Cleavage activation and functional comparison of Manduca sexta serine protease homologs SPH1a, SPH1b, SPH4, and SPH101 in conjunction with SPH2.
Cleavage activation and functional comparison of Manduca sexta serine protease homologs SPH1a, SPH1b, SPH4, and SPH101 in conjunction with SPH2.
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DOI：
10.1016/j.ibmb.2022.103762
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发表时间：
2022-05
影响因子：
3.8
通讯作者：
Jiang, Haobo
中科院分区：
文献类型：
--
作者：
Jin, Qiao;Wang, Yang;Hartson, Steven D.;Jiang, Haobo
酚氧化酶(PO)是昆虫抵御微生物感染的免疫反应的重要组成部分。在烟草角虫Manduca sexta中，PO是由其前体ProPO在两种非催化丝氨酸蛋白酶同源物(SPHs)存在的情况下由酚氧化酶激活酶(PAPs)产生的。CDNA克隆和基因组分析表明，SPH1a(以前称为SPH1)、SPH1b、SPH4、SPH101和SPH2含有一个CLIP结构域、一个连接子和一个蛋白酶样结构域(PLD)。SPH1b、SPH4和SPH101 PLD的前22个残基相同，与SPH1a的不同之处仅在第4位，Thr154被SPH1a中的Asn154取代。虽然来自Edman降解的序列被用来建立PAP辅因子作为SPH1a和SPH2的高MR复合体，但这一分配需要进一步的验证，特别是因为SPH1b的mRNA水平远远高于SPH1a的水平，并且与SPH2的转录更好地相关。因此，在这里，我们使用高度特异的引物，测定了这些SPH基因在不同发育阶段的不同组织中的表达谱。根据胰酶处理的SPH的独特多肽，通过靶向LC-MS/MS分析，在取食期、游走期和条虫期、蛹和成虫的血淋巴中检测到高水平的SPH1b和SPH2蛋白，低SPH4，而不检测到SPH1a和SPH101。我们在杆状病毒感染的Sf9细胞中表达了这五种proSPH，作为在血浆样本中鉴定和定量它们的对应物的标准。此外，我们还测试了它们被PAP3的切割以及SPH1a、1b、4和101作为SPH2伙伴在PAP3介导的ProPO激活中的有效性。PAP3比ProSPH1a和4更容易处理proSPH1b和101；当SPH2与SPH101或SPH1b一起存在时，PAP3比SPH1a或SPH4更有效地激活ProPO。这些结果与它们的出现顺序或序列相似性基本一致：SPH101>；SPH1b(98%)>；SPH1a(90%)>；SPH4(83%)。换句话说，可能是由于正选择，新复制的基因(SPH1b和SPH101)的产物是PAP3更有利的底物，在形成高MR辅助因子方面比SPH1a或SPH4有更好的SPH2伙伴。天然凝胶电泳和免疫印迹分析进一步表明，SPH101或1b比SPH1a或4更容易形成高MR复合体。相比之下，SPH2在PAP3第一位点裂解后，在天然凝胶上的迁移率略有下降，然后又有所增加。由于BAR阶段血淋巴中的天然辅因子是平均MR为790 kDa的SPH1和2的复合体，PAP3激活的SPH2可能与较高MR的SPH1b支架结合形成超复合体。它们的结构和形成与SPH1b在不同位置的裂解有关，有待于进一步研究。
Phenoloxidase (PO) is a crucial component of the insect immune response against microbial infection. In the tobacco hornworm Manduca sexta, PO is generated from its precursor proPO by prophenoloxidase activating proteases (PAPs) in the presence of two noncatalytic serine protease homologs (SPHs). cDNA cloning and genome analysis indicate that SPH1a (formerly known as SPH1), SPH1b, SPH4, SPH101, and SPH2 contain a clip domain, a linker, and a protease-like domain (PLD). The first 22 residues of the SPH1b, SPH4, and SPH101 PLDs are identical, and differ from SPH1a only at position 4, Thr154 substituted with Asn154 in SPH1a. While the sequence from Edman degradation was used to establish PAP cofactor as a high Mr complex of SPH1a and SPH2, this assignment needed further validation, especially because SPH1b mRNA levels are much higher than SPH1a's and better correlate with SPH2 transcription. Thus, here we determined expression profiles of these SPH genes in different tissues from various developmental stages using highly specific primers. High levels of SPH1b and SPH2 proteins, low SPH4, and no SPH1a or SPH101 were detected in hemolymph from larvae in the feeding, wandering and bar stages, pupae, and adults by targeted LC-MS/MS analysis, based on unique peptides from the trypsin-treated SPHs. We expressed the five proSPHs in baculovirus-infected Sf9 cells for use as standards to identify and quantify their counterparts in plasma samples. Moreover, we tested their cleavage by PAP3 and efficacy of the SPH1a, 1b, 4, and 101 as SPH2 partners in PAP3-mediated proPO activation. PAP3 processed proSPH1b and 101 more readily than proSPH1a and 4; PAP3 activated proPO more efficiently in the presence of SPH2 with SPH101 or SPH1b than with SPH1a or SPH4. These results generally agree with their order of appearance or sequence similarity: SPH101 > SPH1b (98%) > SPH1a (90%) > SPH4 (83%). In other words, likely due to positive selection, products of the newly duplicated genes (SPH1b and SPH101) are more favorable substrates of PAP3 and better SPH2 partners in forming a high Mr cofactor than SPH1a or SPH4 is. Electrophoresis on native gel and immunoblot analysis further indicated that SPH101 or 1b form high Mr complexes more readily than SPH1a or 4 does. In comparison, SPH2 showed a small mobility decrease and then increase on native gel after PAP3 cleavage at the first site. Since the natural cofactor in bar-stage hemolymph is complexes of SPH1 and 2 with an average Mr of 790 kDa, PAP3-activated SPH2 may associate with the higher Mr SPH1b scaffolds to form super-complexes. Their structures and formation in relation to cleavage of SPH1b at different sites await further exploration.
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DOI：
10.1046/j.1432-1033.2002.03155.x
发表时间：
2002-09-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Lee, KY;Zhang, R;Lee, BL
通讯作者：
Lee, BL
DOI：
10.1046/j.1432-1327.2000.01695.x
发表时间：
2000-10-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Kwon, TH;Kim, MS;Lee, BL
通讯作者：
Lee, BL
影响因子：
3.8
作者：
Cao X;He Y;Hu Y;Zhang X;Wang Y;Zou Z;Chen Y;Blissard GW;Kanost MR;Jiang H
通讯作者：
Jiang H
影响因子：
3.8
作者：
Cao X;Wang Y;Rogers J;Hartson S;Kanost MR;Jiang H
通讯作者：
Jiang H
影响因子：
4.8
作者：
Kim, MS;Baek, MJ;Lee, BL
通讯作者：
Lee, BL