Complementary Acceptor and Site Specificities of Fuc-TIV and Fuc-TVII Allow Effective Biosynthesis of Sialyl-TriLex and Related Polylactosamines Present on Glycoprotein Counterreceptors of Selectins*

Ritva NiemeläDagger §, Jari NatunenDagger §, Marja-Leena Majuri, Hannu MaaheimoDagger , Jari HelinDagger , John B. Lowepar **, Ossi RenkonenDagger , and Risto RenkonenDagger Dagger

From the Dagger  Institute of Biotechnology, P.O. Box 56 and  Department of Bacteriology and Immunology, Haartman Institute, P.O. Box 21, University of Helsinki, FIN-00014 Helsinki, Finland and par   the Howard Hughes Medical Institute and Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0650

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The P-selectin counterreceptor PSGL-1 is covalently modified by mono alpha 2,3-sialylated, multiply alpha 1,3-fucosylated polylactosamines. These glycans are required for the adhesive interactions that allow this adhesion receptor-counterreceptor pair to facilitate leukocyte extravasation. To begin to understand the biosynthesis of these glycans, we have characterized the acceptor and site specificities of the two granulocyte alpha 1,3-fucosyltransferases, Fuc-TIV and Fuc-TVII, using recombinant forms of these two enzymes and a panel of synthetic polylactosamine-based acceptors. We find that Fuc-TIV can transfer fucose effectively to all N-acetyllactosamine (LN) units in neutral polylactosamines, and to the "inner" LN units of alpha 2,3-sialylated acceptors but is ineffective in transfer to the distal alpha 2,3-sialylated LN unit in alpha 2,3-sialylated acceptors. Fuc-TVII, by contrast, effectively fucosylates only the distal alpha 2,3-sialylated LN unit in alpha 2,3-sialylated acceptors and thus exhibits an acceptor site-specificity that is complementary to Fuc-TIV. Furthermore, the consecutive action of Fuc-TIV and Fuc-TVII, in vitro, can convert the long chain sialoglycan SAalpha 2-3'LNbeta 1-3'LNbeta 1-3'LN (where SA is sialic acid) into the trifucosylated molecule SAalpha 2-3'Lexbeta 1-3'Lexbeta 1-3'Lex (where Lex is the trisaccharide Galbeta 1-4(Fucalpha 1-3)GlcNAc) known to decorate PSGL-1. The complementary in vitro acceptor site-specificities of Fuc-TIV and Fuc-TVII imply that these enzymes cooperate in vivo in the biosynthesis of monosialylated, multifucosylated polylactosamine components of selectin counterreceptors on human leukocytes.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Extravasation of leukocytes is initiated by interactions between the selectin family of cell adhesion molecules and their glycoprotein counterreceptors, leading in turn to vascular shear flow-dependent rolling of leukocytes on endothelial cell surfaces (1-4). E- and P-selectin, expressed by activated endothelium (2, 5), recognize their leukocyte glycoprotein counterreceptors only when the counterreceptors are properly modified by glycosylation. Biosynthesis of counterreceptor glycans thought to be essential for effective recognition by E- and P-selectins includes alpha 1,3-fucosylation and terminal alpha 2,3-sialylation. Of the five human alpha 1,3-fucosyltransferases (Fuc-T) that have been cloned (6-10), only two (Fuc-TIV and Fuc-TVII) are expressed to a significant degree in granulocytes (11-13). Hence, these two enzymes are candidates for participation in the biosynthesis of the fucosylated glycans that decorate selectin counterreceptors on leukocytes.

P-selectin glycoprotein ligand-1 (PSGL-1),1 a mucin-type glycoprotein expressed by human neutrophils and HL-60 cells, can function as a ligand for each of the three selectins (2, 14, 15). The major O-linked alpha 1,3-fucosylated core 2 sialyl glycan expressed by HL-60 cell-derived PSGL-1 contains the decasaccharide sequence SAalpha 2-3'Lexbeta 1-3'Lexbeta 1-3'Lex (where Lex is Galbeta 1-4(Fucalpha 1-3)GlcNAc) (16). This epitope likely contributes to interactions between PSGL-1 and E- or P-selectins, because PSGL-1 recognition by these two selectins requires sialylation and alpha 1,3-fucosylation of its O-linked core 2 glycans. Similar alpha 2,3-sialylated, multifucosylated polylactosamines found in granulocytes have also been identified as E-selectin ligands (17, 18).

To begin to address the relative roles of Fuc-TIV and Fuc-TVII in the biosynthesis of these multifucosylated molecules, we have used recombinant forms of these enzymes (7, 10) and in vitro fucosyltransferase assays to define acceptor site specificities for candidate precursors to sialylated, multifucosylated selectin ligands. Acceptor site specificities derived from a panel of synthetic polylactosamine precursors imply that Fuc-TIV and Fuc-TVII exhibit distinct acceptor specificities, as observed previously (7, 10, 11), and, more importantly, exhibit distinct site-directed preferences for fucosylation among potential lactosamine units in sialyl polylactosamine precursors. Specifically, we find that Fuc-TIV preferentially fucosylates "inner" LN units on alpha 2,3-sialyl polylactosamine chains, whereas Fuc-TVII preferentially fucosylates distal LN units on such alpha 2,3-sialylated polylactosamine precursors. These observations demonstrate alternative, and complementary substrate site-specificities for Fuc-TIV and Fuc-TVII, and imply that this pair of enzymes catalyzes the synthesis of polyfucosylated selectin ligands in leukocytes through complementary catalytic activities.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Transfected Cells and Cell Lysates-- The transfection of Chinese hamster ovary (CHO) cells stably expressing human Fuc-TIV or Fuc-TVII has been described previously (7, 10). For the enzyme assays, the cells were lysed in 1% Triton X-100 on ice in the presence of a mixture of protease inhibitors (16 µg/ml benzamidine HCl, 10 µg/ml phenanthroline, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride, Pharmingen, San Diego, CA).

Oligosaccharide Acceptors-- The structures of the oligosaccharide acceptors are shown in Fig. 1. Glycan 1 was from Sigma and glycan 2 from Oxford Glycosystems (Abingdon, UK). The others, we synthesized enzymatically. Briefly, glycan 11 was constructed from GlcNAcbeta 1-3Galbeta 1-OMe (Sigma) by beta 1,4-galactosylation (19) followed by beta 1,3-N-acetylglucosaminylation (20) and a second round of beta 1,4-galactosylation. The intermediates as well as the final product were isolated in pure form; 1H-NMR-spectrum of 11 at 500 MHz confirmed its structure; matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) revealed that the sample was pure and had the expected molecular weight (M+Na)+ m/z 948.0 (calc. 947.9). Glycan 3 was obtained from glycan 11 by alpha 2,3-sialylation as in (21). Glycan 12 was constructed from the GNbeta 1-3'LNbeta 1-3'LN (22) by beta 1,4-galactosylation, MALDI-TOF MS: (M+Na)+ m/z 1137.1 (calc. 1137.0). Glycan 4 was obtained from glycan 12 by alpha 2,3-sialylation. Glycan 15 was synthesized as in (23), MALDI-TOF MS: (M+Na)+ m/z 1136.9 (calc. 1137.0). Glycan 16 was obtained by alpha 2,3-sialylation of 15. Synthesis of glycan 8 will be described in detail elsewhere (24). Briefly, GNbeta 1-3'LNbeta 1-3'LN was alpha 1,3-14C fucosylated at both LN units with alpha 1,3/4-FucTs of human milk and then beta 1,4-galactosylated. MALDI-TOF MS of the resulting octasaccharide LNbeta 1-3'Lexbeta 1-3'Lex gave (M+Na)+ m/z 1429.3 (calc. 1429.3). The octasaccharide was finally alpha 2,3-sialylated to give glycan 8. The NMR spectrum of glycan 8 is shown in Fig. 4; a comparison with the spectrum of the unsialylated precursor is presented in Table I. Degradation of glycan 8 by sialidase and a mixture of beta -galactosidase and beta -N-acetylhexosaminidase gave the neutral hexasaccharide [14C]Fucalpha 1-3LNbeta 1-3'[14C]Fucalpha 1-3LN, which chromatographed like an authentic marker on paper. Synthesis of glycans 5 and 6, too, will be described elsewhere (24). Briefly, glycan 10 was converted to the pentasaccharide LNbeta 1-3'(GNbeta 1-6')LN as in (25, 26), MALDI-TOF MS: (M+Na)+ m/z 974.8 (calc. 974.9). The product was alpha 2,3-sialylated, was alpha 1,3-fucosylated selectively at the distal, sialylated LN-unit (25), and was treated with beta -N-acetylhexosaminidase for removal of the protecting beta 1-6GN-unit to give 5. Glycan 6, in turn, was obtained by alpha 1,3-fucosylating glycan 9 at the LN unit by human milk alpha 1,3/4-Fuc-Ts. The product was then beta 1,4-galactosylated and finally alpha 2,3-sialylated. Glycan 7 was synthesized by alpha 2,3-sialylation of the heptasaccharide LNbeta 1-3'Lexbeta 1-3'LN, prepared by beta 1,4-galactosylation of GNbeta 1-3'Lexbeta 1-3'LN, which had been isolated from a mixture of monofucosylated isomers by wheat germ agglutinin-agarose chromatography. LNbeta 1-3'Lexbeta 1-3'LN was characterized by 1H-NMR spectroscopy; MALDI-TOF MS: (M+Na)+ m/z 1282.5 (calc. 1282.5).2 Glycan 9 was obtained by beta 1,3-N-acetylglucosaminylation of LN (20), MALDI-TOF MS: (M+Na)+ m/z 609.7 (calc. 609.5). Glycan 10 was synthesized by beta 1,4-galactosylation of 9. Glycans 13 and 14 were obtained by separating monofucosylated derivatives of 10 by wheat germ agglutinin chromatography.2 Glycan 13, MALDI-TOF MS: (M+Na)+ m/z 917.9 (calc. 917.8). Glycan 14, MALDI-TOF MS: (M+Na)+ m/z 917.8 (calc. 917.8).

Fucosyltransferase Reactions-- GDP-[14C]fucose (100 000 cpm, Amersham, UK), GDP-fucose (1 nmol, Sigma) and the individual polylactosamine acceptors (50 nmol) were incubated for 1 h at 37 °C in 10 µl of 50 mM MOPS, pH 7.2, containing 10 mM MnCl2, 10 mM fucose, 5 mM ATP, 0.4% TX-100 and lysates of CHO cells transfected with Fuc-TIV or Fuc-TVII (35-50 µg of protein, assayed by the BCA kit of Pierce). 100 nmol of LN and 100 nmol SAalpha 2-3LN were used as reference acceptors. The reactions were terminated by adding 10 µl of ethanol followed by 100 µl of ice cold water, and the reaction mixtures containing acidic glycans were purified by gel filtration on a Superdex column, subsequently fractionated by anion exchange chromatography on a Mono Q column and finally desalted on a Superdex column. The reaction mixtures obtained from neutral acceptors were desalted in a mixed bed ion exchange resin, after which the mixtures of acceptor and product were isolated by gel filtration. In all cases, the reaction products were quantitated by subjecting aliquots of the purified mixtures of unlabeled surplus acceptor and labeled product to liquid scintillation counting. All reactions were run and analyzed twice.

Methods Used in the Analysis of Fuc-TIV and Fuc-TVII Reactions-- Degradations with a mixture of jack bean beta -galactosidase (EC 3.2.1.23, Sigma), jack bean beta -N-acetylhexosaminidase (EC 3.2.1.30, Sigma) (27), Arthrobacter ureafaciens sialidase (EC 3.2.1.18, Boehringer Mannheim) (28), and Bacteroides fragilis endo-beta -galactosidase (EC 3.2.1.103, Boehringer Mannheim) (22) were carried out as described previously.

Gel filtration was performed in a column of Superdex peptide HR 10/30 (Pharmacia, Sweden), with 50 mM NH4HCO3 as the eluant at a flow rate of 1 ml/min. The eluant was monitored at 205 or 214 nm, and oligosaccharides were quantified against external GN and SA. Neutral oligosaccharides were desalted by filtration in water through AG-50W (H+) and AG-1 (AcO-) (Bio-Rad).

Paper chromatography of radiolabeled oligosaccharides was carried out as described in Ref. 29, using the upper phase of n-butanol:acetic acid:water (4:1:5) (v/v) (solvent A) for the chromatographic runs and Optiscint (Wallac, Finland) for the liquid scintillation counting. Anion exchange chromatography on a Mono Q (5/5) column (Pharmacia) was performed essentially as in (21).

Oligosaccharide Markers-- The hexasaccharide Lexbeta 1-3'Lex and the nonasaccharide Lexbeta 1-3'Lexbeta 1-3'Lex, MALDI-TOF MS: (M+Na)+ m/z 1575.8 (calc. 1575.5) as well as the mixture of the octasaccharide Lexbeta 1-3'Lexbeta 1-3'LN and its isomers, MALDI-TOF MS: (M+Na)+ m/z 1429.5 (calc. 1429.3) were synthesized from fucose-free precursors as will be described.2

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Fuc-TIV and Fuc-TVII Exhibit Shared and Distinct Acceptor Substrate Specificities-- A panel of neutral and sialylated polylactosamine acceptors was synthesized, and individual acceptor substrates were utilized in in vitro fucosyltransferase assays containing radiolabeled GDP-fucose and CHO transfectant-derived recombinant human Fuc-TIV or Fuc-TVII (7, 10). N-Acetyllactosamine (LN; glycan 1) and sialyl N-acetyllactosamine (SAalpha 2-3'LN; glycan 2) served as reference acceptors for Fuc-TIV- and Fuc-TVII-dependent reactions, respectively. Fuc-TIV rapidly fucosylates LN (glycan 1) but is ineffective in its ability to fucosylate the sialylated acceptors 2, 6, and 8, each of which can be fucosylated only at the distal, sialylated LN unit (Fig. 1). By contrast, Fuc-TVII effectively fucosylates all sialopolylactosamines tested except glycan 5, which can be fucosylated only on an internal LN unit (Fig. 1). The neutral glycans 9-15 are efficiently fucosylated by Fuc-TIV but are poor acceptors when tested with Fuc-TVII. Thus, human Fuc-TIV and Fuc-TVII show gross differences in their acceptor specificities as implied from previous studies using much smaller acceptors (7, 10, 11). Fuc-TIV also efficiently fucosylates the sialylated linear acceptors 3 and 4, each of which contain "inner" N-acetyllactosamine residues. Furthermore, Fuc-TIV effectively utilizes sialylated, fucosylated linear acceptors 5 and 7, each of which also contain unoccupied "inner" N-acetyllactosamine residues.


View larger version (46K):
[in this window]
[in a new window]
 
Fig. 1.   Relative initial transfer rates at individual acceptor sites of sialylated and neutral polylactosamines catalyzed by lysates of CHO cells transfected with human Fuc-TIV and Fuc-TVII. *, transfer rate to LN was typically 3.9 pmol/µg protein/h; Dagger , transfer rate to SAalpha 2-3'LN was typically 3.2 pmol/µg protein/h; §, only 0.15 mM acceptor was used, and the reference acceptor was also 0.15 mM; par-bars , only 1 mM acceptor was used, and the reference acceptor was also 1 mM. Glycan 8 was analyzed only once; ¶, the acceptor site specificity was not determined; n.d., not determined.

Fuc-TIV and Fuc-TVII Show Alternative Site Specificities on Sialylated Multisite Acceptors-- To determine which of the different GlcNAc residues were fucosylated in the sialylated multisite acceptors 3 and 4, the products were degraded by sialidase and then by mixed beta -galactosidase and beta -N-acetylhexosaminidase. The latter digestion removes any fucose-free LN units from the nonreducing end of desialylated polylactosamines but is unable to act on distal, alpha 1,3-fucosylated LN residues (30). Hence, the desialylated chains were shortened in a way that established the position of the alpha 1,3-fucosylated LN residue. Products of these digestions were analyzed as described under "Experimental Procedures" and were used to derive the site specificity data displayed in Fig. 1. Fuc-TIV transfers rapidly to sialoglycan 4, at both inner LN units (residues 1 and 2 in Fig. 1) but transfers to the sialylated LN unit (residue 3) at a rate 30-40 times slower (Fig. 1 and Fig. 2A). In contrast, Fuc-TVII transfers preferentially to the sialylated, distal LN residue of acceptor 4 (Fig. 2B); the rate of transfer to the middle LN unit (residue 2) and to the reducing end LN unit (residue 1) were, respectively, 17 and 84 times slower than transfer to the sialylated LN residue (Fig. 1). The structural data inferred from the chromatograms in Fig. 2 were confirmed by degrading fucosylated products derived from glycan 4 by sialidase treatment, followed by digestion with endo-beta -galactosidase, which cleaves internal beta -galactosidic linkages of linear polylactosamines, but is unable to hydrolyze the same bonds of alpha 1,3-fucosylated LN units (31, 32) (data not shown).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   Paper chromatography of oligosaccharides generated by a treatment with sialidase and a mixture of jack bean beta -galactosidase and beta -N-acetylhexosaminidase from Fuc-TIV and Fuc-TVII products of sialoglycan 4. Panel A, the digest of Fuc-TIV products; panel B, the digest of Fuc-TVII products. For both runs: Solvent A, 97 h. Markers: MH, maltoheptaose; MP, maltopentaose; MTe, maltotetraose; MT, maltotriose; Lac, lactose.

The fucosylated products of sialoglycan 3 were analyzed in a similar way to ascertain site specificity of fucosylation. These analyses indicate that Fuc-TIV overwhelmingly fucosylates at the inner LN unit, whereas Fuc-TVII fucosylates preferentially at the distal, sialylated LN unit (Fig. 1; data not shown). Taken together, these results imply that "internal" fucosylation events occurring within sialoglycans are catalyzed by Fuc-TIV, whereas the terminal fucosylation event that creates sialyl Lewis x (sLex) type products is catalyzed by Fuc-TVII.

Fuc-TIV and Fuc-TVII Show Alternative Preferences Among Prefucosylated Acceptors of the VIM-2 and sLex type-- In vitro assays using the prefucosylated glycans 5 and 6 indicate that Fuc-TIV transfers fucose to the inner LN unit and that Fuc-TVII transfers to the sialylated, distal LN unit (Fig. 1). Hence, the two enzymes complement each other efficiently in the synthesis of the sialylated, bifucosylated epitope from the fucose-free precursor via intermediates of VIM-2 and sLex type glycans.

The unlabeled glycan SAalpha 2-3'LNbeta 1-3'Lexbeta 1-3'LN (7) was synthesized in nanomolar amounts as specified under "Experimental Procedures" and was then used as an acceptor in a Fuc-TIV-dependent reaction. Structural characterization of the products (Fig. 3) revealed that 86% of the [14C]fucose was transferred to the "innermost" LN unit (residue 1), generating SAalpha 2-3'LNbeta 1-3'Lexbeta 1-3'[14C]Lex (glycan 8). Less than 5% of the fucose transfer occurred at the sialylated LN unit (residue 3). These observations, together with those displayed in Fig. 2A, demonstrate that Fuc-TIV can convert glycan 4 into the bifucosylated glycan 8.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3.   Paper chromatography of oligosaccharides generated by a treatment with sialidase and a mixture of jack bean beta -galactosidase and beta -N-acetylhexosaminidase from Fuc-TIV products of sialoglycan 7. The major peak at fractions 11-12 co-chromatographed with an authentic sample of the hexasaccharide Lexbeta 1-3'Lex. The small peak at fraction 5 chromatographed like Lexbeta 1-3'Lexbeta 1-3'LN and its isomers. Solvent A, 168 h; markers as in Fig. 2.

Fuc-TVII Catalyzes Rapid Fucosylation of the Monosialyl-Bifucosyl Glycan 8 at the Distal, Sialylated LN Unit-- The nonasaccharide SAalpha 2-3'LNbeta 1-3'Lexbeta 1-3'Lex (8) was synthesized in nanomolar amounts as specified under "Experimental Procedures"; its structure was confirmed by NMR (Fig. 4 and Table I), and the radiolabeled molecule was used as an acceptor in either a Fuc-TVII-dependent reaction or Fuc-TIV-dependent reaction. Fuc-TVII was found to fucosylate glycan 8 nearly as rapidly as glycan 2 (Fig. 1) to yield as a major product SAalpha 2-3'[14C]Lexbeta 1-3'Lexbeta 1-3'Lex (Fig. 5A). Fuc-TIV was found to be competent to construct the identical product but only at a rate that represents 6-7% of the rate catalyzed by Fuc-TVII (Fig. 5B). Considered together, the efficient conversion of glycan 4 to glycan 8 in two steps catalyzed by Fuc-TIV, but not by Fuc-TVII, and the further rapid Fuc-TVII-dependent conversion of glycan 8 to SAalpha 2-3'Lexbeta 1-3'Lexbeta 1-3'Lex, demonstrate that the monosialylated, fucose-free tri-lactosamine chain (4) can be converted into the sialyl-triLex product by the complementary actions of the two alpha 1,3-fucosyltransferases present in human leukocytes.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 4.   1H-NMR spectrum at 500 MHz of glycan 8, SAalpha 2-3'LNbeta 1-3'Lexbeta 1-3'Lex. The resonances marked by an asterisk around 1.3-1.5 ppm originate from unknown contaminants, and the resonances marked by 13C are the 13C satellites of acetone. The numbering of the monosaccharide units is shown in Table I.

                              
View this table:
[in this window]
[in a new window]
 
Table I
1H-NMR chemical shifts (ppm) of structural reporter groups of glycans at 23 °C in reference to internal acetone, 2.225 ppm


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5.   Paper chromatography of oligosaccharides generated by a treatment with sialidase and a mixture of jack bean beta -galactosidase and beta -N-acetylhexosaminidase from Fuc-TVII and Fuc-TIV products of sialoglycan 8. Panel A, the digest of Fuc-TVII products. The major peak at fraction 2 co-chromatographed with an authentic sample of the nonasaccharide [14C]Lexbeta 1-3'[14C]Lexbeta 1-3'[14C]Lex. For comparison, the digest from the original sialoglycan 8 revealed only the peak of the hexasaccharide [14C]Lexbeta 1-3'[14C]Lex at fraction 7. Panel B, the digest of Fuc-TIV products. The small peak at fraction 2 represents [14C]Lexbeta 1-3'[14C]Lexbeta 1-3'[14C]Lex, derived from the trifucosylated sialoglycan product, whereas the peak at fraction 7 is [14C]Lexbeta 1-3'[14C]Lex, which was derived from the radiolabeled acceptor (8) by the exohydrolase treatment. For both runs: Solvent A, 120 h; markers as in Fig. 2.

Site Specificity of Fuc-TIV Reactions with Neutral Linear Acceptors-- Fuc-TIV efficiently fucosylated GNbeta 1-3'LN (9), representing a growing lactosamine chain (Fig. 1), whereas Fuc-TVII did not utilize this acceptor. Fucosylation occurred only at the reducing end N-acetylglucosamine moiety, as the 14C-fucosylated product was cleaved by beta -N-acetylhexosaminidase to [14C]Lex (data not shown). Fuc-TIV also transferred effectively to LNbeta 1-3'LNbeta 1-3Gbeta 1-OMe (11), most rapidly (67%) to the middle LN unit (residue 2 in Fig. 1) and more slowly (33%) to the terminal LN unit (Fig. 6A). For comparison, initial transfer to glycan 10 occurred in 61% of the molecules at residue 1 and in 39% of the molecules at residue 2 (data not shown). When the hexasaccharide LNbeta 1-3'LNbeta 1-3'LN (glycan 12) was used with Fuc-TIV, 28% of the initial fucosylation occurred at the reducing end LN unit, 47% occurred at the middle LN unit, and 25% occurred at the nonreducing end LN unit (Fig. 6B). Fuc-TIV also transferred rapidly to the prefucosylated glycans Lexbeta 1-3'LN (13) and LNbeta 1-3'Lex (14), confirming that fucosylation of vicinal LN units is feasible.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6.   Paper chromatography of oligosaccharides generated by a treatment with a mixture of jack bean beta -galactosidase and beta -N-acetylhexosaminidase from Fuc-TIV products of neutral glycans. Panel A, exoglycosidase digest from products of glycan 11; panel B, exoglycosidase digest from products of glycan 12. For both runs: Solvent A, 99 h; markers as in Fig. 2.

Site Specificity of Fucosylation Reactions with Branched Acceptors-- Fuc-TIV transferred in a slightly preferential manner to the beta 1,3-branch of glycan 15 and Fuc-TVII did likewise with sialyl glycan 16 (Fig. 1; data not shown). Neither enzyme transferred to the branch-bearing LN unit (residue 1 in Fig. 1) in their acceptors (data not shown).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

By using lysates of appropriately transfected CHO cells (7, 10) and a panel of enzymatically synthesized oligosaccharides, we show here that human Fuc-TIV and Fuc-TVII catalyze the transfer of alpha 1,3-bonded fucose units to sialylated linear poly-N-acetyllactosamines in a complementary manner. The two enzymes show alternative acceptor and site specificities such that their concerted action seems to be required for efficient biosynthesis of sialyl-triLex and related sugar epitopes expressed by selectin counterreceptors on leukocytes.

Most of the present experiments were performed at 5 mM acceptor concentrations to give "initial reaction rates at saturating acceptor concentrations." The Km values of the acceptors are not known, but for comparison most reported Km values of purified natural Fuc-Ts are in the range of 0.2-1.0 mM for SAalpha 2-3'LN (33-36). For the cloned human Fuc-TIV, a Km of 3.3 mM has been measured for LN and 27 µM for GDP-Fuc (37). The possibility of using free acceptor saccharides of relatively complex structures is a result of advances made in our program on enzyme-assisted polylactosamine synthesis (20-23, 25-28).

Previously, analogous transferase experiments have been carried out by using small oligosaccharides (7, 10, 11) and glycolipids (38, 39) as acceptors. These early studies have shown that Fuc-TIV prefers neutral LN over SAalpha 2-3'LN, whereas the opposite is true for Fuc-TVII (10-12, 39, 40). Several previous studies on the role of the fucosyltransferases have been performed also by analyzing transfected cells with anti-oligosaccharide antibodies.

Having independently cloned the Fuc-TIV gene, three groups reported initial transfection experiments that yielded conflicting data. One group reported initially anti-sLex reactivity on CHO cells after transfection with Fuc-TIV (41), whereas the other two did not (7, 42). The explanation for this apparent discrepancy probably resides in incompletely characterized differences in the glycosylation status of the CHO sublines (43). By contrast, the Fuc-TIV transfectants have yielded consistently anti-VIM-2 reactivity or chemically identified VIM-2 sequences, implying that an inner LN residue becomes fucosylated (7, 44, 45). In turn, transfection with the Fuc-TVII gene resulted in anti-sLex reactivity as analyzed by antibodies (10, 12, 44), but so far there is no previous knowledge of how Fuc-TVII acts on sialylated and neutral polylactosamine glycans.

The present data show that Fuc-TIV lysates transferred preferentially to the inner LN residues of sialylated linear polylactosamines, whereas Fuc-TVII lysates preferred the distal, sialylated LN units of all acceptors, which contain several potential acceptor sites. The site specificities of the two enzymes were remarkable but not absolute; the "cross-reactivities," which catalyzed the transfer to the nonpreferred acceptor loci, represented generally less than 10% of the preferred activities in both enzymes. The distinct site specificities of Fuc-TIV and Fuc-TVII were true also in reactions involving prefucosylated sialoglycans, implying that the two enzymes may act in concert for efficient biosynthesis of sialylated, multifucosylated polylactosamines. However the present data was obtained under in vitro conditions, which may differ in many ways from those prevailing in vivo in living cells synthesizing selectin ligands. The two transferases may be expressed at unequal levels, and their activities may be affected, e.g. by posttranslational modifications or the lipid microenvironment.

Several observations suggest that monosialylated, multifucosylated polylactosamine sequences are important in selectin recognition. (i) The sequence SAalpha 2-3'Lexbeta 1-3'Lexbeta 1-3'Lex is believed to contribute to P-selectin-PSGL-1 binding (16). (ii) Polylactosamine chains such as SAalpha 2-3'Lexbeta 1-3'Lex on one arm of tetra-antennary N-glycans are high affinity ligands for E-selectin (17). (iii) The divalent glycan SAalpha 2-3'Lexbeta 1-3'Lexbeta 1-3'(SAalpha 2-3'Lexbeta 1-3'Lexbeta 1-6')LN was recently synthesized in our laboratory and shown to be an efficient antagonist of L-selectin.3 (iv) Monosialylated, multifucosylated polylactosamine glycolipids of HL-60 cells are high affinity ligands for E-selectin (18). In myeloid cells, which carry only Fuc-TIV and Fuc-TVII in their alpha 1,3-fucosylation machinery (7, 10, 11, 46), the two transferases may work together in the biosynthesis of multifucosylated sialoglycans. In the present experiments, Fuc-TIV efficiently converted a monosialylated, fucose-free polylactosamine SAalpha 2-3'LNbeta 1-3'LNbeta 1-3'LN (glycan 4) in two steps into a monosialylated, bifucosylated glycan SAalpha 2-3'LNbeta 1-3'Lexbeta 1-3'Lex (glycan 8) that has the two fucose residues at inner, vicinal LN units. The bifucosylated product 8 was then converted by Fuc-TVII into the monosialylated, trifucosylated polylactosamine SAalpha 2-3'Lexbeta 1-3'Lexbeta 1-3'Lex known as sialyl-triLex. Only one pathway leading to this compound was traced completely in the present experiments, but several related pathways were traced almost completely as shown in Fig. 7. The pathways summarized in Fig. 7 are probably not the only ones that lead to the sialyl-triLex sequence. Indeed, the present data show that Fuc-TIV appears to catalyze the fucosylation of sialic acid-free polylactosamine chains even faster than corresponding sialoglycans (Fig. 1, cf. transfer rates at glycan 11 versus 3, 12 versus 4, and 13 versus 5). This suggests that monosialylated, multiply fucosylated polylactosamines in human leukocytes may be generated also by a sequence of reactions consisting of (i) polylactosamine backbone elongation, (ii) multiple stepwise alpha 1,3-fucosylation by Fuc-TIV at vicinal inner LN residues, (iii) alpha 2,3-sialylation at the terminus, and (iv) Fuc-TVII reaction at the distal, sialylated LN residue. Variations of this scheme are also possible in that some of the steps (ii) may occur after the step (iii). It remains to be tested whether or not prefucosylated polylactosamines can be alpha 2,3-sialylated by human leukocyte sialyltransferases. If they can, cellular topography of the alpha 2,3-sialyl- and alpha 1,3-fucosyltransferases, together with the routing of PSGL-1 and other selectin glycoprotein ligands will be the decisive factors in determining whether fucosylation can actually precede sialylation in vivo.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 7.   Biosynthetic pathways leading to multiple fucosylation of a monosialylated fucose-free polylactosamine that has a long chain. The scheme shows the complementary Fuc-TIV and Fuc-TVII reactions traced by the present experiments. The reactions marked by an asterisk were actually demonstrated by using sialofucoglycans from which the reducing end LN unit was missing.

The leukocytes in Fuc-TVII null mice do not express E- or P-selectin counterreceptors (47). This leads to markedly impaired leukocyte rolling and extravasation. However, we note that a substantial amount of residual leukocyte rolling is observed in these mice, suggesting a possible role for Fuc-TIV in contributing to residual selectin ligand "activity" in the absence of Fuc-TVII (47). This possibility is consistent with the observation that the VIM-2 structure has a low affinity for E-selectin (48) and would be predicted to be expressed by Fuc-TVII null neutrophils based on the in vitro results reported here. It will be interesting to learn about the expression of functional selectin counterreceptors and leukocyte extravasation in mice lacking the Fuc-TIV gene.

    FOOTNOTES

* This work was supported by grants from the Academy of Finland (project 101-38042), the Technology Development Center of Finland, the University of Helsinki, and National Institutes of Health Grant P01 CA71932.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

** Investigator of the Howard Hughes Medical Institute.

Dagger Dagger To whom correspondence should be addressed: Dept. of Bacteriology and Immunology, Haartman Institute, P. O. Box 21, University of Helsinki, FIN-00014 Helsinki, Finland. Tel.: 358-9-19126387; Fax: 358-9-19126382; E-mail: Risto.Renkonen@Helsinki.FI.

1 The abbreviations used are: PSGL-1, P-selectin glycoprotein ligand-1; Fuc, L-fucose; Fuc-T, alpha 1,3-fucosyltransferase; G and Gal, D-galactose; GN and GlcNAc, N-acetyl-D-glucosamine; Lex and Lewis x, Galbeta 1-4(Fucalpha 1-3)GlcNAc; LN, N-acetyllactosamine and Galbeta 1-4GlcNAc; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Me, methyl; SA, N-acetylneuraminic acid; sLex and sialyl Lewis x, SAalpha 2-3Galbeta 1-4(Fucalpha 1-3)GlcNAc; CHO, Chinese hamster ovary.

2 R. Niemelä, J. Natunen, L. Penttilä, H. Salminen, J. Helin, H. Maaheimo, C. E. Costello, and O. Renkonen, manuscript in preparation.

3 S. Toppila, L Penttilä, J. Natunen, H. Salminen, J. Helin, H. Maaheimo, R. Renkonen, and O. Renkonen, manuscript in preparation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Ley, K., and Tedder, T. F. (1995) J. Immunol. 155, 525-528[Abstract]
  2. McEver, R. P., Moore, K. L., and Cummings, R. D. (1995) J. Biol. Chem. 270, 11025-11028[Abstract/Free Full Text]
  3. Butcher, E. C., and Picker, L. J. (1996) Science 272, 60-66[Abstract]
  4. Springer, T. A. (1995) Annu. Rev. Physiol. 57, 827-872[CrossRef][Medline] [Order article via Infotrieve]
  5. Bevilacqua, M. P., Nelson, R. M., Mannori, G., and Cecconi, O. (1994) Annu. Rev. Med. 45, 361-378[CrossRef][Medline] [Order article via Infotrieve]
  6. Kukowska-Latallo, J. F., Larsen, R. D., Nair, R. P., Lowe, J. B. (1990) Genes Dev. 4, 1288-1303[Abstract]
  7. Lowe, J. B., Kukowska-Latallo, J. F., Nair, R. P., Larsen, R. D., Marks, R. M., Macher, B. A., Kelly, R. J., Ernst, L. K. (1991) J. Biol. Chem. 266, 17467-17477[Abstract/Free Full Text]
  8. Weston, B. W., Nair, R. P., Larsen, R. D., Lowe, J. B. (1992) J. Biol. Chem. 267, 4152-4160[Abstract/Free Full Text]
  9. Weston, B. W., Smith, P. L., Kelly, R. J., Lowe, J. B. (1992) J. Biol. Chem. 267, 24575-24584[Abstract/Free Full Text]
  10. Natsuka, S., Gersten, K. M., Zenita, K., Kannagi, R., and Lowe, J. B. (1994) J. Biol. Chem. 269, 16789-16794[Abstract/Free Full Text]
  11. Clarke, J. L., and Watkins, W. (1996) J. Biol. Chem. 271, 10317-10328[Abstract/Free Full Text]
  12. Sasaki, K., Kurata, K., Funayama, K., Nagata, M., Watanabe, E., Ohta, S., Hanai, N., and Nishi, T. (1994) J. Biol. Chem. 269, 14730-14737[Abstract/Free Full Text]
  13. Hiraiwa, N., Dohi, T., Kawakami-Kimura, N., Yumen, M., Ohmori, K., Maeda, M., and Kannagi, R. (1996) J. Biol. Chem. 271, 31556-31561[Abstract/Free Full Text]
  14. Sako, D., Chang, X. J., Barone, K. M., Vachino, G., White, H. M., Shaw, G., Veldman, G. M., Bean, K. M., Ahern, T. J., Furie, B., Cumming, D. A., Larsen, G. R. (1993) Cell 75, 1179-1186[Medline] [Order article via Infotrieve]
  15. Guyer, D. A., Moore, K. L., Lynam, E. B., Schammel, C. M. G., Rogelj, S., McEver, R. P., Sklar, L. A. (1996) Blood 88, 2415-2421[Abstract/Free Full Text]
  16. Wilkins, P. P., McEver, R. P., and Cummings, R. D. (1996) J. Biol. Chem. 271, 18732-18742[Abstract/Free Full Text]
  17. Patel, T. P., Goelz, S. E., Lobb, R. R., Parekh, R. B. (1994) Biochemistry 33, 14815-14824[Medline] [Order article via Infotrieve]
  18. Stroud, M. R., Handa, K., Salyan, M. E. K., Ito, K., Levery, S. B., Hakomori, S., Reinhold, B. B., Reinhold, V. N. (1996) Biochemistry 35, 770-778[CrossRef][Medline] [Order article via Infotrieve]
  19. Brew, K., Vanaman, T. C., and Hill, R. L. (1968) Proc. Natl. Acad. Sci. U. S. A. 59, 491-497[Medline] [Order article via Infotrieve]
  20. Seppo, A., Penttilä, L., Makkonen, A., Leppänen, A., Niemelä, R., Jäntti, J., Helin, J., and Renkonen, O. (1990) Biochem. Cell Biol. 68, 44-53[Medline] [Order article via Infotrieve]
  21. Maaheimo, H., Renkonen, R., Turunen, J. P., Penttilä, L., Renkonen, O. (1995) Eur. J. Biochem. 234, 616-625[Abstract]
  22. Leppänen, A., Penttilä, L., Niemelä, R., Helin, J., Seppo, A., Lusa, S., and Renkonen, O. (1991) Biochemistry 30, 9287-9296[Medline] [Order article via Infotrieve]
  23. Renkonen, O., Helin, J., Vainio, A., Niemelä, R., Penttilä, L., and Hilden, P. (1990) Biochem. Cell Biol. 68, 1032-1036[Medline] [Order article via Infotrieve]
  24. Räbinä, J., Natunen, J., Niemelä, R., Salminen, H., Ilves, K., Aitio, O., Maaheimo, H., Helin, J., and Renkonen, O. (1998) Carbohydr. Res., in press
  25. Niemelä, R., Räbinä, J., Leppänen, A., Maaheimo, H., Costello, C. E., Renkonen, O. (1995) Carbohydr. Res. 279, 331-338[CrossRef][Medline] [Order article via Infotrieve]
  26. Maaheimo, H., Räbinä, J., and Renkonen, O. (1997) Carbohydr. Res. 297, 145-151[CrossRef][Medline] [Order article via Infotrieve]
  27. Niemelä, R., Natunen, J., Brotherus, E., Saarikangas, A., and Renkonen, O. (1995) Glycoconj. J. 12, 36-44[Medline] [Order article via Infotrieve]
  28. Seppo, A., Turunen, J. P., Penttilä, L., Keane, A., Renkonen, O., and Renkonen, R. (1996) Glycobiology 6, 65-71[Abstract]
  29. Renkonen, O., Penttilä, L., Makkonen, A., Niemelä, R., Leppänen, A., Helin, J., and Vainio, A. (1989) Glycoconj. J. 6, 129-140[Medline] [Order article via Infotrieve]
  30. Arakawa, M., Ogata, S.-I., Muramatsu, T., and Kobata, A. (1974) J. Biochem. 75, 707-714[Medline] [Order article via Infotrieve]
  31. de Vries, T., Norberg, T., Lönn, H., and van den Eijnden, D. H. (1993) Eur. J. Biochem. 216, 769-777[Abstract]
  32. de Vries, T., and van den Eijnden, D. H. (1994) Biochemistry 33, 9937-9944[Medline] [Order article via Infotrieve]
  33. Johnson, P. H., Donald, A. S. R., Feeney, J., and Watkins, W. M. (1992) Glycoconj. J. 9, 251-264[Medline] [Order article via Infotrieve]
  34. Johnson, P. H., and Watkins, W. M. (1985) Biochem. Soc. Trans. 13, 1119-1120
  35. Jezequel-Cuer, M., N'Guyen-Cong, H., Biou, D., and Durand, G. (1993) Biochim. Biophys. Acta 1157, 252-258[Medline] [Order article via Infotrieve]
  36. Sarnesto, A., Köhlin, T., Hindsgaul, O., Vogele, K., Blaszczyk-Thurin, M., and Thurin, J. (1992) J. Biol. Chem. 267, 2745-2752[Abstract/Free Full Text]
  37. Gersten, K. M., Natsuka, S., Trinchera, M., Petryniak, B., Kelly, R. J., Hiraiwa, N., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., Lowe, J. B. (1995) J. Biol. Chem. 270, 25047-25056[Abstract/Free Full Text]
  38. Holmes, E. H., and Macher, B. A. (1993) Arch. Biochem. Biophys. 301, 190-199[CrossRef][Medline] [Order article via Infotrieve]
  39. de Vries, T., Srnka, C. A., Palcic, M. M., Swiedler, S. J., van den Eijnden, D. H., Macher, B. A. (1995) J. Biol. Chem. 270, 8712-8722[Abstract/Free Full Text]
  40. Koszdin, K. L., and Bowen, B. R. (1992) Biochem. Biophys. Res. Commun. 187, 152-157[Medline] [Order article via Infotrieve]
  41. Goelz, S. E., Hession, C., Goff, D., Griffits, B., Tizard, R., Newman, B., Chi-Rosso, G., and Lobb, R. (1990) Cell 63, 1349-1356[Medline] [Order article via Infotrieve]
  42. Kumar, R., Potvin, B., Muller, W. A., Stanley, P. (1991) J. Biol. Chem. 266, 21777-21783[Abstract/Free Full Text]
  43. Goelz, S., Kumar, R., Potvin, B., Sundaram, S., Brickelmaier, M., and Stanley, P. (1994) J. Biol. Chem. 269, 1033-1040[Abstract/Free Full Text]
  44. Knibbs, R. N., Craig, R. A., Natsuka, S., Chang, A., Cameron, M., Lowe, J. B., Stoolman, L. M. (1996) J. Cell Biol. 133, 911-920[Abstract]
  45. Sueyoshi, S., Tsuboi, S., Sawada-Hirai, R., Dang, U. N., Lowe, J. B., Fukuda, M. (1994) J. Biol. Chem. 269, 32342-32350[Abstract/Free Full Text]
  46. Watkins, W. M., Skacel, P. O., and Clarke, J. L. (1995) Adv. Exp. Med. Biol. 376, 83-93[Medline] [Order article via Infotrieve]
  47. Maly, P., Thall, A. D., Petryniak, B., Rogers, C. E., Smith, P. L., Marks, R. M., Kelly, R. J., Gersten, K. M., Cheng, G., Saunders, T. L., Camper, S. A., Camphausen, R. T., Sullivan, F. X., Isogai, Y., Hindsgaul, O., von Andrian, U. H., Lowe, J. B. (1996) Cell 86, 643-653[Medline] [Order article via Infotrieve]
  48. Tiemeyer, M., Swiedler, S. J., Ishihara, M., Moreland, M., Schweingruber, H., Hirtzer, P., and Brandley, B. K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1138-1142[Abstract]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.