Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, 74078, USA.
Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS, 66506, USA.
Insect Biochem Mol Biol. 2022 Sep;148:103818. doi: 10.1016/j.ibmb.2022.103818. Epub 2022 Aug 23.
Serine protease cascades have evolved in vertebrates and invertebrates to mediate rapid defense responses. Previous biochemical studies showed that in hemolymph of a caterpillar, Manduca sexta, recognition of fungi by β-1,3-glucan recognition proteins (βGRP1 and βGRP2) or recognition of bacteria by peptidoglycan recognition protein-1 (PGRP1) and microbe binding protein (MBP) results in autoactivation of hemolymph protease-14 precursor (proHP14). HP14 then activates downstream members of a protease cascade leading to the melanization immune response. ProHP14 has a complex domain architecture, with five low-density lipoprotein receptor class A repeats at its amino terminus, followed by a Sushi domain, a Sushi domain variant called Wonton, and a carboxyl-terminal serine protease catalytic domain. Its zymogen form is activated by specific proteolytic cleavage at the amino-terminal end of the protease domain. While a molecular mechanism for recognition and triggering the response to β-1,3-glucan has been delineated, it is unclear how bacterial recognition stimulates proHP14 activation. To fill this knowledge gap, we expressed the two domains of M. sexta MBP and found that the amino-terminal domain binds to diaminopimelic acid-peptidoglycan (DAP-PG). ProHP14 bound to both the carboxyl-terminal domain (MBP-C) and amino-terminal domain (MBP-N) of MBP. In the mixture of DAP-PG, MBP, and larval plasma, inclusion of an HP14 fragment composed of LDLa repeats 2-5 (LDLa) or MBP-C significantly reduced prophenoloxidase activation, likely by competing with the interactions of the full-length proteins, and suggesting that molecular interactions involving these regions of proHP14 and MBP take part in proHP14 activation in response to peptidoglycan. Using a series of N-terminally truncated versions of proHP14, we found that autoactivation required LDLa. The optimal ratio of PGRP1, MBP, and proHP14 is close to 3:2:1. In summary, proHP14 autoactivation by DAP-type peptidoglycan requires binding of DAP-PG by PGRP1 and the MBP N-terminal domain and association of the LDLa region of proHP14 with the MBP C-terminal domain. These interactions may concentrate the proHP14 zymogen at the bacterial cell wall surface and promote autoactivation.
丝氨酸蛋白酶级联在脊椎动物和无脊椎动物中进化，以介导快速防御反应。先前的生化研究表明，在毛毛虫 Manduca sexta 的血淋巴中，β-1,3-葡聚糖识别蛋白（βGRP1 和 βGRP2）识别真菌，或肽聚糖识别蛋白-1（PGRP1）和微生物结合蛋白（MBP）识别细菌，导致血淋巴蛋白酶-14 前体（proHP14）的自动激活。HP14 然后激活蛋白酶级联反应的下游成员，导致黑化免疫反应。ProHP14 具有复杂的结构域架构，其氨基末端有五个低密度脂蛋白受体 A 重复序列，后面是 Sushi 结构域、称为 Wonton 的 Sushi 结构域变体和羧基末端丝氨酸蛋白酶催化结构域。其酶原形式通过蛋白酶结构域氨基末端的特异性蛋白水解切割而被激活。虽然已经描述了识别和触发对β-1,3-葡聚糖的反应的分子机制，但尚不清楚细菌识别如何刺激 proHP14 激活。为了填补这一知识空白，我们表达了 M. sexta MBP 的两个结构域，发现氨基末端结构域与二氨基庚二酸-肽聚糖（DAP-PG）结合。ProHP14 与 MBP 的羧基末端结构域（MBP-C）和氨基末端结构域（MBP-N）都结合。在 DAP-PG、MBP 和幼虫血浆的混合物中，包含由 LDLa 重复 2-5 组成的 HP14 片段（LDLa）或 MBP-C 会显著降低原酚氧化酶的激活，可能是通过与全长蛋白的相互作用竞争，这表明涉及 proHP14 和 MBP 这些区域的分子相互作用参与了对肽聚糖的 proHP14 激活。使用一系列 N 端截断版本的 proHP14，我们发现自动激活需要 LDLa。PGRP1、MBP 和 proHP14 的最佳比例接近 3:2:1。总之，DAP 型肽聚糖对 proHP14 的自动激活需要 PGRP1 与 DAP-PG 的结合以及 proHP14 的 MBP N 末端结构域与 MBP C 末端结构域的关联。这些相互作用可能将 proHP14 酶原浓缩在细菌细胞壁表面，并促进自动激活。
