252 Biochimica et Biophysica Acta, 1157(1993) 252 25,~ © 1993 Elsevier Science Publishers B.V. All rights resefvcd [1304-4165/93/$06.1J~

BBAGEN 23814

Oligosaccharide specificity of normal human hepatocyte a l - 3 fucosyltransferase

Maryvonne Jezequel-Cuer a, Han N'Guyen-Cong b Daniel Biou b and Genevi6ve Durand a,b

Laboratoire de Biochimie A, H~pital Bichat, Paris (France) and h Laboratoire de Biochimie, Universitd Paris" X1, Chdtenay-Malabry (France)

(Received 1 December 1992)

Key words: Fucosyltransferase; Hepatocyte; Human; Lewis×; Oligosaccharide

A purified a l - 3 fucosyltransferase ( a l - 3 FT) was recovered in the Golgi fraction of isolated hepatocytes from normal human liver tissue. The efficiency of purification was controled by measurement of fucose transfer to asialotransferrin (for cd-3 FT), to phenyl-/3-D-galactose (for a l - 2 FT) and to 2' fucosyl lactose (for a l - 3 / 4 FT). The initial hepatocyte isolation step got rid of 97% and 94% of eel-2 and a l - 3 / 4 total liver FT, respectively. After Golgi enrichment (26-fold purification and a yield of 7.6%), a l - 3 FT extract expressed a specific activity of 2 pM/min per mg protein. When incubated in optimized conditions with type 1, 2 or 6 oligosaccharidc acceptors (10 mM), hepatocellular a l - 3 FT efficiently transferred fucose to N-acetyllactosamine and its 3'sialylated derivative, but poorly to lactose. When incubated with neutral or sialylated biantennary N-glycans, the enzyme expressed the highest affinity (Km = 0.38 mM) for the 3'bisialylated derivative. This suggests that hepatocellular a l - 3 FT is involved in the synthesis of sialosyl Le X determinants on cirrhotic alAGP.

Introduction

Lewis x (LeX), Gal/31-4 ( F u c a l - 3 ) GlcNac, and sia- lyl Lewis x (Sialyl Le x) antigens have been detected in a number of normal human tissues [1], but their entire carbohydrate structures have been elucidated in only a few membrane glycoconjugates of normal human gran- ulocytes [2] and kidney tissue [3]. In contrast, carbo- hydrate units of normal plasma glycoproteins have been extensively studied but only ceruleoplasmin [4] and a l -ac id glycoprotein ( a l - A G P ) [5] have been found to possess these fucosyl a n d / o r sialylated fucosyl lac- tosamino glycan structures. In human a l - A G P , the fucose residue was reported to be linked at a l - 3 to either of the external GlcNAc residues of biantennary N-glycans [6], or to GlcNAc 7 or 7' of te traantennary N-glycans [7]. In the later case, the a l - 3 fucosylated side chains were terminated with sialic acid through a 2 - 3 linkage.

Addition of fucose to the GlcNac 3 O H of a N- acetyllactosamine may be catalysed by at least two

Correspondence to: M. Jezequel-Cuer, Laboratoire de Biochimie A, H~pital Bichat, 46 rue Huchard, 75877 Paris C~dex 18, France.

different enzymes: a Le gene-encoded a l - 3 / 4 fucosyl- transferase (EC2.4.1.65) and a Le-independent a l - 3 fucosyltransferase (EC2.4.1.152). These fucosyltrans- ferases (FT), which have been characterised in several human tissues [for a review, see 8], are known to be involved in the last steps of the glycosylation process in the Golgi apparatus [9]. In a previous paper, we re- ported that total normal human liver tissue usually expressed a l - 2 , ~1-3 and a l - 3 / 4 fucosyltransferase activities and that the increase levels observed in sev- eral liver diseases might derive partially from non- parenchymal liver cells [10].

The aim of this work was to determine lactosaminyl substrate requirements of the hepatocellular a l - 3 FT involved in the glycosylation of ceruleoplasmin and a l - A G P . The a l - 3 FT activity of a Golgi-enriched fraction from isolated normal human hepatocytes was measured on neutral type 1, 2 and 6 oligosaccharides and on sialylated type 2 oligosaccharides (Table I), the preferential utilisation of which distinguishes several tissular subtypes a l - 3 FT [8]. We also compared ap- parent Michaelis constants of the enzyme for mono- lactosamine unit acceptors to its affinity for bianten- nary complex-type N-glycans which are closer to the endogenous substrates.

Mater ia l s and Methods

Reagents Unlabeled GDP-fucose and 2'fucosyl lactose were

from Biocarb; GDP[14C] fucose was from Amersham and 3' and 6' sialyl N-acetyl lactosamine were from Oxford glycosystem.

Human transferrin from Behring was used to both prepare asialotransferrin as described by Beyer et al. [ l l ] and neutral biantennary N-glycan as described below.

Bisialyl biantennary N-glycan was a generous gift from Strecker et al, who isolated it from the urine of patients with sialidosis [12].

All other acceptors were obtained from Sigma and chemicals from Merck. Protein was determined with the Biorad protein assay reagent.

Isolation of hepatocytes A human liver fragment was obtained from a patient

with a well-delimited hepatic tumour undergoing par- tial hepatectomy. A non-tumourous fragment (8 g) was also resected at the edge of the lobe, placed in ice-cold isotonic NaCI solution and transported to the labora- tory.

The non-recirculating perfusion technique described by Ballet et al. [13] was used to isolate hepatocytes. The first cell pellet collected was resuspended in 25 mM Hepes buffer (pH 7.5) supplemented with 2% bovine albumin, 1.25 mM CaCI2, and 0.75 mM MgSO 4, centrifuged for 2 rain at 50 x g and washed 3 times in Hepes buffer. The last cell pellet (225 X 10 6 cells) was

253

immediately stored at - 80°C until subcellular fraction- ation.

Cell fractionation Cells were lysed using a modification of the method

of Dignam et al. [14]. Briefly, the cell pellets were dispersed in 0.25 mM buffered sucrose solution (25/xl for 10 6 cells) containing 10 mM Mops buffer pH 7.2, 1 mM DTT, 0.5 mM PMSF and 10 /xM pepstatine A. The only lysis buffer also contained 2 mM EDTA.

The cell suspension was first drawn into a 2-ml glass pipette 10 times and the homogenate was sedimented at 750 x g for 10 min. The supernatant was decanted and the pellet was resuspended in lysis buffer then homogenised by 10 strokes of a glass Dounce homoge- nizer.

The 750 g supernatants of the two homogenates were pooled and adjusted to 1.0 M sucrose with 1.25 M buffered sucrose solution.

For subcellular fractionation we used a discontinu- ous sucrose gradient as described by Sandberg et al. [15] with slight modifications: from bottom to top of l l -ml centrifuge tubes, we added 3 ml of 1.25 M buffered sucrose, 5 ml of the 1 M sucrose supernatant and 3 ml of 0.5 M buffered sucrose. The tubes were then centrifuged at 1 2 0 0 0 0 x g (SW 41, Beckman L5/65 ultracentrifuge) for 90 min.

The Golgi apparatus-enriched fraction was collected at the 0.5/1.0 M sucrose interface and the microsomes at the 1.0/1.25 M interface. These two fractions were diluted with 0.25 M unbuffered sucrose and pelleted at 150000 X g (SW 55) for 45 rain. The pellets obtained

TABLE I

Oligosaccharide acceptors

Oligosaccharide structure Type Common denomination

Gal/31 ~ 3GIcNAc

Gal/31 --* 4GIcNAc NeuAc a2 ~ 3 Gal/31 ~ 4GlcNAc NeuAc a2 ~ 6 Gal/31 --* 4GIcNAc

Gal/31 --* 4Glc Fuc al ~ 2 Gal/31 ~ 4Glc

Gal/31 --, 4GIcNAc/31 ~ 2 Man al..~ 3

R1 6

Gal/31 ~ 4GlcNAc/31 ~ 2 Man c~l ''~

NeuAc a2 ~ 3 Gal/31 ~ 4GlcNAc/31 ~ 2 Man al..~ 3

R2

NeuAc a2 --* 3 Gal/31 ~ 4GIcNAc/31 ~ 2 Man a l / ' 6

l Lacto-N-biose I

2 N-acetyl lactosamine 2 3'-Sialyl-N-acetyl lactosamine 2 6'-Sialyl-N-acetyl lactosamine

6 Lactose 6 2' fucosyl lactose

Neutral biantennary N-glycan

3'bisialyl biantennary N-glycan

RI = Man/31 ~ 4GIcNAc/31 ~ 4GlcNAc °l R2 = Man/31 ~ 4GIcNAc.

254

were immediately stored at -80°C for enzyme meas- urements.

Preparation o f biantennary N-glycans We used human serotransferrin, an easily available

source of biantennary N-glycans [16]. OIigosaccharides from 500 mg of human transferrin were obtained by hydrazinolysis, re-N-acetylation and reduction accord- ing to Takasaki et al. [17]. The free oligosaccharides were desalted by gel filtration on a HW-40-S Fractogel (Merck) column (50 × 1.6 cm) equilibrated with 0.5% acetic acid at a flow rate of 1 ml × min - t

Biantennary N-glycans were separated from tri- antennary N-glycans by affinity chromatography on a Con A-Sepharose (Pharmacia) column (10 × 1 cm) ac- cording to Biou et al. [18].

Desialylation of the desalted Con A-reactive oligo- saccharides was carried out with 0.1 N H2SO 4 at 80°C for l b .

The carbohydrate composition of the neutral biantennary N-glycans obtained was determined after methanolysis, re-N-acetylation [19] and GLC [20] of the trimethylsilylated glycosides on a capillary 25 QC3/BP1 0.5 (SGE) column (0.33 m m × 25 m).

Standard a fucosyltransferase assay Liver tissue, isolated hepatocytes and Golgi pellets

were homogenized with appropriate amounts of extrac- tion buffer (1% Triton X 100, 1 mM D T r , 0.5 mM PMSF, 50 mM Mops, pH 7.2) to obtain protein con- centrations of 10, 5 and 1 rag/l , respectively.

We first tested the samples in standard conditions described elsewhere [10]. Phenyl /3-D-galactose (1 ~mol), asialotransferrin (12.5 nmol) and 2'fucosyl lac- tose (0.5/xmol) were used as substrates for the deter- mination of a l - 2 , a l - 3 and a l - 3 / 4 fucosyltrans- ferase activities, respectively. The reaction was started with the addition of 1.45 nmol of GDP [14C] fucose (120000 cpm) in a total volume of 100 Ixl, and contin- ued for 2 h at 37°C.

Optimized a l - 3 fucosyltransferase assay In a first step, incorporation of [14C] fucose into

asialotransferrin was measured with aliquots of the

Golgi-enriched extract under various pH conditions (from 6.72 to 8.12, 0.05 M Mops /Hepes buffer) and concentrations of Mn 2+ (from 0 to 20 mM), CDP choline (from 0 to 1.25 mM), and GDP fucose (from 3.3 × 10 -6 M to 2.2 × 10 4 M).

The saccharide acceptors listed in Table I were then tested under the optimal conditions selected.

The reaction mixture consisted of 25 /zl of Golgi extract and 10 /xl of a specific acceptor in a final volume of 50 tzl containing 1.35 nmol GDP [~4C] fucose (60000 cpm), 2.5/xmol Mops pH 7.5, 0.5 /xmol MnC12, 12.5 nmol CDP choline, 0.5 /xmol fucose and 0.5% Triton X100. Following 120 rain incubation at 37°C, the reaction was stopped by adding 50/xl of cold ethanol.

Aliquots of the centrifuged samples (20 #1) were applied in a thin 0.5-cm-long streak with a Hamilton syringe to Merck silicagel chromatographic plates (20 × 20 cm) without a fluorescent indicator.

Plates containing bi-, tri- or tetra-saccharides were developed in a first solvent mixture (bu tanol / e thanol /pyr id ine /ace t ic a c i d / H 2 0 , 1 0 / 1 0 0 / 1 0 / 3 / 30). After drying, the bottom of the plate was cut off to eliminate non-migrating GDP [14C] fucose. The plates were developed twice in a second solvent mixture (butanol /pyr id ine /1 M acetic acid, 5 / 3 / 2 ) to separate fucosylated oligosaccharides from free [~4C] fucose.

With reference to control sugars revealed by orcinol reaction, 0.5-cm strips were cut and placed in 3 ml of scintillation cocktail (Insta Fluor).

Plates containing larger oligosaccharide acceptors were developed three times in butanol /acet ic ac id / H 2 0 (65 /30 /40 ) as described by Schindler et al. [21].

a L-fucosidase and sialidase activities aL-fucosidase was assayed with 2.0 mM p-

nitrophenyl aL-fucopyranoside (pNP fucose) according to White et al. [22] and sialidase was assayed with 1.0 mM 4 methylumbelliferyl a N-acetylneuraminic acid (4 MU NeuAc) according to Potier et al. [23]. Reactions were run for 1 h in 50 mM Mops buffer (pH 7.5), instead of the usual acidic buffers.

TABLE II

Acceptor specificity pattern of fucosyltransferases expressed by three normal human liver tissue fractions

Acceptors Total liver extract Isolated hepatocytes Golgi fraction

Spec. Relative Spec. Relative Spec. Relative act. a act. b act. a act. b act. a act. b (mU/mg) (%) (mU/mg) (%) (mU/rng) (%)

asialotransferrin 78 100 155 100 2 015 100 2'-fucosyl lactose 30 38.5 21 13.5 220 10.9 P-/3-D-galactose 28 35.9 11 7.1 121 6

a fmol of [14C]fucose transferred to each acceptor/min/mg protein in standard conditions described in 'Materials and Methods'. b Relative activities are expressed as the percentage [I4C]fucose incorporation into the asialotransferrin acceptor.

Results

Fucosyltransferase activities of total liver t&sue and iso- lated hepatocytes

We first isolated hepatocytes in order to eliminate fucosyltransferases derived from non-parenchymal liver cells. Aliquots of each tissue extract were assayed in standard conditions, on phenyl-/3-o-galactose and 2' fucosyl lactose (for contaminating cd-2 and a l - 3 / 4 FT activities, respectively), and using [t4C] fucose transfer to asialotransferrin as a reference for cd-3 FT activity (Table II).

About 250.106 hepatocytes were isolated from 8 g wet liver tissue, i.e., about 20% of the theoretical total content of the liver fragment. Fifteen per cent of the total liver a l - 3 FT activity was recovered in the total isolated hepatocyte extract (Table III), but 97% and 94% of a l - 2 and a l - 3 / 4 total liver FT, respectively, was eliminated. The elimination of contaminating a l - 2 and c d - 3 / 4 FT was thus efficient (relative decreases of 5-fold and 3-fold, respectively).

Golgi enrichment in a 1-3 fucosyltransferase Subcellular fractionation did not give a further sig-

nificant decrease in a l - 2 or a l - 3 / 4 FT (Table II), but the Golgi fraction contained 2 units/rag specific a l - 3 FT activity under standard conditions, after ap- proximately 26-fold purification (Table III).

This last cell fractionation step was performed to eliminate the lysosomal fraction and was controlled by the measurement of contaminating fucosidase and sial- idase activities using the optimized a l - 3 FT assay conditions (Fig. 1). Approx. 32% of total hepatocellular a l - 3 FT was recovered in the microsomal extract, with only a 64% decrease in sialidase activity. The Golgi extract still contained 44% of total hepatocellular a l - 3 FT, negligeable residual sialidase activity (3%) but significant fucosidase activity (7%). We thus used the Golgi fraction, adding a sufficient amount of free fu-

T A B L E III

Partial purification of al-3 fucosyltransferase from normal human licer tissue

Purification Spec. Total Total Yield Total step act. protein act. (%) purification

( m U / m g ) a (mg) (mU) (fold)

total liver tissue (8g) 78 806 62868 100 1

1. isolated hepatocytes 155 61.25 9 494 15.1 1.9

2. golgi fraction 2015 2.37 4775 7.6 25.8

a fmol of [14C]fucose transferred to a s i a l o t r ans f e r r i n / min /mg pro- tein in s tandard conditions described in 'Materials and Methods ' .

255

>-

1 0 0 -

8 0

60

4(/

20

0

A

L tt~

H M G

B

%<

H M G

C

H M G

Fig. 1. Efficiency of subcellular enrichment of normal human hepato- cellular al-3 fucosyl transferase (A) and concomitant decrease in fucosidase (B) and sialidase (C). The subcellular fractions tested were: (H) total hepatocyte extract, (M) microsomal fraction, and (G) Golgi fraction. * Specific activities in units/mg protein, defined as pmol of substrate or product transferred or liberated per minute, in optimal conditions (see 'Materials and Methods'): (A) [lac]fucose to N-acetyl lactosamine, (B) fucose from pNP fucose and (C) sialic acid

from 4 MU NeuAc.

cose (10 raM) to the incubation mixtures to inhibit at least 90% of the residual fucosidase activity, as sug- gested by inhibition studies of Alhadeff et al. [24].

Aliquots of this latter Golgi-enriched a l - 3 FT frac- tion were first used to determine the apparent Krn value for GDP-fucose (about 6.5/zM). Optimisation of the incubation buffer composition was carried out us- ing 27/zM GDP-fucose (representing nearly 4 K m for this donor substrate). Optimal activity of the partially purified a l - 3 FT was obtained in Mops buffer, pH 7.3 to 7.8, in the presence of at least 5 mM Mn 2+. When adding 0.5 mM CDP-choline, we found a 90% inhibi- tion of residual pyrophosphatase activity [25].

Substrate specificity and kinetics of Golgi-enriched a l - 3 fucosyltransferase

The wide variety in the size and polarity of the oligosaccharide substrates used (Table I) required dis- tinct procedures for the separation of radiolabeled products from the unreacted nucleotide sugars and acceptors. Tri- and tetra-oligosaccharides were effi- ciently separated after the third migration step of the first two-solvent procedure. After staining with orcinol, small-oligosaccharide controls displayed relative Rf / fuc values from 0.9 for lacto N biose 1 to 0.43 for 6' sialyl N-acetyl lactosamine. The isocratic procedure used to analyse large-oligosaccharide products gave good separation of neutral and bisialylated biantennary N-glycans from GDP-fucose, with relative Rf/GDP- fucose values of 0.42 and 0.37, respectively.

When added at 10 mM in optimal conditions, NeuAc a2-3 Gal /31-4 GlcNAc (type 2 oligosaccharide) was

256

TABLE IV

Oligosaccharidic substrate requirements of Golgi-enriched human hepa- tocellular ~1-3 fucosyltransferase

Oligosaccharidic acceptors Relative act. (10 mM) (%) a

Ga1131 ~ 3 GlcNAc 14 Gal/31 ~ 4 G|cNAc 98 NeuAc a2 ~ 3 Gal/31 --0 4 GlcNAc 100 NeuAc o<2 ~ 6 Ga1131 ---, 4 GIcNAc 0 Gal/31 ~ 4 Glc 2 Fuc al ~ 2 Ga1131 ~ 4 GIc 8

al-3 fucosyltransferase activities were measured in optimized con- ditions as described in 'Materials and Methods', and expressed as the percentage [I4C]fucose incorporation into the 3'sialyl-N-acetyl lactosamine acceptor.

found to be the best acceptor for the Golgi-enriched normal human liver a l - 3 FT and was thus used as the reference for comparison with other oligosaccharidic acceptors (Table IV). It was confirmed that Gal /31-4 GIcNAc, another type 2 oligosaccharide, was a good acceptor (98% relative activity), whereas the human hepatic a l - 3 FT showed no detectable transfer activity of fucosyl residues to NeuAc a 2 - 6 G a l / 3 1 - 4 GIcNAc. Very low relative activities (2% and 14%) were ob- tained with Gal /31-4 Glc (type 6 oligosaccharide) and

J A " 6 1 6

~ 1 2

0 2 4 6 8 I 0 12 14 16 18 2 0 2 2 2 4

V(pmol[14C]Fuclmin) I S(mM)

12 1

~4

0 . . . . . . 0 2 4 6 8 10 12 14 16 18 2 0 2 2 2 4 2 6

V(pmol[14C]Fuc/min) ! S(rnM)

Fig. 2. Enzyme kinetics of Golgi-enriched normal human hepatocel- lular od-3 fucosyltransferase for small oligosaccharides (A) and biantennary N-glycans (B). The substrates used were N-acetyl lac- tosamine ( ), 3'sialyl lactosamine ( . . . . . ), neutral biantennary N-glycans (o e) and 3'bisialyl biantennary N-glycans

(o o).

Gal /31-3 GIcNAc (type 1 oligosaccharide). These re- sults confirmed the ability of the partially purified a l - 3 FT to preferentially transfer fucose to the 3 - O H position of the GIcNAc unit in a type 2 oligosaccha- ride.

The residual Golgi-enriched extract was then used for the determination of apparent K m values from the initial velocity data for four type 2 oligosaccharide acceptors (Fig. 2). Incorporation of fucose was meas- ured in optimized conditions, with increasing concen- trations of N-acetyl lactosamine and 3'sialyl N-acetyl lactosamine (0.5 mM to 17 raM) and of neutral and bisialyl biantennary N-glycans (0.05 mM to 2.0 raM). The human hepatic a l - 3 FT had a greater affinity for a 2 - 3 bisialylated than unsialylated biantennary gly- cans, with apparent K m values of 0.38 and 0.74 raM, respectively. In the same way, the affinity for 3'sialyl Noacetyl lactosamine (Km = 0.65 raM) was greater than that for N-acetyl lactosamine (K m = 2.60 raM).

Discussion

We report here the acceptor specificity of partially purified c~1-3 fucosyltransferase from isolated normal human hepatocytes. Up to now, fucosyltransferases have essentially been studied in total liver extracts. The normal human liver can express at least four types of fucosyltransferases: two a l - 2 FT encoded by the H and Se genes use phenyl-/3-D-galactose as specific ac- ceptor [26], while a l - 2 / 3 FT and a l - 6 FT can trans- fer fucose to asialofetuin [27] and asialogalactofetuin [27, 28], respectively. In a recent paper [10] we re- ported the existence of a fifth type of fucosyltrans- ferase, a l - 3 / 4 FT, which easily transfers fucose to the 3 - O H position of the Glc residue of 2'fucosyl lactose.

Some mesenchymal cells which are associated with parenchymal ceils in the liver can express several types of fucosyltransferase, notably a l - 3 (for a review, see Ref. 8). We thus isolated hepatocytes to study the only hepatocellular a l - 3 FT implicated in the fucosylation of plasma glycoproteins.

For a hepatocyte isolation yield of 20%, we recov- ered 15%, 3% and 5% of the total liver a l - 3 , a l - 2 and a l - 3 / 4 FT activities, respectively, in the hepato- cyte extract, implying that at least 25%, 85% and 75% of these a l - 3 , a l - 2 and a l - 3 / 4 liver FT derive from non-parenchymal cells.

Fucosidases and sialidases are usually known as acidic hydrolases. However, human liver fucosidase can act on 2'fucosyl lactose in pH 7.5 buffer [24]. We supposed that in incubation conditions chosen for the a l - 3 FT assay, this fucosidase could also hydrolyse GDP-fucose and fucosylated products and that siali- dase could hydrolyse sialylated substrates, albeit to a lesser extent. Subcellular fractionation was thus used to eliminate the lysosomal fraction, but the discontinu-

257

ous sucrose gradient method did not allow us to totally eliminate hepatocellular fucosidase and sialidase activi- ties from the microsomal fraction. We in fact used the Golgi-enriched fraction, which contained a a l - 3 FT that was sufficiently purified (26-fold) to study its oligo- saccharide requirements and kinetics.

The pattern of reactivity with three different types of oligosacharide acceptors showed that the Golgi-en- riched a l - 3 FT purified from normal human hepato- cytes efficiently utilizes neutral and 3'sialylated type 2 acceptors. Lacto N biose I (type 1) is a poor substrate for the enzyme and type 6 the poorest.

The ability of hepatocellular a l - 3 FT to transfer fucose to 3 OH of the GIcNAc residue of N-acetyl lactosamine is enhanced by the 3 OH substitution of the Gal residue by NeuAc and totally abolished by its 6 OH substitution. Structural analysis of type 2 sialosides based on hard-sphere exoanomeric effect calculations demonstrated that NeuAc a 2 - 6 sialosides can exist in several minimum energy conformers in which the NeuAc residue appears to be placed in close proximity to the N-acetyl lactosamine unit, reducing the inter- atomic distance between the 3 OH of the GlcNac residue and the 3 H of the NeuAc residue to 2.6 ,&. In contrast, NeuAc a 2 - 3 sialosides can exist in a more rigid and stable conformer in which the NeuAc residue appears to be far from the GlcNac residue [29]. Thus it is apparent that the binding specificity of a l - 3 FT to its substrate is primarily governed by Gal accessibility, which can be favoured by the stabilizing effect of the NeuAc a 2 - 3 residue.

Hepatocellular a l - 3 FT very weakly transfered fu- cose to the 4 OH position of the GIcNAc residue of lacto N biose I. Another conformational analysis of derivatives of N-acetyl lactosamine and lacto N biose I showed that the 3 OH of the type 2 structure is more sheltered by the N-acetamido group than is the 4 OH of the typel isomer [30]. This suggests that the binding of a l - 3 FT to its substrate is also highly restricted by the N-acetamino group of the GIcNAc unit, which could be involved in hydrophobic interactions.

To our knowledge, only Mollicone et al. [31] have studied the acceptor specificity of isolated human hep- atocyte a l - 3 FT. In their work, enzyme assay was performed with small oligosaccharide acceptors synthe- sized as 8-methoxy-carbonyloctyl glycosides. They ob- served efficient fucose transfer to neutral and 3'sialy- lated type 2 acceptors, very poor transfer to type 1 acceptors and no transfer to type 6 acceptors. They classified this liver a l - 3 FT reactivity as a 'plasma type' acceptor pattern. Our data confirm this pattern but cannot be directly compared to those of Mollicone et al., because we used oligosaccharides with no hy- drophobic core, implying affinity modifications.

We can compare our data with those of Sarnesto et al. [32] who described the acceptor pattern of a human

serum a l - 3 FT using both glycolipid and tree oligo- saccharides. We found that hepatocellular a l - 3 FT shared with serum a l - 3 FT the ability to efficiently transfer fucose to type 2 acceptors and, to a minor degree, to type 6 acceptors. These two enzymes express a similar high affinity for 3'sialyl N-acetyllactosamine and no afinity for 6' sialyl N-acetyllactosamine. Hep- atocellular a l - 3 FT mainly differs from the human serum a l - 3 FT described by Sarnesto et al. in its moderate ability to transfer fucose to lacto N-biose I (a feature of Lewis type a l - 3 / 4 FT).

Further purification steps and competition experi- ments are required to determine if our partially puri- fied hepatocellular a l - 3 FT is a single molecule with specific oligosaccharide affinity, or a mixture of two a l - 3 FT, one similar to the 'plasma-type' enzyme and the other similar to the 'Lewis-type' enzyme.

Most in vitro studies have been performed with small oligosaccharide acceptors but, physiologically, hepatocellular a l - 3 FT fucosylates larger oligosaccha- rides such as complex type N-glycans of a l - A G P and ceruleoplasmin. We thus assessed the reactivity of the enzyme with neutral and sialylated biantennary N gly- cans in order to get closer to endogenous acceptor structures. A comparison of the K m values of human hepatic a l - 3 FT for linear and bisected lactosaminyl structures indicated that the enzyme has the highest affinity for 3'bisialylated biantennary N-glycans.

The Golgi-enriched a l - 3 FT from a cirrhotic liver biopsy specimen gave the same acceptor specificity pattern but with a 10-fold higher initial velocity (data not shown). This is in accordance with the simultane- ous presence of the 3'sialyl Le x determinant on the two antennae (7 and 7 ') of a l - A G P observed in ascitic fluid from a cirrhotic patient [7].

These structural data suggest that human hepatic a l - 3 FT may act preferentially on particular isomeric complex-type N glycan structures. Another hypothesis is that during the hepatocellular glycosylation process, a l - 3 fucosylation of GlcNAc residues occur last and cannot take place on antennae with an a 2 - 6 linked NeuAc.

Acknowledgements

The authors thank Prs Dominique Franco and Guy Lemaigre for their assistance in liver tissue collection and Christophe Lamaze for his kind technical help.

References

1 Howie, A.J. and Brown, G. (1985) J. Clin. Pathol. 38, 409-416. 2 Fukuda, M., Spooncer, E., Oates, J.E., Dell, A. and Klock, J.C.

(1984) J. Biol. Chem. 259, 10925-10935. 3 Yamashita, K., Hitoi, A., Matsuda, Y., Miura, T., Katunuma, N.

and Kobata, A. (1986) J. Biochem. 99, 55-62.

258

4 Endo, M., Suzuki, K., Schmid, K., Fournet, B., Karamanos, Y., Montreuil, J., Dorland, L., Van Halbeek, H. and Vliegenthart, J.F.G. (1982) J. Biol. Chem. 257, 8755-8760.

5 Fournet, B., Montreuil, J., Strecker, G., Dorland, L., Haverkamp, J., Vliegenthart, J.F.G., Binette, J.P. and Schmid, K. (1978) Biochemistry 17, 5206-5214.

6 Cardon, P., Paz Parente, J., Leroy, Y., Montreuil, J. and Fournet, B. (1986) J. Chromatogr. 356, 135-146.

7 Wieruszeski, J.M., Fournet, B., Konan, D., Biou, D. and Durand, G. (1988) FEBS Lett. 238, 390-394.

8 Macher, B.A., Holmes, E.H., Swiedler, S.J., Stults, C.L.M. and Srnka, C.A. (1991) Glycobiology 1,577-584.

9 Hirschberg, C.B. and Snider, M.D. (1987) Ann. Rev. Biochem. 56, 63-87.

10 Jezequel-Cuer, M., Dalix, A.M., Flejou, J.F. and Durand, G. (1992) Liver 12, 140-146.

11 Beyer, T.A., Rearick, J.I., Paulson, J.C., Prieels, J.P., Sadler, J.E. and Hill, R.L. (1979) J. Biol.Chem. 254, 12531-12541.

12 Strecker, G., Peers, M.C., Michalski, J.C., Hondi-Assah, T., Four- net, B., Spik, G., Montreuil, J., Farriaux, J.P., Maroteaux, P. and Durand, P. (1977) Eur. J. Biochem. 75, 391-403.

13 Ballet, F., Bouma, M.E., Wang, S.R., Amit, N., Marais, J. and Infante, R. (1984) Hepatology 4, 849-854.

14 Dignam, J.D., Martin, P.L., Shastry, B.S. and Roeder, R.G. (1983) in Methods in Enzymology, Vol. 101 (Wu, R., Grossman, L . a~d Moldave, K., Eds.), Academic Press, New York, pp. 58~7598.

I~o~dbe rg , P.O., Marzella, L. and Glaumann, H. (1980) Exp. Cell Res. 130, 393-400.

16 Dorland, L., Haverkamp, J., Schut, B.L., Vliegenthart, J.F.G., Spik, G., Strecker, G., Fournet, B. and Montreuil, J. (1977) FEBS Lett. 77, 15-20.

17 Takasaki, S., Mizuochi, T. and Kobata, A. (1982) in Methods in Enzymology, Vol. 83 (Ginsburg, V., Ed.) Academic Press, New York, pp. 263-269.

18 Biou, D., Bauvy, C., N'Guyen, H., Codogno, P., Durand, G. and Aubery, M. (1991) Clin. Chim. Acta 204, 1-12.

19 Zanetta, J.P., Breckenridge, W.C. and Vincendon, G. (1972) J. Chromatogr. 69, 291-304.

20 Kamerling, J.P., Gerwig, G.J., Vliegenthart, J.F.G. and Clamp, J.R. (1975) Biochem. J. 151,491-495.

21 Schindler, D., Kanzaki, T. and Desnick, R.J. (1990) Clin. Chim. Acta 190, 81-92.

22 White, W.J., Schray, K.J. and Alhadeff, J.A. (1985) Biochim. Biophys. Acta 829, 303-310.

23 Potier, M., Mameli, L., Belisle, M., Dallaire, L. and Melan§on, S.B. (1979) Anal. Biochem. 94, 287-296.

24 Alhadeff, J.A., Miller, A.L., Wenaas, H., Vedvick, T. and O'Brien, J.S. (1975)J. Biol. Chem. 250, 7106-7113.

25 Byrd, J.C., Fearney, F.J. and Kim, Y.S. (1985) J. Biol. Chem. 260, 7474-7480.

26 Iizuka, S. and Yoshida, A. (1987) Enzyme 37, 159-163. 27 Hutchinson, W.L., Du, M.Q., Johnson, P.J. and William, R.

(1991) Hepatology 13, 683-688. 28 Kessel, D., Sykes, E. and Henderson, M. (1977) J. Natl. Cancer

Inst. 59, 29-32. 29 Sabesan, S., Bock, K. and Paulson, J.C. (1991) Carbohydr. Res.

218, 27-54. 30 Khare, D.P., Hindsgaul, O. and Lemieux, R.U. (1985) Carbohydr.

Res. 136, 285-308. 31 Mollicone, R., Gibaud, A., Fran~2ois, A., Ratcliffe, M. and Oriol,

R. (1990) Eur. J. Biochem. 191, 169-176. 32 Sarnesto, A,, K6hlin, T., Hindsgaul, O., Vogele, K., Blaszczyk, M.

and Thurin, J. (1992) J. Biol. Chem. 267, 2745-2752.