Type-I-hypersensitivity to 15 kDa, 28 kDa and 54 kDa proteins in vitellogenin specific to Gadus chalcogrammus roe

BackgroundFish roe allergy is a common health problem in countries where sea food is a major part of the diet, such as Japan. β′-component (β′-c) in fish roe has been identified as a major antigen for patients who show hypersensitivity to various fish roes. However, little is known about causative antigens for patients reactive to fish roe of specific species.MethodsSerum and basophils were obtained from patients who had reactivity to roes of Gadus chalcogrammus (GC) and/or other fish species. GC roe specific antigens were analyzed by immunoblotting, histamine release assay (HRA) and mass spectrometry. Recombinant-fragments of vitellogenin (Vg) were obtained by the Escherichia coli expression system.ResultsSerum IgE of a patient with specific reactions to GC roe bound to 15, 28, 40 and 70 kDa-proteins in GC roe extract. Mass spectrometry analysis revealed that proteins in these bands contained fragments corresponding to Vg. Immunoblotting of Vg immunoprecipitated by rabbit anti-Vg antiserum from the extract revealed 15, 28 and 54 kDa fragments bound by the patient's IgE. These bindings were inhibited by the pretreatment of recombinant phosvitin (rPv) and β′-c (rβ′-c). Fractions obtained by native gel electrophoresis containing 15, 28 and 54 kDa proteins, but not the other fractions, induced significant histamine release from the patient's basophils. Sera of the other patients with GC roe specific-IgE showed IgE binding to rPv and/or rβ′-c.ConclusionsThe 15, 28 and 54 kDa-fragments of Vg which include structures of Pv and β′-c, could be antigens for GC roe specific type-I-hypersensitivity.     

Multi-component adsorption model for pellicle formation: the influence of salivary proteins and non-salivary phospho proteins on the binding of histatin 5 onto hydroxyapatite

The acquired enamel pellicle formed by selective adsorption of proteins in whole saliva is a protective integument on the tooth surface. The purpose of the present study was to investigate the formation of human acquired enamel pellicle using an in vitro hydroxyapatite (HA) model and 3H-histatin 5 to allow accurate measurement of histatin 5 binding in a multi-component experimental system. A binary system was employed by mixing 3H-histatin 5 with one unlabeled protein prior to incubation with HA or by first incubating 3H-histatin 5 with the HA which had been pre-coated with one of a panel of unlabeled proteins (human albumin, salivary amylase, lysozyme, acidic PIFs, statherin, the N-terminal fragment of statherin, and egg yolk phosvitin). A ternary system was employed by mixing 3H-histatin 5 with HA sequentially pre-coated with two different unlabeled proteins, including recombinant histatin 1. The results showed that only salivary statherin and egg yolk phosvitin promote histatin 5 adsorption significantly. The amount of histatin 5 adsorbed was also found to increase as a function of the amount of phosvitin and statherin used to pre-coat HA up to a maximum level that was two- to four-fold greater than that observed on untreated HA. These data suggest that specific protein-protein interactions may play important roles in pellicle formation in vivo.     

Type I collagen shows a specific binding affinity for bovine dentin phosphophoryn

Summary Bovine dentin phosphophoryn was iodinated with¹²⁵I, then tested for binding to native monomeric collagen, to collagen fibrils, and to gelatin. The phosphophoryn was found to bind reversibly, but not to denatured collagen (gelatin). Competitive binding studies showed that bovine serum albumin, fibronectin, and bovine bone 34K glycoprotein (osteonectin) did not compete with phosphophoryn and did not inhibit its binding to collagen fibrils. Phosvitin, a phosphoserine-rich protein, did compete, but sixfold higher concentrations of phosvitin than of unlabeled phosphophoryn were required to reduce iodinated phosphophoryn binding to the same extent. Quantitative analyses of the binding showed binding to be limited to the fibril surfaces. Bound phosphophoryn enhanced the uptake of⁴⁵Ca onto collagen fiber surfaces. These data support the hypothesis that, in dentin, the phosphophoryn plays an important role in localizing the calcium binding leading to the growth of collagen-oriented calcium hydroxyapatite crystals.     

Sensitive double antibody sandwich ELISA for the quantification of phosvitin

An effective double antibody sandwich ELISA (DAS-ELISA) method based on monoclonal (mAb) and chicken egg yolk IgY antibodies was developed to determine phosvitin (PV) content in therapeutic and functional products. Leghorn laying hens were immunized with purified PV to produce anti-PV IgY antibody in the egg yolk. High anti-PV IgY titer obtained from the egg yolks collected during 4–10 weeks of the immunization period contained approximately 6.2% of specific anti-PV IgY in total IgY. The PV detection range of the DAS-ELISA and biotinylated DAS-ELISA was 16.8–90 and 7.5–40?ng/mL, respectively. However, biotinylated DAS-ELISA was the better method for PV quantification in terms of accuracy and sensitivity. This highly efficient PV detection method may recuperate the performance of the existing protein assay methods as well as facilitate future research on PV bioactivities and applications.