Recombinant L-selectin Research Articles

Human Synovial Lubricin Expresses Sialyl Lewis x Determinant and Has L-selectin Ligand Activity

Lubricin (or proteoglycan 4 (PRG4)) is an abundant mucin-like glycoprotein in synovial fluid (SF) and a major component responsible for joint lubrication. In this study, it was shown that O-linked core 2 oligosaccharides (Galβ1-3(GlcNAcβ1-6)GalNAcα1-Thr/Ser) on lubricin isolated from rheumatoid arthritis SF contained both sulfate and fucose residues, and SF lubricin was capable of binding to recombinant L-selectin in a glycosylation-dependent manner. Using resting human polymorphonuclear granulocytes (PMN) from peripheral blood, confocal microscopy showed that lubricin coated circulating PMN and that it partly co-localized with L-selectin expressed by these cells. In agreement with this, activation-induced shedding of L-selectin also mediated decreased lubricin binding to PMN. It was also found that PMN recruited to inflamed synovial area and fluid in rheumatoid arthritis patients kept a coat of lubricin. These observations suggest that lubricin is able to bind to PMN via an L-selectin-dependent and -independent manner and may play a role in PMN-mediated inflammation.

Binding of L-selectin to its vascular and extravascular ligands is differentially regulated by pH

Ligands for L-selectin, a leukocyte adhesion molecule, are expressed in high endothelial venules (HEVs) in lymph nodes and extravascular tissues, such as renal tubules. Here, we report that the binding of L-selectin to its vascular and extravascular ligands is differentially regulated by pH. The optimal L-selectin-dependent binding of leukocytes to HEVs was observed at pH 7.4, a physiological pH in the blood. In contrast, the optimal binding of leukocytes to the renal tubules was observed at pH 5.6. Consistently, optimal binding of soluble recombinant L-selectin to a major vascular ligand, 6-sulfo sialyl Lewis X, was observed at pH 7.4. Binding to extravascular ligands, such as chondroitin sulfate (CS) B, CS E and heparan sulfate, occurred at pH 5.6. Under physiological shear stress ranging from 1 to 2dynes/cm2, maximal leukocyte rolling on vascular ligands was observed at pH 6.8 to 7.4, and no rolling was detected at pH conditions below 5.6. These findings suggest that the pH environment is one important factor that determines leukocyte trafficking under physiological and pathological conditions.

Human L-selectin preferentially binds synthetic glycosulfopeptides modeled after endoglycan and containing tyrosine sulfate residues and sialyl Lewis x in core 2 O-glycans

Endoglycan is a mucin-like glycoprotein expressed by endothelial cells and some leukocytes and is recognized by L-selectin, a C-type lectin important in leukocyte trafficking and extravasation during inflammation. Here, we show that recombinant L-selectin and human T lymphocytes expressing L-selectin bind to synthetic glycosulfopeptides (GSPs). These synthetic glycosulfopeptides contain 37 amino acid residues modeled after the N-terminus of human endoglycan and contain one or two tyrosine sulfates (TyrSO(3)) along with a nearby core-2-based Thr-linked O-glycan with sialyl Lewis x (C2-SLe(x)). TyrSO(3) at position Y118 was more critical for binding than at Y97. C2-SLe(x) at T124 was required for L-selectin recognition. Interestingly, under similar conditions, neither L-selectin nor T lymphocytes showed appreciable binding to the sulfated carbohydrate epitope 6-sulfo-SLe(x). P-selectin also bound to endoglycan-based GSPs but with lower affinity than toward GSPs modeled after PSGL-1, the physiological ligand for P- and L-selectin that is expressed on leukocytes. These results demonstrate that TyrSO(3) residues in association with a C2-SLe(x) moiety within endoglycan and PSGL-1 are preferentially recognized by L-selectin.

Sulfation of L-Selectin Ligands by an HEV-Restricted Sulfotransferase Regulates Lymphocyte Homing to Lymph Nodes

Lymphocytes home to lymph nodes, using L-selectin to bind specific ligands on high endothelial venules (HEV). In vitro studies implicate GlcNAc-6-sulfate as an essential posttranslational modification for ligand activity. Here, we show that genetic deletion of HEC-GlcNAc6ST, a sulfotransferase that is highly restricted to HEV, results in the loss of the binding of recombinant L-selectin to the luminal aspect of HEV, elimination of lymphocyte binding in vitro, and markedly reduced in vivo homing. Reactivity with MECA 79, an adhesion-blocking mAb that stains HEV in lymph nodes and vessels in chronic inflammatory sites, is also lost from the luminal aspects of HEV. These results establish a critical role for HEC-GlcNAc6ST in lymphocyte trafficking and suggest it as an important therapeutic target.

Identification of podocalyxin-like protein as a high endothelial venule ligand for L-selectin: parallels to CD34.

The leukocyte adhesion molecule, L-selectin, mediates the recruitment of lymphocytes to secondary lymphoid organs via interactions with specific ligands presented on high endothelial venules (HEV). Although the HEV-derived ligands for L-selectin are still incompletely defined, they share a common sialomucin-like structure which is thought to present clustered oligosaccharides to the lectin domain of L-selectin. Podocalyxin-like protein (PCLP) is a transmembrane sialomucin that is similar in structure to the well-characterized L-selectin ligand CD34. PCLP has been shown previously to be expressed on the foot processes of podocytes in the kidney glomerulus as well as on vascular endothelium at some sites. We have determined that PCLP is present on HEV, where it binds to both recombinant L-selectin and the HEV-specific monoclonal antibody MECA-79. Furthermore, purified HEV-derived PCLP is able to support the tethering and rolling of lymphocytes under physiological flow conditions in vitro. These results suggest a novel function for PCLP as an adhesion molecule and allow the definition of conserved structural features in PCLP and CD34, which may be important for L-selectin ligand function.

Use of receptors immobilized on microspheres to identify ligand binding sites in tissue sections: detection of lymph node ligands for L-Selectin.

Particulate microspheres bearing immobilized probes can be used to identify ligands expressed by cells and require only brightfield microscopy for detection. There are distinct advantages to using microspheres to detect low affinity interactions; microspheres require no secondary amplification or detection procedures subsequent to the binding interaction, reducing opportunities for detachment of bound probe, and concentrating probes on microspheres may greatly increase binding avidity. Selectin leukocyte-endothelial adhesion molecules undergo low affinity binding to ligands, and these interactions may be difficult to detect with standard techniques. The aim of this study was to determine if immobilizing recombinant L-Selectin on microspheres would facilitate detection of specific tissue ligands. Microspheres were incubated with sections of rabbit peripheral lymph node in a modified Stamper-Woodruff assay, and binding was assessed by brightfield microscopy. L-Selectin-IgG microspheres bound to high endothelial venules, known to be sites of expression for L-Selectin ligands. Specificity was indicated by the lack of binding of microspheres coated with control protein, and inhibition of binding by antibody to L-Selectin and by competitive antagonists of L-Selectin ligand interactions.

Characterization of a 180 kDa molecule apparently reactive with recombinant L-selectin.

In the present study we identified a 180 kDa molecule (p180) in rat lymph nodes (LN) apparently reactive with silkworm derived recombinant L-selectin (LEC-IgG) in a Ca(2+)-dependent manner. Analysis of amino acid sequence revealed that p180 has a strong homology to the macrophage mannose receptor (MMR), which was corroborated by the observation that p180 reacted with polyclonal anti-alveolar MMR antibody and mannosyl-BSA-agarose. In agreement with this notion, the binding of p180 to the silkworm LEC-IgG was inhibited by alpha-methyl-D-mannoside. However, in sharp contrast to its reactivity against the silkworm LEC-IgG, p180 failed to bind LEC-IgG produced by COS-7 cells, suggesting that p180 reacted with the silkworm LEC-IgG through the recognition of oligomannose-type oligosaccharides expressed on the silkworm products and that the lectin activity of L-selectin was not involved in the interaction. These results, together with the immunohistochemical studies showing that p180 was absent from the majority of high endothelial venules (HEV) but present in medullary macrophages, led us to conclude that p180 obtained from LN lysates by the use of the silkworm LEC-IgG is not a physiological ligand for L-selectin, warning against the use of recombinant proteins expressed in the baculovirus/ silkworm expression system for the detection of carbohydrate ligands.

Cloning of the cDNA for Rabbit L-Selectin and Expression of Recombinant Protein with a Kinase Target Site to Facilitate Radiolabeling

The cDNA encoding rabbit L-Selectin has been cloned from a cDNA library, utilizing a PCR-derived probe. It encodes a peptide of 377 amino acids, including a signal peptide of 38 amino acids. Sequence analysis demonstrated extensive homology with L-Selectin's from other species. Recombinant rabbit L-Selectin protein was expressed in eukaryotic cells in a chimeric construct incorporating the entire extracellular portion of the protein coding region, a phosphokinase target site to allow high activity radiolabeling with32P, and the constant region of the heavy chain of human IgG1to facilitate purification and detection. Amino-terminal peptide sequencing of recombinant L-Selectin confirmed that the signal peptide had been removed at the expected site.

Calcium-dependent oligonucleotide antagonists specific for L-selectin.

The selectins are calcium-dependent C-type lectins that recognize complex anionic carbohydrate ligands, initiating many cell-cell interactions in the vascular system. Selectin blockade shows therapeutic promise in a variety of inflammatory and postischemic pathologies. However, the available oligosaccharide ligand mimetics have low affinities and show cross-reaction among the three selectins, precluding efficient and specific blockade. The SELEX (systematic evolution of ligands by exponential enrichment) process uses combinatorial chemistry and in vitro selection to yield high affinity oligonucleotides with unexpected binding specificities. Nuclease-stabilized randomized oligonucleotides subjected to SELEX against recombinant L-selectin yielded calcium-dependent antagonists with approximately 10(5) higher affinity than the conventional oligosaccharide ligand sialyl LewisX. Most of the isolated ligands shared a common consensus sequence. Unlike sialyl LewisX, these antagonists show little binding to E- or P-selectin. Moreover, they show calcium-dependent binding to native L-selectin on peripheral blood lymphocytes and block L-selectin-dependent interactions with the natural ligands on high endothelial venules.

L-selectin-carbohydrate interactions: relevant modifications of the Lewis x trisaccharide.

Protein-carbohydrate interactions are known to mediate cell-cell recognition and adhesion events. Specifically, three carbohydrate binding proteins termed selectins (E-, P-, and L-selectin) have been shown to be essential for leukocyte rolling along the vascular endothelium, the first step in the recruitment of leukocytes from the blood into inflammatory sites or into secondary lymphoid organs. Although this phenomenon is well-established, little is known about the molecular-level interactions on which it depends. All three selectins recognize sulfated and sialylated derivatives of the Lewis x [Le(x):Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] and Lewis a [Le(a): Gal beta 1-->3(Fuc alpha 1-->4)GlcNAc] trisaccharide cores with affinities in the millimolar range, and it is believed that variants of these structures are the carbohydrate determinants of selectin recognition. Recently it was shown that the mucin GlyCAM-1, a secreted physiological ligand for L-selectin, is capped with sulfated derivatives of sialyl Lewis x [sLe(x): Sia alpha 2-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] and that sulfation is required for the high-affinity interaction between GlyCAM-1 and L-selectin. To elucidate the important sites of sulfation on Le(x) with respect to L-selectin recognition, we have synthesized six sulfated Le(x) analogs and determined their abilities to block binding of a recombinant L-selectin-Ig chimera to immobilized GlyCAM-1. Our results suggest that 6-sulfo sLe(x) binds to L-selectin with higher affinity than does sLe(x) or 6'-sulfo sLe(x) and that sulfation of sLe(x) capping groups on GlyCAM-1 at the 6-position is important for L-selectin recognition.

Biosynthesis of GlyCAM-1, a Mucin-like Ligand for L-Selectin

L-selectin, a member of the selectin family of leukocyte-endothelial adhesion proteins, mediates the initial attachment of lymphocytes to lymph node high endothelial venules during lymphocyte recirculation. One of the endothelial-associated ligands for L-selectin is GlyCAM-1, a mucin-like glycoprotein, which presents novel sulfated, sialylated and fucosylated O-glycans. In order to understand the generation of these glycans, we have examined the biosynthesis of GlyCAM-1 in lymph node organ culture. Using peptide-specific antibodies, lectins, and recombinant L-selectin, we detected the following species of GlyCAM-1: unglycosylated (< 28 kDa); modified with GalNAc only (28-33 kDa); modified with sialic acid, fucose, and sulfate but lacking L-selectin reactivity (40-50 kDa); and mature (L-selectin-reactive) ligand (50-60 kDa). Pulse-chase labeling at 15 degrees C suggested that GalNAc is added in a pre-Golgi compartment. Treatment with brefeldin A almost completely blocked sulfation, indicating that this modification occurs in the trans-Golgi network. Two distinct sialylation events occurred in the presence of brefeldin A, while fucosylation was partially blocked. We conclude that sialylation precedes both fucosylation and sulfation during biosynthesis. This ordering will help to identify the critical acceptor structures recognized by lymph node glycosyltransferases and sulfotransferases.

Binding of L-selectin to the vascular sialomucin CD34.

The adhesive interactions between leukocyte L-selectin and the endothelium are involved in the migration of lymphocytes through peripheral lymph nodes and of neutrophils to sites of inflammation. A recombinant L-selectin stains high endothelial venules (HEVs) in lymph nodes and recognizes sulfated carbohydrates found on two endothelial glycoproteins, Sgp50 and Sgp90. Amino acid sequencing of purified Sgp90 revealed a protein core identical to that CD34, a sialomucin expressed on hematopoietic stem cells and endothelium. A polyclonal antiserum to recombinant murine CD34 stains peripheral lymph node endothelium and recognizes Sgp90 that is functionally bound by L-selectin. Thus, an HEV glycoform of CD34 can function as a ligand for L-selectin.