Natural forms of shed tumor gangliosides

Yu Kong , Ruixiang Li , Stephan Ladisch
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引用次数: 72

Abstract

Gangliosides shed by tumor cells are immunosuppressive molecules, but the mechanisms of shedding are poorly understood. We therefore conducted a comprehensive study of shedding to identify the natural forms of shed gangliosides. By chemical detection and mass spectrometric analysis of the gangliosides of YAC-1 murine lymphoma cells, we first confirmed that all major ganglioside species are released. Then, by the combination of metabolic and cell surface radiolabeling, we further demonstrated that gangliosides are released directly from the cell plasma membrane, i.e. by shedding. Ultracentrifugation separated the conditioned medium of metabolically radiolabeled cells cultured in either serum-free or serum-containing medium into: (1) a pellet of 100–200 nm membrane vesicles (visualized by electron microscopy) containing nearly one-third of total shed gangliosides; and (2) the supernatant, which contained soluble gangliosides (two-thirds of the total shed gangliosides). Although the ganglioside concentration in the conditioned medium (6–14×10−8 M) was above the critical micelle concentration of purified YAC-1 gangliosides (<1×10−8 M), by gel filtration >90% of the soluble gangliosides were found in monomeric form (MW <2 kDa) and only <10% in micelles (130 kDa). Ultrafiltration of fresh conditioned medium likewise showed the existence of monomers, and the findings were confirmed in human Daoy medulloblastoma and mouse MEB4 melanoma cells. Thus, in their natural states, shed tumor cell gangliosides exist in three forms: membrane vesicles, micelles, and monomers.

脱落瘤神经节苷的天然形式
肿瘤细胞脱落的神经节苷是免疫抑制分子,但脱落的机制尚不清楚。因此,我们进行了一项全面的脱落研究,以确定脱落神经节苷脂的自然形式。通过化学检测和质谱分析YAC-1小鼠淋巴瘤细胞的神经节苷类,我们首次证实了所有主要的神经节苷类都被释放。然后,通过代谢和细胞表面放射性标记的结合,我们进一步证明了神经节苷脂是直接从细胞膜释放的,即通过脱落。在无血清或含血清培养基中培养的代谢放射性标记细胞的条件培养基中,用超离心将其分离成:(1)100-200 nm的膜泡(电子显微镜下可见)小球,其中含有近三分之一的总脱落神经节苷;(2)含有可溶性神经节脂苷的上清(占总脱落神经节脂苷的三分之二)。虽然条件培养基中的神经节苷脂浓度(6-14×10−8 M)高于纯化的YAC-1神经节苷脂的临界胶束浓度(1×10−8 M),但通过凝胶过滤,90%的可溶性神经节苷脂以单体形式存在(MW <2 kDa),只有10%以胶束形式存在(130 kDa)。新鲜条件培养基的超滤同样显示了单体的存在,并且在人道氏髓母细胞瘤和小鼠MEB4黑色素瘤细胞中证实了这一发现。因此,在自然状态下,脱落瘤细胞神经节苷以三种形式存在:膜囊、胶束和单体。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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