Mycotoxin Prevention and Applied Microbiology Research Unit, National Center for Agriculture Utilization Research, Peoria, IL 61604, United States.
Nebraska Center for Biotechnology, Beadle Center, University of Nebraska-Lincoln, Lincoln, NE 68588, United States.
Fungal Genet Biol. 2020 Aug;141:103399. doi: 10.1016/j.fgb.2020.103399. Epub 2020 May 6.
Subtilases are a large family of serine proteases that occur throughout biology. A small subset contain protease-associated (PA) domains that are structurally separate from but encoded within the active site. In bacteria, subtilase PA domains function to recruit specific protein substrates. Here we demonstrate that a protease secreted by the fungal corn pathogen Stenocarpella maydis, which truncates corn ChitA chitinase, is a PA domain subtilase. Protease was purified from S. maydis cultures and tryptic peptides were analyzed by LC-MS/MS. Ions were mapped to two predicted PA domain subtilases. Yeast strains were engineered to express each protease. One failed to produce recombinant protein while the other secreted protease that truncated ChitA. This protease, that we named kilbournase, was purified and characterized. It cleaved multiple peptide bonds in the amino-terminal chitin binding domain of ChitA while leaving the catalytic domain intact. Kilbournase was more active on the ChitA-B73 alloform compared to ChitA-LH82 and did not cleave AtChitIV3, a homolog from Arabidopsis thaliana, indicating a high level of specificity. Truncation of corn ChitA by kilbournase resembles truncation of human C5a by Streptococcus pyogenes ScpA, arguing that PA domain proteases in bacteria and fungi may commonly target specific host proteins.
丝氨酸蛋白酶广泛存在于生物界，是一个大家族。其中一小部分包含蛋白酶相关（PA）结构域，该结构域与活性位点分开，但编码在其中。在细菌中，丝氨酸蛋白酶的 PA 结构域可用于招募特定的蛋白质底物。在这里，我们证明了一种由真菌玉米病原菌稻瘟病菌分泌的蛋白酶能够切割玉米几丁质酶 ChitA，它是一种 PA 结构域丝氨酸蛋白酶。从 S. maydis 培养物中纯化了蛋白酶，并通过 LC-MS/MS 对胰酶肽段进行了分析。离子被映射到两个预测的 PA 结构域丝氨酸蛋白酶上。设计了酵母菌株来表达每种蛋白酶。一个未能产生重组蛋白，而另一个则分泌了切割 ChitA 的蛋白酶。我们将这种蛋白酶命名为 kilbournase，并对其进行了纯化和特性分析。它在 ChitA 的氨基末端几丁质结合域中切割了多个肽键，而保留了催化结构域的完整性。与 ChitA-LH82 相比，kilbournase 在 ChitA-B73 同种型上的活性更高，并且不切割拟南芥的 AtChitIV3 同源物，表明其具有高度的特异性。kilbournase 对玉米 ChitA 的切割类似于化脓性链球菌 ScpA 对人 C5a 的切割，这表明细菌和真菌中的 PA 结构域蛋白酶可能通常靶向特定的宿主蛋白。
