Iozzo R V, Hassell J R
Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Arch Biochem Biophys. 1989 Feb 15;269(1):239-49. doi: 10.1016/0003-9861(89)90105-7.
Human colon carcinoma cells synthesize a high-molecular-weight heparan sulfate proteoglycan which is localized at the cell surface. In this study we have performed a series of immunoprecipitation and pulse-chase experiments associated with various pharmacological agents that interfere with the synthesis and post-translational modification of the proteoglycan. We demonstrate that colon carcinoma cells synthesize the heparan sulfate proteoglycan from a 400-kDa precursor protein that is immunologically related to the Engelbreth-Holm-Swarm (EHS) tumor cell proteoglycan. The cells contain a large pool of precursor protein with a half-life of about 75 min. Most of the precursor protein receives heparan sulfate side chains and is then transported to the cell surface and released into the medium. A portion of the precursor pool, however, does not receive heparan sulfate chains but is secreted into the medium. The glycosylation and subsequent secretion of the 400-kDa precursor protein was inhibited by NH4Cl and even more by monensin, indicating that the transit of precursor from the rough endoplasmic reticulum to the cell surface occurred through the Golgi complex and acidic compartments. The existence of a sizable pool of precursor protein was confirmed by additional experiments using cycloheximide and xyloside. These experiments showed that the half-life of the precursor protein was also 75 min and that stimulation of heparan sulfate synthesis by xyloside was greatly enhanced (about 12-fold) after new protein core synthesis was blocked by cycloheximide. Although the structural models proposed for the EHS and colon carcinoma heparan sulfate proteoglycans differ, the observation that they are derived from a precursor protein with dimensional and immunological similarities suggests that they may be genetically related.
人结肠癌细胞合成一种高分子量硫酸乙酰肝素蛋白聚糖，其定位于细胞表面。在本研究中，我们进行了一系列免疫沉淀和脉冲追踪实验，并结合了各种干扰蛋白聚糖合成和翻译后修饰的药理试剂。我们证明，结肠癌细胞从一种400 kDa的前体蛋白合成硫酸乙酰肝素蛋白聚糖，该前体蛋白与恩格尔布雷特-霍尔姆-斯旺（EHS）肿瘤细胞蛋白聚糖存在免疫相关性。细胞中含有大量半衰期约为75分钟的前体蛋白。大多数前体蛋白接上硫酸乙酰肝素侧链，然后被转运到细胞表面并释放到培养基中。然而，一部分前体蛋白池没有接上硫酸乙酰肝素链，而是分泌到培养基中。NH4Cl抑制了400 kDa前体蛋白的糖基化及随后的分泌，莫能菌素的抑制作用更强，这表明前体从糙面内质网到细胞表面的转运是通过高尔基体复合体和酸性区室进行的。使用环己酰亚胺和木糖苷的额外实验证实了存在相当数量的前体蛋白池。这些实验表明，前体蛋白的半衰期也是75分钟，并且在环己酰亚胺阻断新的蛋白核心合成后，木糖苷对硫酸乙酰肝素合成的刺激作用大大增强（约12倍）。尽管为EHS和结肠癌细胞硫酸乙酰肝素蛋白聚糖提出的结构模型不同，但它们源自具有尺寸和免疫相似性的前体蛋白这一观察结果表明，它们可能在遗传上相关。
