Functional significance of Asn-linked glycosylation of proteinase 3 for enzymatic activity, processing, targeting, and recognition by anti-neutrophil cytoplasmic antibodies.
Functional significance of Asn-linked glycosylation of proteinase 3 for enzymatic activity, processing, targeting, and recognition by anti-neutrophil cytoplasmic antibodies.
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蛋白酶 3 的 Asn 连接糖基化对于抗中性粒细胞胞质抗体的酶活性、加工、靶向和识别的功能意义。
DOI：
10.1093/jb/mvm008
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发表时间：
2007
影响因子：
2.7
通讯作者：
McDonald,CariJ
中科院分区：
文献类型：
--
作者：
Specks,Ulrich;Fass,DavidN;Finkielman,JavierD;Hummel,AmberM;Viss,MargaretA;Litwiller,RobertD;McDonald,CariJ
蛋白酶3 （PR3）是一种储存在中性粒细胞颗粒中的中性丝氨酸蛋白酶。它与弹性酶、组织蛋白酶G和偶氮虫蛋白具有显著的序列同源性。PR3是韦格纳肉芽肿病（一种坏死性血管炎综合征）中自身抗体（ANCA）的靶抗原。ANCA与该病的发病机制有关。PR3有两个潜在的asn连接糖基化位点。本研究旨在确定这些糖基化位点的占用情况，并评估它们对酶功能、细胞内加工、靶向颗粒和ANCA识别的影响。我们发现，在天然中性粒细胞PR3和在hcc -1细胞中表达的野生型重组PR3 （rPR3）中，糖基化发生在两个位点。利用糖基化缺陷的rPR3突变体，我们发现Asn-147而非Asn-102的糖基化对PR3的热稳定性和最佳水解活性至关重要。rPR3高效的氨基端蛋白水解过程依赖于Asn-102的糖基化。靶向颗粒不依赖于糖基化，但未糖基化的rPR3优先分泌到介质上清液中。最后，使用rPR3糖基化变体作为靶抗原的ANCA捕获ELISA检测显示，在大约20%的患者中，ANCA的表位识别受到PR3糖基化状态的影响。
Proteinase 3 (PR3) is a neutral serine protease stored in neutrophil granules. It has substantial sequence homology with elastase, cathepsin G and azurocidin. PR3 is the target antigen for autoantibodies (ANCA) in Wegener's granulomatosis, a necrotizing vasculitis syndrome. ANCA have been implicated in the pathogenesis of this disease. PR3 has two potential Asn-linked glycosylation sites. This study was designed to determine the occupancy of these glycosylation sites, and to evaluate their effect on enzymatic function, intracellular processing, targeting to granules and recognition by ANCA. We found that glycosylation occurs at both sites in native neutrophil PR3 and in wild type recombinant PR3 (rPR3) expressed in HMC-1 cells. Using glycosylation deficient rPR3 mutants we found that glycosylation at Asn-147, but not at Asn-102, is critical for thermal stability, and for optimal hydrolytic activity of PR3. Efficient amino-terminal proteolytic processing of rPR3 is dependent on glycosylation at Asn-102. Targeting to granules is not dependent on glycosylation, but unglycosylated rPR3 gets secreted preferentially into media supernatants. Finally, a capture ELISA for ANCA detection, using rPR3 glycosylation variants as target antigens, reveals that in about 20% of patients, epitope recognition by ANCA is affected by the glycosylation status of PR3.