DISULFIDE BONDS, N-GLYCOSYLATION AND TRANSMEMBRANE TOPOLOGY OF SKELETAL-MUSCLE TRIADIN
DISULFIDE BONDS, N-GLYCOSYLATION AND TRANSMEMBRANE TOPOLOGY OF SKELETAL-MUSCLE TRIADIN
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DOI：
10.1021/bi00045a035
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发表时间：
1995-11-14
期刊：
影响因子：
2.9
通讯作者：
CASWELL, AH
中科院分区：
文献类型：
--
作者：
FAN, HR;BRANDT, NR;CASWELL, AH
天然Triadin是一种亚基数目可变的二硫键连接的均聚物。AE8.91和GE4.90两种单抗分别识别110和163个氨基酸之间的Triadin细胞质区域和C-末端34个氨基酸的区域。在完整的三联体中，三聚氰胺基本上不受胰酶的影响，而其膜被低渗或洗涤剂Triton X-100处理破坏的三联体会产生可溶性和膜结合的片段。在巯基乙醇存在下，在28、16、10和7 kDa的蛋白质降解过程中，mAbGE4.90监测到的可溶性片段似乎是按顺序形成的。在非还原条件下观察到较高的相对分子质量条带。用单抗GE4.90形成的可溶性片段的双向电泳免疫印迹(第一次非还原；第二次还原)显示存在几条带，可被解释为包含由存在于消化中的16、10或7 kDa片段中的任何两个片段组合形成的二聚体。MAb AE8.91没有检测到这些片段。这一观察表明，在半胱氨酸671处，两个Triadin分子的相同结构域之间形成了一个分子间二硫键。用mAbAE8.91对不溶片段进行巯基乙醇免疫印迹表明，在两个亚基为60 kDa的Triadin分子的相同结构域之间存在一个二聚体。该片段未被GE4.90检测到，表明半胱氨酸270处存在分子间二硫键连接。糖苷酶Endo F/N-糖苷酶F改变了单抗GE4.90检测到的TC/Triads及其蛋白水解段中完整Triadin的迁移率。它减少了单抗GE4.90检测到的46、28和16 kDa可溶片段的质量，但不减少10、7和5 kDa物种的质量，证实了625位残基的天冬酰胺是糖化的。单抗的细胞质定位以及胰酶消化条件和N-625处的糖基化位点提供了Triadin跨膜拓扑模型的框架，其中每个二硫键位于跨膜的β片断或类似的扩展结构的单独片段中。一个额外的β片断和一个a螺旋由另外两个跨膜的Triadin结构域组成，从而形成了一个具有四个膜转运区和广泛的细胞质和管腔区域的模型。α螺旋片段与兰尼定受体的M2片段有一些相同之处，而β折叠片段与二氢吡啶受体的序列相似，后者被认为排列在钙通道的孔中。后一种比较表明，Triadin可能是一种通道。
Native triadin is a disulfide linked homopolymer of variable subunit number. Two monoclonal antibodies (mAbs), AE8.91 and GE4.90, recognize cytoplasmic regions of triadin between amino acids 110 and 163 and at the C-terminal 34 amino acids, respectively. Triadin in intact triads is largely unaffected by trypsin, while triads whose membrane has been disrupted by hypotonicity or by treatment with the detergent Triton X-100 yield both soluble and membrane bound fragments. Soluble fragments monitored by mAb GE4.90 appear to be formed sequentially during the course of proteolysis at 28, 16, 10 and 7 kDa in the presence of mercaptoethanol. Higher molecular weight bands are observed under nonreducing, conditions. A two-dimensional electrophoresis immunoblot (first nonreducing; second reducing) of the soluble fragments developed with mAb GE4.90 shows the presence of several bands which can be interpreted as containing a dimer formed by a combination of any two of the fragments of 16, 10, or 7 kDa present in the digest. MAb AE8.91 does not detect these fragments. This observation indicates that one of the intermolecular disulfide bonds is formed between the identical domains of two triadin molecules at cysteine 671. Immunoblots performed with and without mercaptoethanol of the insoluble fragments using mAb AE8.91 indicate the presence of a dimer formed between identical domains of two triadin molecules with a subunit of 60 kDa. This fragment which was not detected with GE4.90 indicates an intermolecular disulfide linkage at cysteine 270. The glycosidase endo F/N-glycosidase F changed the mobility of intact triadin in TC/triads and its proteolytic fragments detected by mAb GE4.90. It decreased the mass of the 46, 28, and 16 kDa soluble fragments, detected by mAb GE4.90, but not the 10, 7, and 5 kDa species confirming that the asparagine at residue 625 is, glycosidated. The cytoplasmic location of the mAbs together with the conditions of tryptic digestion and the glycosylation site at N-625 provides the framework of a model of the transmembrane topology of triadin in which each disulfide resides in a separate segment of a membrane-spanning beta sheet or similar extended structure. An additional beta sheet segment and an a helix comprise two more membrane-spanning domains of triadin giving rise to a model with four membrane transits and extensive cytoplasmic and luminal regions. The alpha helix segment shares some identity with the M2 segment of the ryanodine receptor while the beta sheet segments resemble the sequences of the dihydropyridine receptor which are considered to line the pore of the Ca2+ channel. This latter comparison suggests the possibility that triadin may be a channel.