Rabbit Anti-Human CANX Polyclonal antibody

BackgroundWhile extraocular muscles are affected early in myasthenia gravis (MG), but respond to treatment, we observe a high incidence of treatment-resistant ophthalmoplegia (OP-MG) among MG subjects with African genetic ancestry. Previously, using whole exome sequencing, we reported potentially functional variants which associated with OP-MG. The aim of this study was to profile the expression of genes harbouring the OP-MG associated variants using patient-derived subphenotype-specific myocyte' cultures.MethodsFrom well-characterised MG patients we developed the myocyte' culture models by transdifferentiating dermal fibroblasts using an adenovirus expressing MyoD. These myocyte cultures were treated with homologous acetylcholine receptor antibody-positive myasthenic sera to induce muscle transcripts in response to an MG stimulus. Gene expression in myocytes derived from OP-MG (n=10) and control MG subjects (MG without ophthalmoplegia; n=6) was quantified using a custom qPCR array profiling 93 potentially relevant genes which included the putative OP-MG susceptibility genes and other previously reported genes of interest in MG and experimental autoimmune myasthenia gravis (EAMG).ResultsOP-MG myocytes compared to control MG myocytes showed altered expression of four OP-MG susceptibility genes (PPP6R2, CANX, FAM136A and FAM69A) as well as several MG and EAMG genes (p<0.05). A correlation matrix of gene pair expression levels revealed that 15% of gene pairs were strongly correlated in OP-MG samples (r>0.78, p<0.01), but not in control MG samples. OP-MG susceptibility genes and MG-associated genes accounted for the top three significantly correlated gene pairs (r0.98, p<1x10(-6)) reflecting crosstalk between OP-MG and myasthenia pathways, which was not evident in control MG cells. The genes with altered expression dynamics between the two subphenotypes included those with a known role in gangliosphingolipid biosynthesis, mitochondrial metabolism and the IGF1-signalling pathway.ConclusionUsing a surrogate cell culture model our findings suggest that muscle gene expression and co-expression differ between OP-MG and control MG individuals. These findings implicate pathways not previously considered in extraocular muscle involvement in myasthenia gravis and will inform future studies.

Retention of misfolded proteins by the endoplasmic reticulum (ER) is a quality control mechanism involving the participation of endogenous chaperones such as calnexin (CANX). CANX interacts with and restricts plasma membrane expression (PME) of the I gonadotropin releasing hormone receptor (GnRHR), a G protein-coupled receptor. CANX also interacts with ERP-57 a thiol oxidoreductase chaperone present in the ER. CANX along with ERP-57 promotes the formation of disulfide bond bridges in nascent proteins. The human GnRH receptor (hGnRHR) is stabilized by two disulfide bond bridges (C-14-C-200 and C-114-C-196), that, when broken, lead to a decrease in receptor expression at the plasma membrane. To determine if the presence of chaperones CANX and ERP-57 exerts an influence over membrane routing and second messenger activation, we assessed the effect of various mutants including those with broken disulfide bridges (Cys -> Ala) along with the hGnRHR. The effect of chaperones on mutants was insignificant, whereas the over expression of ERP-57 led to an hGnRHR retention. This effect was further enhanced by cotransfection with cDNA for CANX showing receptor retention by ERP-57 augmented by CANX, suggesting utilization of these chaperones for quality control of the GnRHR. Copyright (C) 2009 John Wiley & Sons, Ltd.