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> 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

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

<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

Proposed model.

<p>Panel A illustrates the following: in epithelial cells, <i>CDH1</i>, <i>Mgat3</i> and <i>Mgat5</i> are transcribed. Partial promoter methylation of <i>Mgat3</i> and no methylation of <i>CDH1</i> and <i>Mgat5</i> promoters were observed. The transcription levels of <i>Mgat3</i> generate sufficient GnT-III enzyme levels that catalyze the addition of bisecting GlcNAc structures, specifically on E-cadherin. No information is available concerning the status of the remaining molecules in the adhesion complex (catenins). Panel B illustrates the following: in mesenchymal cells, <i>Mgat3</i>'s promoter is methylated in some <i>CpG</i> sites which were associated with a significant decrease of <i>Mgat3</i> transcription. No significant changes were observed in terms of both promoter methylation status and transcription of <i>CDH1</i> and <i>Mgat5</i>. There was a significant decrease of GnT-III-mediated E-cadherin glycosylation. In reverted epithelial cells, <i>Mgat3</i>'s promoter methylation status returns to its status in original epithelial cells accompanied with a significant increase of <i>Mgat3</i> transcription, in comparison to mesenchymal cells. Concomitantly there was an increased GnT-III-mediated E-cadherin glycosylation, resembling that observed in original epithelial cells.</p

GnT-III-mediated E-cadherin glycosylation during EMT/MET.

<p>Panel A shows the co-immunofluorescence for E-cadherin and E-PHA (400× for E, M, RE and 630× for M*) illustrating that E-cadherin and bisecting GlcNAc structures co-localize in the cell membrane in E and RE. In mesenchymal cells (M and M*), it was observed a significant decrease in both the expression of E-cadherin and bisecting GlcNAc structures. M cells shows residual E-cadherin expression at the focal points of intercellular contacts (red, E-cadherin) and some green staining (E-PHA reactivity) could be observed in the perinuclear region (Golgi compartment). Immunoprecipitation of E-cadherin followed by E-PHA lectin blot is represented in panel B. Panel C represents the normalization of bisecting GlcNAc structures (E-PHA reactivity) that are modifying E-cadherin. Amounts of N-glycan structures were determined from the ratios of densities of E-PHA reactivity after normalization to E-cadherin. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033191#s2" target="_blank">Results</a> are described as mean ± standard error mean of two biological replicas. Single asterisk corresponds to <i>p</i>≤0.05 and <i>ns</i> stands for non-significant, <i>p</i>>0.05. The modification of E-cadherin with bisecting GlcNAc N-glycan structures in E, M and RE are expressed as the fold increase, compared with the E cells. Panels B and C show that E-cadherin is specifically glycosylated with bisecting GlcNAc structures in E, losing this glycoform in M and recovering again in RE.</p

Expression levels and cellular localization of the product of GnT-III enzyme during EMT/MET induction.

<p>Panel A shows the lectin blot analysis using E-PHA lectin, showing the total expression levels of bisecting GlcNAc structures during EMT/MET. Panel B illustrates the quantification of E-PHA lectin normalized to actin (<i>n</i> = 2 biological replicas). Single asterisk corresponds to <i>p</i>≤0.05 and double asterisks stands for <i>p</i>≤0.001. Bisecting GlcNAc structures significantly decrease when comparing E and M cells and their expression is significantly recovered in RE cells. Panel C represents the immunofluorescence for E-PHA lectin during EMT/MET induction (400×). Bisecting GlcNAc structures are preferentially localized in the cell membrane of E cells. M cells exhibit a clear decrease in expression of the bisecting GlcNAc structures that was only observed in focal areas in the perinuclear region. In RE cells, there was a significant increase in the E-PHA staining showing an increase in the expression levels of bisecting GlcNAc structures that are localized in the cell membrane and in the cytoplasm.</p

<i>Mgat3</i> and Mgat5 RNA expression and methylation status of their predicted promoter-associated CpG islands.

<p>Panel A illustrates the quantification of <i>Mgat3</i> relative mRNA expression (<i>n</i> = 3 biological replicas). Data was normalized for E cells for each biological replica. Single asterisk corresponds to p≤0.05 and double asterisks stands for p≤0.001. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033191#s2" target="_blank">Results</a> are described as mean ± standard error mean of three biological replicas. Panel A shows that <i>Mgat3</i> expression was significantly decreased in M cells (in comparison to E cells) and recovered in RE cells (in comparison to M cells). Schematic representation of <i>Mgat3</i> genomic locus is represented in panel B. White squares correspond to exonic untranslated regions and black squares to exonic translated regions. Black line stands for intronic regions. Grey squares represent the position of the bioinformatically predicted CpG islands (classified as 1 and 2). Panel C and D show the schematic representation of the methylation status of several CpG dinucleotides evaluated within CpG islands 1 (C) and 2 (D) of <i>Mgat3</i> across the EMT/MET experiment (E, M and RE). White circles correspond to unmethylated CpGs, grey circles correspond to partially methylated CpGs, black circles correspond to methylated CpGs, white circles with a question mark correspond to unknown methylation status. Panel C shows methylation pattern alterations across several CpG sites within <i>Mgat3</i>'s CpG island 1 in E, M and RE cells. Panel E illustrates the quantification of Mgat5 relative mRNA expression (<i>n</i> = 3 biological replicas). Same legend as in A applies. No significant variation of Mgat5 RNA expression was observed during EMT/MET. Schematic representation of part of the Mgat5 genomic locus is represented in panel F. Same legend as in panel B applies. Schematic representation of the methylation status of several CpG dinucleotides evaluated within the annotated Mgat5 CpG island is represented in panel G. Same legend as in panels C and D applies. The results showed no variation of the methylation status of Mgat5 promoter during EMT/MET.</p

EMT/MET model validation.

<p>Panel A shows the Western blot for E-cadherin. Panel B illustrates the quantification of E-cadherin across the EMT/MET induction (<i>n</i> = 3 biological replicas). Data was normalized for E cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033191#s2" target="_blank">Results</a> are described as mean±standard error mean of 3 biological replicas. No significant differences were observed concerning E-cadherin expression (<i>ns</i> stands for non-significant, <i>p</i>>0.05). Panels A and B show that E-cadherin expression is decreased in M cells (in comparison to E cells) and partially recovered in RE cells (in comparison to M cells). Panel C represents the immunofluorescence for E-cadherin during EMT/MET induction (200×). <i>NC</i> stands for negative control (no E-cadherin antibody used). Panel C illustrates the variation of E-cadherin localization during the EMT/MET induction: E cells display the classical E-cadherin expression at the cell membrane; M cells show a decreased expression of E-cadherin which is only observed in some points of intercellular contacts and in the cytoplasm; RE cells display E-cadherin expression in the cell membrane.</p