SummaryAcute organ transplant rejection is characterized by a heavy lymphocyte infiltration. We have previously shown that alterations in the graft endothelium lead to increased lymphocyte traffic into the graft. Here, we demonstrate that lymphocytes adhere to the endothelium of rejecting cardiac transplants, but not to the endothelium of syngeneic grafts or normal hearts analyzed with the in vitro Stamper-Woodruff binding assay. Concomitant with the enhanced lymphocyte adhesion, the cardiac endothelium begins to de novo express sialyl Lewis a and sialyl Lewis x (sLea and sLex) epitopes, which have been shown to be sequences of L-selectin counterreceptors. The endothelium of allografts, but not that of syngeneic grafts or normal controls, also reacted with the L-selectin-immunoglobulin G fusion protein, giving further proof of inducible L-selectin counterreceptors. The lymphocyte adhesion to endothelium could be significantly decreased either by treating the iymphocytes with anti-L-selectin antibody HRL-1, or by treating the tissue sections with sialidase or anti-sLea or anti-sLex monoclonal antibodies. Finally, we synthetized enzymatically several members of the sLex family oligosaccharides and analyzed their ability to block lymphocyte adhesion to cardiac endothelium. The monovalent sLex (a tetramer), divalent sLex (a decamer), and tetravalent sLex (a 22-mer) could all significantly reduce lymphocyte binding, but the inhibition by the tetravalent sLex-construct was clearly superior to other members of the sLex family. The crucial control oligosaccharides, sialyl lactosamines lacking fucose but being otherwise similar to the members of sLex family, had no effect on lymphocyte binding.
Lymphocyte infiltration is a hallmark of acute rejections in solid organ transplants, such as cardiac allograft. We have previously shown that lymphocyte extravasation to cardiac grafts undergoing rejection is largely due to interactions between lymphocyte L-selectin and its sialyl Lewis x (sLex) decorated ligands. Our previous work demonstrated further that an enzymatically synthetized tetravalent sLex glycan of a branched polylactosamine backbone is a highly efficient inhibitor of L-selectin-dependent lymphocyte adhesion to graft endothelium. To improve the availability of multivalent sLex glycans for anti-inflammatory indications, we now report enzymatic synthesis of another tetravalent sLex glycan that can be potentially produced on a large scale, and show that even the new saccharide is a nanomolar inhibitor of L-selectin-dependent lymphocyte adhesion. The novel antagonist is sLex beta 1-3' (sLex beta 1-6') LacNAc beta 1-3' (sLex beta 1-6') LacNAc beta 1-3' (sLex beta 1-6') LacNAc (8) (where LacNAc is the disaccharide Gal beta 1-4GlcNac and sLex is the tetrasaccharide Neu5Ac alpha 2-3Gal beta 1-4 (Fuc alpha 1-3) GlcNAc). Its five-step synthesis was started from the octameric polylactosamine LacNAc beta 1-3' (GlcNAc beta 1-6') LacNAc beta 1-3' (GlcNAc beta 1-6') LacNAc (3), which in turn is accessible in one step from the hexasaccharide LacNAc beta 1-3'LacNAc beta 1-3'LacNAc. Importantly, the hexasaccharide primer has been synthesized chemically (Alais and Veyrieres, Tetrahedron Lett., 24, 5223, 1983). Hence, our data outline a route to glycan 8, consisting of a combination of chemical and enzymatic methods of oligosaccharide synthesis. In addition, our data show that polylactosamine backbones are able to present multiple sialyl Lewis x determinants to L-selectin in high-affinity mode, without a requirement for uniqueness in the backbone structure.
The recognition of cell-surface L-selectin by its carbohydrate ligands causes lymphocytes to roll on capillary endothelium at sites of inflammation. As this primary contact is a prerequisite for extravasation of the leukocytes to the tissue, its inhibition by frce oligosaccharides capable of competing with the natural L-selectin ligands is an attractive therapeutic possibility.The exact structures of the biological ligands of L-selectin are not yet known, but the principal carbohydrate epitopcs share some structural features : they are 0-glycosidically linked mucin-type oligosaccharides with N-acetyllactosamine backbone, which is 3'-sialylated or 3'-sulfated, 3-fucosylated and sometimes 6-or 6'hulfated at the distal N-acetyllactosamine termini. Multivalency of the ligand, which is believed to enhance the binding, is achieved by a branched polylactosamine backbone or by a clustered array of 0-glycans.We report here enzymic synthesis of a large oligosaccharide fulfilling several of the features characteristic to the L-selectin ligands : it is a dodecameric 0-glycosidic core-2-type oligosaccharidc alditol with a branched polylactosamine backbone carrying two distal m-2,3'-sialylated and a-1,3-fucosylated N-acetyllactosamine groups (sialyl Lewis x, sialyl Le"). The structure of each saccharide on the synthesis route from disaccharide Galj31-3GalNAc to the dodecasaccharide alditol was established by several methods including one-and two-dimensional 'H-NMR spectroscopy. The last step of the synthesis, the rx-l,3-fucosylation of the 6-linked arm proceeded sluggishly, and was associated with a noticeable shift in H i resonance of the GlcNAc residue of the branch-bearing N-acetyllactosamine unit.The final synthesis product and its analogs lacking one or both of the fucose residues were tested as inhibitors of L-selectin-mediated lymphocyte-cndothelium interaction in vitro in rejecting rat kidney transplant. While the non-fucosylated 0-glycosidic oligosaccharide alditol did not possess any inhibitory activity, the mono-fucosylated one (i.e. monovalent sialyl Le") prevented the binding significantly and the difucosylated dodecasaccharide alditol (i.e. divalent sialyl Le") was a very potent inhibitor (IC,n, inhibitory concentration preventing 50% of binding = 0.3 5 pM). Besides the multivalency, also the GalPl-3GalNAc-ol sequence of the 0-glycosidic core appeared to increase the affinity of the glycan to L-selectin. This was indicated by parallel inhibition experiments, where a disialylated and difucosylated branched polylactosamine decasaccharide, similar to the divalent dodecasaccharide alditol, but lacking the reduced 0-glycosidic core, was a less effective inhibitor (TCso = 0.5 pM) than the 0-glycosidic dodecasaccharide alditol.Krywords: sialyl Lewis x; NMR; in vitro synthesis; 0-linked saccharide; L-selectin.The current model of adhesion and extravasation of lymphocytes from the blood stream into a site of intlammation is a multistep process, which is initiated by rolling and tethering of lymphocytes on the endothelium...
Kidney transplant rejection is an inflammatory process characterized by lymphocyte infiltration. Our earlier observations have shown that peritubular capillary endothelium (PTCE) is the site of lymphocyte entry into the rejecting renal allograft. During rejection, PTCE begins to express sialyl Lewis x de novo, and binds lymphocytes by a mechanism largely dependent on L-selectin. Hence, inhibiting the lymphocyte-endothelial interaction with oligosaccharide ligands of L-selectin offers an attractive possibility to prevent the inflammation and rejection. Here, we report enzyme-assisted synthesis of N-acetyllactosamine-based tetra-, deca-, and docosameric saccharides carrying one, two or four distally located sialyl Lewis x groups [Neu-NAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc] (sLex), respectively. When tested for their ability to inhibit lymphocyte-endothelial interaction during rat kidney transplant rejection, all sLex-saccharides were inhibitors in the Stamper-Woodruff binding assays; the analogues lacking fucose showed no inhibitory potency. The tetravalent sLex glycan proved to be a high-affinity adhesion inhibitor with an IC50 < 50 nM. While less powerful than the tetravalent glycan, also the divalent sLex saccharide was a much better inhibitor than the monovalent glycan. Hence, increasing multivalency and, possibly, increasing chain length of the polylactosamine backbone, enhances the inhibitory potency of sLex bearing glycans in the lymphocyte-endothelial adhesion assay. This suggests that L-selectin behaves as a "functional oligomer" on lymphocyte surfaces.
Kidney allograft rejection is an inflammatory process dominated by lymphocytes. During rejection lymphocytes preferentially adhere to the peritubular capillary endothelium (PTCE), which acquires morphological features common to high endothelium. These observations indicate that PTCE is the site of lymphocyte entry into the rejecting renal allograft. Of the identified endothelial adhesion molecules, ICAM-1 was already expressed on the endothelium of normal kidneys, and its expression was strongly enhanced during rejection without site-specific restriction. VCAM-1 was not expressed on the endothelium of normal or syngeneic kidneys, but its expression was induced during allograft rejection not only in PTCE, but occasionally also on the endothelium of larger vessels. Sialyl Lewisx (sLex) showed a very restricted pattern of expression; endothelium was sLex-negative both in control and syngeneic kidneys. On the other hand, PTCE reacted strongly with anti-sLex antibody in allografts. When kidney frozen sections were treated with sialidase the binding of lymphocytes decreased by 70%. Low-dose chymotrypsin treatment of lymphocytes, known to remove L-selectin from the lymphocyte surface, decreased their binding to PTCE by 60%. Likewise lymphocyte adhesion to PTCE was inhibited by 70% by anti-sLex- and anti-L-selectin-antibodies and by sLex tetrasaccharide. Finally PTCE in the allografts, but not in syngeneic grafts or normal kidneys, bound an L-selectin-IgG fusion protein, indicating that ligands for L-selectin were induced during rejection.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.