BIOSYNTHESIS AND INITIAL PROCESSING OF THE CARDIAC SARCOLEMMAL NA+-CA2+ EXCHANGER
BIOSYNTHESIS AND INITIAL PROCESSING OF THE CARDIAC SARCOLEMMAL NA+-CA2+ EXCHANGER
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DOI：
10.1016/0005-2736(93)90068-b
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发表时间：
1993-09-05
期刊：
影响因子：
--
通讯作者：
PHILIPSON, KD
中科院分区：
文献类型：
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作者：
HRYSHKO, LV;NICOLL, DA;PHILIPSON, KD
根据推导的心脏Na+-Ca 2+交换蛋白的氨基酸序列，有六个潜在的N-连接糖基化位点和一个潜在的切割信号序列。为了研究交换剂的翻译后修饰，在存在和不存在犬胰腺微粒体的情况下检查体外翻译。糖基化，检测为内切糖苷酶H诱导的分子大小的变化，通过使用全长和部分长度的RNA转录物检查具有不同数量的潜在N-连接的糖基化位点的蛋白质。在微粒体的存在下，全长克隆的分子量从110 kDa增加到113 kDa。内切糖苷酶H处理导致降低至108 kDa，表明糖基化使分子量增加约100 kDa。5 kDa和约5 kDa的信号序列。2 kDa在加工过程中裂解。与部分成绩单获得的分子量变化的分析表明，糖基化发生在位置N-9。这是通过定点诱变研究证实的。分子量约为。表达野生型交换蛋白的心肌肌膜或卵母细胞的蛋白质印迹测定为120 kDa。分子量降低了约。对于N9 Y突变体或来自从其中不发生糖基化的杆状病毒感染的昆虫细胞系获得的交换剂，所述交换剂的分子量为10 kDa。巨大的切除补丁技术被用来确定糖基化的功能后果。Na+-Ca 2+交换电流进行了检查补丁从卵母细胞表达野生型或N9 Y突变体。非糖基化突变体在电压、钠依赖性和胰凝乳蛋白酶的影响方面表现出与天然交换剂相同的性质。结果表明，糖基化不影响交换功能，在非洲爪蟾卵母细胞，并帮助定义交换拓扑结构。
Based on the deduced amino-acid sequence of the cardiac Na+-Ca2+ exchanger, there are six potential N-linked glycosylation sites and a potential cleaved signal sequence. To study the post-translational modifications of the exchanger, in vitro translation was examined in the presence and absence of canine pancreatic microsomes. Glycosylation, detected as endoglycosidase H induced shifts in molecular size, was examined for proteins having different numbers of potential N-linked glycosylation sites by using full and partial length RNA transcripts. In the presence of microsomes, the molecular mass of the full-length clone increased from 110 to 113 kDa. Endoglycosidase H treatment led to a reduction to 108 kDa, indicating that glycosylation increases the molecular mass by approx. 5 kDa and a signal sequence of approx. 2 kDa is cleaved during processing. Analysis of molecular-mass shifts obtained with partial transcripts suggested that glycosylation occurs at position N-9. This was confirmed by site-directed mutagenesis studies. A molecular mass of approx. 120 kDa was measured for Western blots of cardiac sarcolemmal membrane or oocytes expressing the wild-type exchanger. The molecular mass was reduced by approx. 10 kDa for the N9Y mutant or from exchanger obtained from a baculovirus-infected insect cell line where glycosylation does not occur. The giant excised patch technique was used to determine the functional consequences of glycosylation. Na+-Ca2+ exchange current was examined in patches from oocytes expressing either the wild-type or N9Y mutant. The non-glycosylated mutant exhibited the same properties as the native exchanger with respect to voltage, sodium dependence, and the effects of chymotrypsin. The results indicate that glycosylation does not affect exchanger function in Xenopus oocytes and help to define exchanger topology.