Susan R. Watson

An endothelial ligand for L-Selectin is a novel mucin-like molecule

The adhesive interaction between circulating lymphocytes and the high endothelial venules (HEV) of lymph nodes (LN) is mediated by lymphocyte L-selectin, a member of the selectin family of cell adhesion proteins. Previous work has identified a sulfated 50 kd glycoprotein (Sgp50) as an HEV ligand for L-selectin. We now report the purification of this glycoprotein and the utilization of the derived N-terminal amino acid sequence to clone a cDNA. The predicted sequence reveals a novel, mucin-like molecule containing two serine/threonine-rich domains. The mRNA encoding this glycoprotein is preferentially expressed in LN. Antibodies against predicted peptides immunoprecipitate Sgp50 and stain the apical surface of LN HEV. These results thus define a tissue-specific mucin-like endothelial glycoprotein that appears to function as a scaffold that presents carbohydrates to the L-selectin lectin domain.

Selective modulation of the expression of L-selectin ligands by an immune response

BACKGROUND The adhesion molecule L-selectin is expressed on the cell surface of lymphocytes and mediates their migration from the bloodstream into lymph nodes. L-selectin is able to recognize four glycoprotein ligands, three of which--Sgp50, Sgp90, and Sgp200--are sulphated, bind specifically to L-selectin and are synthesized by the high endothelial venules of the peripheral and mesenteric lymph nodes. One of these three sulphated L-selectin ligands, Sgp90, has been shown to be identical to the known surface marker CD34 and is expressed on the cell surface of endothelial cells. The cDNA encoding Sgp50 has been cloned, and its product, which has been designated GlyCAM-1, is secreted. The third ligand, Sgp200, is both secreted and cell-associated. We have investigated how the expression of these sulphated glycoproteins is regulated during an immune response. RESULTS Here we demonstrated that, during a primary immune response, the expression and secretion of both GlyCAM-1 and Sgp200 are reduced, recovering to normal levels 7-10 days after antigen stimulation. In contrast, the expression of cell-associated CD34 and Sgp200 is relatively unaffected. These results may account for the modest decreases in the binding of an L-selectin-IgG fusion protein to high endothelial venules of inflamed peripheral lymph nodes that have been observed after antigen exposure. In vivo experiments show that, following the decrease in the levels of secreted GlyCAM-1 and Sgp200, migration of lymphocytes from the blood stream into lymph nodes remains L-selectin-dependent, but more lymphocytes home to antigen-primed than unprimed peripheral lymph nodes. CONCLUSIONS We suggest that the secreted forms of the L-selectin ligands GlyCAM-1 and Sgp200 act as modulators of cell adhesion, and that cell-associated CD34 and Sgp200 are the ligands that mediate the initial loose binding of lymphocytes to high endothelial venules.

A P-selectin-immunoglobulin G chimera is protective in a rabbit ear model of ischemia-reperfusion

BACKGROUND Neutrophils have been shown to play a role in ischemia-reperfusion injury, and the initial interaction of neutrophils with the endothelium is mediated through the selectin family of adhesion molecules. Thus the purpose of these studies was to determine whether a P-selectin-IgG chimera was protective in a model of ischemia-reperfusion injury. METHODS The model used was a rabbit ear model of ischemia-reperfusion. Selectin-IgG chimeras were given at the time of reperfusion of the tissue, and their efficacy was compared with an anti-CD18 antibody (MHM23). RESULTS The P-selectin-IgG was as protective in this model as an anti-CD18 antibody. The chimera did not mediate its effect by causing the animals to become neutropenic. CONCLUSIONS P-selectin plays a role in ischemia-reperfusion injury. This is in agreement with data from other groups. The fact that the chimera was effective in this model suggests that carbohydrates or small molecule mimics of carbohydrates would be effective in this model. Such antiinflammatory agents may have fewer side effects in terms of increased risk of sepsis.

Site-specific Fucosylation of Sialylated Polylactosamines by α1,3/4-Fucosyltransferases-V and -VI Is Defined by Amino Acids Near the N Terminus of the Catalytic Domain

Fucose transfer from GDP-fucose to GlcNAc residues of the sialylated polylactosamine acceptor NeuAcα2-3Galβ1-4Glc-NAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-ceramide leads to two isomeric monofucosyl antigens, VIM2 and sialyl-Lex. Human α1,3/4-fucosyltransferase (FucT)-V catalyzes primarily the synthesis of VIM2, whereas human FucT-VI catalyzes primarily the synthesis of sialyl-Lex. Thus, these two enzymes have distinct “site-specific fucosylation” properties. Amino acid sequence alignment of these enzymes showed that there are 24 amino acid differences in their catalytic domains. Studies were conducted to determine which of the amino acid differences are responsible for the site-specific fucosylation properties of each enzyme. Domain swapping (replacing a portion of the catalytic domain from one enzyme with an analogous portion from the other enzyme) demonstrated that site-specific fucosylation was defined within a 40-amino acid segment containing 8 amino acid differences between the two enzymes. Site-directed mutagenesis studies demonstrated that the site-specific fucosylation properties of these enzymes could be reversed by substituting 4 amino acids from one sequence with the other. These results were observed in both in vitro enzyme assays and flow cytometric analyses of Chinese hamster ovary cells transfected with plasmids containing the various enzyme constructs. Modeling studies of human FucT using a structure of a bacterial fucosyltransferase as a template demonstrated that the amino acids responsible for site-specific fucosylation map near the GDP-fucose-binding site. Additional enzyme studies demonstrated that FucT-VI has ∼12-fold higher activity compared with FucT-V and that the Trp124/Arg110 site in these enzymes is responsible primarily for this activity difference.