摘要
肝脏铁过载是血液系统疾病患者进行骨髓移植后的典型并发症之一,长期铁过载可引发肝脏细胞凋亡和器官损坏,然而铁过载的分子调控机理迄今仍不清楚。以培养的枸橼酸铁铵过载人肝细胞HH4和正常人肝细胞HH4为研究对象,细胞裂解提取总蛋白,分子筛超滤管分离获得总糖肽,PNGase F酶解释放出N-糖链,Sepharose 4B除盐纯化N-糖链,再利用基质辅助激光解析电离飞行时间质谱(MALDI-TOF/TOF-MS)技术比较N-糖链变化情况。同时,采用荧光细胞凝集素免疫组化方法验证糖链分析结果。结果在铁过载人肝细胞和正常细胞中均检测到16种N-糖链,但糖链丰度存在明显差异。与正常人肝细胞相比,铁过载人肝细胞中高甘露糖型糖链的含量降低,而杂合型、复杂型、平分型、岩藻糖化和唾液酸化糖链的含量明显升高。凝集素免疫组化结果显示铁过载后细胞对凝集素Con A的亲和作用减弱,而对PHA-E、AAL、LCA和MAL-II的亲和作用增强,与糖链质谱分析结果相一致。研究为进一步探索人肝细胞铁过载模式下N-糖链表达差异的分子机理提供了技术支持。
Hepatic iron overload is common in patients undergoing hematopoietic cell transplantation and may be associated with hepatic injury. Here iron overload model of HH4 cells induced by FAC （ferric ammonium citrate） was established. The total proteins of HH4 cells treated with or without FAC were extracted, and total glycopeptides were isolated by an ultrafiltration unit. The glycopeptides were enzymatically hydrolyzed by Peptide-N-glycosidase F （PNGase F） and the released N-linked glycans were desalinated using Sepharose 4B. The structures of the purified N-glycans were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry （MALDI-TOF/TOF-MS）. Furthermore, the comparative expression of N-glycans was analyzed by lectin immunohistochemistry. The result indicated that 16 N-glycans were differentially expressed in HH4 cells treated with or without FAC. The levels of high-mannose-type N-glycans were decreased significantly, while the expression of hybrid type, complex type, bisecting type, fucosylation and sialylation of glycans were enhanced markedly in FAC-treated HH4 cells. Consistent with MS analysis, lectin immunohistochemistry study showed that the binding affinity to lectin ConA was reduced in FAC-treated HH4 cells, but the binding affinities to 4 lectins PHA-E, AAL, LCA and MAL-II were significantly increased. The present research provides the fundamental observations for further understanding functional roles of differential expression of N-linked glycans in iron-overload HH4 cells.
出处
《生物学杂志》
CAS
CSCD
2015年第3期9-14,共6页
Journal of Biology
基金
国家自然科学青年基金(31400691)
江苏省自然科学基金(BK20140169)
