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Objectives Autoantibodies against apolipoprotein A1 (anti‐apoA1 IgGs) and its C‐terminal region (cter apoA1) have emerged as an independent biomarker for cardiovascular disease. Cter apoA1 mimetic peptides were shown to reverse the deleterious anti‐apoA1 IgG effects in vitro. We evaluated the association of anti‐cter apoA1 IgGs with overall mortality in the general population and tested the ability of a cter apoA1 mimetic peptide to reverse the anti‐apoA1 IgG‐induced inflammatory response and mortality in vitro and in vivo, respectively. Methods Anti‐cter apoA1 IgGs were measured in serum samples of 6386 participants of the CoLaus study of which 5220 were followed for a median duration of 5.6 years. The primary outcome was overall mortality. The peptide inhibitory concentration 50% (IC50) was determined in vitro on HEK‐Blue‐4 and RAW cells. ApoE−/− mice were exposed to 16 weeks of anti‐apoA1IgG passive immunisation with and without peptide co‐incubation. Results Anti‐cter apoA1 IgGs were associated with higher interleukin 6 levels and independently predicted overall mortality; an increase of one standard deviation of anti‐cter apoA1 IgG level was associated with an 18% increase in mortality risk (hazard ratio: 1.18, 95% confidence interval: 1.04–1.33; P = 0.009). The cterApoA1 analogue reversed the antibody‐mediated inflammatory response with an IC50 of 1 µm in vitro but did not rescue the significant anti‐apoA1 IgG‐induced mortality rate in vivo (69% vs. 23%, LogRank P = 0.02). Conclusion Anti‐cter apoA1 IgG independently predicts overall mortality in the general population. Despite being effective in vitro, our cter apoA1 analogue did not reverse the anti‐apoA1 IgG‐induced mortality in mice. Our data suggest that these autoantibodies are not readily treatable through cognate peptide immunomodulation., Autoantibodies against apolipoprotein A1 (anti‐apoA1 IgGs) have emerged as an independent cardiovascular risk factor in different settings including in the general population and seem to be preferentially oriented against the its C‐terminal region (cterA1) in humans. In this translational work, we extend previous knowledge in the field by demonstrating for the first time that anti‐cterA1 IgGs are independently predictive of premature mortality in a large general population sample of 5220 individuals. Furthermore, we also report that, despite being effective in vitro, our cterA1 analogue did not reverse the significant anti‐apoA1 IgG‐induced mortality in mice.

BACKGROUND AND PURPOSE The µ-conopeptide family is defined by its ability to block voltage-gated sodium channels (VGSCs), a property that can be used for the development of myorelaxants and analgesics. We characterized the pharmacology of a new µ-conopeptide (µ-CnIIIC) on a range of preparations and molecular targets to assess its potential as a myorelaxant. EXPERIMENTAL APPROACH µ-CnIIIC was sequenced, synthesized and characterized by its direct block of elicited twitch tension in mouse skeletal muscle and action potentials in mouse sciatic and pike olfactory nerves. µ-CnIIIC was also studied on HEK-293 cells expressing various rodent VGSCs and also on voltage-gated potassium channels and nicotinic acetylcholine receptors (nAChRs) to assess cross-interactions. Nuclear magnetic resonance (NMR) experiments were carried out for structural data. KEY RESULTS Synthetic µ-CnIIIC decreased twitch tension in mouse hemidiaphragms (IC50= 150 nM), and displayed a higher blocking effect in mouse extensor digitorum longus muscles (IC = 46 nM), compared with µ-SIIIA, µ-SmIIIA and µ-PIIIA. µ-CnIIIC blocked NaV1.4 (IC50= 1.3 nM) and NaV1.2 channels in a long-lasting manner. Cardiac NaV1.5 and DRG-specific NaV1.8 channels were not blocked at 1 µM. µ-CnIIIC also blocked the α3β2 nAChR subtype (IC50= 450 nM) and, to a lesser extent, on the α7 and α4β2 subtypes. Structure determination of µ-CnIIIC revealed some similarities to α-conotoxins acting on nAChRs. CONCLUSION AND IMPLICATIONS µ-CnIIIC potently blocked VGSCs in skeletal muscle and nerve, and hence is applicable to myorelaxation. Its atypical pharmacological profile suggests some common structural features between VGSCs and nAChR channels.

Human blood plasma is a useful source of proteins associated with both health and disease. Analysis of human blood plasma is a challenge due to the large number of peptides and proteins present and the very wide range of concentrations. In order to identify as many proteins as possible for subsequent comparative studies, we developed an industrial-scale (2.5 liter) approach involving sample pooling for the analysis of smaller proteins (M(r) generally < ca. 40 000 and some fragments of very large proteins). Plasma from healthy males was depleted of abundant proteins (albumin and IgG), then smaller proteins and polypeptides were separated into 12 960 fractions by chromatographic techniques. Analysis of proteins and polypeptides was performed by mass spectrometry prior to and after enzymatic digestion. Thousands of peptide identifications were made, permitting the identification of 502 different proteins and polypeptides from a single pool, 405 of which are listed here. The numbers refer to chromatographically separable polypeptide entities present prior to digestion. Combining results from studies with other plasma pools we have identified over 700 different proteins and polypeptides in plasma. Relatively low abundance proteins such as leptin and ghrelin and peptides such as bradykinin, all invisible to two-dimensional gel technology, were clearly identified. Proteins of interest were synthesized by chemical methods for bioassays. We believe that this is the first time that the small proteins in human blood plasma have been separated and analyzed so extensively.

A new side reaction during Native Chemical Ligation (NCL) at aspartic and glutamic acid/cysteine ligation sites is reported. To exploit NCL at these sites, test peptides having either a C-terminal unprotected aspartyl or glutamyl thioester were ligated with a peptide containing an N-terminal free cysteine generating, in each case, two major compounds having equal masses but different retention times by HPLC analysis. Comparison of these ligation products with reference peptides of identical sequence synthesized by total SPPS (solid phase peptide synthesis) with both natural and unnatural backbones at the cysteinyl residue is in agreement with the hypothesis of migration of the thioester moiety on the side chain carboxyl group of both C-terminal Asp and Glu residues. Enzymatic digestion of the purified ligation products with a V8-protease further confirmed the nature of the side reaction, with a total resistance of the non-natural backbone peptide to the protease. To overcome this problem the use of a protecting group stable to both HF cleavage and ligation conditions, but removable after ligation, became a necessity. A series of side-chain protected derivatives were selected, specifically 9-fluorenylmethyl ester (OFm), (phenylsulfonyl)ethyl ester (OPse), 2,2,2-trichloroethyl ester (OTce), and phenacyl esters (OPac). For the glutamic acid, OPse gave the best results, generating the correct peptide and avoiding the isomerization at the γ-carboxyl group. In the case of the aspartic residue, all the protecting groups were removed under basic conditions, worsening the problem and generating, for example, 70% yield of the β-isomer after OFm deprotection. This prompted us to propose a protecting group derived from the phenacyl ester 1-methyl-2-oxo-2-phenylethyl ester (OMop). This protecting group showed greater stability during the ligation reaction than OPac and OTce, and was easily removed by Zn/acetic acid reduction after the ligation step. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)