Peroxynitrous acid (ONOOH) modifies the structure of anastellin and influences its capacity to polymerize fibronectin

Abstract

Anastellin (AN), a fragment of the first type III module in fibronectin (FN), initiates formation of superfibronectin, a polymer which resembles the native cell-derived fibrillar FN found in the extracellular matrix of many tissues, but which displays remarkably different functional properties. Here we demonstrate that exposure of AN to the biologically-important inflammatory oxidant, peroxynitrous acid (ONOOH), either as a bolus or formed at low levels in a time-dependent manner from SIN-1, impairs the capability of AN to polymerize FN. In contrast, exposure of FN to ONOOH does not seem to affect superfibronectin formation to the same extent. This oxidant-induced loss-of-function in AN occurs in a dose-dependent manner, and correlates with structural perturbations, loss of the amino acid tyrosine and tryptophan, and dose-dependent formation of modified amino acid side-chains (3-nitrotyrosine, di-tyrosine and 6-nitrotryptophan). Reagent ONOOH also induces formation of oligomeric species which decrease in the presence of bicarbonate, whereas SIN-1 mainly generates dimers. Modifications were detected at sub-stoichiometric (0.1-fold), or greater, molar excesses of oxidant compared to AN. These species have been localized to specific sites by peptide mass mapping. With high levels of oxidant (>100 times molar excess), ONOOH also induces unfolding of the beta-sheet structure of AN, thermal destabilization, and formation of high molecular mass aggregates. These results have important implications for the understanding of FN fibrillogenesis in vivo , and indicates that AN is highly sensitive to pathophysiological levels of oxidants such as ONOOH.

Elevated levels of iodide promote peroxidase-mediated protein iodination and inhibit protein chlorination

“…In a similar manner to the serum albumin systems described above, treatment of anastellin (which runs as a band at ∼12 kDa, which is slightly higher than the molecular mass for this recombinant protein of 9.4 kDa, but consistent with previous reports [24]) with LPO/H₂O₂/I⁻ resulted in minimal gross structural changes to the parent protein as detected by SDS-PAGE. No evidence was detected for dimers or higher oligomers, or low molecular mass fragments.…”

“…All chemicals were of high purity and purchased from Sigma-Aldrich (Søborg, Denmark) unless otherwise stated. All solutions for SDS-PAGE and LC-MS were prepared with nano-pure H2O sourced from a described previously [24]. Sequencing grade modified trypsin (V5111), rLys-C (V1671), and Glu-C (V1651)…”

Anastellin impacts on the processing of extracellular matrix fibronectin and stimulates release of cytokines from coronary artery smooth muscle cells

“…Fluorescein-conjugated heparin (~ 18 kDa; H7482) and RIPA Lysis Buffer (89,900) was purchased from Thermo Fisher Scientific. Recombinant AN was produced in Escherichia coli essentially as described previously 51 . The L37AY40A mutant of AN (translated sequence in Supplementary Fig.…”

“…CD spectroscopy. CD spectra were acquired on a Jasco-815 CD Spectrometer (Jasco Corporation, Japan), as previously 51 . Far-UV CD spectra of 0.1 mg mL −1 wt and L37AY40A AN was recorded between 190-260 nm, in a 1 mm path length cell at 25 °C, at 1 nm intervals with a 1 nm bandwidth, and a scan speed of 50 nm min −1 , with six scans averaged for each spectrum.…”

“…SAXS analysis. SAXS measurements were performed as previously 51 on an optimized NanoSTAR SAXS instrument 54 , with wt and L37AY40A AN (100 μM; 0.93 mg mL −1 ) in 10 mM phosphate buffer (pH 7.4). The SAXS data is displayed as a function of the scattering vector, q.…”