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.

The P-selectin counterreceptor PSGL-1 is covalently modified by mono alpha2,3-sialylated, multiply alpha1,3-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 alpha1,3-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 alpha2,3-sialylated acceptors but is ineffective in transfer to the distal alpha2,3-sialylated LN unit in alpha2,3-sialylated acceptors. Fuc-TVII, by contrast, effectively fucosylates only the distal alpha2,3-sialylated LN unit in alpha2,3-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 SAalpha2-3'LNbeta1-3'LNbeta1-3'LN (where SA is sialic acid) into the trifucosylated molecule SAalpha2-3'Lexbeta1-3'Lexbeta1-3'Lex (where Lex is the trisaccharide Galbeta1-4(Fucalpha1-3)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.

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)(2)(3)(4). E-and P-selectin, expressed by activated endo-thelium (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 ␣1,3-fucosylation and terminal ␣2,3-sialylation. Of the five human ␣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)(12)(13). Hence, these two enzymes are candidates for participation in the biosynthesis of the fucosylated glycans that decorate selectin counterreceptors on leukocytes.
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 ␣2,3-sialyl polylactosamine chains, whereas Fuc-TVII preferentially fucosylates distal LN units on such ␣2,3sialylated 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
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 g/ml benzamidine HCl, 10 g/ml phenanthroline, 10 g/ml aprotinin, 10 g/ml leupeptin, 10 g/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride, Pharmingen, San Diego, CA).

Methods Used in the Analysis of Fuc-TIV and Fuc-TVII Reactions-
Gel filtration was performed in a column of Superdex peptide HR 10/30 (Pharmacia, Sweden), with 50 mM NH 4 HCO 3 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 ϩ ) and AG-1 (AcO Ϫ ) (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).
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 ␤-galactosidase and ␤-Nacetylhexosaminidase. The latter digestion removes any fucose-free LN units from the nonreducing end of desialylated polylactosamines but is unable to act on distal, ␣1,3-fucosylated LN residues (30). Hence, the desialylated chains were shortened in a way that established the position of the ␣1,3fucosylated 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-␤-galactosidase, which cleaves internal ␤-galactosidic linkages of linear polylactosamines, but is unable to hydrolyze the same bonds of ␣1,3-fucosylated LN units (31, 32) (data not shown).
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 fucosefree precursor via intermediates of VIM-2 and sLex type glycans.
The unlabeled glycan SA␣2-3ЈLN␤1-3ЈLex␤1-3ЈLN (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 [ 14 C]fucose was transferred to the "innermost" LN unit (residue 1), generating

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/g protein/h; ‡, transfer rate to SA␣2-3ЈLN was typically 3.2 pmol/g protein/h; §, only 0.15 mM acceptor was used, and the reference acceptor was also 0.15 mM; ʈ, 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. SA␣2-3ЈLN␤1-3ЈLex␤1-3Ј[ 14 C]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.
Site Specificity of Fucosylation Reactions with Branched Acceptors-Fuc-TIV transferred in a slightly preferential manner to the ␤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
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 ␣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.  Table I. 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 SA␣2-3ЈLN, 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-reactivi-ties," 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.
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.