Genetic Diversity in the Suppressyn Gene Sequence: From Polymorphisms to Loss-of-Function Mutations

Abstract

The suppressive regulator of cell fusion, suppressyn, is specifically expressed in the human placenta and is thought to play a crucial role in trophoblast fusion or syncytialization. Previous studies have suggested that alterations in its expression are associated with aberrant placental development, such as the immature placental morphology observed in Down syndrome, and may contribute to the pathogenesis of fetal growth restriction. While syncytialization in trophoblasts is an essential process for normal placental development, the precise molecular causes of its dysregulation remain poorly understood. In the present study, we aimed to elucidate the potential contribution of genomic variation to the loss of suppressyn function, extending previous analyses of expression abnormalities in perinatal disorders. Through sequence analysis, (1) we identified six polymorphisms within the coding region of the suppressyn gene, and (2) discovered that certain deletions and specific amino acid substitutions result in a complete loss of suppressyn-mediated inhibition of cell fusion. Although these mutations have not yet been reported in disease-associated genomic databases, our findings suggest that comprehensive genomic studies of perinatal and other disorders may reveal pathogenic variants of suppressyn, thereby uncovering novel genetic contributions to placental dysfunction. It is also anticipated that these findings might direct the development of therapeutic strategies targeting loss-of-function mutations.

Keywords: SNP; cell fusion; endogenous retroviruses (ERV); loss-of-function; suppressyn.

Figure 1

Functional analysis of suppressyn single nucleotide variants (SNVs) identified in the 1000 Genomes Project database. (a) Schematic representation of six suppressyn SNVs (c.47, c.79, c.385, c.394, c.427, and c.449) mapped within the coding region, as identified in the 1000 Genomes Project database. The single nucleotide substitutions at c.79 and c.449 have been reported to co-localize on the same allele and are therefore connected by a line and labeled as c.79/c.449 (p.I27L/p.P150L) in this scheme. (b) Western blot analysis of suppressyn protein expression in HTR8 cells stably expressing suppressyn variants containing SNVs (c.79, c.394, and c.449). Cell lysates and conditioned media (after immunoprecipitation with anti-Flag M2 agarose) were analyzed to detect the expected protein bands. The numbers 1 and 2 shown in the figure indicate technical duplicates generated using two independent clones. ‘cell’ indicates the cell lysate, and ‘sup.’ indicates the culture supernatant. (c) Western blot analysis of suppressyn protein expression in HTR8 cells stably expressing suppressyn variants containing SNVs (c.47, c.385, and c.427), as performed in (b). (d) Cell fusion assays were performed using suppressyn variant-expressing cells as in (b), transfected with increasing amounts (25, 50, or 125 ng) of syncytin-1 expression vector. Fusion rates were quantified by FACS and normalized to the untransfected control (set as 1.0). The abbreviation ‘un-T.F.’ refers to untransfected cells. ‘cell’ indicates the cell lysate, and ‘sup.’ indicates the culture supernatant. Error bars represent the standard deviation (SD). (e) Cell fusion assays were performed using suppressyn variant-expressing cells from (c), analyzed as in (d). Statistical significance compared to the corresponding vector-only control was assessed using the Mann–Whitney U test. p < 0.01 by **, and “n.s.” denotes not significant. All experiments were performed in biological duplicates and repeated independently at least three times. Original full-length blots for SDS-PAGE analyses are provided in Supplementary Figure S7.

Figure 2

Functional characterization of the aberrantly secreted suppressyn protein harboring SNV c.427. (a) Western blot analysis comparing the molecular weight of intracellular and secreted suppressyn proteins containing the SNV c.427. Samples from both cell lysates and conditioned media were immunoprecipitated using anti-Flag M2 agarose prior to detection. ‘C’ indicates the cell lysate, and ‘S’ indicates the culture supernatant. (b) Schematic of the transwell-based cell fusion inhibition assay used to evaluate the function of secreted suppressyn containing the c.427 SNV. Suppressyn-expressing cells were cultured in the upper chamber of a 0.4 μm transwell insert, while HTR8 cells transfected with syncytin-1 were placed in the lower chamber. Representative western blot analysis of suppressyn protein expression in both cell lysates and conditioned media, following immunoprecipitation with anti-Flag M2 agarose. ‘cell’ indicates the cell lysate, and ‘sup.’ indicates the culture supernatant. (c) Quantification of cell fusion in the syncytin-1-transfected HTR8 cells cultured in the lower chamber of the transwell plate. Data are presented as mean fusion rates, and statistical significance was assessed using the Mann–Whitney U test. p < 0.01 is indicated by **. All assays were performed in biological duplicates and independently repeated at least three times. Error bars represent the standard deviation (SD). The numbers 1 and 2 (c.427-1, c.427-2) shown in the figure indicate technical duplicates generated using two independent clones. Original full-length SDS-PAGE blots are provided in Supplementary Figure S8.

Figure 3

Functional analysis of suppressyn deletion constructs. (a) Schematic representation of the suppressyn protein deletion constructs. The full-length suppressyn sequence (160 amino acids) was divided into six fragments (F1–F6) based on predicted structural and functional domains. F1 and F2 correspond to the 39-amino acid signal peptide; F3 and F5 encompass putative ASCT2-binding motifs; and F5 also includes a predicted O-glycosylation site. The signal peptide cleavage site is indicated by a triangle. Deleted regions in each construct are indicated by dotted lines. (b) Western blot analysis of suppressyn protein expression in HTR8 cells stably expressing each deletion construct. Immunoprecipitation was performed using anti-Flag M2 agarose prior to detection. Upper panel: intracellular protein; middle panel: secreted protein in conditioned media; lower panel: ASCT2 detection by immunoblotting. Constructs containing F2 fragments retained signal peptide cleavage activity, indicated by asterisks. ‘cell’ indicates the cell lysate, and ‘sup.’ indicates the culture supernatant. (c) Cell fusion assay results using syncytin-1 transiently-transfected HTR8 cells stably expressing each suppressyn deletion mutant. Fusion inhibition activity was evaluated by FACS analysis, and statistical significance was assessed using the Mann–Whitney U test. * p < 0.05; ** p < 0.01. All experiments were performed in duplicate and independently repeated at least three times. The abbreviation ‘un-T.F.’ refers to untransfected cells. Error bars represent the standard deviation (SD). Full-length SDS-PAGE images for all immunoblots are shown in Supplementary Figure S9.

Figure 4

Functional analysis of cysteine residue mutants of suppressyn. (a) Schematic representation of the amino acid sequence of suppressyn, highlighting the positions of the seven cysteine residues (Cys#1–#7, shown in dashed boxes). The boxed region indicates the signal peptide, and the triangle denotes the predicted cleavage site. Underlined sequences represent the putative ASCT2-binding regions. (b) Western blot analysis of suppressyn proteins carrying point mutations at individual cysteine residues expressed in HTR8 cells. Proteins were detected after immunoprecipitation using anti-Flag M2 agarose. Upper panel: intracellular protein; middle panel: secreted protein in conditioned media; lower panel: ASCT2 detection by immunoblotting. ‘cell’ indicates the cell lysate, and ‘sup.’ indicates the culture supernatant. (c) Cell fusion inhibition assay using syncytin-1 transiently transfected HTR8 cells stably expressing each cysteine mutant-containing suppressyn protein. Fusion suppression activity was quantified by FACS analysis. Statistical significance was assessed using the Mann–Whitney U test. * p < 0.05; ** p < 0.01; n.s., not significant. All experiments were performed in duplicate and repeated independently at least three times. The abbreviation ‘un-T.F.’ refers to untransfected cells. Error bars represent the standard deviation (SD). Full-length SDS-PAGE images of the immunoblots are provided in Supplementary Figure S10.