Shworak N W, Shirakawa M, Colliec-Jouault S, Liu J, Mulligan R C, Birinyi L K, Rosenberg R D
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
J Biol Chem. 1994 Oct 7;269(40):24941-52.
L cells and endothelial cells synthesize a heparan sulfate (HS) subpopulation, HSact, that exhibits anticoagulant activity due to a specific monosaccharide sequence; the remaining heparan sulfate, HSinact, lacks this region of defined structure and is anticoagulantly inactive. HSact biosynthesis was examined in these two cell types by stably expressing epitope-tagged rat ryudocan (ryudocan12CA5), which possesses three glycosaminoglycan (GAG) acceptor sites. Both HSact and HSinact were present on ryudocan12CA5 isolated from L cells and endothelial cells; thus, a core protein with a unique primary sequence initiates the synthesis of both GAGs. The expression in L cells of ryudocan12CA5 variants containing a single functional GAG acceptor site demonstrated that each of the three acceptor regions initiates the synthesis of both types of GAGs to a similar extent. Most importantly, in both cell types total HSact generation declined as a function of ryudocan12CA5 overexpression even though HSinact production increased linearly as a function of this variable. This discordant relationship is a general property of the biosynthetic machinery since in both cell types HSact production was reduced to an equal extent on protein cores of either exogenous or endogenous origins. The suppression of HSact generation was also observed with a secreted form of core protein lacking transmembrane and cytoplasmic domains or by a GAG acceptor site mutated form of core protein incapable of augmenting GAG synthesis. These results suggest that elevated intracellular levels of core protein saturate the capacity of a critical component of the HSact biosynthetic machinery. This critical component is not a member of the common set of biosynthetic enzymes involved in the production of HSact and HSinact since no structural changes were observed in either GAG during overexpression of core protein. Based upon the above data, we conclude that increased intracellular levels of ryudocan probably act by saturating the capacity of components which regulate HSact production by coordinating the function of biosynthetic enzymes.
L细胞和内皮细胞合成一种硫酸乙酰肝素（HS）亚群，即HSact，由于特定的单糖序列而具有抗凝活性；其余的硫酸乙酰肝素HSinact缺乏这一特定结构区域，无抗凝活性。通过稳定表达带有表位标签的大鼠ryudocan（ryudocan12CA5）来研究这两种细胞类型中的HSact生物合成，ryudocan12CA5具有三个糖胺聚糖（GAG）受体位点。从L细胞和内皮细胞中分离出的ryudocan12CA5上同时存在HSact和HSinact；因此，具有独特一级序列的核心蛋白启动了两种GAG的合成。含有单个功能性GAG受体位点的ryudocan12CA5变体在L细胞中的表达表明，三个受体区域中的每一个都在相似程度上启动了两种类型GAG的合成。最重要的是，在两种细胞类型中，尽管HSinact的产生随该变量呈线性增加，但总HSact的产生却随着ryudocan12CA5的过表达而下降。这种不一致的关系是生物合成机制的一个普遍特性，因为在两种细胞类型中，HSact的产生在来自外源或内源的蛋白质核心上都同等程度地减少。在缺乏跨膜和细胞质结构域的核心蛋白分泌形式中，或在不能增强GAG合成的GAG受体位点突变形式的核心蛋白中，也观察到了HSact产生的抑制。这些结果表明，核心蛋白细胞内水平的升高使HSact生物合成机制中一个关键成分的能力饱和。这个关键成分不是参与HSact和HSinact产生的常见生物合成酶中的一员，因为在核心蛋白过表达期间，两种GAG均未观察到结构变化。基于上述数据，我们得出结论，ryudocan细胞内水平的升高可能是通过使通过协调生物合成酶功能来调节HSact产生的成分的能力饱和而起作用的。
