Biosynthesis of Sulfated L-Selectin Ligands in Human High Endothelial Venules (HEV)
High endothelial venules (HEVs) are specialized post-capillary venules found in lymphoid tissues, that support high levels of lymphocyte extravasation from the blood. Lymphocyte L-selectin plays a key role in the initial interaction of lymphocytes with HEVs by recognizing sulfated carbohydrate ligands on HEV mucin-like glycoproteins, GlyCAM-1, CD34 and MAdCAM-1. Sulfation is key to the uniqueness of the HEV ligands since 6 or 6'-sulfated-sLe(x) isoforms have recently been identified as major capping groups of GlyCAM-1 and sulfation of both GlyCAM1 and CD34 has been shown to be required for high-affinity L-selectin binding and recognition by the HEV-specific monoclonal antibody MECA-79. To characterize the molecular mechanisms involved in the biosynthesis of sulfated L-selectin ligands in HEVs, we have started to isolate genes that play a role in sulfate metabolism in HEVs. Studies with chlorate, a selective inhibitor of the synthesis of the high energy donor of sulfate, PAPS (3'-phosphoadénosine 5'-phosphosulfate), had previously revealed that PAPS synthesis is required for sulfation of HEV ligands and recognition by L-selectin. Therefore, we screened an HEV cDNA library in order to isolate cDNAs encoding enzymes involved in PAPS synthesis. This strategy allowed us to isolate a novel cDNA encoding the PAPS synthetase from human HEVs. The molecular characteristics of PAPS synthetase and its role in biosynthesis of sulfated L-selectin ligands in HEVs are discussed.
Available from: Jean-Michel Girardet
- "In lymph nodes, Gly- CAM-1 is indirectly involved in the immune response by acting as an endothelial cell surface ligand for Lselectin , a lymphocyte homing receptor (Girard and Almaric, 1998). This function requires the presence of sulfated and sialylated Lewis × antigen bound to the glycan moiety of GlyCAM-1 (Girard and Almaric, 1998), but sulfatation has not been demonstrated in mammary gland (Dowbenko et al., 1993). Despite the large homology observed between PP3 and GlyCAM-1, the exact function in vivo of bovine PP3 still remains unknown. "
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ABSTRACT: A synthetic peptide of 23 residues corresponding to the carboxyterminal 113 to 135 region of component-3 of proteose peptone (PP3) has been investigated with regard to its antibacterial properties. This cationic amphipathic peptide that we refer to as lactophoricin, displayed a growth-inhibitory activity against both gram-positive and gram-negative bacteria. For most of the strains tested, bacterial growth was observed in the presence of lactophoricin except for Streptococcus thermophilus. In that case, lactophoricin exhibited a minimum inhibitory concentration of 10 microM and a minimum lethal concentration of 20 microM. No hemolysis of human red blood cells was detected for peptide concentrations between 2 to 200 microM, indicating that lactophoricin would be noncytotoxic when used in this concentration range.
Available from: Espen Baekkevold
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ABSTRACT: High endothelial venules (HEVs) are specialized postcapillary venules, found in lymphoid organs and chronically inflamed tissues, that support high levels of lymphocyte extravasation from the blood. Molecular characterization of HEV endothelial cells (HEVECs) has been hampered by difficulties in their purification and in vitro maintenance. To overcome these limitations, we developed a strategy combining the use of freshly purified HEVECs ( approximately 98% positive for the HEV-specific marker MECA-79) and the recently described polymerase chain reaction (PCR)-based cDNA subtraction cloning procedure called suppression subtractive hybridization (SSH). Subtracted probes prepared by SSH from small amounts of total RNA were used to screen a HEVEC cDNA library. This resulted in cloning of 22 cDNAs preferentially expressed in HEVECs, which encode the promiscuous chemokine receptor DARC, mitochondrial components, and matricellular proteins. The latter included hevin, thrombospondin-1, and mac25/IGFBP-rP1, which is a secreted growth factor-binding protein previously found to accumulate specifically in tumor blood vessels. Biochemical and histochemical analysis confirmed the identification of mac25 and DARC as novel markers of the HEVECs. Ultrastructural immunolocalization revealed a noticeable association of mac25 and MECA-79 antigens with microvillous processes near the endothelial cell junctions, suggesting a role for mac25 in the control of lymphocyte emigration. This study shows that PCR-based SSH is useful for cloning of differentially expressed genes in very small samples.
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