Human Copper Transporter 1 Lacking O-Linked Glycosylation Is Proteolytically Cleaved in a Rab9-positive Endosomal Compartment
Human Copper Transporter 1 Lacking O-Linked Glycosylation Is Proteolytically Cleaved in a Rab9-positive Endosomal Compartment
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DOI：
10.1074/jbc.m109.044925
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发表时间：
2009-10-09
影响因子：
4.8
通讯作者：
Kaplan, Jack H.
中科院分区：
文献类型：
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作者：
Maryon, Edward B.;Zhang, Jing;Kaplan, Jack H.
人铜转运蛋白hCTR 1是由190个氨基酸的质膜蛋白组成的同源三聚体，其含有三个跨膜区段。hCTR 1的细胞外65个氨基酸的氨基末端含有N-连接（在Asn（15））和O-连接（在Thr（27））的糖基化位点。如果在Thr（27）处的O-糖基化被阻止，则hCTR 1被有效地切割，从氨基末端去除近似30个氨基酸。我们现在已经研究了（i）这种切割的位点，确定哪些肽键被切割，（ii）糖基化阻止切割的机制，以及（iii）在细胞中蛋白水解切割发生的位置。切割发生在序列Ala-Ser-His-Ser-His（残基29-33）中，其不包含先前识别的蛋白酶切割位点。使用一系列hCTR 1突变体，我们表明切割优先发生在残基Ala（29）-Ser（30）-His（31）之间。我们还表明，O-连接的多糖在Thr（27）块蛋白水解，由于其接近切割位点。通过插入少至5个氨基酸使切割位点远离Thr（27）多糖，允许在糖基化存在下发生切割。在固定细胞中使用免疫荧光和在活细胞中使用功能性绿色荧光蛋白标记的hCTR 1转运蛋白的成像研究表明，裂解的肽在细胞质中以点状结构积累。这些斑点重叠区室被Rab 9染色，表明hCTR 1裂解发生在转运蛋白递送至质膜之前的晚期内体区室中。
The human copper transporter hCTR1 is a homotrimer composed of a plasma membrane protein of 190 amino acids that contains three transmembrane segments. The extracellular 65-amino acid amino terminus of hCTR1 contains both N-linked (at Asn(15)) and O-linked (at Thr(27)) sites of glycosylation. If O-glycosylation at Thr(27) is prevented, hCTR1 is efficiently cleaved, removing similar to 30 amino acids from the amino terminus. We have now investigated (i) the site of this cleavage, determining which peptide bonds are cleaved, (ii) the mechanism by which glycosylation prevents cleavage, and (iii) where in the cell the proteolytic cleavage takes place. Cleavage occurs in the sequence Ala-Ser-His-Ser-His (residues 29-33), which does not contain previously recognized protease cleavage sites. Using a series of hCTR1 mutants, we show that cleavage occurs preferentially between residues Ala(29)-Ser(30)-His(31). We also show that the O-linked polysaccharide at Thr(27) blocks proteolysis due to its proximity to the cleavage site. Moving the cleavage site away from the Thr(27) polysaccharide by insertion of as few as 5 amino acids allows cleavage to occur in the presence of glycosylation. Imaging studies using immunofluorescence in fixed cells and a functional green fluorescent protein-tagged hCTR1 transporter in live cells showed that the cleaved peptide accumulates in punctate structures in the cytoplasm. These puncta overlap compartments were stained by Rab9, indicating that hCTR1 cleavage occurs in a late endosomal compartment prior to delivery of the transporter to the plasma membrane.