Reilly P L, Woska J R, Jeanfavre D D, McNally E, Rothlein R, Bormann B J
Department of Immunologic Disease, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT 06877, USA.
J Immunol. 1995 Jul 15;155(2):529-32.
In solution, intercellular adhesion molecule-1 (ICAM-1) exhibits extremely low affinity for its receptor, LFA-1, as direct binding to LFA-1 has not been reported. Furthermore, there are conflicting reports on the ability of ICAM-1 in solution to inhibit cell adhesion events. These differences could be due to the valency or an oligomeric native biochemical form of membrane-bound and soluble ICAM-1, which may correlate with its ability to bind to integrins. To test this, stimulated adenocarcinoma (A549) cells or HUVEC were labeled with 35S-methionine/cysteine and treated with a chemical cross-linker. A high m.w. form (200 kDa) of ICAM-1 but not ICAM-2 was specifically immunoprecipitated from cross-linked cell lysates and supernatants. Affinity purification of crosslinked supernatants revealed that the majority of ICAM-1 was dimeric as opposed to recombinant soluble ICAM-1, which contains a minor fraction of dimer. Gel filtration chromatography was used to isolate monomeric and dimer-rich fractions of recombinant soluble ICAM-1, and tested for direct binding to affinity-purified LFA-1. Dimer-rich fractions demonstrated an enhanced ability and estimated affinity, compared with monomeric protein, to bind to purified LFA-1. These data suggest that ICAM-1 exists in its native membrane-bound and shed form as a non-covalent dimer, and that dimerization directly correlates with enhanced binding to LFA-1.
在溶液中，细胞间粘附分子-1（ICAM-1）对其受体淋巴细胞功能相关抗原-1（LFA-1）的亲和力极低，因为尚未有直接结合LFA-1的报道。此外，关于溶液中的ICAM-1抑制细胞粘附事件的能力，存在相互矛盾的报道。这些差异可能归因于膜结合型和可溶性ICAM-1的价态或寡聚天然生化形式，这可能与其结合整合素的能力相关。为了验证这一点，用35S-甲硫氨酸/半胱氨酸标记受刺激的腺癌（A549）细胞或人脐静脉内皮细胞（HUVEC），并用化学交联剂处理。从交联的细胞裂解物和上清液中特异性免疫沉淀出高分子量形式（200 kDa）的ICAM-1，而非ICAM-2。对交联上清液进行亲和纯化显示，与重组可溶性ICAM-1（其中二聚体占一小部分）相反，大部分ICAM-1是二聚体。使用凝胶过滤色谱法分离重组可溶性ICAM-1的单体和富含二聚体的组分，并测试其与亲和纯化的LFA-1的直接结合。与单体蛋白相比，富含二聚体的组分显示出更强的结合能力和估计亲和力，能够结合纯化的LFA-1。这些数据表明，ICAM-1以非共价二聚体的形式存在于其天然的膜结合和脱落形式中，并且二聚化与增强的LFA-1结合直接相关。
