Protein and Peptide Letters - Volume 14, Issue 10, 2007

Small insects pose certain health hazards in households. For example, the ingestion of fresh vegetables containing small worms and eggs can lead to the transmission and spreading of diseases. Therefore pest control is an integral component of successful crop production. Even the dead bodies and excreted materials of infesting arthropods have an impact on our lives; the number of patients suffering from allergic diseases due to arthropod allergens is continuously increasing. A number of arthropod allergens have been identified and characterized during the last two decades and many studies are in progress to identify new allergens. The World Health Organization and the International Union of Immunological Societies Allergen Nomenclature Subcommittee, which is maintained by several experts in the field, provide systematic nomenclature and assistance for the clear identification of allergens. Investigators who identify new allergens should submit the data to the official web site (http://www.allergen.org). One can also access current references and a list of allergen sources from the allergome (http://www.allergome.org). Our goal is to gain valuable insights on allergens from this wealth of information in order to improve the indoor environment for patients with allergies. I had the honorable opportunity to organize a Hot Topic issue for Protein and Peptide Letters and to work with experts to review allergens from domestic arthropods. This issue includes articles on the following topics: domestic arthropod allergens written by myself, house dust mite allergens written by Dr. Wayne R. Thomas, allergens of Blomia tropicalis, a dominant species of storage mite in tropical and subtropical regions written by Dr. Kaw Yan Chua (this article is published separately: PPL; 2007: 14 (4): 325-333), storage mite allergens written by Dr. Enrique Fernandez-Caldas, cockroach allergens written by Dr. Anna Pomes, silverfish allergens written by Dr. Bianca Barletta, mosquito salivary allergens written by Zhikang Peng, other allergens from miscellaneous arthropods written by Dr. Cheol Woo Kim and the application of recombinant allergens for the diagnosis and immunotherapy of allergic diseases written by Dr. John Donnie A. Ramos. I hope that readers enjoy these topics and that the ideas and information available here contribute to their own research.

Allergy prevalence has increased worldwide over the last 25 years along with industrialization and westernized lifestyles. Indoor allergens are primarily responsible for the sensitization and development of atopic diseases. The main indoor allergens are known to be derived from various arthropods which account for up to 80% of the kingdom Animalia. The two classes of arthropods are Insecta, which includes cockroaches, flies, mosquitoes, ants and silverfishes, and Arachnida, which includes mites, spiders, ticks, and scorpions and are the main sources of the allergens. Excreted materials, cast-overs from skin-molting, and dead debris are sources of allergens that can sensitize genetically predisposed individuals and elicit allergic disorders. The use of molecular biology techniques has contributed to the identification and characterization of an ever-increasing number of allergens. However, key determinants and allergen properties that drive allergic responses are poorly understood. The biological characterization of allergens will provide an understanding of the pathogenesis of allergic diseases and contribute to the development of new diagnostic and therapeutic approaches.

Mites of the family Pyroglyphidae are the most important source of house dust mite allergens. A small number of allergens, namely those of groups 1, 2, 4, 5 and 7 constitute the known major and mid-potency specificities, with possible important contributions of the groups 11, 14 and 15 requiring further definition. Most of the allergens can be identified by sequence homologies and the structures of the major allergens have been solved. There are however challenges in determining the nature of the group 5 and 7 allergens and in obtaining detailed structures of the significant allergens to be used for genetic engineering.

In recent years, the allergological importance of different mite species not belonging to the family Pyroglyphidae has been demonstrated. These mites, commonly named storage mites, include Lepidoglyphus destructor, Glycyphagus domesticus, Tyrophagus putrescentiae, Acarus siro, Aleuroglyphus ovatus, Suidasia medanensis and Thyreophagus entomophagus. Several allergens from these species have been purified, sequenced and cloned. Many of these allergens have shown sequence homology and a biological function similar to those previously described in Blomia tropicalis and the Dermatophagoides spp. The main allergens described in storage mites include fatty acid binding proteins, tropomysin and paramyosin homologues, apoliphorine like proteins, alfa-tubulines and other, such as group 2, 5 and 7 allergens, which definitive biological function has not been described yet. Besides the purification and characterization of allergens, the allergenicity of other species such as Acarus farris, Austroglycyphagus malaysiensis, Blomia kulagini and B. tjibodas, Cheyletus eruditus, Chortoglyphus arcuatus, Gohieria fusca, Thyreophagus entomophagus and Tyrophagus longior has been investigated. Research has also been conducted to identify allergens in parasitic mites, such as Psoroptes ovis, Sarcoptes scabiei, Varroa jacobsoni, Diplaegidia columbae and Hemisarcoptes cooremani. The allergenicity of mites present in agricultural environments has been investigated. Crossreactivity studies have also been performed to elucidate to what extent all these mites share common, or species specific epitopes. Herein we present a comprehensive review of the allergenicity of mite species which have been implicated in human respiratory and/or dermatological diseases.

Cockroach allergy is a widespread health problem in the world, associated with the development of asthma. The German and American cockroach species are important producers of a wide variety of allergens. Knowledge of their structure and function contributes to understand their role in allergy and to design tools for diagnosis and immunotherapy.

Insects and insect-derived materials have been implicated as a risk factor for sensitization and subsequent elicitation of allergic rhinitis and allergic bronchial asthma. During the last decades, insects other than those known as allergenic, were investigated for their potential role in inducing and triggering an IgE immune response. Among these, the silverfish, an insect belonging to the Thysanura order, appeared to be of particular interest. Silverfish (Lepisma saccharina) is the most primitive living insect, and represents a descendent of the ancestral wingless insects. They are 3-12 mm long, have three tail feelers and are covered with shiny scales. They shun light and need a humid environment and their diet consists of carbohydrate materials such as paper and book-binding glue, crumbs of bread and flour. Because of these features, silverfish finds an optimal habitat both in dwellings and workplaces and in spite of its antiquity, silverfish has succeeded in exploiting the new opportunity created by man. Although its importance significantly increased when it has been demonstrated that house dust contains significant silverfish levels even in houses where the inhabitants were unaware of its presence, no silverfish extract for diagnosis of allergic diseases is commercially available yet. Identification of optimal extraction conditions and characterization of allergenic extracts are the first steps to obtain an effective allergen preparation suitable for diagnosis and therapy, and will be useful as a reference preparation for assessing silverfish exposure in different indoor environments. It has been cloned and characterized a silverfish tropomyosin, named Lep s 1, which represents the first allergen identified in silverfish extract and can be regarded as a molecule cross-reactive among inhalant and edible invertebrates allergenic sources. rLep s 1 displayed biological activity, suggesting that it could be regarded as a useful tool to study the role of silverfish tropomyosin in the sensitization to invertebrate allergic sources.

Allergic reactions to mosquito bites are caused by allergens in mosquito saliva. In this review, allergic reactions to mosquito salivary allergens, and characteristics of salivary allergens and their recombinant forms are described. The use of the recombinant allergens in the diagnosis of mosquito allergy is discussed.

Of the various arthropods, humans have the greatest contact with mites and cockroaches, and as a result, allergies to these two groups have been the most frequently reported. Changes in lifestyle and living environment have encouraged the growth of arthropods other than mites and cockroaches, and consequently, human exposure to antigens derived from the new arthropods has increased. Although systematic approaches to and immunobiochemical studies of these arthropods are relatively sparse compared with those of mites and cockroaches, recent reports have shown that many species of household arthropods can elicit IgE-mediated reactions via inhalation, biting, or stinging. In this chapter, we review the other arthropods that can induce allergic reaction to human beings. Where the information exists, the frequency of sensitivity and clinical manifestation, and the characterization of the allergens are also reviewed. Virtually all species of arthropods may be sources of allergens that can sensitize and induce IgE-mediated allergic reactions in humans.

Domestic arthropods are chief sources of potent allergens that trigger sensitization and stimulate IgE-mediated allergies. Diagnosis and immunotherapy of arthropod allergies rely on the use of natural allergen extracts which are associated with low specificity and efficacy, the risk of anaphylactic reactions, and the extended period of treatment. Most of the problems associated with natural allergen extracts for allergy diagnosis and immunotherapy can be circumvented with the use of recombinant allergens and peptides. Recombinant allergens are recently developed for microarray-based multiallergen tests which provide component-resolved diagnosis (CRD) of the patient's sensitization profile. Moreover, recombinant protein technology and peptide chemistry have been used to construct isoallergens, allergen mutants, allergoids, T and B cell peptides, hypoallergens, and mimotopes with reduced allergenicity but enhanced immunogenicity for allergenspecific immunotherapy (SIT) and vaccination. The basics of recombinant arthropod allergen technology are in place providing a lucid future for the advancement of diagnosis and immunotherapy of arthropod allergies.

To develop a novel cell-selective antimicrobial peptide with potent anti-inflammatory activity as well as high bacterial cell selectivity, we synthesized a Leu/Lys-rich model peptide, KLW-f (KWKKLLKKfLKLfKKLLK-NH2) containing two Phe-peptoid residues in its middle position. KLW-f exhibited high antimicrobial activity (the MIC range: 0.5∼ 2.0μM) against the tested six bacterial cells. In contrast, KLW-f was no cytotoxic to human red blood cells and HeLa and NIH-3T3 cells. KLW-f caused no or little dye leakage from EYPE/EYPG (7:3, w/w) vesicles (bacterial membranemimicking environments), indicating its bacterial-killing action is probably not due to permeabilization/disruption of bacterial cytoplasmic membranes. Furthermore, KLW-f induced a significant inhibition in LPS-stimulated NO production from mouse macrophage RAW264.7 cells at 10μg/ml. Taken together, our results suggest that KLW-f appear to have promising therapeutic potential for future development as a novel antisepsis agent as well as antimicrobial agent.

This paper studies the β-glucuronidase in the mollusk Pomacea sp. The β-glucuronidase was isolated 206-fold with a 1,5% yield and the cinetc parameters was: pH 5.0, 65°C, Km of 72 x 10-2 mM and molecular mass of 116 kDa. HPLC confirmed the purity. BaCl2 increased β-glucuronidase activity and SDS and NaH2PO4 inhibited completely.

The reaction between Arg-Gly-Asp (RGD) and Lys-Gly-Asp (KGD) derivatives with 3,4',5-trihydroxy-transstilbene (resveratrol) was investigated. Knowing that resveratrol, RGD as well as KGD analogues inhibit human platelet aggregation in vitro, it was tempting for us to examine whether their coupling products present enhanced biological activity. Here, we report on the synthesis and identification of these coupling products. The N-protected peptides were synthesized by solid phase technique, using the 2-chlorotrityl-chloride resin, by the method of carbodiimides. Coupling reactions with resveratrol took place in solution using N,N-dicyclohexylcarbodiimide (DCC) as coupling reagent and 4- dimethylaminopyridine (DMAP) as catalyst. The reaction products were purified by reversed phase HPLC and identified by ESI-MS. The mono-esterified resveratrol derivative was the main (or only) reaction product, whereas the di- and the tri-ester (to a less extent) formation was noticed in some cases.

To elucidate potential biological roles of human β-synuclein, we studied its conformational changes under various conditions. The structural and functional properties of β-synuclein were characterized using biochemical and biophysical methods including: a functional assay, mass spectrometry, size exclusion chromatography, circular dichroism (CD), and fluorescence spectroscopy. The results showed β-synuclein has a high proportion of random coil in solution.

In this study, we investigated inhibitory effects of some metal ions on human erythrocyte glutathione reductase. For this purpose, initially human erythrocyte glutathione reductase was purified 1051-fold in a yield of 41% by using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis was done in order to control the purification of enzyme. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. A constant temperature (4°C) was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. Hg2+, Cd2+, Pb2+, Cu2+, Fe3+ and Al3+ exhibited inhibitory effects on the enzyme in vitro. Ki constants and IC50 values for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I]. IC50 values of Pb2+, Hg2+, Cu2+, Cd2+ , Fe3+ and Al3+ were 0.011, 0.020, 0.0252, 0.0373, 0.209 and 0.229 mM, and the Ki constants 0.0254±0.0027, 0.0378±0.0043, 0.0409±0.0048, 0.0558±0.0083, 0.403±0.043 and 1.137±0.2 mM, respectively. While Pb2+, Hg2+, Cd2+ and Fe3+ showed competitive inhibition, others displayed noncompetitive inhibition.

We demonstrate feasibility of biophysical characterization of the peripheral cannabinoid receptor CB2 produced by heterologous expression in E. coli membranes. Recombinant receptor was purified by affinity chromatography, and NMR diffusion experiments performed on CB2 solubilized in dodecylphosphocholine (DPC) micelles. Circular dichroism spectroscopy indicated high α-helical content (49 %) of CB2.

Cytochrome c from Rhodothermus marinus has been crystallized using the hanging-drop vapor-diffusion method in 30 % (w/v) polyethylene glycol 8K, 0.2 M ammonium sulfate, 8 % hexanediol and 50 mM sodium citrate pH 2.2. The crystals belong to space group P21. X-ray diffraction data were collected to 1.23 Å resolution using synchrotron radiation and a wavelength of 0.93 Å.