverse-phase HPLC, a similar finding has been made with chito-oligosaccharides (Blumberg ef a!. 1982); the peak doubling was thought to represent anomeric separation and the reestablishment of the equilibrium was taken as mutarotation.

GlcNAc/31-3(GlcNAc~ 1-6)Galp 1 -4GlcNAc was bound to WGA-agarose to a much lesser degree than GlcNAcfll-3- (GlcMAcfll-6)Gal. However, even the branched tetrasac- charide was retarded approximately 0.4 column volumes. Considering the low WGA content in our experiments, this retardation may be approximately equivalent with that observed recently for a novel sidylhexasaccharide (Tarrago et al. 1988). In an earlier report (Wenkonen et al. 1988) we showed that GlcNAcP 1 -6Gal0 1 -4GlcNAc bound less strongly than GlcNAcfll-dGal. Hence, the sequence GlcNAcol -6Gal at the reducing end of oligosaccharides seemed to be associated with a particularly high binding affinity.

Intact poly-N-acetyllactosaminoglycans bind to WGA with high affinity (Ivatt et al. 1986; Gallagher et sl. 198%). The strong binding of sequences that carry galactose at the reducing end may not be responsible for this; intact poly- N-acetyllacto~inoglycans have no reducing end galactose. Instead, we think that despite their weaker affinity, the bind- ing of several GlcNAc~l-3(GlcNAcfll-6)Gal~ 1 -4GlcNAc, GlcNAcfl 1 -6(Ga43 1 -4GlcNAc, and GlcNAcP 1 -3Galp 1-4-

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G ~ C N A ~ units may be a major cause of the high WGA affinity of the polysaccharides.

Acknowledgements Support by grants (011548 and 101 1027) from the Finnish

Academy (to 0 . R . ) and by a grant from Oscar &lunds ~oundation, Helsinki, Finland (to O.R.) is acknowledged.

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BLUMBERG, K.* LINIERE, F., PUSTILNIK, L., and BUSH, C.A. 1982. Fractionation of oligosaccharides containing N-acetyl amino sugars by reverse-phase high-pressure liquid chromatog- raphy. Anal. Biochem. 119: 407-412. ,

BREW, K., VANAMAN, T.C., and HILL, R.L. 1968. The role of a-lactalbumin and the A protein in lactose synthetase: A unique mechanism for the control of a biological reaction. Proc. Natl. Acad. Sci. U.S.A. 59: 491-497.

BRWKHAUSEN, I., WILLIAMS, D., MATTA, K.L., ORR, J., and SCHACHTER, W. 1983. Mucin synthesis. 111. UDP-GlcNAc: Gal01 -3(GlcNAcfil-6)GaINAc-R (GlcNAc to Gal) @3-N- acetylglucosaminyltransferase, an enzyme in porcine gastric mucosa involved in the elongation of mucin-type oligosac- charides. Can. J. Biochem. Cell Biol. 61: 1322-1333.

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USILO, M. -L . , and RENKONEN, 0. 19820. Cell-associated glycosaminoglycans of human teratocarcinoma-derived cells of line PA 1. Eur . J. Biochem. 123: 397-405. - 1982b. Analysis s f linkage monosaccharides in human

teratocarcinoma cell glycopeptides: Paper chromatographic separation of N-acetylglucosaminitol and N-acetylgalactos- aminitol. Hoppe-Seyler's Z. Physiol. Chem. 363: 89-93.

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RENKONEN, O., MAKINEN, P., WARD, K., HELIN, J., and PENTTILA, L. 1988. Immobilized wheat germ agglutinin sepa- rates small oligosaccharides derived from poly-N-acetyllactos- aminoglycms of embryond carcinoma cells. Biochem. Cell Biol. 66: 449-453.

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