Gilbert C W, Zaroukian M H, Esselman W J
Department of Microbiology, Michigan State University, East Lansing 48824.
J Immunol. 1988 Apr 15;140(8):2821-8.
Cell-surface murine T200 glycoprotein has been implicated in the binding of NK cells to certain susceptible tumor targets. The existence of poly-N-acetyllactosamine structures on T200 glycoprotein and the ability of lactosamine-type oligosaccharides to inhibit NK cell-mediated cytotoxicity suggest that these structures may also be important in NK-target binding. To further identify and characterize these structures, relevant saccharides and reconstituted membrane liposomes containing fractionated effector cell membrane proteins were tested for their ability to block conjugate formation. Under base line conditions, the majority of plastic-non-adherent, Percoll-fractionated, NK-enriched splenocytes that formed conjugates with NK-susceptible YAC-1 targets functioned as lytic effectors in a single-cell cytotoxicity assay. These effectors were blocked in their ability to bind to YAC-1 targets by the addition of N-acetyllactosamine [Gal(beta 1,4)-GlcNAc] and chitobiose [GlcNAc(beta 1,4)GlcNAc], but not by saccharides lacking lactosamine-type linkages. Liposomes prepared from octyl-beta-D-glucopyranoside-extracted YAC-1 and NK-enriched effector cell membranes interfered with conjugate formation, whereas liposomes prepared from NK-insensitive P815 cells were inconsequential. Surface radiolabeled effector cell membrane proteins were fractionated by tomato lectin-Sepharose 4B (poly-N-acetyllactosamine-specific) column chromatography. Tomato lectin-bound material was enriched in a glycoprotein identical with T200, which, when incorporated into liposomes, was a potent inhibitor of effector-target binding. This inhibitory capacity was abrogated by treatment of liposomes with Ly-5 mAb (T200 mAb) or the lactosamine-specific enzyme endo-beta-galactosidase. When T200 was purified by mAb affinity chromatography and incorporated into liposomes, it was a potent inhibitor of conjugate formation, an effect that was blocked by pretreatment of T200-containing liposomes with Ly-5 mAb or endo-beta-galactosidase. These data provide additional evidence that T200 can mediate binding of NK cells to YAC-1 targets, and that poly-N-acetyllactosamine-type structures on NK cell surface T200 glycoprotein are important in the binding process.
细胞表面的小鼠T200糖蛋白被认为与自然杀伤细胞（NK细胞）和某些敏感肿瘤靶标的结合有关。T200糖蛋白上存在多聚N-乙酰乳糖胺结构，以及乳糖胺型寡糖抑制NK细胞介导的细胞毒性的能力，表明这些结构在NK细胞与靶标的结合中可能也很重要。为了进一步鉴定和表征这些结构，测试了相关糖类以及含有分离的效应细胞膜蛋白的重组膜脂质体阻断结合物形成的能力。在基线条件下，大多数与NK敏感的YAC-1靶标形成结合物的塑料非黏附性、经Percoll分离的、富含NK细胞的脾细胞在单细胞细胞毒性试验中作为溶细胞效应细胞发挥作用。通过添加N-乙酰乳糖胺[Gal(β1,4)-GlcNAc]和壳二糖[GlcNAc(β1,4)GlcNAc]，这些效应细胞与YAC-1靶标的结合能力受到阻断，但缺乏乳糖胺型连接的糖类则无此作用。由辛基-β-D-吡喃葡萄糖苷提取的YAC-1和富含NK细胞的效应细胞膜制备的脂质体干扰结合物的形成，而由NK不敏感的P815细胞制备的脂质体则无影响。表面放射性标记的效应细胞膜蛋白通过番茄凝集素-Sepharose 4B（多聚N-乙酰乳糖胺特异性）柱色谱进行分离。番茄凝集素结合的物质富含一种与T200相同的糖蛋白，当将其掺入脂质体时，是效应细胞与靶标结合的有效抑制剂。用Ly-5单克隆抗体（T200单克隆抗体）或乳糖胺特异性酶内切β-半乳糖苷酶处理脂质体可消除这种抑制能力。当通过单克隆抗体亲和色谱法纯化T200并将其掺入脂质体时，它是结合物形成的有效抑制剂，用Ly-5单克隆抗体或内切β-半乳糖苷酶预处理含T200的脂质体可阻断这种效应。这些数据提供了额外的证据，表明T200可介导NK细胞与YAC-1靶标的结合，并且NK细胞表面T200糖蛋白上的多聚N-乙酰乳糖胺型结构在结合过程中很重要。
