L-selectin from human, but not from mouse neutrophils binds directly to E-selectin.
L-selectin from human, but not from mouse neutrophils binds directly to E-selectin.
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DOI：
10.1083/jcb.136.3.707
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发表时间：
1997-02-10
期刊：
影响因子：
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通讯作者：
Vestweber D
中科院分区：
文献类型：
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作者：
Zöllner O;Lenter MC;Blanks JE;Borges E;Steegmaier M;Zerwes HG;Vestweber D
中性粒细胞上的L-选择素以及内皮上的诱导型E-和P-选择素参与中性粒细胞向发炎组织中的募集。基于细胞粘附试验，L-选择素被认为是E-和P-选择素的碳水化合物呈递配体。然而，先前用E-选择素-IG融合蛋白的亲和分离实验未能在从小鼠嗜中性粒细胞分离的E-选择素配体中检测到L-选择素。我们在这里表明，L-选择素从人类中性粒细胞，在小鼠中性粒细胞相反，可以亲和分离作为一个主要的配体从总细胞提取物使用E-选择素-IG作为亲和探针。人L-选择素与E-选择素的结合是直接的，因为纯化的L-选择素可以用E-选择素-IG再沉淀。L-选择素的识别被唾液酸酶处理所消除，需要Ca 2+，并且对糖苷内切酶F的处理具有抗性。未观察到L-选择素与P-选择素-IG融合蛋白的结合。与生物化学数据一致，抗L选择素mAb DREG 56抑制人中性粒细胞在固定化的E-选择素-IG上的滚动，但不抑制P-选择素-IG上的滚动。抗小鼠L-选择素mAb MEL 14对小鼠嗜中性粒细胞没有观察到这种抑制作用。通过mAb DREG 56抑制E-选择素转染子在纯化和固定化的人L-选择素上的滚动。我们的结论是，L-选择素对人类中性粒细胞是一个主要的糖蛋白配体之间的非常少的糖蛋白，可以通过E-选择素亲和矩阵分离。人和小鼠L-选择素之间的明显差异表明，E-选择素结合碳水化合物部分连接到不同物种的不同蛋白质支架上。
L-Selectin on neutrophils as well as inducible E- and P-selectin on endothelium are involved in the recruitment of neutrophils into inflamed tissue. Based on cell attachment assays, L-selectin was suggested to function as a carbohydrate presenting ligand for E- and P-selectin. However, previous affinity isolation experiments with an E-selectin–Ig fusion protein had failed to detect L-selectin among the isolated E-selectin ligands from mouse neutrophils. We show here that L-selectin from human neutrophils, in contrast to mouse neutrophils, can be affinity-isolated as a major ligand from total cell extracts using E-selectin–Ig as affinity probe. Binding of human L-selectin to E-selectin was direct, since purified L-selectin could be reprecipitated with E-selectin–Ig. Recognition of L-selectin was abolished by sialidase-treatment, required Ca2+, and was resistant to treatment with endoglycosidase F. Binding of L-selectin to a P-selectin–Ig fusion protein was not observed. In agreement with the biochemical data, the anti–Lselectin mAb DREG56 inhibited rolling of human neutrophils on immobilized E-selectin–Ig but not on P-selectin–Ig. No such inhibitory effect was seen with the anti–mouse L-selectin mAb MEL14 on mouse neutrophils. Rolling of E-selectin transfectants on purified and immobilized human L-selectin was inhibited by mAb DREG56. We conclude that L-selectin on human neutrophils is a major glycoprotein ligand among very few glycoproteins that can be isolated by an E-selectin affinity matrix. The clear difference between human and mouse L-selectin suggests that E-selectin–binding carbohydrate moieties are attached to different protein scaffolds in different species.