cDNA cloning, purification, properties, and function of a β-1,3-glucan recognition protein from a pyralid moth, Plodia interpunctella
cDNA cloning, purification, properties, and function of a β-1,3-glucan recognition protein from a pyralid moth, Plodia interpunctella
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DOI：
10.1016/s0965-1748(03)00029-8
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发表时间：
2003-06-01
影响因子：
3.8
通讯作者：
Kanost, MR
中科院分区：
文献类型：
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作者：
Fabrick, JA;Baker, JE;Kanost, MR
微生物在其细胞表面具有独特的生化或分子模式，例如由细菌的脂多糖、脂质胆酸和/或肽聚糖和真菌的-1,3-葡聚糖形成的模式。结合这些表面片段的模式识别蛋白与昆虫和其他无脊椎动物的先天免疫反应的激活有关。报道了一段53-kDa的β -1,3-葡聚糖识别蛋白（betaGRP） cDNA的纯化和克隆。betaGRP cDNA包含一个开放阅读框，编码488个氨基酸，其中前17个残基包含分泌信号肽。471残基成熟蛋白的计算分子质量为53311 Da。该蛋白由一个羧基末端结构域组成，该结构域与其他来自无脊椎动物的识别蛋白、来自细菌的-1,3-葡聚糖酶和来自紫棱海胆的-1,3-葡聚糖酶类似。betaGRP的氨基端与其他无脊椎动物识别分子和紫癜菌的-1,3-葡聚糖酶具有序列相似性。对53 kda蛋白的亲和纯化和随后对胰蛋白酶裂解产生的肽的测序证实了betaGRP在p.a interpantella幼虫血淋巴中的存在。RT-PCR分析表明，betaGRP在所有生命阶段均有组成性表达。流浪幼虫暴露于微生物激发子后没有可检测的诱导。Northern blot分析表明，1.8 kb的betaGRP转录本在脂肪体中转录。重组betaGRP保留β -1,3-葡聚糖结合活性，在体外与脂多糖和脂质胆酸结合，引起微生物聚集，并在可溶性β -1,3-葡聚糖存在下激活酚氧化酶原级联反应。这些数据支持了53-kDa betaGRP的功能，即识别病原体表面分子是非自身的，随后激活昆虫的先天免疫反应。2003爱思唯尔科学有限公司版权所有。
Microorganisms possess distinctive biochemical or molecular patterns on their cell surfaces, such as those formed by the lipopolysaccharides, lipoteichoic acids, and/or peptidoglycans of bacteria and the beta-1,3-glucans of fungi. Pattern recognition proteins that bind to these surface moieties have been implicated in the activation of the innate immune response in insects and other invertebrates. We report the purification and cloning of a cDNA for a 53-kDa beta-1,3-glucan recognition protein (betaGRP) from the Indianmeal moth, Plodia interpunctella (Hubner) (Lepidoptera: Pyralidae). betaGRP cDNA contains an open reading frame that encodes 488 amino acids, of which the first 17 residues comprise the secretion signal peptide. The calculated molecular mass of the 471-residue mature protein is 53,311 Da. The protein consists of a carboxyl-terminal domain that is similar to other recognition proteins from invertebrates, beta-1, 3-glucanases from bacteria, and a beta-1,3-glucanase from the sea urchin, Strongylocentrotus purpuratus. The amino-terminus of betaGRP shares sequence similarity with other invertebrate recognition molecules and the beta-1,3-glucanase from S. purpuratus. Affinity purification of a 53-kDa protein and subsequent sequencing of a peptide produced by tryptic cleavage confirmed the presence of the betaGRP in P. interpunctella larval hemolymph. RT-PCR analysis indicates that betaGRP is constitutively expressed in all life-stages. with no detectable induction following exposure of wandering larvae to microbial elicitors. Northern blot analysis indicates that the 1.8-kb betaGRP transcript is transcribed within the fat body. Recombinant betaGRP retains beta-1,3-glucan-binding activity, binds to lipopolysaccharide and lipoteichoic acid in vitro, causes aggregation of microorganisms, and activates the prophenoloxidase cascade in the presence of soluble beta-1,3-glucan. These data support the hypothesis that the 53-kDa betaGRP functions to recognize pathogen surface molecules as nonself and subsequently activates insect innate immune responses. (C) 2003 Elsevier Science Ltd. All rights reserved.