From the 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
the Howard Hughes Medical Institute and Department
of Pathology, University of Michigan Medical School, Ann Arbor,
Michigan 48109-0650
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ABSTRACT |
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The P-selectin counterreceptor PSGL-1 is
covalently modified by mono 2,3-sialylated, multiply
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
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
2,3-sialylated acceptors but is ineffective in transfer to the
distal
2,3-sialylated LN unit in
2,3-sialylated acceptors. Fuc-TVII, by contrast, effectively fucosylates only the distal
2,3-sialylated LN unit in
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
SA
2-3'LN
1-3'LN
1-3'LN (where SA is sialic acid) into the
trifucosylated molecule SA
2-3'Lex
1-3'Lex
1-3'Lex (where Lex
is the trisaccharide Gal
1-4(Fuc
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.
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INTRODUCTION |
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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
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 (11-13). 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 1,3-fucosylated core 2 sialyl glycan
expressed by HL-60 cell-derived PSGL-1 contains the decasaccharide
sequence SA
2-3'Lex
1-3'Lex
1-3'Lex (where Lex is
Gal
1-4(Fuc
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
1,3-fucosylation of its O-linked core 2 glycans. Similar
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 2,3-sialyl polylactosamine chains, whereas Fuc-TVII preferentially fucosylates distal LN units on such
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.
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EXPERIMENTAL PROCEDURES |
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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 GlcNAc1-3Gal
1-OMe (Sigma) by
1,4-galactosylation (19) followed by
1,3-N-acetylglucosaminylation (20) and a second round of
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
2,3-sialylation as in (21).
Glycan 12 was constructed from the GN
1-3'LN
1-3'LN
(22) by
1,4-galactosylation, MALDI-TOF MS: (M+Na)+ m/z
1137.1 (calc. 1137.0). Glycan 4 was obtained from glycan
12 by
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
2,3-sialylation of 15. Synthesis of glycan 8 will be described in detail elsewhere (24). Briefly, GN
1-3'LN
1-3'LN was
1,3-14C fucosylated at both LN units with
1,3/4-FucTs
of human milk and then
1,4-galactosylated. MALDI-TOF MS of the
resulting octasaccharide LN
1-3'Lex
1-3'Lex gave
(M+Na)+ m/z 1429.3 (calc. 1429.3). The octasaccharide was
finally
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
-galactosidase and
-N-acetylhexosaminidase gave the
neutral hexasaccharide
[14C]Fuc
1-3LN
1-3'[14C]Fuc
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 LN
1-3'(GN
1-6')LN as in (25, 26), MALDI-TOF MS:
(M+Na)+ m/z 974.8 (calc. 974.9). The product was
2,3-sialylated, was
1,3-fucosylated selectively at the distal,
sialylated LN-unit (25), and was treated with
-N-acetylhexosaminidase for removal of the protecting
1-6GN-unit to give 5. Glycan 6, in turn, was
obtained by
1,3-fucosylating glycan 9 at the LN unit by
human milk
1,3/4-Fuc-Ts. The product was then
1,4-galactosylated
and finally
2,3-sialylated. Glycan 7 was synthesized by
2,3-sialylation of the heptasaccharide LN
1-3'Lex
1-3'LN, prepared by
1,4-galactosylation of GN
1-3'Lex
1-3'LN, which
had been isolated from a mixture of monofucosylated isomers by wheat germ agglutinin-agarose chromatography. LN
1-3'Lex
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
1,3-N-acetylglucosaminylation of LN (20), MALDI-TOF
MS: (M+Na)+ m/z 609.7 (calc. 609.5). Glycan 10 was synthesized by
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 SA2-3LN 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
-galactosidase (EC 3.2.1.23, Sigma), jack bean
-N-acetylhexosaminidase (EC 3.2.1.30, Sigma) (27),
Arthrobacter ureafaciens sialidase (EC 3.2.1.18, Boehringer
Mannheim) (28), and Bacteroides fragilis
endo-
-galactosidase (EC 3.2.1.103, Boehringer Mannheim) (22) were
carried out as described previously.
Oligosaccharide Markers--
The hexasaccharide Lex1-3'Lex
and the nonasaccharide Lex
1-3'Lex
1-3'Lex, MALDI-TOF MS:
(M+Na)+ m/z 1575.8 (calc. 1575.5) as well as the mixture of
the octasaccharide Lex
1-3'Lex
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
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RESULTS |
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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 (SA2-3'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.
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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
-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,
1,3-fucosylated LN
residues (30). Hence, the desialylated chains were shortened in a way
that established the position of the
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-
-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).
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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 SA
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Fuc-TVII Catalyzes Rapid Fucosylation of the Monosialyl-Bifucosyl
Glycan 8 at the Distal, Sialylated LN Unit--
The
nonasaccharide SA2-3'LN
1-3'Lex
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
SA
2-3'[14C]Lex
1-3'Lex
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
SA
2-3'Lex
1-3'Lex
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
1,3-fucosyltransferases present in human leukocytes.
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Site Specificity of Fuc-TIV Reactions with Neutral Linear
Acceptors--
Fuc-TIV efficiently fucosylated GN1-3'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
-N-acetylhexosaminidase to [14C]Lex (data
not shown). Fuc-TIV also transferred effectively to LN
1-3'LN
1-3G
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 LN
1-3'LN
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 Lex
1-3'LN (13) and
LN
1-3'Lex (14), confirming that fucosylation of vicinal
LN units is feasible.
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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).
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DISCUSSION |
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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." 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 SA2-3'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 SA2-3'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 SA2-3'Lex
1-3'Lex
1-3'Lex is believed to
contribute to P-selectin-PSGL-1 binding (16). (ii) Polylactosamine
chains such as SA
2-3'Lex
1-3'Lex on one arm of tetra-antennary
N-glycans are high affinity ligands for E-selectin (17).
(iii) The divalent glycan
SA
2-3'Lex
1-3'Lex
1-3'(SA
2-3'Lex
1-3'Lex
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
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
SA
2-3'LN
1-3'LN
1-3'LN (glycan 4) in two steps
into a monosialylated, bifucosylated glycan
SA
2-3'LN
1-3'Lex
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
SA
2-3'Lex
1-3'Lex
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
1,3-fucosylation by Fuc-TIV at
vicinal inner LN residues, (iii)
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
2,3-sialylated by human
leukocyte sialyltransferases. If they can, cellular topography of the
2,3-sialyl- and
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.
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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.
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FOOTNOTES |
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* 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.
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,
1,3-fucosyltransferase; G and Gal, D-galactose; GN and
GlcNAc, N-acetyl-D-glucosamine; Lex and Lewis x,
Gal
1-4(Fuc
1-3)GlcNAc; LN, N-acetyllactosamine and
Gal
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,
SA
2-3Gal
1-4(Fuc
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.
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REFERENCES |
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