Kimura S, Cheng J, Toyoshima K, Oda K, Saku T
Department of Pathology Department of Biology, Niigata University School of Science, Niigata, 951-8514, Japan.
J Biochem. 1999 Feb;125(2):406-13. doi: 10.1093/oxfordjournals.jbchem.a022301.
The biosynthesis of basement membrane heparan sulfate proteoglycan (HSPG), known as perlecan, in ACC3 cells established from a adenoid cystic carcinoma of the human salivary gland was studied using metabolic labeling and immunoprecipitation with discriminative antibodies specific for HSPG core protein. Treatment of immunoprecipitated HSPG with HNO2, heparitinase, and chondroitinase ABC revealed that ACC3 cells synthesized HSPG molecules composed of 470-kDa core protein and heparan sulfate but not of chondroitin sulfate. The core protein was shown to contain complex type N-linked oligosaccharides by digestion with N-glycanase and endoglycosidase H. Pulse-chase experiments showed that the mature form of HSPG was formed in the cells in 30 min and released into the medium thereafter. Degradation of HSPG was also found in the chase period of 3 h. In time course experiments, HSPG was found to be synthesized maximally at day 4 after plating, deposited in the cell layer maximally at day 6, and secreted maximally at day 8. This was also confirmed by immunofluorescence, Northern blotting, and in-situ hybridization. The results indicate that ACC3 cells synthesize, secrete and degrade basement membrane type HSPG, which is analogous to those produced by other cell types, and that the biosynthesis and secretion of HSPG in ACC3 cells are strictly regulated by the cell growth, that may be reflected in the characteristic histology of adenoid cystic carcinomas.
利用代谢标记和针对硫酸乙酰肝素蛋白聚糖（HSPG）核心蛋白的特异性鉴别抗体进行免疫沉淀，研究了从人涎腺腺样囊性癌建立的ACC3细胞中称为基底膜硫酸乙酰肝素蛋白聚糖（HSPG）即基底膜蛋白聚糖的生物合成。用亚硝酸、肝素酶和软骨素酶ABC处理免疫沉淀的HSPG，结果显示ACC3细胞合成的HSPG分子由470 kDa的核心蛋白和硫酸乙酰肝素组成，而非硫酸软骨素。通过用N - 聚糖酶和内切糖苷酶H消化表明核心蛋白含有复合型N - 连接寡糖。脉冲追踪实验表明，HSPG的成熟形式在细胞内30分钟内形成，随后释放到培养基中。在3小时的追踪期内也发现了HSPG的降解。在时间进程实验中，发现HSPG在接种后第4天合成量最大，在第6天最大量沉积在细胞层，在第8天最大量分泌。这也通过免疫荧光、Northern印迹和原位杂交得到证实。结果表明，ACC3细胞合成、分泌和降解基底膜型HSPG，这与其他细胞类型产生的HSPG类似，并且ACC3细胞中HSPG的生物合成和分泌受到细胞生长的严格调控，这可能反映在腺样囊性癌的特征性组织学中。
