Weersink A J, van Kessel K P, van den Tol M E, van Strijp J A, Torensma R, Verhoef J, Elsbach P, Weiss J
Eijkman-Winkler Institute of Medical and Clinical Microbiology, University of Utrecht, The Netherlands.
J Immunol. 1993 Jan 1;150(1):253-63.
Several LPS-binding proteins have been identified on the surface of human granulocytes (polymorphonuclear leukocyte (PMN)). We describe a plasma-membrane associated ca. 55-kDa LPS-binding protein of human PMN that is indistinguishable from the bactericidal/permeability-increasing protein (BPI). To detect LPS-binding proteins on the cell surface, PMN were biotinylated before detergent solubilization and incubation with LPS-coated beads. Several biotinylated proteins bound to LPS-coated beads but not to uncoated beads and were characterized after elution with detergent by SDS-PAGE and western blotting using streptavidin-horseradish peroxidase. The spectrum of biotinylated proteins binding to and eluting from LPS-coated beads increased as the number of beads incubated with PMN lysate increased. However, at all concentrations of beads a 55-kDa protein was a dominant component of the eluate. Binding of the 55-kDa protein to LPS-coated beads was inhibited by lipid A, and both homologous and heterologous LPS, but not by peptidoglycan. Similar amounts of biotinylated 55-kDa LPS-binding protein were detected on PMN from patients with paroxysmal nocturnal hemoglobinuria who lacked membrane bound CD14, a known ca. 55-kDa plasma membrane-associated LPS-binding protein, indicating that the recovered biotinylated protein is not CD14. Several pieces of evidence, however, do indicate that the 55-kDa surface protein is BPI: 1) flow cytometry of PMN after labeling with rabbit anti-BPI serum and FITC-labeled goat anti-rabbit IgG revealed immunoreactive surface molecules on resting PMN and, in increased amounts, on PMN stimulated with FMLP or TNF; 2) This antiserum specifically and quantitatively inhibited binding of the biotinylated 55-kDa species to LPS-coated beads; 3) both BPI and the 55-kDa protein migrated as a doublet during SDS-PAGE and were both converted to single migrated species after N-glycosidase F treatment; 4) chemical cleavage of the biotinylated protein and native BPI with N-chlorosuccinimide yielded the same fragments. Thus, we have positively identified BPI as a LPS-binding protein on the surface of PMN. The role of this potent antibacterial, endotoxin neutralizing protein on the surface of PMN remains to be established.
已在人粒细胞（多形核白细胞（PMN））表面鉴定出几种脂多糖结合蛋白。我们描述了一种与人PMN的质膜相关的约55 kDa脂多糖结合蛋白，它与杀菌/通透性增加蛋白（BPI）无法区分。为了检测细胞表面的脂多糖结合蛋白，在去污剂溶解和与脂多糖包被的珠子孵育之前，先对PMN进行生物素化。几种生物素化蛋白与脂多糖包被的珠子结合，但不与未包被的珠子结合，并在用去污剂洗脱后通过SDS-PAGE和使用链霉亲和素-辣根过氧化物酶的western印迹进行表征。随着与PMN裂解物孵育的珠子数量增加，与脂多糖包被的珠子结合并从其洗脱的生物素化蛋白的谱带增加。然而，在所有珠子浓度下，一种55 kDa的蛋白都是洗脱物的主要成分。脂多糖包被的珠子与55 kDa蛋白的结合受到脂多糖A、同源和异源脂多糖的抑制，但不受肽聚糖的抑制。在缺乏膜结合CD14（一种已知的约55 kDa质膜相关脂多糖结合蛋白）的阵发性夜间血红蛋白尿患者的PMN上，检测到了相似量的生物素化55 kDa脂多糖结合蛋白，这表明回收的生物素化蛋白不是CD14。然而，有几条证据确实表明55 kDa的表面蛋白是BPI：1）用兔抗BPI血清和FITC标记的山羊抗兔IgG标记后，PMN的流式细胞术显示静息PMN表面有免疫反应性分子，在用FMLP或TNF刺激的PMN上，其数量增加；2）这种抗血清特异性地、定量地抑制生物素化的55 kDa物质与脂多糖包被的珠子的结合；3）BPI和55 kDa蛋白在SDS-PAGE期间均以双峰形式迁移，在N-糖苷酶F处理后均转化为单迁移形式；4）用N-氯代琥珀酰亚胺对生物素化蛋白和天然BPI进行化学裂解产生相同的片段。因此，我们已明确鉴定出BPI是PMN表面的一种脂多糖结合蛋白。这种强效抗菌、内毒素中和蛋白在PMN表面的作用仍有待确定。
