Heme Binding Properties of Glyceraldehyde-3-phosphate Dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that also functions in transcriptional regulation, oxidative stress, vesicular trafficking, and apoptosis. Because GAPDH is required for the insertion of cellular heme into inducible nitric oxide synthase [Chakravarti, R., et al. (2010) <i>Proc. Natl. Acad. Sci. U.S.A. 107</i>, 18004–18009], we extensively characterized the heme binding properties of GAPDH. Substoichiometric amounts of ferric heme bound to GAPDH (one heme per GAPDH tetramer) to form a low-spin complex with UV–visible maxima at 362, 418, and 537 nm and when reduced to ferrous gave maxima at 424, 527, and 559 nm. Ferric heme association and dissociation rate constants at 10 °C were as follows: <i>k</i>_on = 17800 M^–1 s^–1, <i>k</i>_off1 = 7.0 × 10^–3 s^–1, and <i>k</i>_off2 = 3.3 × 10^–4 s^–1 (giving approximate affinities of 19–390 nM). Ferrous heme bound more poorly to GAPDH and dissociated with a <i>k</i>_off of 4.2 × 10^–3 s^–1. Magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopic data on the ferric, ferrous, and ferrous–CO complexes of GAPDH showed that the heme is bis-ligated with His as the proximal ligand. The distal ligand in the ferric complex was not displaced by CN^– or N₃^– but in the ferrous complex could be displaced by CO at a rate of 1.75 s^–1 (for >0.2 mM CO). Studies with heme analogues revealed selectivity toward the coordinating metal and porphyrin ring structure. The GAPDH–heme complex was isolated from bacteria induced to express rabbit GAPDH in the presence of δ-aminolevulinic acid. Our finding of heme binding to GAPDH expands the protein’s potential roles. The strength, selectivity, reversibility, and redox sensitivity of heme binding to GAPDH are consistent with it performing heme sensing or heme chaperone-like functions in cells

The effect of NO is rapid.

<p>(A, B) SMCs were treated with 500 μM NOC-18 for up to 8 h. The cell layers were rinsed and stained for HA (green) and nuclei (blue). Arrows point to HA-containing cables. Original magnification 20x, 40x (A), or 63x with a 2x zoom (B). Images shown are representative of experiments performed at least three times. (C) SMCs were treated with 500 μM NOC-18 for up to 6 h. Cell layers were rinsed in PBS, lysed and analyzed by Western blotting for HAS2 and GAPDH. Blots shown are representative of three experiments.</p

sGC does not mediate the effects of NO on HA.

<p>A) A model showing how sGC mediates the effects of NO in SMCs and compounds that can modify or mimic the effects. B) SMCs were treated with 300 μM NOC-18 alone for 6 h; pretreated with 5 μM sildenafil for 30 min and then with 5 μM sildenafil and 300 μM NOC-18 for 6 h, 20 μM BAY 41–2272 or BAY 60–2770 for 6 h; or 250 μg/ml 8-bromoguanosine 3’5’cyclic monophosphate for 6 h. At the end of the incubation the cell layers were rinsed with PBS and fixed in cold methanol, and stained for HA (green) and nuclei (blue). Original magnification - 20x. Images shown represent three separate experiments.</p

NOC-18 promotes the deposition of HA cables.

<p>(A) Wild type Balb/c airway SMCs were treated for 23 h at 37°C with or without 500 μM NOC-18, NaNO₂ or NaNO₃. The cell layer was rinsed in PBS, fixed in cold methanol and stained for HA (green) and nuclei (blue). Original magnification– 20X. (B) Wild type Bl/6 airway SMCs were treated for 23 h at 37°C with 500 μM NOC-18 and then without or with 200 μg/ml bovine testicular hyaluronidase type IV-S for 3–5 minutes. The cell layer was rinsed in PBS, fixed in cold methanol and stained for HA (green) and nuclei (blue). Original magnification– 20X. Images shown are representative of experiments with at least three separate isolates of SMCs.</p

NO promotes the deposition of HA cables.

<p>SMCs were treated for 23 h at 37°C with or without 50 μM SNAP, 500 μM NOC-12, or 500 μM GSNO. The cell layer was rinsed in PBS, fixed in cold methanol and stained for HA (green) and nuclei (blue). Original magnification– 20X. Images shown are representative of experiments with three separate isolates of SMCs.</p

Reduced HAS2 expression limits HA cable formation.

<p>SMCs from HAS2 conditional knockout mice were cultured without or with 2 micromolar hydroxy-tamoxifen for 10 days. The cells were rested for two days and then plated on coverslips. Cultures were treated with 500 μM NOC-18 for 23 h. The cultures were rinsed, fixed in cold methanol, air dried and stained for HA (green) and nuclei (blue). Original magnifications –20x. Images shown represent experiments from three different isolates of SMCs.</p

NO increases HA production.

<p>SMCs were treated with 500 μM NOC-18 for 6 h. Conditioned medium was collected and HA was quantified with a solid phase binding assay. Data represent the mean +/- S.D. n = 6, <i>P</i> < 0.0001.</p

NO promotes HA-mediated leukocyte binding.

<p>SMCs were treated with 500 μM NOC-18 or 100 μM SNAP for 23 h. The cultures were rinsed with PBS and unlabeled, fixed, or fluorescently labeled murine splenocytes were added for 30 min at 4°C and nonadherent cells were removed. (A) The SMC layer with unlabeled, adherent leukocytes was fixed, and stained for HA (green) and nuclei (blue). Original magnification 20x (left) or 40x (right). Images shown are representative of experiments with at least three separate isolates of SMCs. (B) The SMC layer with formalin-fixed, adherent leukocytes was fixed in formalin, rinsed, the coverslip was mounted onto a slide and examined by phase contrast microscopy. Original magnification 40x. The image shown is representative of experiments with two isolates of SMCs and two isolates of leukocytes. For each experiment, SMCs on three coverslips were used. (C) The SMC layer with adherent fluorescent leukocytes was freeze-thawed and the bound leukocytes were quantified. In some wells, the cell layer was treated with hyaluronidase after the attachment of splenocytes, and released leukocytes were removed by rinsing. Data represent the mean +/- S.D. n = 6, <i>P</i> < 0.05.</p

HAS 2 is sufficient for HA cable production in response to NO.

<p><i>HAS 1</i>,<i>3</i> double knockout SMCs were treated with 500 μM NOC-18 for various times. The cultures were rinsed, fixed in cold methanol, air dried and stained for HA (green) and nuclei (blue). Original magnifications –20x. Images shown represent more than three independent experiments.</p

SMCs co-cultured with induced RAW cells produce HA cables.

<p>RAW cells were grown on trans-well inserts and treated with 1 ng/ml LPS and 10 U/ml IFN-γ for 6–8 h to induce iNOS expression. Some cultures were also treated with L-NAME. The inserts were rinsed and transferred to plates containing coverslips with confluent SMCs. The RAW cells and SMCs were grown in co-culture for 13 h and the SMCs were then fixed and stained for HA (green) and nuclei (blue). Original magnification 20x (left panels) or 40x (right panels). Images shown are representative of three separate experiments.</p