Glucose/Ribitol Dehydrogenase and 16.9 kDa Class I Heat Shock Protein 1 as Novel Wheat Allergens in Baker's Respiratory Allergy.

Wheat allergens are responsible for symptoms in 60-70% of bakers with work-related allergy, and knowledge, at the molecular level, of this disorder is progressively accumulating. The aim of the present study is to investigate the panel of wheat IgE positivity in allergic Italian bakers, evaluating a possible contribution of novel wheat allergens included in the water/salt soluble fraction. The water/salt-soluble wheat flour proteins from the Italian wheat cultivar Bolero were separated by using 1-DE and 2-DE gel electrophoresis. IgE-binding proteins were detected using the pooled sera of 26 wheat allergic bakers by immunoblotting and directly recognized in Coomassie stained gel. After a preparative electrophoretic step, two enriched fractions were furtherly separated in 2-DE allowing for detection, by Coomassie, of three different proteins in the range of 21-27 kDa that were recognized by the pooled baker's IgE. Recovered spots were analyzed by nanoHPLC Chip tandem mass spectrometry (MS/MS). The immunodetected spots in 2D were subjected to mass spectrometry (MS) analysis identifying two new allergenic proteins: a glucose/ribitol dehydrogenase and a 16.9 kDa class I heat shock protein 1. Mass spectrometer testing of flour proteins of the wheat cultivars utilized by allergic bakers improves the identification of until now unknown occupational wheat allergens.

Small Heat Shock Proteins, Amyloid Fibrils, and Nicotine Stimulate a Common Immune Suppressive Pathway with Implications for Future Therapies.

The α7 nicotinic acetylcholine receptor (α7nAChR) is central to the anti-inflammatory function of the vagus nerve in a physiological mechanism termed the inflammatory reflex. Studies on the inflammatory reflex have been instrumental for the current development of the field of bioelectronic medicine. An independent investigation of the biological role of αB-crystallin (HspB5), the most abundant gene transcript present in active multiple sclerosis lesions in human brains, also led to α7nAChR. Induction of experimental autoimmune encephalomyelitis (EAE) in HspB5-/- mice results in greater paralytic signs, increased levels of proinflammatory cytokines, and T-lymphocyte activation relative to wild-type animals. Administration of HspB5 was therapeutic in animal models of multiple sclerosis, retinal and cardiac ischemia, and stroke. Structure-activity studies established that residues 73-92 were as potent as the parent protein, but only when it formed amyloid fibrils. Amyloid fibrils and small heat shock proteins (sHsps) selectively bound α7nAChR on peritoneal macrophages (MΦs) and B lymphocytes, converting the MΦs to an immune suppressive phenotype and mobilizing the migration of both cell types from the peritoneum to secondary lymph organs. Here, we review multiple aspects of this work, which may be of interest for developing future therapeutic approaches for multiple sclerosis and other disorders.

Small heat shock protein speciation: novel non-canonical 44 kDa HspB5-related protein species in rat and human tissues.

When analyzing small stress proteins of rat and human tissues by electrophoretic methods followed by western blotting, and using the anti-HspB1/anti-HspB5 antibody clone 8A7, we unexpectedly found a protein with a molecular mass of ~44 kDa. On two-dimensional gels, this protein resolved into four distinct species. Electrophoretic and immunological evidence suggests that this 44 kDa protein is a derivative of HspB5, most likely a covalently linked HspB5 dimer. This HspB5-like 44 kDa protein (HspB5L-P44) is particularly abundant in rat heart, brain, and renal cortex and glomeruli. HspB5L-P44 was also found in human brains, including those from patients with Alexander disease, a condition distinguished by cerebral accumulation of HspB5. Gray matter of such a patient contained an elevated amount of HspB5L-P44. A spatial model of structurally ordered dimeric HspB5 α-crystallin domains reveals the exposed and adjacent position of the two peptide segments homologous to the HspB1-derived 8A7 antigen determinant peptide (epitope). This explains the observed extraordinary high avidity of the 8A7 antibody towards HspB5L-P44, as opposed to commonly used HspB5-specific antibodies which recognize other epitopes. This scenario also explains the remarkable fact that no previous study reported the existence of HspB5L-P44 species. Exposure of rat endothelial cells to UV light, an oxidative stress condition, temporarily increased HspB5L-P44, suggesting physiological regulation of the dimerization. The existence of HspB5L-P44 supports the protein speciation discourse and fits to the concept of the protein code, according to which the expression of a given gene is reflected only by the complete set of the derived protein species.

A small heat shock protein 21 (sHSP21) mediates immune responses in Chinese oak silkworm Antheraea pernyi.

Small heat shock proteins (sHSPs) are conserved among insects and play an important role in the regulation of many biological processes, including temperature stress, abiotic stress, immune responses, metamorphosis, and embryo development. Antheraea pernyi is an economically valuable silk-producing moth and source of insect food containing high-quality protein. The aim of this study was to quantify expression of the ApsHSP21 gene in response to pathogen-associated molecular patterns (PAMPs) and nucleopolyhedrovirus (NPV) challenge. The deduced ApsHSP21 protein sequence consists of 186 residues with a calculated molecular mass of 21.0 kDa and an isoelectronic point (pI) of 6.63. The protein contains a conserved α-crystallin domain (ACD), and includes two casein kinase II phosphorylation sites, a protein kinase C phosphorylation site, two tyrosine kinase phosphorylation sites, and various polypeptide binding sites. Phylogenetic analysis revealed that ApsHSP21 is closely related to homologs from other insects. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis revealed that expression of ApsHSP21 was significantly up-regulated at different timepoints following simulated pathogen challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), glucan, and NPV. The results suggest sHSP21 is involved in innate immune responses in A. pernyi.

Antibodies against small heat-shock proteins in Alzheimer's disease as a part of natural human immune repertoire or activation of humoral response?

Characterization of autoantibodies specific for some disease-related proteins, would allow to better assess their role as diagnostic and prognostic markers. In the light of increasing evidence for both humoral and cellular adaptive immune responses in the pathophysiology of Alzheimer's disease (AD), and data on the increased small heat-shock proteins (sHSP) expression in this disease, it seemed justified to assess humoral response against sHSP in AD patients. The aim of the study was to check whether AD has the ability to elicit immune response against small HSP, which could also serve as disease biomarkers. IgG and IgM autoantibodies against alpha B-crystallin and anti-HSP 60 IgG autoantibodies were assessed in 59 AD patients and 59 healthy subjects. Both IgM and IgG autoantibodies against alpha B-crystallin in AD patients were significantly higher compared to healthy controls (p < 0.05). No statistically significant differences were found between AD patients and healthy subjects were found in anti-HSP60 IgG autoantibody titers (p = 0.29). Anti-HSP60 antibodies present in AD patients may indeed belong to natural human immune repertoire, and chronic neurodegenerative process does not have significant inducing effect on the systemic immunoreactivity against HSP60. Increased titers of IgM and IgG autoantibodies against alpha B-crystallin in AD patients may reflect activation of humoral immune response in the course of this chronic disease, probably secondary to its increased expression. Further prospective studies, on larger group of AD patients and measuring a change in antibodies titers with disease progression are necessary to assess the exact role of these antibodies in AD.

Humoral response against small heat shock proteins in Parkinson's disease.

INTRODUCTION: In the light of evidence for the increased heat shock proteins (HSP) expression in neurodegenerative disorders, the presence of the adaptive humoral response of the immune system can be expected. The aim of the study was to check whether Parkinson's disease (PD) has the ability to elicit immune response against small heat shock proteins. METHODS: IgG and IgM autoantibodies against alpha B-crystallin were assessed in 26 PD patients 26 healthy subjects. For the assessment of anti-HSP IgG autoantibodies serum samples from 31 parkinsonian patients and 31 healthy control subjects were collected. Serum samples from PD patients and healthy control subjects were collected twice, at baseline and after mean of 13 months follow up. RESULTS: Both IgM and IgG autoantibodies against alpha ß-crystallin in PD patients were significantly higher compared to healthy controls (p<0.05). We also found statistically significant increase in antibodies titers against alpha ß-crystallin over the time of 13 months, both for IgG (p = 0.021) and for IgM (p<0.0001). Additionally, PD patients presented higher levels of anti-HSP IgG autoantibodies than healthy controls (p = 0.02). CONCLUSIONS: Increase of IgG and IgM autoantibodies against alpha B-crystallin in PD patients over time may suggest their involvement in the disease pathogenesis and progression. Further studies are required to confirm the role of this antibody as a biomarker of the disease progression.

Eicosanoids mediate sHSP 20.8 gene response to biotic stress in larvae of the Chinese oak silkworm Antheraea pernyi.

Small heat shock proteins (sHSPs) can regulate protein folding and protect cells from stress. To investigate the role of sHSPs in the silk-producing insect Antheraea pernyi (A. pernyi; Lepidoptera: Saturniidae), cDNA encoding HSP20.8 in A. pernyi, termed Ap-sHSP20.8, was identified as a 564 bp ORF. The translated amino acid sequence encoded 187 residues with a calculated molecular mass of 20.8 kDa and an isoelectronic point (pI) of 5.98; the sequence showed homology to sHSP chaperone proteins from other insects. Ap-sHSP20.8 mRNA transcript expression was abundant in the midgut and fat body and found to be both constitutive and inducible by infectious stimuli. Therefore, Ap-sHSP20.8 may play important roles in A. pernyi immune responses under biotic stress. Furthermore, we found that eicosanoids could mediate the induction of Ap-sHSP20.8 in the fat body and midgut. Our findings show that sHSPs may be promising molecules to target in order to cripple immunity in insect pests.

Late onset of the serological response against the 18 kDa small heat shock protein of Mycobacterium ulcerans in children.

A previous survey for clinical cases of Buruli ulcer (BU) in the Mapé Basin of Cameroon suggested that, compared to older age groups, very young children may be less exposed to Mycobacterium ulcerans. Here we determined serum IgG titres against the 18 kDa small heat shock protein (shsp) of M. ulcerans in 875 individuals living in the BU endemic river basins of the Mapé in Cameroon and the Densu in Ghana. While none of the sera collected from children below the age of four contained significant amounts of 18 kDa shsp specific antibodies, the majority of sera had high IgG titres against the Plasmodium falciparum merozoite surface protein 1 (MSP-1). These data suggest that exposure to M. ulcerans increases at an age which coincides with the children moving further away from their homes and having more intense environmental contact, including exposure to water bodies at the periphery of their villages.

Activation of an immune-regulatory macrophage response and inhibition of lung inflammation in a mouse model of COPD using heat-shock protein alpha B-crystallin-loaded PLGA microparticles.

As an extracellular protein, the small heat-shock protein alpha B-crystallin (HSPB5) has anti-inflammatory effects in several mouse models of inflammation. Here, we show that these effects are associated with the ability of HSPB5 to activate an immune-regulatory response in macrophages via endosomal/phagosomal CD14 and Toll-like receptors 1 and 2. Humans, however, possess natural antibodies against HSPB5 that block receptor binding. To protect it from these antibodies, we encapsulated HSPB5 in porous PLGA microparticles. We document here size, morphology, protein loading and release characteristics of such microparticles. Apart from effectively protecting HSPB5 from neutralization, PLGA microparticles also strongly promoted macrophage targeting of HSPB via phagocytosis. As a result, HSPB5 in porous PLGA microparticles was more than 100-fold more effective in activating macrophages than free soluble protein. Yet, the immune-regulatory nature of the macrophage response, as documented here by microarray transcript profiling, remained the same. In mice developing cigarette smoke-induced COPD, HSPB5-loaded PLGA microparticles were selectively taken up by alveolar macrophages upon intratracheal administration, and significantly suppressed lung infiltration by lymphocytes and neutrophils. In contrast, 30-fold higher doses of free soluble HSPB5 remained ineffective. Our data indicate that porous HSPB5-PLGA microparticles hold considerable promise as an anti-inflammatory biomaterial for humans.

Biochemical characterization and evaluation of a Brugia malayi small heat shock protein as a vaccine against lymphatic filariasis.

Filarial nematodes enjoy one of the longest life spans of any human pathogen due to effective immune evasion strategies developed by the parasite. Among the various immune evasion strategies exhibited by the parasite, Interleukin 10 (IL-10) productions and IL-10 mediated immune suppression has significant negative impact on the host immune system. Recently, we identified a small heat shock protein expressed by Brugia malayi (BmHsp12.6) that can bind to soluble human IL-10 receptor alpha (IL-10R) and activate IL-10 mediated effects in cell lines. In this study we show that the IL-10R binding region of BmHsp12.6 is localized to its N-terminal region. This region has significant sequence similarity to the receptor binding region of human IL-10. In vitro studies confirm that the N-terminal region of BmHsp12.6 (N-BmHsp12.6) has IL-10 like activity and the region containing the alpha crystalline domain and C-terminus of BmHsp12.6 (BmHsp12.6αc) has no IL-10 like activity. However, BmHsp12.6αc contains B cell, T cell and CTL epitopes. Members of the sHSP families are excellent vaccine candidates. Evaluation of sera samples from putatively immune endemic normal (EN) subjects showed IgG1 and IgG3 antibodies against BmHsp12.6αc and these antibodies were involved in the ADCC mediated protection. Subsequent vaccination trials with BmHsp12.6αc in a mouse model using a heterologous prime boost approach showed that 83% protection can be achieved against B. malayi L3 challenge. Results presented in this study thus show that the N-BmHsp12.6 subunit of BmHsp12.6 has immunoregulatory function, whereas, the BmHsp12.6αc subunit of BmHsp12.6 has significant vaccine potential.

Molecular cloning of a small heat shock protein (sHSPII) from the cattle tick Rhipicephalus (Boophilus) annulatus salivary gland.

Immunoscreening of a cDNA expression library of the Rhipicephalus (Boophilus) annulatus tick with purified rabbit anti-R annulatus salivary glands antigens polyclonal antibodies led to the identification of a 661bp sequence. The sequence includes an open reading frame of 543bp encoding a protein of 180 amino acids with calculated molecular weight of 20.51kDa, isoelectric point of 9.071 and with no signal sequence. Comparison of the deduced amino acids with protein data bank showed that the identified polypeptide belongs to the alpha crystallin small heat shock proteins superfamily and shows sequence similarity of 62% and 55% to Ixodes scapularis fed tick salivary gland protein and Ornithodoros parkeri alpha-crystallin protein, respectively. Accordingly, this protein was called Ra-sHSPII. The Ra-sHSPII protein was expressed in E. coli under T7 promotor of the pET-30b vector, purified under denaturation conditions and the immunogenicity and cross-reactivity of the recombinant Ra-sHSPII were evaluated. Direct ELISA showed that the Ra-sHSPII is a strong immunogen. In immunoblotting assay the anti-rRa-sHSPII antisera reacted specifically with purified rRa-sHSPII, with several proteins in R. annulatus whole tick, larval and gut protein extracts in addition to Hyalomma dromedarii and Ornithodoros moubata whole tick protein extracts, as examples of hard and soft tick species, respectively. The rRa-sHSPII protein confers thermal protection to other proteins in vitro as found in other sHSPs. E. coli cell extracts containing the protein were protected from heat-denatured precipitation when heated up to 100°C, whereas extracts from cells not expressing the protein were heat-sensitive at 60°C.

ArHsp22, a developmentally regulated small heat shock protein produced in diapause-destined Artemia embryos, is stress inducible in adults.

Diapause embryos of the crustacean Artemia franciscana exhibit extreme stress tolerance, a property thought to involve molecular chaperones known as small heat shock proteins. To further explore this idea, the structure, function and synthesis of ArHsp22, an Artemia small heat shock protein, were characterized. ArHsp22 contains amino-terminal WXDPF motifs, an alpha-crystallin domain with a highly conserved arginine, and a carboxy-terminal I/VXI/V motif, all typical of small heat shock proteins. ArHsp22 formed large oligomers and exhibited molecular chaperone activity in vitro, protecting citrate synthase and insulin from denaturation. Quantitative PCR and immunoprobing of western blots revealed that ArHsp22 synthesis is restricted to diapause-destined Artemia embryos and that the protein is degraded during post-diapause development. ArHsp22 was observed in cyst nuclei, a location shared by p26 but not ArHsp21, which are two other diapause-specific Artemia small heat shock proteins. ArHsp22 production was enhanced by thermal stress, but only in adults, thus representing the first crustacean small heat shock protein whose synthesis is known to be both developmentally regulated and stress inducible. The results demonstrate that expression of the gene for ArHsp22 is modulated by multiple cues that vary with life history stage. Such findings are of importance in understanding diapause maintenance in Artemia embryos and the survival of adult animals experiencing environmental insult.

A novel small heat shock protein 12.6 (HSP12.6) from Brugia malayi functions as a human IL-10 receptor binding protein.

Phage display cDNA expression library of the third stage larvae (L3) of Brugia malayi was screened for identifying target(s) that bound to the human interleukin-10 receptor (huIL10R). This iterative screening identified an insert that showed significant homology to Caenorhabditis elegans HSP12.6. The gene was designated B. malayi HSP12.6 (BmHSP12.6) and has orthologues in several gastrointestinal nematode genome (Ancylostoma caninum, Ascaris lumbricoides and Ascaris suum) but the gene or gene product has not been studied further in these parasites. Structural analyses of BmHSP12.6 showed that it has a highly conserved alpha-crystallin central domain that is characteristic of other small heat shock proteins (HSPs). BmHSP12.6 has a short N-terminal domain and an unusually small C-terminal domain flanking the crystallin domain suggesting that this protein belongs to a novel class of small HSPs. BmHSP12.6 appears to be differentially transcribed with highest expression in the vertebrate stages of the parasite (L4, adult and mf) compared to its mosquito vector stage (L3). More importantly recombinant BmHSP12.6 bound to huIL10R in a dose dependent fashion and inhibited the binding of human IL-10 (huIL10) to huIL10R in vitro. rBmHSP12.6 also enhanced the growth and proliferation of MC/9 mast cells in vitro similar to huIL10. This study thus describes a novel small HSP from B. malayi that has the capacity to bind to huIL10R, block binding of huIL10 to huIL10R and function similar to huIL10.

Use of the immunodominant 18-kiloDalton small heat shock protein as a serological marker for exposure to Mycobacterium ulcerans.

While it is well established that proximity to wetlands is a risk factor for contracting Buruli ulcer, it is not clear what proportion of a population living in an area where the etiologic agent, Mycobacterium ulcerans, is endemic is actually exposed to this disease. Immunological cross-reactivity among mycobacterial species complicates the development of a specific serological test. Among immunodominant proteins recognized by a panel of anti-M. ulcerans monoclonal antibodies, the M. ulcerans homologue of the M. leprae 18-kDa small heat shock protein (shsp) was identified. Since this shsp has no homologues in M. bovis and M. tuberculosis, we evaluated its use as a target antigen for a serological test. Anti-18-kDa shsp antibodies were frequently found in the sera of Buruli ulcer patients and of healthy household contacts but rarely found in controls from regions where the infection is not endemic. The results indicate that only a small proportion of M. ulcerans-infected individuals contract the clinical disease.

A small heat shock protein of Ostertagia ostertagi: stage-specific expression, heat inducibility, and protection trial.

In this study, we isolated and analyzed a small heat shock protein (HSP) of Ostertagia ostertagi (Oo-HSP18). Oo-hsp18 is encoded by a single-copy gene and the full-length cDNA represents an 18-kDa protein. The expression of Oo-hsp18 is highly stage specific and restricted to the adult stage. The protein is synthesized in a tissue-specific manner and localized in the body muscle layer. The levels of Oo-hsp18 mRNAs are sharply induced by heat shock but not by other stressors such as levamisole and H2O2. A vaccination trial with recombinant Oo-HSP18 failed to protect calves against a challenge infection.