Shworak N W, Fritze L M, Liu J, Butler L D, Rosenberg R D
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
J Biol Chem. 1996 Oct 25;271(43):27063-71.
LTA cells synthesize a minor population of heparan sulfate proteoglycans (HSPGact) bearing anticoagulant heparan sulfate (HSact) with a specific monosaccharide sequence that accelerates the action of antithrombin (AT). LTA cells also synthesize a major population of heparan sulfate proteoglycans endowed with nonanticoagulant heparan sulfate (HSinact) lacking the AT-binding site. To investigate the pathway-specific features of HSPGact generation, we established a novel detergent-containing cell-free system with unlabeled and labeled microsomes from wild-type and variant LTA cells, respectively. The unlabeled microsomes provide "HSact conversion activity" that requires 3'-phosphoadenosine 5'-phosphosulfate to convert [35S]HSPGinact into [35S] HSPGact, presumably by sulfation. The reaction kinetics demonstrate that the rate of HSact synthesis is constant over the first 4 h of incubation. During this time, the rate of HSact production is linearly dependent on the amount of unlabeled LTA microsomal protein over a range of 10 to 50 microg as well as on the level of [35S]HS substrate over a range of 0.4 to 4.0 microg, microsomal protein. Compared with labeled microsomes, equivalent or slightly greater levels of HSact were generated from 35S-labeled HSPG, microsomal HS, or cell surface HS, which demonstrates that HSinact is the minimal substrate and that large amounts of HSact precursor exit the Golgi apparatus. Indeed, extensive modification of wild-type LTA cell surface [35S]HS elevated HSact content from 9 to 35%. The hypothesis that microsomal HSact conversion activity predicts the cellular rate of HSact generation was tested with wild-type or variant LTA cells in which production of HSact has been significantly altered by mutagenesis or overexpression of core protein or growth conditions. The data demonstrate that microsomal HSact conversion activity accurately reflects the cellular rate of HSact synthesis over a very wide range of conditions. The possibility that the reduced HSact generation is due to an inhibitor was excluded by mixing experiments. The possibility that reduced HSact generation is caused by decreased levels of HSact precursor was excluded as equivalent levels of HSact were formed from wild-type and variant [35S]HS. Based upon the above data, the LTA cell microsomal HSact conversion activity contains one or more limiting components that kinetically regulate the rate of cellular HSact generation and the levels of HSact precursor in HS greatly exceed HSact production.
LTA细胞合成少量带有抗凝血硫酸乙酰肝素（HSact）的硫酸乙酰肝素蛋白聚糖（HSPGact），其具有特定的单糖序列，可加速抗凝血酶（AT）的作用。LTA细胞还合成大量具有非抗凝血硫酸乙酰肝素（HSinact）的硫酸乙酰肝素蛋白聚糖，这些硫酸乙酰肝素缺乏AT结合位点。为了研究HSPGact生成的途径特异性特征，我们建立了一种新型的无细胞体系，分别使用来自野生型和变异型LTA细胞的未标记和标记的微粒体，并含有去污剂。未标记的微粒体提供“HSact转化活性”，该活性需要3'-磷酸腺苷5'-磷酸硫酸酯将[35S]HSPGinact转化为[35S]HSPGact，推测是通过硫酸化作用。反应动力学表明，在孵育的前4小时内，HSact的合成速率是恒定的。在此期间，HSact的产生速率在10至50微克范围内与未标记的LTA微粒体蛋白量呈线性相关，并且在0.4至4.0微克微粒体蛋白范围内与[35S]HS底物水平呈线性相关。与标记的微粒体相比，从35S标记的HSPG、微粒体HS或细胞表面HS产生的HSact水平相当或略高，这表明HSinact是最小底物，并且大量的HSact前体从高尔基体中排出。实际上，野生型LTA细胞表面[35S]HS的广泛修饰使HSact含量从9%提高到35%。我们用野生型或变异型LTA细胞对微粒体HSact转化活性预测HSact细胞生成速率这一假设进行了测试，在这些细胞中，HSact的产生已通过核心蛋白的诱变或过表达或生长条件而显著改变。数据表明，在非常广泛的条件下，微粒体HSact转化活性准确反映了HSact的细胞合成速率。混合实验排除了HSact生成减少是由于抑制剂的可能性。由于从野生型和变异型[35S]HS形成的HSact水平相当，因此排除了HSact生成减少是由HSact前体水平降低引起的可能性。基于上述数据，LTA细胞微粒体HSact转化活性包含一个或多个限制成分，这些成分在动力学上调节细胞HSact的生成速率，并且HS中HSact前体的水平大大超过HSact的产生。
