Protein disulfide isomerase may facilitate the efflux of nitrite derived S-nitrosothiols from red blood cells

Abstract

Protein disulfide isomerase (PDI) is an abundant protein primarily found in the endoplasmic reticulum and also secreted into the blood by a variety of vascular cells. The evidence obtained here, suggests that PDI could directly participate in the efflux of NO(+) from red blood cells (RBC). PDI was detected both in RBC membranes and in the cytosol. PDI was S-nitrosylated when RBCs were exposed to nitrite under ∼50% oxygen saturation but not under ∼100% oxygen saturation. Furthermore, it was observed that hemoglobin (Hb) could promote PDI S-nitrosylation in the presence of ∼600 nM nitrite. In addition, three lines of evidence were obtained for PDI-Hb interactions: (1) Hb co-immunoprecipitated with PDI; (2) Hb quenched the intrinsic PDI fluorescence in a saturable manner; and (3) Hb-Fe(II)-NO absorption spectrum decreased in a [PDI]-dependent manner. Finally, PDI was detected on the surface RBC under ∼100% oxygen saturation and released as soluble under ∼50% oxygen saturation. The soluble PDI detected under ∼50% oxygen saturation was S-nitrosylated. Based on these data it is proposed that PDI is taken up by RBC and forms a complex with Hb. Hb-Fe(II)-NO that is formed from nitrite reduction under ∼50% O2, then transfers NO(+) to either Hb-Cys β93 or directly to PDI resulting in S-nitroso-PDI which transverses the RBC membrane and attaches to the RBC surface. When RBCs enter tissues the S-nitroso-PDI is released from the RBC-surface into the blood where its NO(+) is transferred into the endothelium thereby inducing vasodilation, suggesting local oxygen-dependent dynamic interplays between nitrite, NO and S-nitrosylation.

Keywords: BCA, bicinchoninic acid; EDTA, ethylenediaminetetraacetic acid; Hb, hemoglobin; Hypoxic vasodilation; NOx, nitric oxide related species; NP-40, nonyl phenoxypolyethoxylethanol; Nitrite reductase; PDI, protein disulfide isomerase; PMSF, penylmethylsulfenylfluoride; Protein disulfide isomerase; RBC, red blood cells; Red blood cells; S-nitroso-protein disulfide isomerase; S-nitrosohemoglobin; SDS-PAGE, sodium dodecyl sulfate, poly acrylamide gel electrophoresis; SNO, S-nitrosothiol; SNO-Hb, S-nitrosohemoglobin.

Graphical abstract

Fig. 1

Western immunoblots of: RBC membrane fraction-Lane 1; RBC homogenate-Lane 2; and standard human PDI-Lane 3, all probed with anti-PDI primary antibodies.

Fig. 2

Nitrite promotes RBC-PDI S-nitrosylation under normoxia but not under hypoxia: (A) Freshly isolated RBCs were equilibrated with either 4% O₂ (Lane 1) or 16% O₂ (Lane 3) in septa sealed vials nitrite (600 nM) was introduced and incubated for 10 min. The RBCs were lysed and the S-nitrosylation status of RBC–PDI was determined by the biotin switch assay visualized with streptavidin-HRP. The band corresponding to PDI was identified from the electrophoretic mobility of standard human PDI subjected to SDS-PAGE under identical conditions (Lane 2). (B) Digitized blot densities (ImageJ) of the bands obtained from 3 different experiments with conditions identical to A in each lane. Error bars represent standard deviation (n=3).

Fig. 3

Hb promotes NO₂⁻-dependent nitrosylation of PDI under normoxic conditions (16%–O₂): (A) These experiments were performed using constant PDI (1 μM), Hb (0.6 mM) and varying amounts of nitrite (78 nM–5 μM) in PBS-Lanes 1–7. The headspace of the vial contained 16% O₂. The mixtures were incubated at 37 °C for 10 min. Aliquots were then removed and added to cold acetone and prepared for either the SNO–PDI determination by the biotin switch assay-visualized by streptavidin-HRP or detecting HB or PDI by Western immunoblots utilizing anti-Hb or anti-PDI, respectively as the primary antibodies. Lane 8-only contained PDI (1 μM) plus nitrite (5 μM) and no Hb. (B) Digitized blot densities (ImageJ) of SNO-PDI as a function of [NO₂⁻] in the presence of Hb (red circles) and absence of Hb (black circles). Error bars represent standard deviation (n=3).

Fig. 4

Hb co-immunoprecipitates with PDI: RBC were immunoprecipitated with anti-PDI:ProteinA/G-Agarose beads and immunoprecipited proteins and various controls were immunoblotted with either anti-PDI (A) or anti-Hb (B) primary antibodies: (A) Lane 1: PDI control; Lane 2: anti-PDI:protein A/G agarose; Lane 3: protein A/G agarose; Lane 4: immunoprecipitation product; (B) Lane 1: Hb control; Lane 2: anti-PDI:protein A/G agarose; Lane 3: protein A/G agarose; Lane 4: immunoprecipitation product.

Fig. 5

PDI intrinsic fluorescence is quenched by Hb: The intrinsic Trp fluorescence (λ_ex 278 nm, λ_em 339 nm) of PDI (0.75 μM) was monitored as a function of [Hb(III)]. The fluorescence was corrected for inner-filter effects by measuring the absorbance of the solution after each addition of Hb and using the equation. $F_{\mathrm{corrected}}=F/1-10^{\mathrm{A}(280\mathrm{nm})}$.

Fig. 6

PDI denitrosates Hb-NO: (A) Hb-(II)–NO was formed by incubating dithionite-reduced Hb(II) (10.8 μM) with nitrite resulting in the characteristic Hb-(II)–NO UV/vis spectrum; (B) The Hb(II)–NO was monitored spectrophotometrically, with respect to time, at 418 nm under Ar (blue circles); or in the presence of air (500 μL ) (red circles); or in the presence of PDI (3.4 μM) plus of air (500 μL) (purple circles). The Hb-NO extinction coefficient used to convert ΔA(418 nm) to [Hb-NO] was 130,000 M⁻¹ cm⁻¹; (C) Hb-(II)–NO (10.8 μM) spectrum in the presence of air (500 μL ) was recorded 10 min after incubation with and varying concentration of PDI to give Hb-NO:PDI ratio between 0 and 10; (D) The re-plot of ΔA(418 nm) from C, corrected for the absorbance decrease by air alone, as a function of PDI:Hb-(II)-NO ratio.

Fig. 7

PDI associates with the RBC surface in an O₂ and nitrite-dependent manner: (A) Representative flow cytometer RBC population distributions, probed for extracellular PDI. The RBCs (∼10⁶ cells/mL) suspended in PBS were equilibrated in 16% O₂ with either no nitrite (blue circles) or 50 μM nitrite (red circles) for 30 min then probed with mouse monoclonal anti-PDI antibody and sheep anti-mouse IgG-FITC and analyzed by flow cytometry; The results obtained for 4% O₂ (not graphically displayed) were −nitrite 0.40%±0.10%, +nitrite 0.35%±0.12%; for 16% O₂ were −nitrite 0.70%±0.085%, +nitrite 8.6%±3.8% (S.D., n=4); (B) Representative immunoblots of soluble PDI detected in the RBC suspension buffer by immunoprecipitation of the RBCs exposed to either 16% or 4% O₂±nitrite. The immunoprecipitation product was subjected to SDS-PAGE and immunoblotted with anti-PDI primary antibodies. (D) Digitized blot densities (ImageJ) of the immunoblots (B) error bars represent S.D. (n=4).

Scheme 1

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