Structure of proteins under pressure: Covalent binding effects of biliverdin on β-lactoglobulin

International audienceHigh pressure (HP) is a particularly powerful tool to study protein folding/unfolding, revealing subtle structural rearrangements. Bovine β-lactoglobulin (BLG), a protein of interest in food science, exhibits a strong propensity to bind various bioactive molecules. We probed the effects of the binding of biliverdin (BV), a tetrapyrrole linear chromophore, on the stability of BLG under pressure, by combining in situ HP-small-angle neutron scattering (SANS) and HP-UV absorption spectroscopy. Although BV induces a slight destabilization of BLG during HP-induced unfolding, a ligand excess strongly prevents BLG oligomerization. Moreover, at SANS resolution, an excess of BV induces the complete recovery of the protein “native” 3D structure after HP removal, despite the presence of the BV covalently bound adduct. Mass spectrometry highlights the crucial role of cysteine residues in the competitive and protective effects of BV during pressure denaturation of BLG through SH/S-S exchange

Mass spectrometry detection of G3m and IGHG3 alleles and follow-up of differential mother and neonate IgG3.

International audienceMass spectrometry (MS) analysis for detection of immunoglobulins (IG) of the human IgG3 subclass is described that relies on polymorphic amino acids of the heavy gamma3 chains. IgG3 is the most polymorphic human IgG subclass with thirteen G3m allotypes located on the constant CH2 and CH3 domains of the gamma3 chain, the combination of which leads to six major G3m alleles. Amino acid changes resulting of extensive sequencing previously led to the definition of 19 IGHG3 alleles that have been correlated to the G3m alleles. As a proof of concept, MS proteotypic peptides were defined which encompass discriminatory amino acids for the identification of the G3m and IGHG3 alleles. Plasma samples originating from ten individuals either homozygous or heterozygous for different G3m alleles, and including one mother and her baby (drawn sequentially from birth to 9 months of age), were analyzed. Total IgG3 were purified using affinity chromatography and then digested by a combination of AspN and trypsin proteases, and peptides of interest were detected by mass spectrometry. The sensitivity of the method was assessed by mixing variable amounts of two plasma samples bearing distinct G3m allotypes. A label-free approach using the high-performance liquid chromatography (HPLC) retention time of peptides and their MS mass analyzer peak intensity gave semi-quantitative information. Quantification was realized by selected reaction monitoring (SRM) using synthetic peptides as internal standards. The possibility offered by this new methodology to detect and quantify neo-synthesized IgG in newborns will improve knowledge on the first acquisition of antibodies in infants and constitutes a promising diagnostic tool for vertically-transmitted diseases

Characteristics of the thirty-two proteotypic peptides for <i>Homo sapiens</i> G3m and IGHG3 alleles.

<p>The proteotypic peptides correspond to an enzymatic AspN and trypsin digestion of the constant region of the IG gamma3 chains encoded by the <i>Homo sapiens</i> IGHG3 gene.</p>a<p>Partial.</p>b<p>Unusual G3m allele <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046097#pone.0046097-Lefranc3" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046097#pone.0046097-Dard1" target="_blank">[12]</a>. This corresponds to the IGHG3*08 allele. Allotypes G3m10, G3m11 and G3m13 are not expressed owing to the presence of CH3 Asn N44, instead of the CH3 Ser S44 usually present in the other G3m5* alleles <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046097#pone.0046097-Lefranc3" target="_blank">[8]</a>.</p>c<p>The IGHG3*11 and IGHG3*12 alleles differ by the number of hinge exons, 4 and 3, respectively (IMGT Repertoire, Gene table <a href="http://www.imgt.org" target="_blank">http://www.imgt.org</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046097#pone.0046097-Lefranc3" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046097#pone.0046097-Giudicelli1" target="_blank">[18]</a>.</p>d<p>Expression of the allotype G3m15 is dependent, in addition to CH3 Met M39, on the presence of CH3 His H115 and Tyr Y116 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046097#pone.0046097-Lefranc3" target="_blank">[8]</a>.</p>e<p>Expression of the allotype G3m13 is dependent, in addition to CH3 Gln Q98, on the presence of CH3 Ser 44 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046097#pone.0046097-Lefranc3" target="_blank">[8]</a>.</p>f<p>Expression of the allotype G3m10 is dependent, in addition to CH3 Ile I101, on the presence of CH3 Ser 44 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046097#pone.0046097-Lefranc3" target="_blank">[8]</a>.</p>g<p>Expression of the allotype G3m15 is dependent, in addition to CH3 His H115 and Tyr Y116, on the presence of CH3 Met M39 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046097#pone.0046097-Lefranc3" target="_blank">[8]</a>.</p>h<p>Expression of the allotype G3m14 is dependent, in addition to CH3 Arg R115 and Phe F116, on the presence of CH3 Met M84 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046097#pone.0046097-Lefranc3" target="_blank">[8]</a>.</p>i<p>Expression of the allotype G3m6 is dependent, in addition to CH3 Glu E98, on the presence of CH3 Ser S44 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046097#pone.0046097-Lefranc3" target="_blank">[8]</a>.</p>j<p>Expression of the allotype G3m24 is dependent, in addition to CH3 Val V101, on the presence of CH3 Ser S44 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046097#pone.0046097-Lefranc3" target="_blank">[8]</a>.</p><p>Amino acids in bold are implicated in the discrimination between IGHG3 alleles. “.” : site of enzymatic cut.</p><p>Amino acids characteristic of the G3m allotypes and IGHG3 alleles are from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046097#pone.0046097-Lefranc3" target="_blank">[8]</a>. They are illustrated in the ‘IMGT G3m allele butterfly’ representation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046097#pone.0046097-Lefranc3" target="_blank">[8]</a>. Amino acid sequences are available in the IMGT Repertoire (<a href="http://www.imgt.org" target="_blank">http://www.imgt.org</a>), IMGT/DomainDisplay and IMGT/GENE-DB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046097#pone.0046097-Giudicelli1" target="_blank">[18]</a>. Positions in the CH domains are according to the IMGT unique numbering for C domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046097#pone.0046097-Lefranc6" target="_blank">[21]</a>.</p

Mass-to-charge ratios (m/z) of thirty-two G3m and IGHG3 allele peptides after AspN and trypsin digestion.

<p>The proteotypic peptides correspond to an enzymatic AspN and trypsin digestion of the constant region of the IG gamma3 chains encoded by the <i>Homo sapiens</i> IGHG3 gene. Masses are determined for detection on the MALDI TOF and ESI Orbitrap mass spectrometers. The methionine (M) could be oxidized (+16 Da). m/z : mass-to-charge ratio; +1, +2, +3, +4 represent the peptide charge state; amino acids in bold are implicated in the discrimination between G3m and IGHG3 alleles (detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046097#pone-0046097-t002" target="_blank">Table 2</a>); «.» : site of enzymatic cut; C^c : carbamidomethylated cysteine.</p

Relative abundance of a volume/volume mixture from BEC1 (G3m5*) and BEC2 (G3m5*/G3m24*) plasma samples.

<p>The relative abundance was calculated by the Progenesis LC-MS software for label-free semi-quantitative data analysis (detailed in 2.7); +2: dicharged peptides; the light grey bars represent the measure of the WQ<b>E</b>GN<b>V</b>FSCVMHEALHNR (#26, G3m24*) peptide (only brought by BEC2), the dark grey bars taken all together represent the measure of the WQ<b>Q</b>GN<b>I</b>FSCVMHEALHNR (#24, G3m5*) peptide (brought by both BEC1 and BEC2). The dark grey bars were divided into a hatched part (for the deduced signal attributable to BEC2, calculated from the 0∶1 ratio) and a non-hatched part (for the deduced signal attributable to BEC1).</p

Protein-A and Protein-G purification fractions from the EUA1 plasma sample on an acrylamid gel.

<p>A. 12% SDS-PAGE in non-reducing conditions: lines AF1 to AF3: consecutive filtrate fractions of a Protein A column containing plasma proteins including IgG3; line AE: Elution fraction of a Protein A column containing IgG1, IgG2, IgG4; lines GE1 and GE2: consecutive elution fractions of a Protein G column containing IgG3. B. 12% SDS-PAGE in reducing conditions: lines GE1 and GE2: consecutive elution fractions of a Protein G column containing IgG3.</p

Transitions used in SRM experiments for peptides WQQGNIFSCSVMHEALHNR (#24, G3m5*) and WQEGNVFSCSVMHEALHNR (#26, G3m24*).

<p>3+: The precursor ions were in triply-charged form; m/z: mass to charge ratio; ox: oxidized methionine (M), accepted nomenclature for fragment ions as proposed by Roepstorff and Fohlman <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046097#pone.0046097-Roepstorff1" target="_blank">[25]</a>; bolded are the heavy arginine.</p