Proteomic approach for acute-phase proteins of hemolymph and muscles in Scylla serrata challenged by a pathogenic bacterium

Acute-phase response is documented to be a significant mechanism of innate immunity in vertebrates and invertebrates. In this study, proteomic methodologies were applied for different protein expressions in hemolymph of Scylla serrata challenged by Vibrio parahaemolyticus after immunization, and in muscles of the crabs separately challenged by V. parahaemolyticus, V. anguillarum and Aeromonas hydrophila. Up-regulated cryptocyanin is documented in the hemolymph and up-regulated calexcitin, wingless (fragment) and tachykinin-related peptide in the muscle as acute-phase proteins. All the four altered proteins were responsible for bacterial stress, but cryptocyanin seemed to be a memory response protein against the challenge by a live bacterium after immunization of the live cells. These up-regulated proteins can be indicative of an understanding of immunity of a crab. __________ Translated from Journal of Xiamen University (Natural Science), 2005, 44(4): 559–562, 44(Sup.): 191–194 [译自: 厦门大学学报(自然科学版), 2005, 44(4): 559–562, 44(增刊): 191–194]     

WSSV对锯缘青蟹的致病性及血清酶指标影响

锯缘青蟹(Scylla serrata)俗称青蟹,是我国重要的海水养殖蟹类.近年来,浙江、福建、广东等青蟹主要养殖地区出现了严重的青蟹病害.对浙江省养殖青蟹的发病原因和流行病学调查发现,白斑综合征病毒(white spotsyndrome virus,WSSV)与青蟹发病存在较大相关性.为进一步研究WSSV对青蟹的致病性和发病机理,作者采用白斑综合征病毒的除菌过滤液,以1:10-1:10000稀释度注射感染青蟹,结果表明1:10、1:100感染组的青蟹死亡率达100%,1:1000感染组死亡率为66.7%,1:10000感染组死亡率为38.9%.根据攻毒悬液的病毒浓度计算出WSSV对青蟹的LD50为1.19×104拷贝/只(7.93×103拷贝/g组织);取WSSV感染青蟹血淋巴进行PCR检测,攻毒死亡青蟹的WSSV检出率为100%,表明WSSV对青蟹有很强的致病力.分析病毒感染濒死蟹的血清酚氧化酶(PO)、过氧化物酶(POD)、超氧化物歧化酶(SOD)、碱性磷酸酶(ALP)、谷丙转氨酶(GPT)、符草转氨酶(GOT)等主要酶指标,发现病毒感染青蟹的PO、POD和SOD活力明显低于对照组,而ALP、GPT和GOT的活力则明显高于对照组;用WSSV单克隆抗体对感染蟹进行免疫组化分析,发现WSSV主要侵染青蟹的鳃、甲壳下表皮、心脏、肠、胃等组织的上皮细胞,尤其以鳃上皮细胞损害最为严重.     

锯缘青蟹复眼的形态和超微结构

对锯缘青蟹(Scylla serrata)的复眼做了电镜观察。扫描电镜下,半球形复眼的背面有一拇指状的无小眼区。透射电镜下,小眼为十足目短尾类特有的长六边形;复眼内小眼的感光系统包括了11个小网膜细胞(RCs),4个RCs位于感光部分的远端,7个RCs构成了感光系统的近端主体;上下两群细胞连接处清晰显示“4 7”且有局部交错的结构。这与三疣梭子蟹和罗氏沼虾溞状幼体的文献记述相似,而与蜘蛛蟹和中华绒螯蟹的“1 7”结构不同,可能与它们对视觉依赖程度差异有关。     

DNA damage and cell necrosis induced by naphthalene due to the modulation of biotransformation enzymes in an estuarine crab Scylla serrata

The sublethal effect of naphthalene (2.5, 5, and 10 mg L(-1)) was studied in an estuarine crab Scylla serrata with reference to macromolecular changes. Biotransformation enzymes such as cytochrome P450, cytochrome b(5), NADPH cytochrome P450 reductase, aryl hydrocarbon hydroxylase, glutathione-S-transferase, and UDP-glucuronyl transferase were elevated in the hepatopancreas of naphthalene-exposed crabs in comparison with control. Remarkable amount of DNA damage and cell necrosis was observed in hepatopancreas, hemolymph, and ovary of the crabs exposed to naphthalene, when compared with control. For all the parameters studied, a concentration-dependent gradient of the changes was observed. The expression of DNA damage and cell necrosis suggests an increased production of oxidants during naphthalene metabolism.     

Immune-related proteins induced in the hemolymph after aseptic and septic injury differ in honey bee worker larvae and adults

We have employed the proteomic approach in combination with mass spectrometry to study the immune response of honey bee workers at different developmental stages. Analysis of the hemolymph proteins of noninfected, mock-infected and immune-challenged individuals by polyacrylamide gel electrophoresis showed differences in the protein profiles. We present evidence that in vitro reared honey bee larvae respond with a prominent humoral reaction to aseptic and septic injury as documented by the transient synthesis of the three antimicrobial peptides (AMPs) hymenoptaecin, defensin1, and abaecin. In contrast, young adult worker bees react with a broader spectrum of immune reactions that include the activation of prophenoloxidase and humoral immune responses. At least seven proteins appeared consistently in the hemolymph of immune-challenged bees, three of which are identical to the AMPs induced also in larvae. The other four, i.e., phenoloxidase (PO), peptidoglycan recognition protein-S2, carboxylesterase (CE), and an Apis-specific protein not assigned to any function (HP30), are induced specifically in adult bees and, with the exception of PO, are not expressed after aseptic injury. Structural features of CE and HP30, such as classical leucine zipper motifs, together with their strong simultaneous induction upon challenge with bacteria suggest an important role of the two novel bee-specific immune proteins in response to microbial infections.     

Proteomic studies of human and other vertebrate muscle proteins

This review summarizes results of some systemic studies of muscle proteins of humans and some other vertebrates. The studies, started after introduction of two-dimensional gel electrophoresis of OFarrell, were significantly extended during development of proteomics, a special branch of functional genomics. Special attention is paid to analysis of characteristic features of strategy for practical realization of the systemic approach during three main stages of these studies: pre-genomic, genomic (with organizational registration of proteomics), and post-genomic characterized by active use of structural genomics data. Proteomic technologies play an important role in detection of changes in isoforms of various muscle proteins (myosins, troponins, etc.). These changes possibly reflecting tissue specificity of gene expression may underline functional state of muscle tissues under normal and pathological conditions, and such proteomic analysis is now used in various fields of medicine.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1574–1591.Original Russian Text Copyright © 2004 by Shishkin, Kovalyov, Kovalyova     

双向电泳-质谱技术筛选肝癌血清标记物

采用双向电泳 - 质谱技术筛选肝癌特异的血清蛋白标记物,以利于肝癌的早期诊断和治疗 . 肝癌、肝炎和正常三组各 20 例血清先采用超声、高丰度蛋白去除、脱盐预处理以优化双向电泳,图像分析三组血清图谱寻找差异点,基质辅助激光解吸飞行时间质谱对差异点进行鉴定 . 结果显示,通过样品预处理,血清上样体积平均增加 3 ～ 4 倍,参考胶点数由 218 个增至 332 个,白蛋白和免疫球蛋白明显减弱,水平条带明显减少 . 图谱比较所得 37 个差异点,经鉴定为 7 种蛋白 . 与正常组比较,转铁蛋白、甲状腺素运载蛋白在肝炎和肝癌组低表达,α-1 抗胰蛋白酶、凝聚素、铜蓝蛋白、触珠蛋白在肝炎和肝癌组均高表达 . α-1 抗胰蛋白酶在肝癌组较肝炎组高表达,而热休克蛋白 27 只在肝癌组表达 . 上述结果提示,双向电泳-质谱技术可发现肝癌发生发展过程中血清蛋白表达谱质或量的变化,从而为肝癌的早期诊断及治疗奠定基础 .     

Xenobiotic absorption and binding by proteins in hemolymph of the weevil Diaprepes abbreviatus

A synthetic coumarin, 7-amino-3-phenyl coumarin (coumarin-10), was used to study the uptake of ingested xenobiotics into hemolymph. Larvae were forcefed coumarin-10 in peanut oil, and hemolymph was extracted and analyzed by fluorescence spectroscopy. Coumarin-10 entered hemolymph within 5 min, reaching a steady state of concentration within 1 h. Assayed 2 h after feeding, hemolymph titers of 1–5 ng/μl were proportional to log dose between 10 and 100 ng/mg body weight; hemolymph did not reach saturation. Fluorescence spectra of hemolymph in saline revealed that energy was readily transferred from hydrophobic residues of hemolymph proteins to coumarin-10. Ultracentrifugal density gradients revealed that 94% of absorbed coumarin-10 was bound to sedimenting proteins while 6% bound to lipophorin. Native polyacrylamide gel electrophoresis (N-PAGE) on minigels identified two major proteins responsible for binding. Though readily separated by native electrophoresis, these proteins were not fully separable by HPLC using a wide variety of columns. Gel permeation-HPLC of the sedimenting proteins from hemolymph revealed a single major peak of 480,000 M_r. When upper and lower electrophoretic bands were isolated by preparative N-PAGE, the upper band (band I) yielded subunits of 75,000 and 71,000 M_r, while the lower band (band II) yielded only one size subunit of 75,000 M_r on denaturing (SDS) PAGE. The fluorescent products bound by sedimenting proteins were identified by thin-layer chromatography and scanning fluorescence densitometry as coumarin-10 (80% of total) and a polar metabolite (20%). In addition, lipophorin-containing fractions contained an apolar metabolite (3% of total fluorescence). In vitro binding studies utilizing fluorescent energy transfer demonstrated saturation binding with a K_D of 1.5 μM.     

Proteomic approach to probe for larval proteins of the mussel Mytilus galloprovincialis

A proteomic approach was used to search for larval proteins specific to the mussel Mytilus galloprovincialis from Galicia in northwest Spain. The study included both a comparative analysis, through two-dimensional electrophoresis, of protein expression maps of the larvae of the mussel and of 5 abundant and commercially important bivalve species from the region (Ostrea edulis, Cerastoderma edule, Pecten maximus, Tapes decussatus, and Venarupis pullastra) and subsequent mass spectrometric analysis of some of the protein spots. A total of 18 spots were selected and isolated from gels of M. galloprovincialis larvae. From their relative position on the electrophoresis gels, 6 of these were clearly exclusive to the mussel species. However, it was not clear whether the other spots were shared by other species. To overcome this ambiguity, first an analysis using matrix assisted laser desorption ionization with time-of-flight (MALDI-TOF) was conducted on the 6 spots of Mytilus that could possibly be shared with only one species. The peptide mass fingerprinting was completely different for the proteins compared. This result confirmed that the 6 proteins were exclusively mussel proteins, but demonstrated the utility of this approach when working with species that are poorly represented at the protein level in databases.     

Alteration of hemolymph polypeptides in Manduca sexta larvae parasitized by Cotesia congregata: A two-dimensional electrophoretic analysis and comparison with major bacteria-induced proteins

Analyses using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) previously demonstrated that parasitization by the braconid wasp Cotesia congregata significantly alters the normal hemolymph polypeptide profile of host Manduca sexta larvae. In the present study two-dimensional gel analyses corroborated our earlier findings and provided additional evidence that multiple parasitism-specific polypeptides were induced, which varied according to the stage of development of the wasps. Parasitization additionally elicited changes in the total protein concentration detected in the blood. Initially an elevation was observed, with newly parasitized larvae exhibiting a twofold elevation in hemolymph protein concentration by 12–24 h postoviposition. In contrast, terminal-stage hosts with second instar parasites had significantly less protein in the hemolymph, likely due to reduced growth and inhibition of arylphorin synthesis by the fat body during the final stages of parasitism. Comparison of the array of hemolymph polypeptides produced in unparasitized larvae injected with 10⁶cells of the gram-negative bacterium Enterobacter cloacae with those proteins induced by parasitization indicated the two classes are different. Our findings confirm that the hostresponse to parasitism is a specific one, and not mimicked by bacterial challenge. Duringshort-term in vitro culture of wasp larvae dissected from the host hemocoel, several proteins were detected in the medium using SDS - PAGE, with their appearance in vitro suggestive of secretion by the wasps in vivo. Moreover, hemolymph from the parasites had significant amounts of putative host proteins, including an arylphorin - like polypeptide and a protein with a mobility similar to that of insecticyanin. Thus, a dynamic interchange of proteins may occur, with the parasites accumulating host proteins while simultaneously secreting a variety of factors into the host hemocoel.     

Pig acute-phase protein levels after stress induced by changes in the pattern of food administration

A total of 240 pigs, 74 days old, half boars and half females, were included in a trial designed to assess the effect of the stress caused by changes in the pattern of food administration on the concentration of acute phase proteins (APP) and productive performance parameters. Half of the animals (pigs fed ad libitum, AL group) had free access to feed, while the rest were fed following a disorderly pattern (DIS group), in which animals had alternating periods of free access to feed and periods of no feeding, when food was removed from the feeder. The periods of free access to feed (two daily periods of 2-h duration) were randomly assigned, and varied from day to day. Total feed supplied per day was identical in both groups, and exceeded the minimal amount required for animals of these ages. Pen feed intake, individual body weights and the main positive pig APP pig major acute phase protein (Pig-MAP), haptoglobin, serum amyloid A (SAA), C-reactive protein (CRP), and the negative APP apolipoprotein A-I (ApoA-I) and transtherytin were determined every 2 weeks during the period 76 to 116 days of age. Animals fed ad libitum had better average daily gain (ADG) than DIS animals in the whole experimental period (P < 0.01) but the differences in ADG were only produced in the two first experimental sub-periods (60 to 74 and 74 to 116 days of age), suggesting that the stress diminished when the animals get used to the DIS feeding. Interestingly differences in ADG between DIS and AL pigs were due to males, whereas no differences were observed between females. The same differences observed for ADG were found for APP. DIS males had higher Pig-MAP concentration than AL males at 74 and 116 days of age, lower ApoA-I concentration at 74 days of age and higher haptoglobin and CRP concentration at 116 days of age (P < 0.05). The results obtained in this trial show an inverse relationship between weight gain and APP levels, and suggest that APP may be biomarkers for the evaluation of distress and welfare in pigs.     

Drosophila hemolymph proteins: Purification,characterization, and genetic mapping of larval serum protein 2 in D. melanogaster

Three of the major protein species present in the hemolymph of Drosophila melanogaster larvae just prior to pupation are absent from second instar larvae but accumulate rapidly during the third instar. This article describes the purification and characterization of one of these, larval serum protein (LSP) 2, using an immunological assay. It is a homohexamer of molecular weight about 450,000, with a polypeptide molecular weight of 78,000–83,000. Fast and slow electrophoretic variants of this protein map between the markers vin and gs, at 36–37 on chromosome 3.This work was partially supported by M.R.C. Research Studentships to J.W. and M.E.A.     

A proteomic approach to identification of transmembrane proteins and membrane-anchored proteins of Arabidopsis thaliana by peptide sequencing.

A proteomic approach was developed for the identification of membrane-bound proteins of Arabidopsis thaliana. A subcellular fraction enriched in vacuolar membranes was prepared from 4-week-old plants and was washed with various agents to remove peripheral membrane proteins and contaminating soluble proteins. The remaining membrane-bound proteins were then subjected to proteomic analysis. Given that these proteins were resolved poorly by standard two-dimensional gel electrophoresis, we subjected them instead to SDS-polyacrylamide gel electrophoresis and to protein digestion within gel slices with lysylendopeptidase. The resulting peptides were separated by reverse-phase high-performance liquid chromatography and subjected to Edman sequencing. From the 163 peptide peaks analyzed, 69 peptide sequences were obtained, 64 of which were informative. The proteins corresponding to these peptide sequences were identified as belonging to 42 families, including two subfamilies, by comparison with the protein sequences predicted from annotation of the A. thaliana genome. A total of 34 proteins was identified definitively with protein-specific peptide sequences. Transmembrane proteins detected in the membrane fraction included transporters, channels, receptors, and unknown molecules, whereas the remaining proteins, categorized as membrane-anchored proteins, included small GTPases, GTPase binding proteins, heat shock protein 70-like proteins, ribosomal proteins, and unknown proteins. These membrane-anchored proteins are likely attached to membranes by hydrophobic anchor molecules or through tight association with other membrane-bound proteins. This proteomic approach has thus proved effective for the identification of membrane-bound proteins.     

短时高温暴露下东亚飞蝗雌虫血淋巴蛋白表达分析及质谱鉴定

为研究短时高温对东亚飞蝗Locusta migratoria manilensis Meyen血淋巴蛋白的影响,采用Bradford法、SDSPAGE电泳和质谱等方法,对东亚飞蝗雌虫血淋巴样品进行检测。结果表明:短时高温对血淋巴蛋白含量有显著影响(P0.01),36℃-42℃范围内,随温度升高,血淋巴蛋白浓度亦升高,其中39℃、42℃处理组与对照组差异显著(P0.01);短时高温对血淋巴蛋白种类存在一定影响,对照组雌虫血淋巴中存在11种蛋白,高温处理后,4种蛋白含量逐渐增加,6种蛋白含量没有明显变化,1种蛋白消失;经质谱检测,鉴定了5种蛋白,分别为载脂蛋白前体、酚氧化酶原、2个储存蛋白和19 kDa血淋巴蛋白,另外6条蛋白未被鉴定。推测载脂蛋白前体、酚氧化酶原、储存蛋白在东亚飞蝗应对高温胁迫过程中发挥重要作用。     

Proteomic analysis of metabolic, cytoskeletal and stress response proteins in human heart failure

Human heart failure is a complex syndrome and a primary cause of morbidity and mortality in the world. However, the molecular pathways involved in the remodelling process are poorly understood. In this study, we performed exhaustive global proteomic surveys of cardiac ventricle isolated from failing and non-failing human hearts, and determined the regulatory pathway to uncover the mechanism underlying heart failure. Two-dimensional gel electrophoresis (2-DE) coupled with tandem mass spectrometry was used to identify differentially expressed proteins in specimens from failing (n = 9) and non-failing (n = 6) human hearts. A total of 25 proteins with at least 1.5-fold change in the failing heart were identified; 15 proteins were up-regulated and 10 proteins were down-regulated. The altered proteins belong to three broad functional categories: (i) metabolic [e.g. NADH dehydrogenase (ubiquinone), dihydrolipoamide dehydrogenase, and the cytochrome c oxidase subunit]; (ii) cytoskeletal (e.g. myosin light chain proteins, troponin I type 3 and transthyretin) and (iii) stress response (e.g. αB-crystallin, HSP27 and HSP20). The marked differences in the expression of selected proteins, including HSP27 and HSP20, were further confirmed by Western blot. Thus, we carried out full-scale screening of the protein changes in human heart failure and profiled proteins that may be critical in cardiac dysfunction for future mapping.     

Purification and characterization of chitin-binding proteins from the hemolymph of sweet potato hornworm, Agrius convolvuli

Three chitin-binding proteins (CBPs: CBP9, CBP15, CBP66) were identified from the larval hemolymph of sweet potato hornworm, Agrius convolvuli.Two (CBP9 and CBP15) of them have been isolated and purified by gel filtration (Superdex HR 75), cation-exchange chromatography (Mono S), and reverse-phase chromatography (μRPC PC 2.1/3). In experiments to detect CBPs in hemolymph, we examined whether ionic strength and existence of bovine serum albumin in the incubation solution influenced binding affinity of CBPs to chitin. The N-terminal sequences of three CBPs were determined by the automated Edman degradation and showed the sequence homology in basic local alignment search tool search. CBP15 and CBP66 were quite similar to lysozymes and bovine serum albumins, respectively. In contrast, CBP9 is not similar to any other known protein, as judged from databank comparisons. Therefore, we concluded that CBP9 is a novel protein with binding capacity to chitin that is a component of the fungal cell wall. CBP9 has no antibacterial activity against Escherichia coli and Micrococcus luteus, and also showed negative response in hemagglutination assay. CBP9 is confirmed as a monomer with a molecular mass of 9.14 kDa by electron spray ionization and matrix-assisted laser desorption ionization mass spectrometry.     

Identification,characterization, and developmental profile of a high molecular weight,juvenile hormone-binding protein in the hemolymph of the migratory grasshopper,Melanoplus sanguinipes

In the hemolymph of Melanoplus sanguinipes, a high molecular weight juvenile hormone binding protein (JHBP) was identified by photoaffinity labelling and found to have a M_r of 480,000. The JHBP, purified using native gel electrophoresis followed by electroelution, has an equilibrium dissociation constant for JH III of 2.1 nM and preferentially binds JH III over JH I. Antibody raised against JHBP recognized only the 480,000 band. Under denaturing conditions the native JHBP gave a single band with a M_r 78,000. The antibody against native JHBP recognized only the 78,000 protein in SDS-treated hemolymph samples, indicating that JHBP is a hexamer in this species. The concentration of JHBP fluctuates in both the sexes during nymphal and adult development in parallel with total protein content of hemolymph. © 1995 Wiley-Liss, Inc.