Post-translational Modification of Thrombospondin Type-1 Repeats in ADAMTS-like 1/Punctin-1 by C-Mannosylation of Tryptophan*
Post-translational Modification of Thrombospondin Type-1 Repeats in ADAMTS-like 1/Punctin-1 by C-Mannosylation of Tryptophan*
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DOI：
10.1074/jbc.m109.038059
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发表时间：
2009-08
期刊：
影响因子：
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通讯作者：
Lauren W. Wang;Christina Leonhard-Melief;R. Haltiwanger;S. Apte
中科院分区：
文献类型：
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作者：
Lauren W. Wang;Christina Leonhard-Melief;R. Haltiwanger;S. Apte
蛋白质C-甘露糖基化是α-吡喃甘露糖通过C-C键与色氨酸连接。这种翻译后修饰通常发生在序列基序WXXW内，其经常存在于血小板反应蛋白1型重复序列（TSR）中。TSR在ADAMTS超家族中特别多，并且是ADAMTS超家族的定义特征。我们研究了ADAMTS-like 1/punctin-1的C-甘露糖基化的存在和功能意义，其中包含四个TSR（两个具有预测的C-甘露糖基化位点），使用质谱，代谢标记，定点突变和C-甘露糖基化缺陷的中国仓鼠卵巢细胞变体中的表达。通过质谱法分析重组人点蛋白-1的胰蛋白酶消化片段，鉴定出源自TSR 1的肽，其含有用两个甘露糖残基和Glc-Fuc二糖（O-岩藻糖基化）修饰的36WDAWGPWSECSRTC 49感兴趣序列。串联质谱（MS/MS）和MS/MS/MS分析证明了C-甘露糖的特征性交叉环切割，并将修饰的残基鉴定为Trp39和Trp42。还鉴定了相关蛋白酶ADAMTS5的TSR 1的C-甘露糖基化。CHO-K1细胞或Lec35.1细胞的代谢标记证实了d-[2，6 - 3H]甘露糖掺入CHO-K1细胞而非Lec35.1细胞分泌的点蛋白-1中。Lec35.1细胞相对于CHO-K1细胞中的点蛋白-1分泌的定量表明Lec35.1细胞中的分泌减少。用Ala或Phe取代TSR1中的甘露糖基化Trp残基影响斑蛋白的分泌效率。这些数据表明，来自点蛋白-1的TSR1携带与O-连接岩藻糖非常接近的C-甘露糖基化。总之，这些修饰似乎为点蛋白-1分泌提供了质量控制机制。
Protein C-mannosylation is the attachment of α-mannopyranose to tryptophan via a C-C linkage. This post-translational modification typically occurs within the sequence motif WXXW, which is frequently present in thrombospondin type-1 repeats (TSRs). TSRs are especially numerous in and a defining feature of the ADAMTS superfamily. We investigated the presence and functional significance of C-mannosylation of ADAMTS-like 1/punctin-1, which contains four TSRs (two with predicted C-mannosylation sites), using mass spectrometry, metabolic labeling, site-directed mutagenesis, and expression in C-mannosylation-defective Chinese hamster ovary cell variants. Analysis of tryptic fragments of recombinant human punctin-1 by mass spectrometry identified a peptide derived from TSR1 containing the 36WDAWGPWSECSRTC49 sequence of interest modified with two mannose residues and a Glc-Fuc disaccharide (O-fucosylation). Tandem mass spectrometry (MS/MS) and MS/MS/MS analysis demonstrated the characteristic cross-ring cleavage of C-mannose and identified the modified residues as Trp39 and Trp42. C-Mannosylation of TSR1 of the related protease ADAMTS5 was also identified. Metabolic labeling of CHO-K1 cells or Lec35.1 cells demonstrated incorporation of d-[2,6-3H]mannose in secreted punctin-1 from CHO-K1 cells but not Lec35.1 cells. Quantitation of punctin-1 secretion in Lec35.1 cells versus CHO-K1 cells suggested decreased secretion in Lec35.1 cells. Replacement of mannosylated Trp residues in TSR1 with either Ala or Phe affected punctin secretion efficiency. These data demonstrate that TSR1 from punctin-1 carries C-mannosylation in close proximity to O-linked fucose. Together, these modifications appear to provide a quality control mechanism for punctin-1 secretion.