Biochemistry and Molecular Biology, Institute of Theoretical Medicine, Faculty of Medicine, University of Augsburg, Universitätstrasse 2, 86159, Augsburg, Germany.
Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354, Freising, Germany.
Sci Rep. 2022 Dec 5;12(1):20987. doi: 10.1038/s41598-022-24772-8.
Signal-Peptide Peptidase Like-3 (SPPL3) is an intramembrane cleaving aspartyl protease that causes secretion of extracellular domains from type-II transmembrane proteins. Numerous Golgi-localized glycosidases and glucosyltransferases have been identified as physiological SPPL3 substrates. By SPPL3 dependent processing, glycan-transferring enzymes are deactivated inside the cell, as their active site-containing domain is cleaved and secreted. Thus, SPPL3 impacts on glycan patterns of many cellular and secreted proteins and can regulate protein glycosylation. However, the characteristics that make a substrate a favourable candidate for SPPL3-dependent cleavage remain unknown. To gain insights into substrate requirements, we investigated the function of a GxxxG motif located in the transmembrane domain of N-acetylglucosaminyltransferase V (GnTV), a well-known SPPL3 substrate. SPPL3-dependent secretion of the substrate's ectodomain was affected by mutations disrupting the GxxxG motif. Using deuterium/hydrogen exchange and NMR spectroscopy, we studied the effect of these mutations on the helix flexibility of the GnTV transmembrane domain and observed that increased flexibility facilitates SPPL3-dependent shedding and vice versa. This study provides first insights into the characteristics of SPPL3 substrates, combining molecular biology, biochemistry, and biophysical techniques and its results will provide the basis for better understanding the characteristics of SPPL3 substrates with implications for the substrates of other intramembrane proteases.
信号肽肽酶 3（SPPL3）是一种跨膜天冬氨酸蛋白酶，可导致 II 型跨膜蛋白的细胞外结构域分泌。许多高尔基定位糖苷酶和葡萄糖基转移酶已被鉴定为生理 SPPL3 底物。通过 SPPL3 依赖性加工，糖基转移酶在细胞内失活，因为它们的包含活性位点的结构域被切割并分泌。因此，SPPL3 影响许多细胞内和分泌蛋白的聚糖模式，并可调节蛋白质糖基化。然而，使底物成为 SPPL3 依赖性切割的有利候选物的特征尚不清楚。为了深入了解底物要求，我们研究了位于 N-乙酰氨基葡萄糖转移酶 V（GnTV）跨膜域中的 GxxxG 基序的功能，GnTV 是一种众所周知的 SPPL3 底物。破坏 GxxxG 基序的突变影响了底物的细胞外结构域的 SPPL3 依赖性分泌。使用氘/氢交换和 NMR 光谱学，我们研究了这些突变对 GnTV 跨膜域螺旋灵活性的影响，观察到增加的灵活性促进了 SPPL3 依赖性脱落，反之亦然。这项研究首次提供了关于 SPPL3 底物特征的见解，结合了分子生物学、生物化学和生物物理技术，其结果将为更好地理解 SPPL3 底物的特征提供基础，这对其他跨膜蛋白酶底物具有启示意义。
