<i>In silico</i> analysis of the impact of germline E-cadherin missense mutations.

<p>A) Schematic representation of E-cadherin domains, mapping all the modelled germline mutations found in the setting of HDGC or EODGC. Above the scheme are the mutations that resulted in destabilization, as predicted by FoldX (ΔΔG>0.8 kcal/mol) and below the scheme all the non-destabilizing mutations (ΔΔG<0.8 kcal/mol). The newly identified mutations are underlined. B) FoldX and SIFT were used to evaluate the impact of the mutations present in A) and the predictions were classified as: True Positive (TP) when the software predicts high impact and the mutants exhibit in vitro loss of function; True Negative (TN) when the software predicts no impact and the mutant is functional in vitro; False Positive (FP) when the software predicts high impact but the mutants is functional in vitro; and False Negative (FN) when the software predicts no impact and the mutants exhibits in vitro loss of function. The results from both predictors result in 70% overlap with E-cadherin protein function tested in vitro (TP+TN). C) Box-plot representing the median and interquartile ranges of the native-state stability changes (ΔΔG) of the Destabilizing and Non-destabilizing mutations, as predicted by FoldX. D) Box-plot representing the median and interquartile ranges of ages of Gastric Cancer detection or associated death, corresponding to the Destabilizing and Non-destabilizing mutations carriers. All the data was collected from the literature. The group of patients harbouring destabilizing mutations is characterized by a clear younger age of diagnosis or death, suggesting the contribution of E-cadherin destabilization for the disease phenotype.</p

Functional impact of three new HDGC-associated E-cadherin missense mutations: E185V, S232C and L583R.

<p>CHO cells were transiently (A) or stably (B–C) transfected with an empty vector (Mock) or WT, E185V, S232C, L583R E-cadherin cDNA. A) Functional aggregation assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033783#s2" target="_blank">Material and Methods</a>. L583R cells show E-cadherin loss of function, resulting in a scattered pattern, resembling Mock cells. ΔΔG was calculated using FoldX algorithm and is 0 for the WT reference; B) Total cell lysates were prepared and E-cadherin was detected by Western Blot using anti-E-cadherin antibody. Anti-α-Tubulin antibody was used as a loading control. The expression of L583R is reduced and shifted to higher molecular weight, indicative of being retained as immature (approximately 130 kDa). C) E-cadherin expression in the Plasma Membrane (PM) was evaluated using Flow Cytometry, after staining with an extracellular anti-human E-cadherin antibody. L583R is less expressed in the PM.</p

ERAD is involved in the regulation of E-cadherin destabilizing mutations.

<p>CHO cells were stably (A, C) or transiently (B, D, E) transfected with an empty vector (Mock) or WT, E185V, S232C, L583R, L583I E-cadherin cDNA. A) E-cadherin and Calnexin immunofluoresce was performed in stable CHO cells expressing WT and L583R. Calnexin was used as an ER marker. L583R is retained in the ER, as evaluated by the colocalization with calnexin (yellow and arrows). B) Protein synthesis was blocked with Cicloheximide for 8 h and 16 h, to analyse E-cadherin turnover. E-cadherin was detected by Western Blot using anti-E-cadherin antibody and anti-α-Tubulin antibody was used as a loading control. L583R exhibits higher turnover. C) Cells were incubated with proteasome inhibitor MG132 for 16 h, and total cell lysates were prepared and analyzed. Proteasomal degradation results on the accumulation of L583R to levels similar to WT, indicating that the proteasome is necessary for the mutant downregulation. D) Functional aggregation assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033783#s2" target="_blank">Material and Methods</a>. Cells expressing the artificial mutant L583I recover E-cadherin adhesive function, resembling WT cells, in contrast to L583R, which are not able to perform adhesion. E) Protein synthesis was blocked with Cicloheximide for 8 h and 16 h, to analyse E-cadherin turnover. In contrast to the unstable L583R, the stable mutation (L583I) is resistant to protein synthesis blockage, exhibiting lower turnover, comparable to the WT protein. The two bands of E-cad in B) and E) correspond to mature (lower, 120 kDa) and immature (upper, 130 kDa) forms of the protein, and result from the overload of protein commonly observed upon transient transfections.</p

<i>In silico</i> based analysis of the impact of HDGC-associated E-cadherin missense mutations.

<p>Only mutations that localize in the domains covered by the structural models are listed. FoldX calculations are reflected by the value of native-state stability changes (ΔΔG = ΔG_WT−ΔG_Mut), expressed in kcal/mol. Mutations associated to structural impact present ΔΔG>0,8 kcal/mol in the FoldX column, and values below 0,05 in the SIFT column are considered to be intolerant due to high conservation. Predictions were scored as: True Positive (TP) when the software predicts high impact and the mutants exhibits in vitro loss of function; True Negative (TN) when the software predicts no impact and the mutant is functional in vitro; False Positive (FP) when the software predicts high impact but the mutant is functional in vitro; and False Negative (FN) when the software predicts no impact and the mutant exhibits in vitro loss of function. Only mutations that have been functionally characterized in vitro are classified. The mutations that have been described to impact the splicing pattern are depicted with (a). Mutations found at a frequency higher than 1% in one control population are considered polymorphisms, and marked with (b). Mutations published as personal communications are referenced with (c). The newly identified mutations are listed in the bottom of the table, unpublished are marked with a (d). ND – Not Determined.</p

E-cadherin structural models.

<p>A) Sequence alignment of the extracellular domains of human E-cad and xenopus EP-cad. The extracellular sequences were obtained in Uniprot with the corresponding references (human E-cadherin, P12830; xenopus EP-cadherin, P33148). M-Coffee regular was used to perform the alignment, a package that combines different alignment methods. Red brick regions are in perfect agreement across all the methods, green and yellow regions are regions of no agreement between the different alignment methods. The average consistency score obtained was 98, confirming the reliability of the alignment. The blue stars identify the aminoacids that were removed from the 1L3W structure before humanizing. The black arrow indicates the end of the structural model obtained. B) The human structure of domains EC1-EC2 (PDB 2O72, blue) was aligned with the same domains of the human model generated from the xenopus structure (PDB 1L3W, red). Image created with Pymol. C) Schematic representation of human E-cadherin domains, highlighting the coverage of the three different structural models obtained with FoldX (models of prodomain, extracellular domain and the Catenin Binding Domain).</p