From the Department of Genetics, Cell Biology and Development, University of Minnesota, 6-160 Jackson Hall, 321 Church St SE, Minneapolis, MN, USA.
Department of Medical Biochemistry and Microbiology, Husargatan 3, 75123 Uppsala University, Uppsala, Sweden.
Glycobiology. 2019 Jun 1;29(6):479-489. doi: 10.1093/glycob/cwz020.
A class of carbohydrate-modified proteins, heparan sulfate proteoglycans (HSPGs), play critical roles both in normal development and during disease. Genetic studies using a model organism, Drosophila, have been contributing to understanding the in vivo functions of HSPGs. Despite the many strengths of the Drosophila model for in vivo studies, biochemical analysis of Drosophila HS is somewhat limited, mainly due to the insufficient amount of the material obtained from the animal. To overcome this obstacle, we generated mutant cell lines for four HS modifying enzymes that are critical for the formation of ligand binding sites on HS, Hsepi, Hs2st, Hs6st and Sulf1, using a recently established method. Morphological and immunological analyses of the established lines suggest that they are spindle-shaped cells of mesodermal origin. The disaccharide profiles of HS from these cell lines showed characteristics of lack of each enzyme as well as compensatory modifications by other enzymes. Metabolic radiolabeling of HS allowed us to assess chain length and net charge of the total population of HS in wild-type and Hsepi mutant cell lines. We found that Drosophila HS chains are significantly shorter than those from mammalian cells. BMP signaling assay using Hs6st cells indicates that molecular phenotypes of these cell lines are consistent with previously known in vivo phenomena. The established cell lines will provide us with a direct link between detailed structural information of Drosophila HS and a wealth of knowledge on biological phenotypic data obtained over the last two decades using this animal model.
一类碳水化合物修饰蛋白，即硫酸乙酰肝素蛋白聚糖（HSPGs），在正常发育和疾病过程中都发挥着关键作用。利用模式生物果蝇进行的遗传研究，有助于理解 HSPGs 的体内功能。尽管果蝇模型在体内研究方面有许多优势，但对果蝇 HS 的生化分析有些受限，主要是由于从动物中获得的物质数量不足。为了克服这一障碍，我们利用最近建立的方法，针对四个对 HS 上配体结合位点形成至关重要的 HS 修饰酶（Hsepi、Hs2st、Hs6st 和 Sulf1）生成了突变细胞系。所建立的细胞系的形态和免疫分析表明，它们是来源于中胚层的梭形细胞。这些细胞系的 HS 二糖图谱特征表明，每种酶的缺乏以及其他酶的补偿修饰。HS 的代谢放射性标记使我们能够评估野生型和 Hsepi 突变细胞系中 HS 总群体的链长和净电荷。我们发现，果蝇 HS 链明显短于哺乳动物细胞中的 HS 链。使用 Hs6st 细胞进行的 BMP 信号转导测定表明，这些细胞系的分子表型与过去二十年来利用该动物模型获得的已知体内现象一致。所建立的细胞系将为我们提供果蝇 HS 的详细结构信息与利用该动物模型获得的丰富生物学表型数据之间的直接联系。
