DataSheet1_Polymorphism Pro64His within galectin-3 has functional consequences at proteome level in thyroid cells.ZIP

IntroductionThe single nucleotide polymorphism (SNP) rs4644 at codon 64 of galectin-3 (gal-3, gene name: LGALS3), specifying the variant proline (P64) to histidine (H64), is known to affect the protein’s functions and has been associated with the risk of several types of cancer, including differentiated thyroid carcinoma (DTC).Materials and methodsTo deepen our understanding of the biological effects of this SNP, we analyzed the proteome of two isogenic cell lines (NC-P64 vs. NA-H64) derived from the immortalized non-malignant thyrocyte cell line Nthy-Ori, generated through the CRISPR-Cas9 technique to differ by rs4644 genotype. We compared the proteome of these cells to detect differentially expressed proteins and studied their proteome in relation to their transcriptome.ResultsFirstly, we found, consistently with previous studies, that gal-3-H64 could be detected as a monomer, homodimer, and heterodimer composed of one cleaved and one uncleaved monomer, whereas gal-3-P64 could be found only as a monomer or uncleaved homodimer. Moreover, results indicate that rs4644 influences the expression of several proteins, predominantly upregulated in NA-H64 cells. Overall, the differential protein expression could be attributed to the altered mRNA expression, suggesting that rs4644 shapes the function of gal-3 as a transcriptional co-regulator. However, this SNP also appeared to affect post-transcriptional regulatory mechanisms for proteins whose expression was oppositely regulated compared to mRNA expression. It is conceivable that the rs4644-dependent activities of gal-3 could be ascribed to the different modalities of self-dimerization.ConclusionOur study provided further evidence that rs4644 could affect the gal-3 functions through several routes, which could be at the base of differential susceptibility to diseases, as reported in case-control association studies.

引言在编码3-半乳糖苷（gal-3，基因名：LGALS3）的64位密码子处，单核苷酸多态性（SNP）rs4644指定了由脯氨酸（P64）至组氨酸（H64）的变异，已知该变异会影响蛋白质的功能，并与多种类型癌症的风险相关，包括分化型甲状腺癌（DTC）。研究方法为深入探究该SNP的生物学效应，我们分析了源自永生化非恶性甲状腺滤泡细胞系Nthy-Ori的两个同源细胞系（NC-P64与NA-H64）的蛋白质组。这些细胞系通过CRISPR-Cas9技术产生，在rs4644基因型上存在差异。我们比较了这些细胞的蛋白质组，以检测差异表达的蛋白质，并研究了它们的蛋白质组与其转录组的关系。结果首先，我们发现，与先前研究一致，gal-3-H64可以作为单聚体、同源二聚体和异源二聚体检测到，其中异源二聚体由一个切割和一个未切割的单聚体组成，而gal-3-P64只能以单聚体或未切割的同源二聚体的形式存在。此外，结果表明rs4644影响了多种蛋白质的表达，其中在NA-H64细胞中主要上调。总体而言，差异蛋白质表达可归因于改变后的mRNA表达，表明rs4644塑造了gal-3作为转录共调节因子的功能。然而，该SNP似乎还影响了与mRNA表达相反调节的蛋白质的转录后调节机制。有理由认为，gal-3的rs4644依赖性活性可以归因于不同的自二聚化模式。结论本研究进一步提供了证据，表明rs4644可以通过多种途径影响gal-3的功能，这些途径可能是病例对照关联研究中报道的疾病易感性的基础。