Bowman K G, Hemmerich S, Bhakta S, Singer M S, Bistrup A, Rosen S D, Bertozzi C R
Department of Chemistry, University of California, Berkeley 94720, USA.
Chem Biol. 1998 Aug;5(8):447-60. doi: 10.1016/s1074-5521(98)90161-2.
The leukocyte adhesion molecule L-selection participates in the initial attachment of blood-borne lymphocytes to high endothelial venules (HEVs) during lymphocyte homing to secondary lymphoid organs, and contributes to leukocyte adhesion and extravasation in HEV-like vessels at sites of chronic inflammation. The L-selection ligands on lymph mode HEVs are mucin-like glycoproteins adorned with the unusual sulfated carbohydrate epitope, 6-sulfo sialyl Lewis x. Sulfation of this epitope on the N-acetylglucosamine (GlcNAc) residue confers high-avidity L-selection binding, and is thought to be restricted in the vasculature to sites of sustained lymphocyte recruitment. The GlcNAc-6-0 sulfotransferase that installs the sulfate ester may be a key modulator of lymphocyte recruitment to secondary lymphoid organs and sites of chronic inflammation and is therefore a potential target for anti-inflammatory therapy.
A GlcNAc-6-0-sulfotransferase activity was identified within porcine lymph nodes and characterized using a rapid, sensitive, and quantitative assay. We synthesized two unnatural oligosaccharide substrates, GlcNAc beta 1-->6Gal alpha-R and Gal beta 1-->4GlcNAc beta 1-->6Gal alpha-R, that incorporate structural motifs from the native L-selection ligands into an unnatural C-glycosyl hydrocarbon scaffold. The sulfotransferase incorporated greater than tenfold more sulfate into the disaccharide than the trisaccharide, indicating a requirement for a terminal GlcNAc. Activity across tissues was highly restricted to the HEVs within peripheral lymph node.
The restricted expression of the GlcNAc-6-0-sulfotransferase activity to lymph node HEVs strongly suggestions a role in the biosynthesis of L-selection ligands. In addition, similar sulfated epitopes are known to be expressed on HEV-like vessels of chronically inflamed tissues; indicating that this sulfotransferase may also contribute to inflammatory lymphocyte recruitment. We identified a concise disaccharide motif, GlcNAc beta 1-->6Gal alpha-R, that preserved both recognition and specificity determinants for the GlcNAc-6-0-sulfotransferase. The absence of activity on the trisaccharide Gal beta 1-->6Gal alpha-R indicates a requirement for a substrate with a terminal GlcNAc residue, suggesting that sulfation precedes further biosynthetic assembly of L-selection ligands.
白细胞黏附分子L-选择素在淋巴细胞归巢至二级淋巴器官的过程中参与血源淋巴细胞与高内皮微静脉（HEV）的初始黏附，并在慢性炎症部位的类HEV血管中促进白细胞黏附和外渗。淋巴模式HEV上的L-选择素配体是带有异常硫酸化碳水化合物表位6-磺基唾液酸化路易斯x的黏蛋白样糖蛋白。该表位在N-乙酰葡糖胺（GlcNAc）残基上的硫酸化赋予L-选择素高亲和力结合，并且被认为在脉管系统中局限于持续淋巴细胞募集的部位。安装硫酸酯的GlcNAc-6-O-磺基转移酶可能是淋巴细胞募集至二级淋巴器官和慢性炎症部位的关键调节因子，因此是抗炎治疗的潜在靶点。
在猪淋巴结内鉴定出GlcNAc-6-O-磺基转移酶活性，并使用快速、灵敏和定量测定法对其进行了表征。我们合成了两种非天然寡糖底物，GlcNAcβ1→6Galα-R和Galβ1→4GlcNAcβ1→6Galα-R，它们将天然L-选择素配体的结构基序整合到非天然C-糖基烃支架中。磺基转移酶掺入二糖的硫酸盐比掺入三糖的多十倍以上，表明需要一个末端GlcNAc。组织间的活性高度局限于外周淋巴结内的HEV。
GlcNAc-6-O-磺基转移酶活性在淋巴结HEV中的局限性表达强烈提示其在L-选择素配体生物合成中的作用。此外，已知类似的硫酸化表位在慢性炎症组织的类HEV血管上表达；这表明该磺基转移酶也可能有助于炎症性淋巴细胞的募集。我们鉴定出一个简洁的二糖基序，GlcNAcβ1→6Galα-R，并保留了GlcNAc-6-O-磺基转移酶的识别和特异性决定簇。三糖Galβ1→6Galα-R上无活性表明需要具有末端GlcNAc残基的底物，这表明硫酸化先于L-选择素配体的进一步生物合成组装。
