Vitellin/Vitellogenin Is an Important Allergen in German Cockroach.

INTRODUCTION: German cockroach (GCr) aeroallergens are associated with allergic rhinitis and asthma. Vitellogenin (Vg) and vitellin (Vn) are abundant proteins in GCr blood and eggs (including egg cases), respectively, and are possible high molecular mass allergens. Prior efforts to purify Vg/Vn yielded amounts too small for subsequent studies. In this study, we report the affinity purification of Vg/Vn from whole-body defatted GCr powder and determination of the binding of Vg/Vn to anti-GCr IgE. METHOD: New Zealand white rabbits were immunized with pure Vg/Vn in Freund's adjuvant, and IgG was purified from the rabbit sera and conjugated to cyanogen bromide (CNBr)-activated Sepharose. Aqueous extracts from GCr powder were passed over the column. After extensive washing, putative Vg/Vn was eluted in low-pH buffer, neutralized, and analyzed by SDS-PAGE and liquid chromatography high-resolution mass spectrometry (LC-HRMS). IgE binding of Vg/Vn was evaluated by inhibition of IgE binding to GCr-ImmunoCAP(I6) in sera from 10 GCr-allergic individuals. In addition, Vg/Vn was biotinylated and bound to ImmunoCAP-streptavidin, and direct IgE antibody binding to the immobilized Vg/Vn was determined in sera from 26 GCr-allergic individuals. RESULTS: Vg/Vn isolated by affinity chromatography was 91% pure by LC-HRMS; contaminants included Bla g 3 (0.9%), human keratin (6%), and rabbit IgG. Vg/Vn inhibited IgE binding to GCr-ImmunoCAP(I6) in 8 of 10 sera. In direct-binding experiments, 21/26 (80%) sera had anti-Vg/Vn IgE at >0.10 kUA/L, while 11/26 (42%) sera were >0.35 kUA/L. CONCLUSIONS: We affinity-purified Vg/Vn and demonstrated that Vg/Vn-specific IgE antibody is a major component of GCr-specific IgE.

Comparison of the oxidative reactivity of recombinant fetal and adult human hemoglobin: implications for the design of hemoglobin-based oxygen carriers.

Hemoglobin (Hb)-based oxygen carriers (HBOCs) have been engineered to replace or augment the oxygen carrying capacity of erythrocytes. However, clinical results have generally been disappointing, in part due to the intrinsic oxidative toxicity of Hb. The most common HBOC starting material is adult human or bovine Hb. However, it has been suggested that fetal Hb may offer advantages due to decreased oxidative reactivity. Large-scale manufacturing of HBOC will likely and ultimately require recombinant sources of human proteins. We, therefore, directly compared the functional properties and oxidative reactivity of recombinant fetal (rHbF) and recombinant adult (rHbA) Hb. rHbA and rHbF produced similar yields of purified functional protein. No differences were seen in the two proteins in: autoxidation rate; the rate of hydrogen peroxide reaction; NO scavenging dioxygenase activity; and the NO producing nitrite reductase activity. The rHbF protein was: less damaged by low levels of hydrogen peroxide; less damaging when added to human umbilical vein endothelial cells (HUVEC) in the ferric form; and had a slower rate of intrinsic heme loss. The rHbA protein was: more readily reducible by plasma antioxidants such as ascorbate in both the reactive ferryl and ferric states; less readily damaged by lipid peroxides; and less damaging to phosphatidylcholine liposomes. In conclusion in terms of oxidative reactivity, there are advantages and disadvantages to the use of rHbA or rHbF as the basis for an effective HBOC.

Dissecting the biochemical architecture and morphological release pathways of the human platelet extracellular vesiculome.

Platelet extracellular vesicles (PEVs) have emerged as potential mediators in intercellular communication. PEVs exhibit several activities with pathophysiological importance and may serve as diagnostic biomarkers. Here, imaging and analytical techniques were employed to unveil morphological pathways of the release, structure, composition, and surface properties of PEVs derived from human platelets (PLTs) activated with the thrombin receptor activating peptide (TRAP). Based on extensive electron microscopy analysis, we propose four morphological pathways for PEVs release from TRAP-activated PLTs: (1) plasma membrane budding, (2) extrusion of multivesicular α-granules and cytoplasmic vacuoles, (3) plasma membrane blistering and (4) "pearling" of PLT pseudopodia. The PLT extracellular vesiculome encompasses ectosomes, exosomes, free mitochondria, mitochondria-containing vesicles, "podiasomes" and PLT "ghosts". Interestingly, a flow cytometry showed a population of TOM20+LC3+ PEVs, likely products of platelet mitophagy. We found that lipidomic and proteomic profiles were different between the small PEV (S-PEVs; mean diameter 103 nm) and the large vesicle (L-PEVs; mean diameter 350 nm) fractions separated by differential centrifugation. In addition, the majority of PEVs released by activated PLTs was composed of S-PEVs which have markedly higher thrombin generation activity per unit of PEV surface area compared to L-PEVs, and contribute approximately 60% of the PLT vesiculome procoagulant potency.

The effect of protein corona composition on the interaction of carbon nanotubes with human blood platelets.

Carbon nanotubes (CNT) are one of the most promising nanomaterials for use in medicine. The blood biocompatibility of CNT is a critical safety issue. In the bloodstream, proteins bind to CNT through non-covalent interactions to form a protein corona, thereby largely defining the biological properties of the CNT. Here, we characterize the interactions of carboxylated-multiwalled carbon nanotubes (CNTCOOH) with common human proteins and investigate the effect of the different protein coronas on the interaction of CNTCOOH with human blood platelets (PLT). Molecular modeling and different photophysical techniques were employed to characterize the binding of albumin (HSA), fibrinogen (FBG), γ-globulins (IgG) and histone H1 (H1) on CNTCOOH. We found that the identity of protein forming the corona greatly affects the outcome of CNTCOOH's interaction with blood PLT. Bare CNTCOOH-induced PLT aggregation and the release of platelet membrane microparticles (PMP). HSA corona attenuated the PLT aggregating activity of CNTCOOH, while FBG caused the agglomeration of CNTCOOH nanomaterial, thereby diminishing the effect of CNTCOOH on PLT. In contrast, the IgG corona caused PLT fragmentation, and the H1 corona induced a strong PLT aggregation, thus potentiating the release of PMP.

Determination and comparison of the baseline proteomes of the versatile microbe Rhodopseudomonas palustris under its major metabolic states.

Rhodopseudomonas palustris is a purple nonsulfur anoxygenic phototrophic bacterium that is ubiquitous in soil and water. R. palustris is metabolically versatile with respect to energy generation and carbon and nitrogen metabolism. We have characterized and compared the baseline proteome of a R. palustris wild-type strain grown under six metabolic conditions. The methodology for proteome analysis involved protein fractionation by centrifugation, subsequent digestion with trypsin, and analysis of peptides by liquid chromatography coupled with tandem mass spectrometry. Using these methods, we identified 1664 proteins out of 4836 predicted proteins with conservative filtering constraints. A total of 107 novel hypothetical proteins and 218 conserved hypothetical proteins were detected. Qualitative analyses revealed over 311 proteins exhibiting marked differences between conditions, many of these being hypothetical or conserved hypothetical proteins showing strong correlations with different metabolic modes. For example, five proteins encoded by genes from a novel operon appeared only after anaerobic growth with no evidence of these proteins in extracts of aerobically grown cells. Proteins known to be associated with specialized growth states such as nitrogen fixation, photoautotrophic, or growth on benzoate, were observed to be up-regulated under those states.

Intact protein analysis for site-directed mutagenesis overexpression products: plasmid-encoded R67 dihydrofolate reductase.

Mass spectrometry is currently the method of choice for the analysis of recombinant protein expression products. By combining proteolytic digestion with peptide mapping and tandem mass spectrometry techniques, verification of site-directed mutagenesis products can be obtained. The proteolytic digestion step converts a purified recombinant protein into a mixture that must be reseparated, thus greatly increasing the analysis time associated with the confirmation of site-directed mutagenesis products. Ion/ion reaction chemistry combined with quadrupole ion trap mass spectrometry provides a fast and efficient way to analyze intact proteins for the correct site-directed mutagenesis products, without heavy reliance on the proteolytic digestion step. Analysis of a series of protein variants (I68M, I68Q, Y69F, and Q67Y) from plasmid-encoded R67 dihydrofolate reductase using ion/ion reaction chemistry confirmed the presence of the correct site-directed mutagenesis products. For the I68M mutant, ion/ion separations detected the presence of extensive degradation from the N-terminal end of the protein. In the case of the Q67Y mutant, a mixture of Q67Y and Q67C species was detected by employing tandem mass spectrometry combined with ion/ion reactions. The ion/ion reaction technique was also performed on a partially purified lysate of the Q67Y/C mixture and successfully screened for the presence of both components in a complex mixture. The ion/ion reaction approach achieved the same results as the proteolytic-digestion-based methodology in a much shorter analysis time.