PSGL-1, a ho-modimeric sialomucin expressed on most leukocytes, also functions as an important capture ligand for E-selectin, while the characteristically slow E-selectin mediated rollin
Trang 1Selectins and glycosyltransferases in leukocyte rolling
in vivo
Markus Sperandio
University Children’s Hospital Heidelberg, Division of Neonatal Medicine, University of Heidelberg, Germany
Leukocyte recruitment is a crucial immunological
pro-cess that enables leukocytes to leave the intravascular
compartment and transmigrate into tissue where they
fulfill their task as immune cells [1,2] Recruitment of
leukocytes proceeds along a cascade of events,
begin-ning with the capture of free flowing leukocytes to the
vessel wall This is followed by leukocyte rolling along
the endothelium Capture and rolling are mediated by
a group of glycoproteins, called selectins, which bind
to carbohydrate determinants on selectin ligands
During rolling, leukocytes are intimately engaged with
the endothelium, which gives endothelial bound
chemo-kines the opportunity to bind to their respective chemokine receptors on leukocytes This triggers the activation of integrins, leading to firm leukocyte adhe-sion to the endothelium and transmigration into tissue [3] A detailed online illustration of the leukocyte recruitment cascade can be found at http://www bme.virginia.edu/ley/
Leukocyte rolling
Leukocyte rolling is mediated by selectins and is consid-ered an important step for the successful recruitment of
Keywords
glycosylation; glycosyltransferases;
leukocyte rolling; selectin ligand; selectin
Correspondence
M Sperandio, Children’s Hospital, Division
of Neonatal Medicine, University of
Heidelberg, INF 150, 69120 Heidelberg,
Germany
Fax: +49 622156 4208
Tel: +49 622156 1759
E-mail: markus.sperandio@med.
uni-heidelberg.de
(Received 15 May 2006, accepted 3 July
2006)
doi:10.1111/j.1742-4658.2006.05437.x
Leukocyte rolling is an important step for the successful recruitment of leu-kocytes into tissue and occurs predominantly in inflamed microvessels and
in high endothelial venules of secondary lymphoid organs Leukocyte roll-ing is mediated by a group of C-type lectins, termed selectins Three differ-ent selectins have been iddiffer-entified – P-, E- and L-selectin – which recognize and bind to crucial carbohydrate determinants on selectin ligands Among selectin ligands, P-selectin glycoprotein ligand-1 is the main inflammatory selectin ligand, showing binding to all three selectins under in vivo condi-tions Functional relevant selectin ligands expressed on high endothelial venules of lymphoid tissue are less clearly defined at the protein level However, high endothelial venule-expressed selectin ligands were instru-mental in uncovering the crucial role of post-translational modifications for selectin ligand activity Several glycosyltransferases, such as core 2 b1,6-N-acetylglucosaminyltransferase-I, b1,4-galactosyltransferases, a1,3-fucosyl-transferases and a2,3-sialyla1,3-fucosyl-transferases have been described to participate
in the synthesis of core 2 decorated O-glycan structures carrying the tetra-saccharide sialyl Lewis X, a carbohydrate determinant on selectin ligands with binding activity to all three selectins In addition, modifications, such
as carbohydrate or tyrosine sulfation, were also found to contribute to the synthesis of functional selectin ligands
Abbreviations
CHO, Chinese hamster ovary; CLA, cutaneous lymphocyte-associated antigen; core 2 GlcNAcT, core 2 b1,6 N-acetylglucosaminyltransferase; FucT, a1,3-fucosyltransferase; HEC, high endothelial cell; PNAd, peripheral node addressin; ppGalNAcT, polypeptide galactosaminyl-transferase; PSGL-1, P-selectin glycoprotein ligand-1; sLex, sialyl Lewis X; ST3Gal, a2,3 sialyltransferase; TNF-a, tumor necrosis factor-a; TPST, tyrosylprotein sulfotransferase.
Trang 2leukocytes into tissue Three different selectins are
known: P-, E- and L-selectin They are all members of a
family of glycoproteins called C-type lectins [4]
Accord-ingly, the characteristic feature of selectins is their
abil-ity to recognize and bind to specific carbohydrate
determinants on selectin ligands in a calcium-dependent
manner [5] Binding takes place under dynamic
condi-tions, where continuous shear forces, exerted by the
flowing blood, act on leukocytes rolling along the
endo-thelium at rolling velocities 100–1000-fold slower than
the mean blood flow velocity To achieve controlled and
stable leukocyte rolling under these conditions, selectin
binding to selectin ligands needs to comply with the
fol-lowing three requirements (a) rapid bond formation at
the leading edge, (b) high tensile strength during
bind-ing and (c) fast dissociation rates Interestbind-ingly, recent
reports have revealed that selectin binding duration
(bond lifetime) adjusts to increasing forces by
decreas-ing off-rates [i.e the bond locks more tightly when
blood flow is increased (catch bonds)] This enables
leu-kocytes to roll even at high shear rates [6,7] However,
after reaching a certain shear rate threshold, the bond
properties change towards a slip bond behaviour, which
eventually leads to breakage of the bond [8] These
properties contribute significantly to the creation of
an effective breaking system that recruits free
flow-ing leukocytes to the endothelial wall and prepares
them (during rolling) for subsequent adhesion and
transmigration
Functionally, leukocyte rolling serves two main
pur-poses First, leukocyte rolling participates in the
successful recruitment of neutrophils, monocytes,
eos-inophils, some effector T cells and dendritic cells to
sites of acute and chronic inflammation This requires
the up-regulation of P- and E-selectin and of
endothel-ial L-selectin ligands on inflamed endothelium In
rest-ing vascular endothelial cells, P-selectin is stored in
secretory granules, called Weibel-Palade bodies In
addition, P-selectin is found in a-granules of platelets
Upon stimulation with pro-inflammatory mediators,
including histamine, tumor necrosis factor-a (TNF-a),
lipopolysaccharide, thrombin, complement C5a and
calcium ionophores, P-selectin can be rapidly
mobil-ized to the cell surface [9] P-selectin is the
predominant leukocyte rolling receptor on acutely
inflamed endothelial cells in vivo This has been
dem-onstrated by intravital microscopy studies of inflamed
mouse cremaster muscle venules from P-selectin
defici-ent mice, where leukocyte rolling was almost
com-pletely absent shortly after exteriorization of the
cremaster muscle [10] Except for skin microvessels,
E-selectin is not constitutively expressed on resting
vas-cular endothelium Expression has to be stimulated
with TNF-a, lipopolysaccharide, interleukin-1, or other pro-inflammatory mediators involving transcriptional mechanisms [11] Kraiss and colleagues recently showed that fluid flow reduces E-selectin expression by inhibiting E-selectin translation [12] In collaboration with P-selectin, E-selectin shares distinct, as well as overlapping, functions as rolling receptor [13] In addi-tion, E-selectin co-operates with the chemokine recep-tor CXCR-2 in mediating the transition from slow rolling to firm leukocyte arrest [14]
During inflammation, E- and P-selectin bind to selectin ligands expressed on rolling leukocytes (Table 1) In vivo studies using mice deficient in P-se-lectin glycoprotein ligand-1 (PSGL-1) have shown that PSGL-1 is the predominant, if not the only, P-selectin ligand during inflammation [15,16] PSGL-1, a ho-modimeric sialomucin expressed on most leukocytes, also functions as an important capture ligand for E-selectin, while the characteristically slow E-selectin mediated rolling velocity, as well as the E-selectin dependent transition from slow rolling to firm arrest,
is not dependent on PSGL-1 [14,16]
Besides PSGL-1, many other E- as well as P-selectin ligands have been identified under in vitro conditions (Table 1), but most of these selectin ligand candidates failed to demonstrate relevance under in vivo condi-tions Recently, CD44 and CD43 have been proposed
to be functionally relevant E-selectin ligands
Katayam-a Katayam-and colleKatayam-agues showed thKatayam-at immunopurified CD44 from peripheral blood polymorphonuclear cells binds
to E-selectin [20] Tunicamycin and O-sialoglycoprotein endopeptidase treatment of myeloid cells revealed that N-linked, but not O-linked, glycans on CD44 con-tribute to the observed binding of CD44 to E-selectin, suggesting that distinct N-glycan-modified CD44 glycoforms exist for binding to E-selectin [20] To test,
in greater detail, the in vivo relevance of CD44 as an E-selectin ligand, additional intravital microscopy experiments were conducted in TNF-a stimulated cremaster muscle venules where E- and P-selectin medi-ated leukocyte rolling occurs Similarly to a1,3-fucosyl-transferase (FucT)-IV deficient mice [27], CD44–⁄ – mice exhibited a significant increase in rolling velocity without affecting the number of rolling leukocytes [20] This provides indirect evidence that CD44 may be an E-selectin ligand in vivo Using E-selectin transfected Chinese hamster ovary (CHO) cells and recombinant murine CD43 immobilized on the surface of glass capil-laries, Matsumoto et al demonstrated that CD43 supports rolling of E-selectin transfected CHO cells, but not of control CHO cells [25] In another report, the core 2 decorated glycoform of CD43 isolated from cutaneous lymphocyte-associated antigen (CLA)+
Trang 3human T cells supported rolling via E-selectin, but not
via P-selectin Interestingly, the same study identified
that the CLA epitope recognized by mAb high
endo-thelial cell (HEC) A-452 is not restricted to PSGL-1
but also found on the core 2 modified glycoform of
CD43 from CLA+ human T cells [28] Both studies on CD43 clearly demonstrate that CD43 interacts with E-selectin under static and dynamic conditions in vitro However, the role of CD43 as a relevant E-selectin lig-and in vivo remains to be determined
Table 1 Relevant selectin ligands for leukocyte rolling under in vivo conditions ESL-1, E-selectin ligand-1; GlyCAM, glycosylation-dependent cell adhesion molecule; HEV, high endothelial venule; MAdCAM-1, mucosal addressin cell adhesion molecule-1; PNAd, peripheral node addressin; PSGL-1, P-selectin glycoprotein ligand-1.
During inflammation
chronically inflamed endothelium in a spontaneous model
of chronic ileitis
Predominant inflammatory selectin ligand in vivo Mediates P-selectin-dependent rolling [17]
Probably the only relevant P-selectin ligand during inflammation [15,16] Mediates L -selectin dependent secondary and primary
tethering events in inflamed venules [18]
Endothelial expressed PSGL-1 mediates L -selectin dependent recruitment of T cells into chronically inflamed ileum [19]
Capture ligand for E-selectin [16]
No influence on slow E-selectin-dependent rolling velocity or E-selectin-mediated arrest [14,16]
erythrocytes and in the brain
Strong indirect evidence that CD44 functions as E-selectin ligand during inflammation from in vivo studies in CD44-deficient mice [20] Binding to E-selectin via specific N-glycan decorated
glycoform of CD44 [20]
Mediates L -selectin-dependent rolling in a flow chamber assay [21] During lymphocyte homing
MAdCAM-1 Constitutive expression in
Peyer’s patch HEV and in intestinal lamina propria vessels; induced expression
in chronically inflamed venules
Mediates L -selectin-dependent leukocyte rolling in Peyer’s patch HEV [22]
The only relevant L -selectin homing ligand in vivo identified at present
PNAd (GlyCAM-1,
CD34, podocalyxin
and endomucin)
Constitutive expression in HEV
of peripheral lymph nodes;
induced expression in chronically inflamed venules
No functional evidence that single members of the group are relevant
L -selectin ligands in vivo Normal lymphocyte homing in GlyCAM-1 – ⁄ – and CD34 – ⁄ – mice [23,24] Probably overlapping L -selectin ligand function by all members of the PNAd group
Other selectin ligands
identified under in vitro
conditions with no proven
relevance for leukocyte
rolling in vivo
neutrophils, B lymphocytes, immature thymocytes, erythrocytes
Mediates tumor metastasis in different mouse models Mediates P-selectin-dependent tumor cell rolling in vitro
hematopoietic cells
Mediates E-selectin-dependent rolling in vitro [25]
surface, but abundantly expressed in the Golgi apparatus
Supports leukocyte rolling in vitro
Heparin derivatives Ubiquitously expressed Contribution to leukocyte rolling in vivo unknown
leukocyte surface
Binds to L -selectin in static in vitro assays [26]
Trang 4L-selectin mediated rolling, observed during acute
inflammation in vivo, is independent of endothelial
L-selectin ligands but dependent on PSGL-1 This has
been shown in PSGL-1 deficient mice, where L-selectin
dependent leukocyte rolling was completely absent in
two models of acute inflammation, suggesting that
PSGL-1 is the main (if not the only) inflammatory
L-selectin ligand [18] Using the same in vivo models in
control mice, it was noted that L-selectin dependent
rolling occurred mostly via interactions between free
flowing and adherent leukocytes (secondary tethering)
and, to a lesser degree, between free flowing leukocytes
and leukocyte fragments deposited on the inflamed
endothelium (primary tethering) [18] In contrast to
acute inflammation, endothelial L-selectin ligand
acti-vity has been reported during chronic inflammatory
states in several disease models, including multiple
sclerosis and rheumatoid arthritis The induction of
endothelial L-selectin ligand activity is frequently
accompanied by the development of inflammatory
infiltrates that exhibit lymphoid organ characteristics,
suggesting that the molecular structure of these
endo-thelial L-selectin ligands are similar to those L-selectin
ligands constitutively expressed on high endothelial
venules (HEVs) of secondary lymphoid organs [29,30]
However, a recent study identified PSGL-1 expression
on chronically inflamed microvessels of the small
intes-tine and on mesenteric lymph node HEV in a
sponta-neous model of chronic ileitis [19] Additional
intravital microscopy studies revealed that blockade of
PSGL-1, using the monoclonal mAb, 4RA10, led to a
significant reduction in rolling leukocytes on inflamed
serosal venules of the terminal ileum, suggesting a
crucial role of PSGL-1 in leukocyte recruitment to
inflamed small intestine in chronic ileitis [19] These
results may stimulate follow-up studies to evaluate
PSGL-1 as a potential target for the treatment of
human chronic inflammatory bowel disease
Apart from its function as a rolling receptor,
L-selectin also influences leukocyte adhesion and
trans-migration during inflammation (reviewed in [31])
In vitro studies revealed that the cross-linking of
L-selectin on neutrophils induces Mac-1 up-regulation
followed by an increase in firm adhesion and
transmigration under shear flow [32,33] In addition,
Hickey and colleagues investigated leukocyte
recruit-ment in response to chemokines and chemotactic
fac-tors in the mouse cremaster muscle [34] The authors
found that superfusion of keratinocyte-derived
chemo-kine or platelet-activating factor over the cremaster
muscle of L-selectin-deficient mice did not alter
leuko-cyte rolling or adhesion, but led to a significant
decrease in the number of emigrated leukocytes when
compared with control mice Furthermore, the authors demonstrated that directed leukocyte migration towards a keratinocyte-derived chemokine-containing gel within the cremaster muscle tissue was significantly impaired in L-selectin deficient mice [34]
Besides the important role of leukocyte rolling dur-ing inflammation, the second major purpose of leuko-cyte rolling involves the successful exit of T- and B lymphocytes from HEV into the parenchyma of secon-dary lymphoid organs Leukocyte rolling on HEV is almost exclusively mediated by L-selectin and an essen-tial step for the effective transmigration of lympho-cytes into secondary lymphoid organs [35] L-selectin is expressed on the microtips of most leukocytes, inclu-ding all myeloid cells, naı¨ve T- and B cells, as well as some activated T cells and memory T cells Therefore, leukocyte rolling on HEV is not restricted to lympho-cytes but also involves other leukocyte populations This explains the observation that more than 50% of leukocytes passing through HEVs of secondary lym-phoid organs are rolling [36] It is obvious that most rolling leukocytes will eventually detach from the sur-face of HEV and return into free flow because they lack the proper signals from specific chemokines neces-sary to trigger the activation of integrins, which leads
to firm leukocyte arrest Successful leukocyte adhesion and consecutive transmigration is only possible for those lymphocytes expressing the appropriate chemo-kine receptors, which then interact with their respective chemokines immobilized on the surface of high endo-thelial cells [37]
In HEVs, L-selectin interacts with HEV-expressed L-selectin ligands, which have been mainly defined as a group of heterogeneous glycoproteins recognized by mAb MECA-79 and termed peripheral node addressins (PNAd) [38] The PNAd group includes glycosylation-dependent cell adhesion molecule-1, CD34, sgp200, HEV-expressed podocalyxin, and a recently identified glycoprotein called endomucin (Table 1) [39] To fur-ther investigate the contribution of the different PNAd members for selectin ligand function in vivo, lympho-cyte homing was investigated in glycosylation-depend-ent cell adhesion molecule-1–⁄ –mice that demonstrated normal lymphocyte trafficking [23] Similarly, CD34–⁄ – mice had no defect in lymphocyte trafficking [24] sug-gesting that L-selectin ligand activity on HEV is not dependent on a single member of the PNAd family, but comprises a redundant system where the loss of one member is compensated by the presence of the others In addition, it indicates that other regulatory mechanisms, such as post-translational modifications, contribute to cell-specific and activation-specific expression of functional selectin ligands in vivo
Trang 5Post-translational glycosylation of
selectin ligands
Selectin ligands belong to a growing number of
glyco-proteins where protein function is closely linked to
its proper post-translational glycosylation
Posttransla-tional glycosylation is mainly performed in the Golgi
apparatus, involving a group of Golgi resident enzymes
termed glycosyltransferases Glycosyltransferases are
type II transmembrane proteins that specifically
trans-fer activated sugar nucleotide donors, including
UDP-N-acetylgalactosamine, UDP-N-acetylglucosamine,
UDP-galactose, GDP-fucose, and CMP-sialic acid to
glycoconjugate acceptors [40] In general, each
glycosyl-transferase recognizes only one type of sugar
nucleo-tide Furthermore, transfer of the sugar nucleotide is
restricted to specific acceptor molecules and glycosidic
bonds formed Additional factors, such as the
expres-sion level of specific glycosyltransferases and the
loca-tion of glycosyltransferases along the different Golgi
compartments, add to the complex machinery necessary
for the synthesis of specific carbohydrate determinants
on glycoproteins Characterization of the carbohydrate
epitopes crucial for selectin ligand activity revealed that
selectins are low affinity receptors to a2,3-sialylated
and a1,3-fucosylated core 2 decorated O-glycans
carry-ing the sialyl Lewis X (sLex) motif as capping group
(Fig 1) [41] Several glycosyltransferases, including
core 2 b1,6-N-acetylglucosaminyltransferase [42,43],
b1,4-galactosyltransferases (Gal-T)-I and -IV [44,45],
FucT-VII and -IV [27,46], and a2,3-sialyltransferase
(ST3Gal)-IV [47] have been identified to participate
directly in the synthesis of functional selectin ligands
in vivo (Table 2) In addition, several other modifica-tions have been described to contribute to selectin ligand function (Table 2) Two enzymes catalyzing carbohydrate sulfation [N-acetylglucosamine 6-O-sulfo-transferase (GlcNAc6ST)-1 and -2] were found to be involved in the generation of 6-sulfo sLex which is important for l-selectin ligand activity on HEV (Fig 2) [48,49] Furthermore, sulfation of tyrosine residues at the N-terminus of PSGL-1 has been reported to signifi-cantly influence binding of selectins to PSGL-1 (Fig 1) [50]
Figure 1 gives an overview on the biosynthetic path-way of core 2 modified O-glycans terminated with sLex O-glycan biosynthesis is initiated with the addi-tion of galactosamine to serine or threonine residues at the protein backbone [61] This step is catalysed by UDP-GalNAc:polypeptide GalNAcT (ppGalNAcT) Twenty-four different ppGalNAcT have been described
in humans [51] No data are available on the role of ppGalNAcT on selectin ligand activity However, in view of the abundance of different isoenzymes it seems likely that a high degree of redundancy exists which may be an indication that ppGalNAcT is not rate-limiting in the synthesis of functional selectin ligands After the addition of galactose to GalNAc in b1,3 linkage, which gives rise to the core 1 extension, core 2 b1,6 N-acetylglucosaminyltransferase (core 2 NAcT-I) initiates the core 2 extension by adding Glc-NAc to GalGlc-NAc in b1,6 linkage This is followed by the alternate action of b1,4-galactosyltransferase (b1,4-GalT) and b1,3-GlcNAcT, which elongate the core 2 branch by forming a polylactosamine chain of various length During elongation, a1,3-fucosylation of Glc-NAc residues by FucT-IV may occur within the poly-lactosamine chain Elongation of core 2 branches is terminated by the addition of sialic acid, in a2,3 link-age, to galactose (Fig 1) This is followed by the addi-tion of fucose to the penultimate GlcNAc, in a1,3 linkage, resulting in the formation of sLex at the end
of core 2 decorated O-glycans (Fig 1) In the following section, the contribution of glycosyltransferases involved in the synthesis of functional selectin ligands
in vivoare discussed
Core 2 GlcNAcT-I
Core 2 GlcNAcT-I is the key branching enzyme in the synthesis of core 2 decorated O-glycans Core 2 Glc-NAcT-I catalyzes the addition of N-acetylglucosamine
to N-acetylgalactosamine in b1,6 linkage, which initi-ates the core 2 extension (Fig 1) Direct evidence that core 2 GlcNAcT-I is important for leukocyte rolling
in vivocomes from mice deficient in core 2 GlcNAcT-I,
Fig 1 Biosynthetic pathway for the synthesis of core 2 decorated
O-glycans carrying the sialyl Lewis X (sLe x ) determinant During
inflammation, the main inflammatory selectin ligand, P-selectin
gly-coprotein ligand-1 (PSGL-1), interacts with P- and L-selectin under
in vivo conditions via core 2 decorated sLe x , in co-operation with
nearby sulfated tyrosines located at the N-terminus of PSGL-1.
Trang 6Table 2 Enzymes involved in the post-translational modification of selectin ligands CDG, congenital deficiency of glycosylation; CHST-2, car-bohydrate sulfotransferase 2; core 2 GlcNAcT, core 2 b1,6 N-acetylglucosaminyltransferase; FucT, a1,3 fucosyltransferase; GalT, galactosyl-transferase; GlcNAc6ST, N-acetylglucosamine 6-O-sulfogalactosyl-transferase; GST, Gal ⁄ GalNAc ⁄ GlcNAc 6-O-sulfotransferase; HEC, high endothelial cell; HEV, high endothelial venule; LSST, L-selectin sulfotransferase; ppGalNAcT, polypeptide galactosaminyltransferase; PSGL-1, P-selectin glycoprotein ligand-1; ST3Gal, a2,3 sialyltransferase; TPST, tyrosylprotein sulfotransferase.
Glycosyltransferases
Probably overlapping function of different ppGalNAcT
in the initiation of O-glycan biosynthesis
[51]
Core 1 b1,3-GalT Initiates the core 1 extension
MECA-79 recognizes GlcNAc-6-O-sulfate on core 1 branch
[52]
Sialylates core 1 extensions Competes with core 2 GlcNAcT-I for substrate
[53]
Core2 b1,6-GlcNAcT-I P- and L -selectin-dependent rolling strongly reduced in core 2
GlcNAcT-I–⁄ –during inflammation in vivo Regulates capture ligand for E-selectin during inflammation
No influence on E-selectin-dependent slow rolling velocity Lymphocyte homing to Peyer’s patches unaffected in core 2 GlcNAcT-I – ⁄ –
Reduced lymphocyte homing to peripheral lymph nodes of core 2 GlcNAcT-I–⁄ –
Reduced lymphocyte rolling on HEV of peripheral lymph nodes
in core 2 GlcNAcT-I – ⁄ –
Increased rolling velocity on HEV of peripheral lymph nodes in core 2 GlcNAcT-I–⁄ –
[36,42,43,54]
FucT-VII P- and E-selectin ligand-dependent rolling dramatically reduced in
FucT-VII – ⁄ –
during inflammation in vivo
L -selectin-dependent rolling almost completely absent in peripheral lymph node HEV of FucT-VII–⁄ –
[46]
P- and L-selectin ligand function unaffected in FucT-IV – ⁄ –
[27] ST3Gal-IV Influences slow E-selectin-dependent rolling velocity
P-selectin-dependent rolling unaffected in ST3Gal-IV – ⁄ –
[47] b1,4GalT-I Influence on leukocyte rolling unknown at present
Binding of soluble P-selectin to b1,4GalT-I – ⁄ – neutrophils impaired Normal lymphocyte homing to peripheral lymph nodes
Deficiency of b1,4GalT-I described in humans (CDG IId)
[44,55]
b1,4GalT-IV Influence on leukocyte rolling unknown at present
Acts specifically on core 2 linked GlcNAc 6-O-sulfate Participates in the synthesis of 6-sulfo sialyl Lewis x
[45]
Sulfotransferases
GlcNAc6ST-1
(also called GST-2 or CHST-2)
Moderate reduction in lymphocyte homing to peripheral lymph nodes Modest increase in rolling velocity of B- and T cells on HEV of peripheral lymph nodes
Overlapping and distinct function with GlcNAcT6ST-2 on L -selectin ligand activity in HEV of lymphoid tissue
Contributes to abluminal MECA-79 staining in HEV
[48,56]
GlcNAc6ST-2
(also called HEC-GlcNAc6ST,
GST-3, LSST and CHST-4)
Marked reduction in lymphocyte homing to peripheral lymph nodes Number of rolling cells on HEV not affected in GlcNAc6ST-2 – ⁄ –
Significant increase in rolling velocity in HEV of GlcNAc6ST-2 – ⁄ –
Reduced leukocyte adhesion in HEV of GlcNAc6ST-2 – ⁄ –
Highly restricted expression on HEV of lymphoid tissue and lymphoid-like aggregates of chronically inflamed tissue Not expressed on Peyer’s patch HEV
Overlapping and distinct function with GlcNAcT6ST-1 on L-selectin ligand activity in HEV of lymphoid tissue
crucial for MECA-79 reactivity on the luminal side of HEV
[57–59]
Trang 7which have been generated recently [42] Intravital
microscopy studies, conducted in untreated and TNF-a
pretreated cremaster muscle venules of core 2
Glc-NAcT-I deficient mice, revealed a dramatic reduction
in P- and L-selectin mediated rolling, and a less
pro-nounced reduction in E-selectin dependent rolling
[36,43] In contrast, leukocyte rolling was unchanged
in Peyer’s patch HEV, where rolling is predominantly
mediated by L-selectin and, to a lesser degree, by a4b7
-integrin and P-selectin [36], suggesting that core 2
GlcNAcT-I is dispensable for L-selectin ligand
func-tion on HEV This was confirmed, in part, by Yeh and
colleagues who identified 6-sulfo sLex on core 1
exten-ded O-glycans of core 2 GlcNAcT-I deficient mice [52]
Core 1 decorated 6-sulfo sLex serve, in collaboration
with core 2 decorated 6-sulfo sLex, as L-selectin
lig-ands on HEV [62] However, subsequent studies of
lymphocyte trafficking to peripheral lymph nodes of
core 2 GlcNAcT-I–⁄ – mice revealed a defect in B-cell
(and less pronounced in T-cell) homing, which
consis-ted of reduced B- and T-cell rolling on peripheral
lymph node HEV accompanied by increased rolling
velocities [54] The difference in B- and T-cell homing
observed in core 2 GlcNAcT-I–⁄ – mice was mainly
attributed to a lower expression of L-selectin on B
cells, which led to a further, functionally relevant,
decrease in L-selectin mediated interactions [54]
b1,4-GalT
To date, seven b1,4-GalT have been identified [63]
Two of them – b1,4-GalT-I and b1,4-GalT-IV – have
been implicated in the synthesis of functional selectin
ligands b1,4-GalT-I catalyzes the addition of
UDP-galactose to terminal N-acetylgalactosamine and acts
in concert with b1,3-N-acetyl-glucosaminyltransferase
to synthesize polylactosamine extensions of core 2
dec-orated O-glycans In addition, b1,4-GalT-I also
partici-pates in the generation of sLex Using b1,4-GalT-I
deficient mice, Asano and colleagues investigated the
contribution of b1,4-GalT-I on selectin ligand activity
They found that binding of soluble P-selectin to
neu-trophils and monocytes of b1,4-GalT-I– ⁄ – mice was
significantly impaired [44], suggesting a role of
b1,4-GalT-I in P-selectin mediated rolling in vivo Although
not formally investigated, a putative P-selectin depend-ent rolling defect in b1,4-GalT-I deficidepend-ent mice would
be sufficient to explain the observed increase in leuko-cyte and neutrophil counts, as well as the significant reduction of recruited neutrophils into zymosan treated earlobes [44] Lymphocyte homing to peripheral lymph nodes, which requires L-selectin ligand activity on HEVs, was not affected in the absence of b1,4-GalT-I, suggesting that b1,4-GalT-I does not contribute to the biosynthesis of HEV-expressed L-selectin ligands
in vivo [44] Recently, the first patient, a 16-month-old boy, with a deficiency in b1,4-GalT-I, has been des-cribed and was designated as having congenital defici-ency of glycosylation IId [55] The little boy suffers from mental retardation, Dandy-Walker malformation with hydrocephalus, myopathy and blood clotting problems [55]
Among the seven b1,4-GalTs, b1,4-GalT-IV is the only b1,4-GalT that specifically acts on core 2 linked 6-sulfo GlcNAc, which is further processed to 6-sulfo sLex [45], a carbohydrate determinant found on L-selectin ligands in HEVs of secondary lymphoid organs and crucial for binding to L-selectin Co-expression profiles of b1,4-GalT-IV and 6-sulfo sLex revealed no correlation in expression, suggesting that b1,4-GalT-IV is not rate limiting for the synthesis of 6-sulfo sLex[45]
Fucosyltransferases
Transfer of the monosaccaride fucose to core 2 decorated O-glycans is dependent on two a1,3-fucosyl-transferases, namely FucT-VII and FucT-IV [41] Expression of a1,3-fucosyltransferases (similarly to other glycosyltransferases) is primarily regulated at the transcriptional level Both FucT-VII and FucT-IV, are expressed in leukocytes FucT-VII has also been identi-fied in murine high endothelial cells of secondary lym-phoid organs, suggesting a role of FucT-VII in the synthesis of functional L-selectin ligands on HEV [64] Direct evidence for a role of FucT-VII and FucT-IV in selectin ligand function in vivo comes from intravital microscopy studies conducted in mice deficient in FucT-VII [46] and FucT-IV [27] FucT-VII–⁄ – mice, which have a significantly increased leukocyte count,
Table 2 (Continued).
TPST-1 and -2 Catalyze sulfation of crucial tyrosines at the N-terminus of PSGL-1
Important for P- and L -selectin ligand function Contribution of different TPSTs on leukocyte rolling unknown TPST-1 – ⁄ –
and TPST-2 – ⁄ –
with no reported defect in PSGL-1 function
[60]
Trang 8demonstrate an almost complete absence of leukocyte
rolling in inflamed venules of the ear and the cremaster
muscle, suggesting a dramatic reduction in E- and
P-selectin ligand function in FucT-VII–⁄ – mice
Leuko-cyte rolling in lymph node HEV of FucT-VII–⁄ – mice
was also dramatically impaired and accompanied by
small hypocellular lymph nodes and a severe defect in lymphocyte homing to secondary lymphoid organs [46] FucT-IV–⁄ –mice appear healthy and show leuko-cyte counts within the normal range Analysis of leu-kocyte rolling in inflamed venules of the ear revealed a similar number of rolling leukocytes when compared
Fig 2 L-selectin ligand activity on high endothelial venules (HEV) of secondary lymphoid organs is predominantly mediated by 6-sulfo sialyl Lewis X (sLe x ), which can be found as a capping group on core 2 extensions, core 1 extensions or on biantennary (core 2 and core 1) exten-sions.
Trang 9with control mice However, leukocyte rolling
veloci-ties were significantly increased, suggesting that
FucT-IV contributes to E-selectin dependent rolling, distinct
from FucT-VII [27]
Sialyltransferases
Sialylation was the first post-translational glycosylation
reported to be crucial for functional L-selectin ligands
on HEV [65] Subsequent studies identified the
tetrasac-charide sLexon selectin ligands to show binding affinity
to all three selectins Sialylation of Lex is catalyzed by
a2,3-sialyltransferases From the six different
a2,3-sial-yltransferases (ST3GalI-VI) described to date,
ST3Gal-IV, ST3Gal-VI and, to a lesser degree, ST3Gal-III,
transfer sialic acid residues to terminal galactose
resi-dues of type II oligosaccharides on core 2 decorated
O-glycans [66] Recently, mice deficient in ST3Gal-IV
have been generated [67] In vivo studies investigating
P- and E-selectin mediated leukocyte rolling in inflamed
cremaster muscle venules of ST3Gal-IV–⁄ – mice
revealed no defect in P-selectin dependent rolling [47]
However, E-selectin dependent leukocyte rolling
velo-city was significantly increased, with no defect in
E-selectin mediated leukocyte capture, suggesting that
ST3Gal-IV regulates E-selectin dependent rolling
velo-city while it does not affect the efficiency of E-selectin
to attract free flowing leukocytes to inflamed
endothe-lium [47] These results imply that PSGL-1, which
mediates P-selectin dependent rolling and functions as
a capture ligand for E-selectin, is not strictly dependent
on ST3Gal-IV, but may also be sialylated by another
a2,3-sialyltransferase, probably ST3Gal-VI
Although ST3Gal-I is not directly involved in the
synthesis of selectin ligands, it is worth mentioning
that ST3Gal-I may exhibit indirect influence on
selec-tin ligand function, and hence leukocyte rolling, by
competing with core 2 GlcNAcT-I for the same
substrate ST3Gal-I specifically catalyzes the
sialyla-tion of core 1 extensions
(NeuAca2,3Galb1,3GalNAc-Ser⁄ Thr) [68] In ST3Gal-I deficient mice, the
expres-sion of Galb1,3GalNAc-Ser⁄ Thr is significantly
increased [53] This is accompanied by strong
up-regu-lation of core 2 decorated O-glycans, which may lead
to enhanced binding of selectins to selectin ligands
[53]
Carbohydrate sulfotransferases
GlcNAc-6-O-sulfation of HEV-expressed L-selectin
lig-ands is an important post-translational modification,
leading to enhanced binding of L-selectin to its ligands
under in vitro and in vivo conditions [30] Five different
GlcNAc-6-O-sulfotransferases (GlcNAc6ST1-5) exist Two of them – GlcNAc6ST-1 and GlcNAc6ST-2 – contribute to the elaboration of 6-sulfo sLex (Fig 2), the most important sulfate modification of functional L-selectin ligands GlcNAc6ST-1, also known as Gal⁄ GalNAc ⁄ GlcNAc 6-O-sulfotransferase-2 or carbo-hydrate sulfotransferase-2, is broadly expressed and demonstrates some overlapping, as well as distinct, functions with GlcNAc6ST-2 [48,49] Mice deficient in GlcNAc6ST-1 show a moderate reduction in lympho-cyte homing to peripheral lymph nodes, mesenteric lymph nodes and Peyer’s patches [56] Intravital micro-scopy studies revealed no defect in lymphocyte rolling flux in HEV of peripheral lymph nodes However, roll-ing velocities of B- and T cells were modestly increased [48] Expression of GlcNAc6ST-2 (also known as HEC-GlcNAc6ST, Gal⁄ GalNAc ⁄ GlcNAc 6-O-sulfo-transferase-3, L-selectin sulfotransferase, and carbohy-drate sulfotransferase-4) is highly restricted to HEVs
of lymphoid tissue and lymphoid-like aggregates in chronically inflamed tissue [30,59] In contrast to Glc-NAc6ST-1, GlcNAc6ST-2 is not expressed on Peyer’s patch HEV: this may indicate a distinct role of Glc-NAc6ST-1 in the synthesis of functional selectin lig-ands on Peyer’s patch HEV GlcNAc6ST-2 leads predominantly to GlcNAc-6-O-sulfation of extended core 1 structures (Fig 2), which is recognized by mAb MECA-79 [52] Accordingly, absence of GlcNAc6ST-2 dramatically reduced the binding of MECA-79 to HEV Interestingly, MECA-79 staining in Glc-NAc6ST-2–⁄ – mice was only reduced at the luminal site Abluminal staining was found to be mainly dependent on GlcNAc6ST-1 [56] Functional assays revealed that lymphocyte homing was reduced by 50%
in GlcNAc6ST-2 deficient mice, whereas leukocyte roll-ing flux on HEV was not affected in GlcNAc6ST-2–⁄ – mice However, rolling velocities were significantly increased and accompanied by a marked reduction in leukocyte adhesion [69] To further investigate the con-tribution of sulfation on L-selectin ligand activity, mice deficient in GlcNAc6ST-1 and -2 have been generated recently [48,49] These mice showed a dramatic reduc-tion in lymphocyte homing to peripheral lymph nodes MECA-79 staining, as a reporter for PNAd activity, was completely absent Intravital analysis revealed that leukocyte rolling flux was significantly, but not com-pletely, reduced In addition, rolling velocity was substantially increased Residual leukocyte rolling observed in the double knockout mouse was com-pletely abolished by the addition of the L-selectin blocking mAb, MEL-14, suggesting that sulfation-independent L-selectin ligands (probably decorated by sLex) exist
Trang 10Tyrosylprotein sulfotransferases
In mice and humans, two tyrosylprotein
sulfotrans-ferases (TPST-1 and -2) have been identified to
medi-ate tryrosine O-sulfation [60] Tyrosine O-sulfation is
an important post-translational modification of critical
tyrosine residues at the N-terminus of PSGL-1, leading
to enhanced binding of P- and L-selectin to PSGL-1
[50] Functional studies revealed that both
tyrosyl-protein sulfotransferases contribute equally to the
sulf-ation of peptides modelled on the N-terminus of
PSGL-1 [70], suggesting a role for both enzymes in the
synthesis of functional PSGL-1 However,
investiga-tions in TPST-1–⁄ – or TPST-2–⁄ – mice have not
repor-ted any decrease in binding activity of P- or L-selectin
to PSGL-1, suggesting that either enzyme is able to
compensate for the loss of the other [71,72]
Conclusion
Leukocyte rolling is an important step in the
recruit-ment of leukocytes into tissue and has been considered
to be a rather nonspecific process, allowing leukocytes
to obtain intimate contact with the vascular wall
Dur-ing rollDur-ing, leukocytes have the opportunity to screen
the endothelial surface for specific trigger signals, which
brings about a decision for extravasation into tissue
Recent advancements in the elucidation of
post-transla-tional modifications relevant for selectin ligand
func-tion in vivo challenge this view and indicate that subtle
differences in the post-translational glycosylation⁄
sulfa-tion of endothelium- or leukocyte-expressed selectin
lig-ands might constitute an important early determinant
for the successful recruitment of leukocytes
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