Cloning and characterisation of the Anisakis simplex allergen Ani s 4 as a cysteine-protease inhibitor

Anisakis simplex is a nematode that can parasitise humans who eat raw or undercooked fish containing live L3s. Larvae invading the gastrointestinal mucosa excrete/secrete proteins implicated in the pathogenesis of anisakiasis that can induce IgE mediated symptoms. Misdiagnosis of anisakiasis, due to cross-reactivity, makes it necessary to develop new diagnostic tools. Recombinant allergens have proved to be useful for diagnosis of other parasitoses. Among the Anisakis allergens, Ani s 4 was considered to be a good potential diagnostic protein because of its heat resistance and its importance in the clinical history of sensitised patients. Therefore, the objective of this study was to clone and characterise the cDNA encoding this allergen. The Ani s 4 mRNA sequence was obtained using a PCR-based strategy. The Ani s 4 amino acid sequence contained the characteristic domains of cystatins. Mature recombinant Ani s 4 was expressed in a bacterial system as a His-tagged soluble protein. The recombinant Ani s 4 inhibited the cleavage of a peptide substrate by papain with a Ki value of 20.6 nM. Immunobloting, ELISA, a commercial fluorescence-enzyme-immunoassay and a basophil activation test were used to study the allergenic properties of rAni s 4, demonstrating that the recombinant allergen contained the same IgE epitopes as the native Ani s 4, and that it was a biologically active allergen since it activated basophils from patients with allergy to A. simplex in a specific concentration-dependent manner. Ani s 4 was localised by immunohistochemical methods, using a polyclonal anti-Ani s 4 anti-serum, in both the secretory gland and the basal layer of the cuticle of A. simplex L3. In conclusion, we believe that Ani s 4 is the first nematode cystatin that is a human allergen. The resulting rAni s 4 retains all allergenic properties of the natural allergen, and can therefore be used in immunodiagnosis of human anisakiasis.     

Ani s 10, a new Anisakis simplex allergen: cloning and heterologous expression

Anisakiasis is a human disease caused by accidental ingestion of larval nematodes belonging to the Anisakidae family. Anisakiasis is often associated with a strong allergic response. Diagnosis of A. simplex allergy is currently carried out by test based on the IgE reactivity to a complete extract of L3 Anisakis larvae although the specificity of these diagnostic tests is poor. Improving the specificity of the diagnostic test is possible using purified recombinant allergens. A new Anisakis allergen, named Ani s 10, was detected by immunoscreening an expression cDNA library constructed from L3 Anisakis simplex larvae. The new allergen was overproduced in Escherichia coli; it is a protein of 212 amino acids and it was localized as a 22 kDa protein band in an ethanol fractionated extract from the parasite. Ani s 10 has no homology with any other described protein, and its sequence is composed by seven almost identical repetitions of 29 amino acids each. A total of 30 of 77 Anisakis allergic patients (39%) were positive both to rAni s 10 and natural Ani s 10 by immunoblotting. The new allergen could be useful in a component-resolved diagnosis system for Anisakis allergy.     

Identification of novel three allergens from Anisakis simplex by chemiluminescent immunoscreening of an expression cDNA library

Anisakis simplex is a representative nematode parasitizing marine organisms, such as fish and squids, and causes not only anisakiasis but also IgE-mediated allergy. Although 10 kinds of proteins have so far been identified as A. simplex allergens, many unknown allergens are considered to still exist. In this study, a chemiluminescent immunoscreening method with higher sensitivity than the conventional method was developed and used to isolate IgE-positive clones from an expression cDNA library of A. simplex. As a result, three kinds of proteins, Ani s 11 (307 amino acid residues), Ani s 11-like protein (160 residues) and Ani s 12 (295 residues), together with three known allergens (Ani s 5, 6 and 9), were found to be IgE reactive. Furthermore, ELISA data showed that both recombinant Ani s 11 and 12 expressed in Escherichia coli are recognized by about half of Anisakis-allergic patients. Ani s 11 and Ani s 11-like protein are characterized by having six and five types of short repetitive sequences (5-16 amino acid residues), respectively. Both proteins share as high as 78% sequence identity with each other and also about 45% identity with Ani s 10, which includes two types of short repetitive sequences. On the other hand, Ani s 12 is also structurally unique in that it has five tandem repeats of a CX(13-25)CX(9)CX(7,8)CX(6) sequence, similar to Ani s 7 having 19 repeats of a CX(17-25)CX(9-22)CX(8)CX(6) sequence. The repetitive structures are assumed to be involved in the IgE-binding of the three new allergens.     

An antibody-based ELISA for quantification of Ani s 1, a major allergen from Anisakis simplex

Anisakis simplex is a nematode parasite that can infect humans who have eaten raw or undercooked seafood. Larvae invading the gastrointestinal mucosa excrete/secrete proteins that are implicated in the pathogenesis of anisakiasis and can induce IgE-mediated symptoms. Since Ani s 1 is a potent secreted allergen with important clinical relevance, its measurement could assess the quality of allergenic products used in diagnosis/immunotherapy of Anisakis allergy and track the presence of A. simplex parasites in fish foodstuffs. An antibody-based ELISA for quantification of Ani s 1 has been developed based on monoclonal antibody 4F2 as capture antibody and biotin-labelled polyclonal antibodies against Ani s 1 as detection reagent. The dose-response standard curves, obtained with natural and recombinant antigens, ranged from 4 to 2000 ng/ml and were identical and parallel to that of the A. simplex extract. The linear portion of the dose-response curve with nAni s 1 was between 15 and 250 ng/ml with inter-assay and intra-assays coefficients of variation less than 20% and 10%, respectively. The assay was specific since there was no cross-reaction with other extracts (except Ascaris extracts) and was highly sensitive (detection limit of 1.8 ng/ml), being able to detect Ani s 1 in fish extracts from codfish and monkfish.     

Production of recombinant gG-1 protein of herpes simplex virus type 1 in a prokaryotic system in order to develop a type-specific enzyme-linked immunosorbent assay kit

The herpes simplex viruses are important causes of disease worldwide. Herpes simplex virus type 1 (HSV-1) is the primary cause of oral-facial and pharyngeal infections and may cause herpetic whitlow, eye infections as well as severe and sometimes dangerous infections of the eyes and brain. HSV-1 also accounts for 10-15% of all genital herpetic infections. Therefore, laboratory diagnosis of this virus and development of diagnostic serological techniques for HSV-1 is of particular importance. In the present study, pTrc His2A-gG1 plasmid, containing the full-length glycoprotein G (gG) protein, was produced in a prokaryotic system for the first time. Upon confirmation of a 37-kDa gG-1 protein production in a prokaryotic system based on western blotting and monoclonal antibodies, the protein was produced at a large scale and purified by ion-exchange chromatography using DEAE-sepharose. An HSV-1 type-specific diagnostic kit was designed and developed and the specificity and sensitivity of this kit were demonstrated to be 89.5% and 100%, respectively, as compared with a commercially available kit. A significant correlation was shown between the developed kit and the commercial kit.     

Relationships between IgE/IgG4 Epitopes,Structure and Function in Anisakis simplex Ani s 5, a Member of the SXP/RAL-2 Protein Family

Background

Anisakiasis is a re-emerging global disease caused by consumption of raw or lightly cooked fish contaminated with L3 Anisakis larvae. This zoonotic disease is characterized by severe gastrointestinal and/or allergic symptoms which may misdiagnosed as appendicitis, gastric ulcer or other food allergies.The Anisakis allergen Ani s 5 is a protein belonging to the SXP/RAL-2 family; it is detected exclusively in nematodes. Previous studies showed that SXP/RAL-2 proteins are active antigens; however, their structure and function remain unknown.The aim of this study was to elucidate the three-dimensional structure of Ani s 5 and its main IgE and IgG₄ binding regions.

Methodology/Principal Findings

The tertiary structure of recombinant Ani s 5 in solution was solved by nuclear magnetic resonance. Mg²⁺, but not Ca²⁺, binding was determined by band shift using SDS-PAGE. IgE and IgG₄ epitopes were elucidated by microarray immunoassay and SPOTs membranes using sera from nine Anisakis allergic patients.The tertiary structure of Ani s 5 is composed of six alpha helices (H), with a Calmodulin like fold. H3 is a long, central helix that organizes the structure, with H1 and H2 packing at its N-terminus and H4 and H5 packing at its C-terminus. The orientation of H6 is undefined. Regarding epitopes recognized by IgE and IgG4 immunoglobulins, the same eleven peptides derived from Ani s 5 were bound by both IgE and IgG₄. Peptides 14 (L40-K59), 26 (A76-A95) and 35 (I103-D122) were recognized by three out of nine sera.

Conclusions/Significance

This is the first reported 3D structure of an Anisakis allergen. Magnesium ion binding and structural resemblance to Calmodulin, suggest some putative functions for SXP/RAL-2 proteins. Furthermore, the IgE/IgG₄ binding regions of Ani s 5 were identified as segments localized on its surface. These data will contribute towards a better understanding of the interactions that occur between immunoglobulins and allergens and, in turn, facilitate the design of novel diagnostic tests and immunotherapeutic strategies.     

Development and preparation of recombinant gD antigen of the herpes simplex type 1 (HSV-1) virus]

The most potent antigen among HSV-1 proteins are glycoproteins gB(UL27) and gD(US6). Multiple amino acid sequence alignment of these proteins shows that gD protein is the most specific for HSV-1. Analysis of gD protein epitopes detected the main antigenic determinants not cross-reactive with antigens of other viruses. Virus was isolated and genome DNA was prepared from morphological elements of a patient with herpes simplex infection. US6 gene fragment was cloned in pUC19 vector. Cloning in bacterial expression vectors helped obtain beta-galactosidase-fused recombinant HSV-1 gD protein with 6-histidines affine target for high-performance chromatography purification. ELISA with a set of HSV-1-positive and negative donor sera and a commercial panel of HSV-1 sera (Vektor-Best) showed that recombinant gD can be used as an antigen to HSV-1-specific IgG.     

Purification and characterization of arginine kinase from sea-urchin eggs

In most invertebrates, creatine kinase is replaced by arginine kinase, which catalyzes reversibly the transfer of a phosphate group between adenosine triphosphate and arginine. In sea-urchin larvae, arginine kinase only is expressed whereas in adult sea-urchins both arginine kinase and creatine kinase can be found in the same tissue. In order to study their developmental regulation and properties, we have purified arginine kinase to homogeneity from the eggs of the sea-urchin Paracentrotus lividus. The purification involves ethanol and ammonium sulfate precipitations, followed by an anion-exchange chromatography, an affinity chromatography and a gel filtration. A 500-fold increase in specific activity leads to a specific activity of 360 IU/mg protein at 25 degrees C. Arginine kinase (pI = 5.7) is rapidly and irreversibly inactivated at 45 degrees C. Amino acid composition and Km values (2.08 mM for phospho-L-arginine and 1.25 mM for ADP) are also given. Determination of molecular mass by gel filtration and separation by SDS/polyacrylamide gel electrophoresis indicate that the enzyme is an 81-kDa dimer of two subunits of 42 kDa.     

Purification and Characterization of Vitellin from the Egg of the Suminoe Oyster <Emphasis Type="Italic">Crassostrea ariakensis</Emphasis> and Cross-Reactivity of Anti-vitellin Antibody with Other Marine Invertebrate Egg Proteins

A polyclonal antibody specific to an egg protein of Suminoe oyster Crassostrea ariakensis was previously developed in our laboratory to assess the reproductive life cycle of the oyster. The present study was undertaken to investigate vitellin of C. ariakensis (CAVt). Vitellin is an essential component of egg proteins in marine invertebrates as it provides energy and nutrients to the embryo and larvae. CAVt was purified from eggs of the oyster using ammonium sulfate precipitation followed by affinity chromatography with Concanavalin A-agarose. Native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE showed that CAVt is a high molecular weight [532 kiloDaltons (kDa)] protein, with multiple subunits. Similar to other vitellin proteins, it is a phospholipoglycoprotein composed of phospholipids (12.06%), carbohydrates (mannose, 10.08% or glucose, 9.84%), and alkali-labile phosphates (4.16%). Affinity chromatography, enzyme-linked immunosorbent aasay (ELISA) and western blot analysis revealed that CAVt is only present in the ovary, and two subunits of CAVt (72 and 35 kDa) are believed to be incorporated from the hemolymph into the oocyte. The antibody specific to CAVt (anti-CAVt), raised in rabbit, strongly cross reacted with the egg proteins of oyster species and scallops, suggesting that the antigenic epitopes are highly conserved among species. Our results suggest that the anti-CAVt antibody can be used to develop a tool similar to ELISA or western blotting for investigation of the effect of microorganisms on reproduction as well as the effect of chemicals on the endocrine system in C. ariakensis.     

H1亚型猪流感病毒抗体间接ELISA检测方法的建立及其应用

为建立H1亚型猪流感病毒抗体检测方法,扩增了H1N1亚型猪流感病毒流行株的血凝素基因HA1部分,构建原核表达载体pET30a-HA1,并转化大肠杆菌BL21表达重组蛋白。对重组蛋白包涵体进行变性、复性和Ni-NTA亲和层析纯化。以纯化后的蛋白作包被抗原,建立间接ELISA检测方法。利用该检测方法检测了2008?2009年采集的猪血清785份,阳性率为15.54％,不同省份的阳性率存在差异 (8％~47％)。以IDEXX相关试剂盒检测结果作为参照,该方法的诊断特异性达到91％,诊断敏感性达到95％。     

Molecular and immunological characterization of arginine kinase from the Indianmeal moth, Plodia interpunctella, a novel cross-reactive invertebrate pan-allergen.

IgE recognition of indoor allergens represents a major cause of allergic asthma in atopic individuals. We found that 52 of 102 patients suffering from allergic symptoms indoors contained IgE Abs against allergens from the Indianmeal moth (Plodia interpunctella), a ubiquitous food pest. Using serum IgE from a moth-sensitized patient we screened an expression cDNA library constructed from P. interpunctella larvae. cDNAs coding for arginine kinase (EC 2.7.3.3), a 40-kDa enzyme commonly occurring in invertebrates that is involved in the storage of such high-energy phosphate bonds as phosphoarginine, were isolated. Recombinant moth arginine kinase, designated Plo i 1, was expressed in Escherichia coli as a histidine-tagged protein with enzymatic activity, and purified to homogeneity by nickel chelate affinity chromatography. Purified recombinant arginine kinase induced specific basophil histamine release and immediate as well as late-phase skin reactions. It reacted with serum IgE from 13 of the 52 (25%) moth-allergic patients and inhibited the binding of allergic patients' IgE to an immunologically related 40-kDa allergen present in house dust mite, cockroach, king prawn, lobster, and mussel. Our results indicate that arginine kinases represent a new class of cross-reactive invertebrate pan-allergens. Recombinant arginine kinase may be used to identify a group of polysensitized indoor allergic patients and for immunotherapy of these individuals.     

Effective elution of antibodies by arginine and arginine derivatives in affinity column chromatography

It has been shown that the recovery of monomeric antibodies from protein A affinity chromatography is enhanced significantly by using arginine as an eluent. To extend the applications of arginine to antibody purification and obtain an insight into the mechanism of arginine elution, we compared arginine with citrate, guanidine hydrochloride (GdnHCl), arginine derivatives, and other amino acids in protein A chromatography. We also applied arginine to elution of polyclonal antibodies (pAbs) in antigen affinity chromatography. As described previously, arginine was effective in eluting monoclonal antibodies IgG1 and IgG4. Two arginine derivatives, acetyl-arginine and agmatine, resulted in efficient elution at pH 4.0 or higher, and this was comparable to arginine. On the other hand, other amino acids, such as glycine, proline, lysine, and histidine, are much less effective than arginine under identical pH conditions. Whereas elution increased with arginine concentration, elution with citrate was insignificant in excess of 1 M at pH 4.3. Arginine was also effective in fractionation of pAbs using antigen-conjugated affinity columns. Although GdnHCl was also effective under similar conditions, the eluted material showed more aggregation than did the protein eluted by arginine.     

Degradation of Manduca sexta allatostatin and allatotropin by proteases associated with the foregut of Lacanobia oleracea larvae

The degradation of synthetic Manduca sexta allatostatin (Manse-AS) and allatotropin (Manse-AT), by enzymes of the foregut of larvae of the tomato moth, Lacanobia oleracea was investigated using reversed-phase high performance liquid chromatography (RP-HPLC) together with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and Edman sequencing. Metabolism of 1nmol Manse-AS by foregut extract (1microg protein) was rapid, t(1/2) approximately 5min, with two major products produced. Mass spectrometry of HPLC fractions identified cleavage products Manse-AS-(4-15) and Manse-AS-(6-15), which indicates enzymatic cleavage at the C-terminal side of arginine residues (R(3) and R(5)). This degradation of Manse-AS could be inhibited by up to 80% by the serine protease inhibitor aprotinin, but not PMSF, pepstatin, E64, EDTA, or 1,10-phenanthroline.M. sexta allatotropin was also rapidly degraded when incubated with foregut extract, t(1/2) approximately 8min, producing two metabolic products, one of which was identified as Manse-AT-(1-11), showing enzymatic cleavage at the C-terminal side of arginine (R(11)). The second product was identified as Manse-AT-(1-8). Hydrolysis of Manse-AT could only be partially inhibited by high doses of aprotinin (30%).     

Comparative structural analysis of glycoprotein gD of herpes simplex virus types 1 and 2.

We studied the synthesis and processing of the type-common glycoprotein gD in herpes simplex virus type 2 (HSV-2) and compared it structurally to glycoprotein gD of herpes simplex virus type 1 (HSV-1). We demonstrated that in HSV-2, gD undergoes posttranslational processing from a lower-molecular-weight precursor (pgD51) to a higher-molecular-weight product (gD56). Tryptic peptide analysis by cation-exchange chromatography indicated that this processing step altered neither the methionine nor the arginine tryptic peptide profile of gD of HSV-2. Comparative tryptic peptide analysis of gD of HSV-1 and HSV-2 showed that the methionine and arginine tryptic peptide profiles of these two proteins were very similar, but not identical. Some of the resolved peptides coeluted from the cation-exchange column, suggesting that some amino acid sequences of the two proteins might be very similar. However, each protein also appeared to possess several type-specific tryptic peptides. The structural similarity of these two glycoproteins correlates well with their antigenic cross-reactivity since monoprecipitin antibody to gD of HSV-1 also immunoprecipitates gD of HSV-2 and neutralizes the infectivity of both viruses to approximately the same extent.     

Arginine methylation of the RGG box does not appear to regulate ICP27 import during herpes simplex virus infection

Arginine methylation can regulate protein import and export and can modulate protein interactions. Herpes simplex virus 1 (HSV-1) ICP27 is a shuttling protein involved in viral mRNA export. We previously reported that ICP27 is methylated on three arginines within its RGG box and that arginine methylation regulates ICP27 export and its interaction with SRPK1 and Aly/REF. Here, we report that ICP27 was efficiently imported into the nucleus when hypomethylated as determined by Fluorescence Recovery After Photobleaching (FRAP). Furthermore, coimmunoprecipitation of ICP27 with β-importin was not significantly affected by ICP27 hypomethylation. Thus, ICP27 import does not appear to be regulated by arginine methylation.     

Production of a recombinant antibody fragment in whole insect larvae

Infection of insect cells with baculovirus expression constructs is commonly used to produce recombinant proteins that require post-translational modifications for their activity, such as mammalian proteins. However, technical restraints limit the capacity of insect cell-based culture systems to be scaled up to produce the large amounts of recombinant protein required for human pharmaceuticals. In this study, we designed an automated insect rearing system and whole insect baculovirus expression system (PERLXpress™) for the expression and purification of recombinant proteins on a large scale. As a test model, we produced a recombinant mouse anti-botulinum antibody fragment (Fab) in Trichoplusia ni larvae. A recombinant baculovirus co-expressing the Fab heavy and light chains together with N-terminal sequences from the silkworm hormone bombyxin, to direct proteins into the secretory pathway, was constructed. Fifth instar larvae were reared and infected orally with recombinant (pre- occluded) baculovirus using the automated system and harvested approximately after 4 days. The total yield of recombinant Fab was 1.1 g/kg of larvae, resulting in 127 mg of pure Fab in one production run. The Fab was purified to homogeneity using immobilized metal affinity chromatography, gel filtration, and anion exchange chromatography. The identity of the purified protein was verified by Western blots and size-exclusion chromatography. Purified recombinant Fab was used to detect botulinum toxin in ELISA experiments, demonstrating that the heavy and light chains were properly assembled and folded into functional heterodimers. We believe that this is the first demonstration of the expression of a recombinant antibody in whole insect larvae. Our results demonstrate that a baculovirus-whole larvae expression system can be used to express functionally active recombinant Fab fragments. As the PERLXpress™ system is an automated and linearly scalable technology, it represents an attractive alternative to insect cell culture for the production of large amounts of human pharmaceuticals.     

Purification, characterization, and complete amino acid sequence of a trypsin inhibitor from amaranth (Amaranthus hypochondriacus) seeds.

A protein proteinase inhibitor was purified from a seed extract of amaranth (Amaranthus hypochondriacus) by precipitation with (NH4)2SO4, gel-filtration chromatography, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography. It is a 69-amino acid protein with a high content of valine, arginine, and glutamic acid, but lacking in methionine. The inhibitor has a relative molecular weight of 7400 and an isoelectric point of 7.5. It is a serine proteinase inhibitor that recognizes chymotrypsin, trypsin, and trypsin-like proteinase activities extracted from larvae of the insect Prostephanus truncatus. This inhibitor belongs to the potato-I inhibitor family, showing the closest homology (59.5%) with the Lycopersicum peruvianum trypsin inhibitor, and (51%) with the proteinase inhibitor 5 extracted from the seeds of Cucurbita maxima. The position of the lysine-aspartic acid residues present in the active site of the amaranth inhibitor are found in almost the same relative position as in the inhibitor from C. maxima.     

Production of monoclonal antibodies against grouper nervous necrosis virus (GNNV) and development of an antigen capture ELISA

Five (2 IgG, 3 IgM) monoclonal antibodies (MAbs) against the G9508KS strain of grouper nervous necrosis virus (GNNV) were produced and characterized. All 5 MAbs showed positive signals in the retina of GNNV-infected grouper larvae and in the cytoplasm of GNNV-infected GF-1 cells using immunohistochemistry staining. Two MAbs reacted with the denatured capsid protein derived from GNNV-infected GF-1 cells in Western blot analysis, but did not react with the GNNV recombinant capsid protein expressed by E. coli in an indirect immnunosorbent assay (ELISA). All 5 MAbs were able to neutralize GNNV, tiger puffer NNV (TPNNV) and barfin flounder NNV (BFNNV), while only 2 of the MAbs neutralized striped jack NNV (SJNNV). A capture ELISA system based on the use of MAbs for capture and a rabbit polyclonal antibody for detection was developed. When absorbance values higher than 0.5 were judged to be positive, the sensitivity of the capture ELISA system was 2.5 ng per well of purified GNNV protein or 6.5 x 10(4) TCID50 per well of GNNV supernatant from culture cells. This capture ELISA system could become a more specific and sensitive tool for NNV diagnosis in the field and in routine laboratories.     

Restriction fragment length polymorphisms of Anisakinae larvae

The analysis of restriction fragment length polymorphisms (RFLPs) was applied to distinguish several kinds of Anisakinae larvae, Anisakis larvae (type I) collected from two different paratenic hosts, Anisakis larvae (type II) and Contracaecum larvae. The patterns of the two different paratenic host-derived DNA of Anisakis larva (I) were exactly the same in hybridized fragments generated by six endonucleases. The quite different patterns in RFLPs of genomic DNA were observed among the Anisakis larva (I), Anisakis larva (II) and Contracaecum larvae. The results suggest that the RFLPs analysis may be useful for distinguishing Anisakinae larvae and clarifying the relationships between Anisakis larvae and their adult worms.