Insect immunity. Isolation and sequence of two cDNA clones corresponding to acidic and basic attacins from Hyalophora cecropia

The Cecropia moth has three known classes of antibacterial immune proteins, attacins, lysozyme and cecropins (earlier referred to as P5, P7 and P9, respectively). Six attacins with different isoelectric points have been purified. The N-terminal sequences for five of these forms imply that only two different genes exist. We have now isolated and sequenced two cDNA clones, one for the basic attacin and one for the acidic form. The two mature proteins show 76% homology at the nucleotide level, while the regions beyond the stop codons are 36% homologous. The differences in the content of aspartic acid accounts for the difference in net charge between the acidic and basic attacin. Further differences in charge can be obtained by post-translational removal of a lysine-containing tetrapeptide at the C-terminal end of the two proteins. Evidence for a prepro form of the basic attacin is presented.     

Cloning of cDNAs for Cecropins A and B,and Expression of the Genes in the Silkworm,Bombyx mori

cDNA clones coding cecropins A and B were isolated from a cDNA library constructed from the fat body of immunized Bombyx mori larvae. The cloned cDNAs had an open reading frame of 63 amino acids, indicating the primary translated peptides were processed to form mature cecropins of 35 amino acid residues. The homology in the coding regions of cecropins A and B was 73%.

In immunized fat body, the expression of both cecropin A and B genes reached the maximal level 5 h after the injection of soluble peptidoglycan, and the high level was maintained until 9 h after immunization. The cecropin A and B genes were expressed at high levels in fat body and hemocytes, at lower but significant levels in malpighian tube, slightly in midgut, and none in silk gland.     

Insect immunity. The primary structure of the antibacterial protein attacin F and its relation to two native attacins from Hyalophora cecropia

The attacins are antibacterial proteins present in the hemolymph of the pupae of the silk moth Hyalophora cecropia after bacterial infection. We present the primary structure of one attacin, the F form. We show that this protein is derived by proteolysis from the native protein, attacin E. Using a method for rapid purification from the hemolymph of immunized pupae of the neutral attacin E and a basic attacin, both proteins were found in freshly collected immune hemolymph. We conclude that they are the native products of two attacin genes, the existence of which was inferred from the isolation of two cDNA clones as described in the accompanying paper. The two proteins, which differed in their pIs (7 and 9), were found to have similar mol. wts. (20 000) and closely related primary structures, displaying a total of 40 amino acid substitutions, 12 of which were of a non-conservative nature.     

Insect immunity. Attacins,a family of antibacterial proteins from Hyalophora cecropia.

Six closely related antibacterial proteins, attacins A-F, were isolated from the hemolymph of immunized pupae of the Cecropia moth, Hyalophora cecropia. Chromatofocusing separated attacins A-F, with isoelectric points between 5.7 and 8.3. Immunological experiments show that the attacins constitute antibacterially active forms of the previously isolated inducible immune protein P5. Their mol. wts., 20-23 K, are similar to that of protein P5, but significantly lower than 28 K found for preP5 synthesized in vitro (see accompanying paper). The six attacins can be divided into two groups according to their amino acid composition and amino-terminal sequences, attacins A-D constitute a basic group and attacins E and F an acidic one. Within each group the forms are very similar. The attacins efficiently killed Escherichia coli and two other Gram-negative bacteria isolated from the gut of a silk worm but they did not act on other Gram-positive and Gram-negative bacteria tested. Only growing cells of E. coli were attacked; cells suspended in phosphate buffer were inert. Besides the cecropins and lysozyme, the attacins represent a third class of antibacterial proteins in the humoral immune system of H. cecropia.     

Purification of antibacterial attacins induced in Hyalophora cecropia pupae immunized with Enterobacter cloacae C7-501 using ion-exchange chromatography,hydrophobic interaction chromatography,and Rotofor® isoelectric focusing

Hyalophora cecropia pupae were infected by Enterobacter cloacae C7-501 to induce antibacterial attacins for purification. The induction of attacins in immunized pupae was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), and Rotofor® isoelectric focusing (ISEF) were applied to isolate attacins from the hemolymph. IEC separated attacins from most hemolymph proteins, but the fractions containing attacins also had other proteins of 20 and 64 kDa in length. In IEC, attacin was eluted with ~0.2 M NaCl. The best conditions for IEC were pH 9, flow rate of 2 mL/min, with step elution (0.025, 0.05, 0.075, 0.1, 0.2, 0.4 and 1.0 M NaCl). In HIC, most other proteins were eluted with the ammonium persulfate treatment. HIC isolated attacin proteins under hydrophobic conditions, at ~50% EtOH. However, the fraction with attacins also contained other proteins. The Rotofor® ISEF produced fractions containing attacins at isoelectric points ranging between 5.7 and 8.3. However, non-specific proteins were detected in the fraction samples, and the recovery of attacins was low. The purification efficiency of ISEF was lower than IEC and HIC. In this study, the expression of attacins was induced in H. cecropia pupae infected with E. cloacae C7-501, and attacins could be purified by IEC and ISEF. Overall, IEC provided better separation of attacins from the hemolymph of H. cecropia pupae immunized with E. cloacae bacteria than HIC and Rotofor® ISEF.     

Three peptidoglycan recognition protein (PGRP) genes encoding potential amidase from eri-silkworm, Samia cynthia ricini

Three cDNA clones encoding peptidoglycan recognition proteins (PGRP-B, -C and -D) were isolated from larval fat body of immunized Samia cynthia ricini. The deduced amino acid sequences show high homology to each other and also to Drosophila PGRP-LB, but rather lower homology to all of the known lepidopteran PGRPs including Samia PGRP-A, a receptor-type PGRP. The three PGRPs conserve the five amino acid residues which form the catalytic site of N-acetylmuramoyl L-alanine amidase as in Drosophila LB. The PGRP-C and -D genes were silent in naive larvae, but strongly induced in fat body by an injection of peptidoglycan. PGRP-B gene, in contrast, constitutively expressed at high levels in naive midgut, and the gene was weakly induced in fat body after injection of peptidoglycan.     

荒漠昆虫小胸鳖甲抗菌肽Attacin基因的克隆及低温表达模式分析

昆虫在低温冷驯化过程中会发生很多基因表达的改变, 从转录组层面上开展广泛的研究有助于全面认识昆虫对冷响应的分子机制。为了对荒漠昆虫小胸鳖甲Microdera punctipennis的 4℃低温转录组数据中一个上调表达的Attacin基因（MpAttacin1）进行深入了解并分析其对低温诱导的响应情况, 本研究通过生物信息学分析对该基因进行了鉴定, 并利用实时荧光定量PCR对其在低温下的mRNA水平进行了检测。结果表明： 获得的MpAttacin1 cDNA长度为523 bp, 包含一个456 bp的开放阅读框和67 bp的5′端非翻译区。MpAttacin1编码含有151个氨基酸残基的多肽, N端含有17个氨基酸的信号肽序列。同源性分析表明, 其氨基酸序列与其他鳞翅目、 双翅目和鞘翅目昆虫的抗菌肽Attacin具有30%~40%的一致性。以邻接法（neighbor-joining, NJ）构建的系统进化树表明, 小胸鳖甲Attacin1与其他鞘翅目昆虫的Attacin起源于共同的祖先, 属于Attacin_C超家族。实时荧光定量PCR分析结果显示, 在4℃与-4℃低温胁迫时, MpAttacin1基因的转录都呈现先升高后降低的应激反应趋势, 但在对低温的响应时间和强度上有所不同。在4℃处理5 h和9 h后, MpAttacin1的表达量分别为对照组的2.3和3.8倍, 而在-4℃处理7 h和9 h后, 分别为对照组的2.4和1.5倍。这些研究表明, 除已知的抗菌功能外, Attacin在昆虫的低温适应过程中可能也发挥着重要的作用。     

Cloning and expression of a putative transferrin cDNA of the spruce budworm, Choristoneura fumiferana

A spruce budworm (Choristoneura fumiferana) transferrin cDNA (CfTf) was isolated and cloned from a cDNA library that was constructed using mRNA from fifth to sixth instar larvae. CfTf cDNA encoded a predicted protein of 681 amino acids with a molecular mass of approximately 76 kDa. CfTf shared 72% and 74% identities at the amino acid level with transferrins of Manduca sexta and Bombyx mori, respectively. Like other transferrins, CfTf retains most of the N-terminal, iron-binding amino acid residues. Northern blot analyses indicated that CfTf mRNA was present at high levels after ecdysis, but that the expression level was low prior to ecdysis at the fourth-sixth instar stages. The highest level of CfTf expression was detected in the fat body. Relatively low levels of expression were detected in the epidermis and no expression was found in the midgut. Expression of CfTf mRNA could be induced by bacteria but not fungi. Expression of CfTf mRNA was suppressed by iron load.     

cDNA cloning and deduced amino acid sequence of microvitellogenin, a female specific hemolymph and egg protein from the tobacco hornworm, Manduca sexta

Microvitellogenin is a female-specific yolk protein from the tobacco hornworm moth Manduca sexta. A cDNA library was constructed from poly(A)+ RNA isolated from adult female fat body. cDNA clones of mRNA for microvitellogenin were isolated by using antiserum against microvitellogenin. Northern blot analysis of poly(A)+ RNA isolated from different life stages and sexes reveals that mRNA coding for microvitellogenin is only present in adult female fat body. Immunoprecipitation of the protein product translated from hybrid selected mRNA indicates that the cDNA clone is specific for microvitellogenin. The complete nucleotide sequence of the 834-base pair cDNA insert has been determined by the dideoxy chain termination method. The cDNA sequence predicts that microvitellogenin is a protein of 232 residues with a calculated molecular weight of 26,201. The cDNA also predicts an amino-terminal extension of 17 residues which are not present in the mature form. This sequence appears to be a signal peptide. A comparison of the translated amino acid sequence with the sequences in the National Biomedical Foundation protein library did not establish any sequence homology with other known proteins.     

Molecular cloning and sequence analysis of aggretin, a collagen-like platelet aggregation inducer.

A cDNA library derived from the Malayan-pit-viper (Calloselasma rhodostoma) venom gland was constructed in the phagemid vector. Using the information of the N-terminal amino acid sequences of two subunits of aggretin, synthetic mixed-base oligonucleotides were employed as a screening probe for colony hybridization. Separate cDNA clones encoding for the alpha and beta chains of aggretin were isolated and sequenced. The results revealed that mature alpha and beta chains contain 136 and 123 amino acid residues, respectively. Aggretin subunits show high degrees of identity with respective subunits (50-60% for alpha, 49-58% for beta) of C-type lectin-like snake venoms. The identity to rattlesnake lectin is relatively lower (i.e., 39 and 30%). All cysteine residues in each chain of aggretin are well conserved and located at the positions corresponding to those of C-type lectins. Thus, three intracatenary disulfide bridges and an interchain disulfide bond between Cys83(alpha) and Cys75(beta) may be allocated. This is the first report regarding the entire sequence of venom GPIa/IIa agonist. According to the alignment of amino acid sequences, hypervariable regions among these C-type lectin-like proteins were revealed. These hypervariable regions are proposed to be the counterparts directly interacting with different receptors or different domains of a receptor on the surface of platelet.     

Induction of hemolin gene expression by bacterial cell wall components in eri-silkworm, Samia cynthia ricini

A cDNA clone encoding hemolin was isolated from fat body of immunized Samia cynthia ricini larvae based on subtractive suppression hybridization method. The cDNA encodes 413 amino acid residue open reading frame with an 18 residue predicted signal peptide. The expression of the gene was strongly induced in fat body and midgut by an injection of bacterial cells or peptidoglycans, but very weakly by lipopolysaccharide. The mRNA expression in the fat body was detected as early as 3 h post-injection, and reached the peak level at 12 h.     

cDNA cloning and gene expression of cecropin D, an antibacterial protein in the silkworm, Bombyx mori

We have isolated a cDNA clone encoding a cecropin D precursor from the fat body of Bombyx mori larvae immunized with bacteria by means of differential display. The cDNA contains 298 bp with a coding region of 183 bp for 61 amino acids plus a termination codon (TAG), a 5′-untranslated region of 36 bp, and a 3′-untranslated region of 79 bp including the poly(A) tail. There is a polyadenylation signal sequence of AATAAA at position 266, 43 nucleotides downstream from the termination codon TAG. The homology of the deduced amino acids is greater to the cecropin D precursor from Hyalophora cecropia (67% identity) than to the precursors of cecropins A and B from B. mori (49% to both). Northern blotting analyses reveal that the gene expression of cecropin D is detectable by 4 h after the bacterial injection and reaches the maximal level at 24 h. That high level is maintained up to 48 h post-immunization. Additionally, the gene is expressed mainly in the fat body and slightly in hemocytes, but it is undetectable in other tissues such as the midgut, the Malpighian tubule and silk gland.     

Molecular cloning and characterization of a cDNA encoding a novel antibacterial peptide, defensin, from the mulberry longicorn beetle, Apriona germari.

A full-length cDNA clone with high homology (62% mature peptide sequence identity) to an Acalolepta luxuriosa antibacterial gene, possessing a conserved cysteine-stabilized alphabeta motif, was cloned by screening an Apriona germari cDNA library. This gene (AgCRP) had a total length of 360 bp with an open reading frame of 207 bp, and encoded a predicted peptide of 69 amino acid residues. The mature AgCRP peptide was 27 amino acid residues long and had a cysteine-stabilized alphabeta motif of C...CXXXC...C...CXC consensus sequence, similar to insect defensins. Northern blot analysis revealed that the AgCRP exhibited fat body-specific expression and was up-regulated by wounding, bacterial or fungal challenge.