Structural biological analysis of activation mechanism of hematocyte by ENF peptide family

生长阻断肽（growth -blocking peptide， GBP）是一种小型昆虫细胞因子，具有多种活性：调节幼虫生长、细胞增殖和刺激一类鳞翅目昆虫血细胞（浆细胞）。GBP最初被鉴定为一种25个氨基酸的肽激素（1-25 GBP），在被寄生的粘虫的血淋巴中发现，可以抑制宿主粘虫的生长。而在未被寄生的粘虫幼虫中，血浆中表达23个氨基酸GBP。随后的研究表明，被黄蜂寄生导致GBP的c端氨基酸序列（1-28 GBP）伸长。在这项研究中，我们通过比较它们的生物活性和三维结构来表征由不同长度的c端区组成的GBP类似物。结果表明，1-28 GBP对幼虫的抑制作用最强。核磁共振分析表明，这两种肽具有基本相同的三级结构，^-由非有序的n端区、由二硫键稳定的结构核和水溶液中反平行的β-片组成。然而，在十二烷基磷酸胆碱（DPC）胶束的存在下，1-28 GBP的c端区域经历了从随机盘绕状态到非螺旋状态的构象转变。这表明与膜结合的c端区对幼虫的生长活性有影响。我们从鳞翅目中分离、鉴定并确定了一种新型肽的溶液结构，该肽可诱导快速血细胞聚集。经n端氨基酸测序和3′-RACE分析，从粗被提取物中分离得到的AGF肽含有32个氨基酸残基（h_2n - svqilrcpdgmqmlrsgqcvatteppfdpdsy_ cooh）。在283 ~ 313 K和pH 5.0条件下，记录了^<15> n标记AGF的核磁共振波谱。使用^<15> n编辑的TOCSY和NOESY光谱实现了所有非脯氨酸残基的完整序列分配。用CYANA软件计算解的结构，并结合AGF的功能特征进行讨论。少

Growth-blocking peptide (GBP) is a small insect cytokine with various activities: larval growth regulation, cell proliferation, and stimulation of a class of lepidopteran insect blood cells (plasmatocytes). GBP was initially identified as a 25 amino acid peptide hormone (1-25 GBP), which suppress the growth of host armyworm, found in the hemolymph of the parasitized armyworm. However, in non-parasitized armyworm larvae, 23 amino acid GBP is expressed in insect plasma. Subsequent studies revealed that the parasitization by wasp induced an elongation of the C-terminal amino acid sequence of GBP (1-28 GBP). In this study, we characterized the GBP analogs, which consist of various length of C-terminal region, by comparison of their biological activities and three-dimensional structures. The results indicate that 1-28 GBP exhibits the strongest activity to suppress the larval growth. NMR analysis shows that these peptides have the same tertiary structures basically, ^-which consist of a dis … More ordered N-terminal region, a structured core stabilized by a disulfide bond and an antiparallel β-sheet in aqueous solution. However, the C-terminal region of 1-28 GBP undergoes conformational transition from a random-coiled state to an a-helical state in the presence of dodecylphosphocholine (DPC) micelles. It suggests that the C-terminal region bound to membrane would have an affects on the larval growth activity.We isolated, identified, and determined the solution structure of a novel peptide from lepidopteran, Pseudaletia separata that induces rapid hemocyte aggregation. The isolated peptide from crude integument extract, designated hemocyte-aggregation factor (AGF), was revealed to contain 32 amino acid residues (H_2N-SVQILRCPDGMQMLRSGQCVATTEPPFDPDSY-COOH) by N-terminal amino acid sequencing and 3'-RACE. NMR spectra of ^<15>N-labelled AGF were recorded at 283-313 K and pH 5.0. The full sequential assignment for all non-proline residues was achieved using ^<15>N-edited TOCSY and NOESY spectra. The solution structure was calculated by CYANA and discussed in relevance to the functional feature of AGF. Less