Selectins and glycosyltransferases in leukocyte rolling in vivo

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1 MINIREVIEW Selectins and glycosyltransferases in leukocyte rolling in vivo Markus Sperandio University Children s Hospital Heidelberg, Division of Neonatal Medicine, University of Heidelberg, Germany Keywords glycosylation; glycosyltransferases; leukocyte rolling; selectin ligand; selectin Correspondence M. Sperandio, Children s Hospital, Division of Neonatal Medicine, University of Heidelberg, INF 150, Heidelberg, Germany Fax: Tel: markus.sperandio@med. uni-heidelberg.de (Received 15 May 2006, accepted 3 July 2006) doi: /j x Leukocyte rolling is an important step for the successful recruitment of leukocytes into tissue and occurs predominantly in inflamed microvessels and in high endothelial venules of secondary lymphoid organs. Leukocyte rolling is mediated by a group of C-type lectins, termed selectins. Three different selectins have been identified 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 conditions. 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 instrumental 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-fucosyltransferases and a2,3-sialyltransferases have been described to participate in the synthesis of core 2 decorated O-glycan structures carrying the tetrasaccharide 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. Leukocyte recruitment is a crucial immunological process 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, beginning 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 chemokines the opportunity to bind to their respective chemokine receptors on leukocytes. This triggers the activation of integrins, leading to firm leukocyte adhesion to the endothelium and transmigration into tissue [3]. A detailed online illustration of the leukocyte recruitment cascade can be found at bme.virginia.edu/ley/ Leukocyte rolling Leukocyte rolling is mediated by selectins and is considered an important step for the successful recruitment of 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 galactosaminyltransferase; PSGL-1, P-selectin glycoprotein ligand-1; sle x, sialyl Lewis X; ST3Gal, a2,3 sialyltransferase; TNF-a, tumor necrosis factor-a; TPST, tyrosylprotein sulfotransferase. FEBS Journal 273 (2006) ª 2006 The Author Journal compilation ª 2006 FEBS 4377

2 Leukocyte rolling and glycosyltransferases M. Sperandio leukocytes 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]. Accordingly, the characteristic feature of selectins is their ability to recognize and bind to specific carbohydrate determinants on selectin ligands in a calcium-dependent manner [5]. Binding takes place under dynamic conditions, where continuous shear forces, exerted by the flowing blood, act on leukocytes rolling along the endothelium at rolling velocities 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 following three requirements (a) rapid bond formation at the leading edge, (b) high tensile strength during binding and (c) fast dissociation rates. Interestingly, recent reports have revealed that selectin binding duration (bond lifetime) adjusts to increasing forces by decreasing off-rates [i.e. the bond locks more tightly when blood flow is increased (catch bonds)]. This enables leukocytes 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 flowing leukocytes to the endothelial wall and prepares them (during rolling) for subsequent adhesion and transmigration. Functionally, leukocyte rolling serves two main purposes. First, leukocyte rolling participates in the successful recruitment of neutrophils, monocytes, eosinophils, 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 endothelial L-selectin ligands on inflamed endothelium. In resting 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 mobilized to the cell surface [9]. P-selectin is the predominant leukocyte rolling receptor on acutely inflamed endothelial cells in vivo. This has been demonstrated by intravital microscopy studies of inflamed mouse cremaster muscle venules from P-selectin deficient mice, where leukocyte rolling was almost completely absent shortly after exteriorization of the cremaster muscle [10]. Except for skin microvessels, E-selectin is not constitutively expressed on resting vascular 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 addition, E-selectin co-operates with the chemokine receptor 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-selectin 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 homodimeric 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 conditions. Recently, CD44 and CD43 have been proposed to be functionally relevant E-selectin ligands. Katayama and colleagues showed that 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 contribute 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 mediated leukocyte rolling occurs. Similarly to a1,3-fucosyltransferase (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 capillaries, 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) FEBS Journal 273 (2006) ª 2006 The Author Journal compilation ª 2006 FEBS

3 M. Sperandio Leukocyte rolling and glycosyltransferases 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. Selectin ligand Expression Function During inflammation PSGL-1 CD44 During lymphocyte homing MAdCAM-1 PNAd (GlyCAM-1, CD34, podocalyxin and endomucin) Other selectin ligands identified under in vitro conditions with no proven relevance for leukocyte rolling in vivo CD24 CD43 ESL-1 Most leukocytes, chronically inflamed endothelium in a spontaneous model of chronic ileitis Expressed on leukocytes, erythrocytes and in the brain Constitutive expression in Peyer s patch HEV and in intestinal lamina propria vessels; induced expression in chronically inflamed venules Constitutive expression in HEV of peripheral lymph nodes; induced expression in chronically inflamed venules Different tumor cells, neutrophils, B lymphocytes, immature thymocytes, erythrocytes Expressed on most hematopoietic cells Low expression on neutrophil surface, but abundantly expressed in the Golgi apparatus 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] 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] Mediates L-selectin-dependent leukocyte rolling in Peyer s patch HEV [22] The only relevant L-selectin homing ligand in vivo identified at present 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 Mediates tumor metastasis in different mouse models Mediates P-selectin-dependent tumor cell rolling in vitro Mediates E-selectin-dependent rolling in vitro [25] Supports leukocyte rolling in vitro Heparin derivatives Ubiquitously expressed Contribution to leukocyte rolling in vivo unknown Versican Renal tubular cells Binds to L-selectin in vitro Nucleolin Weakly expressed on leukocyte surface Binds to L-selectin in static in vitro assays [26] human 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 endothelial 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 ligand in vivo remains to be determined. FEBS Journal 273 (2006) ª 2006 The Author Journal compilation ª 2006 FEBS 4379

4 Leukocyte rolling and glycosyltransferases M. Sperandio L-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 activity 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 endothelial 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 intestine and on mesenteric lymph node HEV in a spontaneous 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 transmigration 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 recruitment in response to chemokines and chemotactic factors in the mouse cremaster muscle [34]. The authors found that superfusion of keratinocyte-derived chemokine or platelet-activating factor over the cremaster muscle of L-selectin-deficient mice did not alter leukocyte 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 during inflammation, the second major purpose of leukocyte rolling involves the successful exit of T- and B lymphocytes from HEV into the parenchyma of secondary lymphoid organs. Leukocyte rolling on HEV is almost exclusively mediated by L-selectin and an essential step for the effective transmigration of lymphocytes into secondary lymphoid organs [35]. L-selectin is expressed on the microtips of most leukocytes, including all myeloid cells, naïve T- and B cells, as well as some activated T cells and memory T cells. Therefore, leukocyte rolling on HEV is not restricted to lymphocytes but also involves other leukocyte populations. This explains the observation that more than 50% of leukocytes passing through HEVs of secondary lymphoid organs are rolling [36]. It is obvious that most rolling leukocytes will eventually detach from the surface of HEV and return into free flow because they lack the proper signals from specific chemokines necessary 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 chemokine receptors, which then interact with their respective chemokines immobilized on the surface of high endothelial 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 glycosylationdependent cell adhesion molecule-1, CD34, sgp200, HEV-expressed podocalyxin, and a recently identified glycoprotein called endomucin (Table 1) [39]. To further investigate the contribution of the different PNAd members for selectin ligand function in vivo, lymphocyte homing was investigated in glycosylation-dependent cell adhesion molecule-1 mice that demonstrated normal lymphocyte trafficking [23]. Similarly, CD34 mice had no defect in lymphocyte trafficking [24] suggesting 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 FEBS Journal 273 (2006) ª 2006 The Author Journal compilation ª 2006 FEBS

5 M. Sperandio Leukocyte rolling and glycosyltransferases Post-translational glycosylation of selectin ligands Selectin ligands belong to a growing number of glycoproteins where protein function is closely linked to its proper post-translational glycosylation. Posttranslational 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 transfer 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 glycosyltransferase recognizes only one type of sugar nucleotide. Furthermore, transfer of the sugar nucleotide is restricted to specific acceptor molecules and glycosidic bonds formed. Additional factors, such as the expression level of specific glycosyltransferases and the location 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 carrying the sialyl Lewis X (sle x ) 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 modifications have been described to contribute to selectin ligand function (Table 2). Two enzymes catalyzing carbohydrate sulfation [N-acetylglucosamine 6-O-sulfotransferase (GlcNAc6ST)-1 and -2] were found to be involved in the generation of 6-sulfo sle x 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 significantly influence binding of selectins to PSGL-1 (Fig. 1) [50]. Figure 1 gives an overview on the biosynthetic pathway of core 2 modified O-glycans terminated with sle x. O-glycan biosynthesis is initiated with the addition 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 ratelimiting 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 Glc- NAcT-I) initiates the core 2 extension by adding Glc- NAc to GalNAc 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 polylactosamine chain. Elongation of core 2 branches is terminated by the addition of sialic acid, in a2,3 linkage, to galactose (Fig. 1). This is followed by the addition of fucose to the penultimate GlcNAc, in a1,3 linkage, resulting in the formation of sle x 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 vivo are discussed. 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 glycoprotein 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. 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 initiates the core 2 extension (Fig. 1). Direct evidence that core 2 GlcNAcT-I is important for leukocyte rolling in vivo comes from mice deficient in core 2 GlcNAcT-I, FEBS Journal 273 (2006) ª 2006 The Author Journal compilation ª 2006 FEBS 4381

6 Leukocyte rolling and glycosyltransferases M. Sperandio Table 2. Enzymes involved in the post-translational modification of selectin ligands. CDG, congenital deficiency of glycosylation; CHST-2, carbohydrate sulfotransferase 2; core 2 GlcNAcT, core 2 b1,6 N-acetylglucosaminyltransferase; FucT, a1,3 fucosyltransferase; GalT, galactosyltransferase; GlcNAc6ST, N-acetylglucosamine 6-O-sulfotransferase; 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. Suspected identified leukocyte rolling defect Reference Glycosyltransferases ppgalnact Core 1 b1,3-galt ST3Gal-I Influence on leukocyte rolling unknown at present Probably overlapping function of different ppgalnact in the initiation of O-glycan biosynthesis Initiates the core 1 extension MECA-79 recognizes GlcNAc-6-O-sulfate on core 1 branch Indirect influence on leukocyte rolling Sialylates core 1 extensions Competes with core 2 GlcNAcT-I for substrate 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 FucT-VII FucT-IV ST3Gal-IV b1,4galt-i b1,4galt-iv Sulfotransferases GlcNAc6ST-1 (also called GST-2 or CHST-2) GlcNAc6ST-2 (also called HEC-GlcNAc6ST, GST-3, LSST and CHST-4) 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 P- and E-selectin ligand-dependent rolling dramatically reduced in [46] FucT-VII during inflammation in vivo L-selectin-dependent rolling almost completely absent in peripheral lymph node HEV of FucT-VII Influences slow E-selectin-dependent rolling velocity [27] P- and L-selectin ligand function unaffected in FucT-IV Influences slow E-selectin-dependent rolling velocity [47] P-selectin-dependent rolling unaffected in ST3Gal-IV Influence on leukocyte rolling unknown at present [44,55] 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) Influence on leukocyte rolling unknown at present [45] Acts specifically on core 2 linked GlcNAc 6-O-sulfate Participates in the synthesis of 6-sulfo sialyl Lewis x 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 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 [51] [52] [53] [36,42,43,54] [48,56] [57 59] 4382 FEBS Journal 273 (2006) ª 2006 The Author Journal compilation ª 2006 FEBS

7 M. Sperandio Leukocyte rolling and glycosyltransferases Table 2. (Continued). TPST-1 and -2 Suspected identified leukocyte rolling defect 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 Reference [60] which 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 pronounced 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 a 4 b 7 - integrin and P-selectin [36], suggesting that core 2 GlcNAcT-I is dispensable for L-selectin ligand function on HEV. This was confirmed, in part, by Yeh and colleagues who identified 6-sulfo sle x on core 1 extended O-glycans of core 2 GlcNAcT-I deficient mice [52]. Core 1 decorated 6-sulfo sle x serve, in collaboration with core 2 decorated 6-sulfo sle x, as L-selectin ligands 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 consisted 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]. not formally investigated, a putative P-selectin dependent rolling defect in b1,4-galt-i deficient mice would be sufficient to explain the observed increase in leukocyte 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 described and was designated as having congenital deficiency 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 sle x [45], a carbohydrate determinant found on L-selectin ligands in HEVs of secondary lymphoid organs and crucial for binding to L-selectin. Coexpression profiles of b1,4-galt-iv and 6-sulfo sle x revealed no correlation in expression, suggesting that b1,4-galt-iv is not rate limiting for the synthesis of 6-sulfo sle x [45]. 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 UDPgalactose to terminal N-acetylgalactosamine and acts in concert with b1,3-n-acetyl-glucosaminyltransferase to synthesize polylactosamine extensions of core 2 decorated O-glycans. In addition, b1,4-galt-i also participates in the generation of sle x. 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 neutrophils 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 Fucosyltransferases Transfer of the monosaccaride fucose to core 2 decorated O-glycans is dependent on two a1,3-fucosyltransferases, 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 identified in murine high endothelial cells of secondary lymphoid 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, FEBS Journal 273 (2006) ª 2006 The Author Journal compilation ª 2006 FEBS 4383

8 Leukocyte rolling and glycosyltransferases M. Sperandio 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) extensions. demonstrate 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. Leukocyte 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 leukocyte counts within the normal range. Analysis of leukocyte rolling in inflamed venules of the ear revealed a similar number of rolling leukocytes when compared 4384 FEBS Journal 273 (2006) ª 2006 The Author Journal compilation ª 2006 FEBS

9 M. Sperandio Leukocyte rolling and glycosyltransferases with control mice. However, leukocyte rolling velocities 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 tetrasaccharide sle x on selectin ligands to show binding affinity to all three selectins. Sialylation of Le x is catalyzed by a2,3-sialyltransferases. From the six different a2,3-sialyltransferases (ST3GalI-VI) described to date, ST3Gal- IV, ST3Gal-VI and, to a lesser degree, ST3Gal-III, transfer sialic acid residues to terminal galactose residues 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 velocity was significantly increased, with no defect in E-selectin mediated leukocyte capture, suggesting that ST3Gal-IV regulates E-selectin dependent rolling velocity while it does not affect the efficiency of E-selectin to attract free flowing leukocytes to inflamed endothelium [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 selectin ligand function, and hence leukocyte rolling, by competing with core 2 GlcNAcT-I for the same substrate. ST3Gal-I specifically catalyzes the sialylation of core 1 extensions (NeuAca2,3Galb1,3GalNAc- Ser Thr) [68]. In ST3Gal-I deficient mice, the expression of Galb1,3GalNAc-Ser Thr is significantly increased [53]. This is accompanied by strong up-regulation 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 ligands 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 sle x (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 carbohydrate 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 lymphocyte homing to peripheral lymph nodes, mesenteric lymph nodes and Peyer s patches [56]. Intravital microscopy studies revealed no defect in lymphocyte rolling flux in HEV of peripheral lymph nodes. However, rolling velocities of B- and T cells were modestly increased [48]. Expression of GlcNAc6ST-2 (also known as HEC-GlcNAc6ST, Gal GalNAc GlcNAc 6-O-sulfotransferase-3, L-selectin sulfotransferase, and carbohydrate 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 ligands 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 rolling 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 contribution 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 reduction 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 completely, reduced. In addition, rolling velocity was substantially increased. Residual leukocyte rolling observed in the double knockout mouse was completely abolished by the addition of the L-selectin blocking mab, MEL-14, suggesting that sulfationindependent L-selectin ligands (probably decorated by sle x ) exist. FEBS Journal 273 (2006) ª 2006 The Author Journal compilation ª 2006 FEBS 4385

10 Leukocyte rolling and glycosyltransferases M. Sperandio Tyrosylprotein sulfotransferases In mice and humans, two tyrosylprotein sulfotransferases (TPST-1 and -2) have been identified to mediate 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 tyrosylprotein sulfotransferases contribute equally to the sulfation 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, investigations in TPST-1 or TPST-2 mice have not reported 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 recruitment of leukocytes into tissue and has been considered to be a rather nonspecific process, allowing leukocytes to obtain intimate contact with the vascular wall. During rolling, 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-translational modifications relevant for selectin ligand function in vivo challenge this view and indicate that subtle differences in the post-translational glycosylation sulfation of endothelium- or leukocyte-expressed selectin ligands might constitute an important early determinant for the successful recruitment of leukocytes. References 1 Springer TA (1995) Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu Rev Physiol 57, Butcher EC (1991) Leukocyte-endothelial cell recognition Three (or more) steps to specificity and diversity. Cell 67, Hamann A & Engelhardt B (2005) Leukocyte Trafficking. Wiley-VCH, Weinheim, Germany. 4 Zelensky AN & Gready JE (2005) The C-type lectin-like domain superfamily. FEBS J 272, Vestweber D & Blanks JE (1999) Mechanisms that regulate the function of the selectins and their ligands. Physiol Rev 79, Marshall BT, Long M, Piper JW, Yago T, McEver RP & Zhu C (2003) Direct observation of catch bonds involving cell-adhesion molecules. Nature 423, Yago T, Wu J, Wey CD, Klopocki AG, Zhu C & McEver RP (2004) Catch bonds govern adhesion through L-selectin at threshold shear. J Cell Biol 166, Smith ML, Smith MJ, Lawrence MB & Ley K (2002) Viscosity-independent velocity of neutrophils rolling on p-selectin in vitro or in vivo. Microcirculation 9, Sperandio M & Ley K (2005) The physiology and pathophysiology of P-selectin. Mod Asp Immunobiol 15, Ley K, Bullard DC, Arbones ML, Bosse R, Vestweber D, Tedder TF & Beaudet AL (1995) Sequential contribution of L- and P-selectin to leukocyte rolling in vivo. J Exp Med 181, Bevilacqua MP, Stengelin S, Gimbrone MA Jr & Seed B (1989) Endothelial leukocyte adhesion molecule-1: An inducible receptor for neutrophils related to complement regulatory proteins and lectins. Science 243, Kraiss LW, Alto NM, Dixon DA, McIntyre TM, Weyrich AS & Zimmerman GA (2003) Fluid flow regulates E-selectin protein levels in human endothelial cells by inhibiting translation. J Vasc Surg 37, Ley K (2001) Pathways and bottlenecks in the web of inflammatory adhesion molecules and chemoattractants. Immunol Rev 24, Smith ML, Olson TS & Ley K (2004) CXCR2- and E-selectin-induced neutrophil arrest during inflammation in vivo. J Exp Med 200, Yang J, Hirata T, Croce K, Merrill-Skoloff G, Tchernychev B, Williams E, Flaumenhaft R, Furie B & Furie BC (1999) Targeted gene disruption demonstrates that PSGL-1 is required for P-Selectin mediated but not E-Selectin mediated neutrophil rolling and migration. J Exp Med 190, Xia L, Sperandio M, Yago T, McDaniel JM, Cummings RD, Pearson-White S, Ley K & McEver RP (2002) P-selectin glycoprotein ligand-1-deficient mice have impaired leukocyte tethering to E-selectin under flow. J Clin Invest 109, Mayadas TN, Johnson RC, Rayburn H, Hynes RO & Wagner DD (1993) Leukocyte rolling and extravasation are severely compromised in P selectin-deficient mice. Cell 74, Sperandio M, Smith ML, Forlow SB, Olson TS, Xia L, McEver RP & Ley K (2003) P-selectin glycoprotein ligand-1 mediates L-selectin-dependent leukocyte rolling in venules. J Exp Med 197, Rivera-Nieves J, Burcin TL, Olson TS, Morris MA, McDuffie M, Cominelli F & Ley K (2006) Critical role of endothelial P-selectin glycoprotein ligand 1 in chronic murine ileitis. J Exp Med 203, Katayama Y, Hidalgo A, Chang J, Peired A & Frenette PS (2005) CD44 is a physiological E-selectin ligand on neutrophils. J Exp Med 201, FEBS Journal 273 (2006) ª 2006 The Author Journal compilation ª 2006 FEBS

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