ИСТИНА

Molecular Structures of Orthologous Transcripts: Full-Length T-Cadherin and T-Cadherin Lacking the 3rd Exon in Mice and Humans M. Arbatskiy, V. Sysoeva, E. Semina, K. Rubina Faculty of Medicine, Lomonosov Moscow State University, 119192 Moscow, Russia Background/Objectives: We have previously generated Cdh13∆Exon3 mice lacking exon 3 in the Cdh13 gene (T-cadherin) and described their phenotype. While these mice exhibited normal gross morphology, life expectancy, and breeding capacity, their body weight was considerably lower than of WT mice. Upon intensive physical training the systolic blood pressure was significantly elevated. Moreover, the adiponectin plasma level and the level of AMPK phosphorylation in the skeletal muscles and myocardium of Cdh13∆Exon3 mice were significantly elevated as compared to WT. Herein, we aimed to investigate the relevance of the truncated form of murine T-cadherin lacking the 3rd exon in the context of human T-cadherin. Methods: mRNA and amino acid sequences for mouse and human T-cadherin were obtained from the NCBI database. Subsequently, using the Comparative Genome Viewer we confirmed the presence of orthologous transcripts for both mouse and human sequences, despite their different genomic structure (mouse - GRCm39, human - GRCh38.p14). The sequence of the 3rd exon of the CDH13 gene in mice was obtained using the Genome Data Viewer. The sequence alignment of the mouse 3rd exon to the transcripts of the mouse and human full-length CDH13 genes was carried out using the MUSCLE algorithm in the Unipro UGENE program. Open reading frames (ORFs) were identified, and similarity scores were computed using the Hamming distance tool. Domain and signal analysis of the truncated and full-length forms of mouse T-cadherin were conducted using the InterPro web server. To predict the protein folding, we applied the ColabFold with the AlphaFold2 neural network using MMseqs2 for sequence searching. The best model was refined by relaxation based on the Local Distance Difference Test score (pLDDT) using AMBER (Assisted Model Building with Energy Refinement). Results: A complete amino acid sequence of the truncated mouse T-cadherin lacking the 3rd exon was generated in FASTA format. The similarity between the truncated mouse isoform lacking the 3rd exon and the related isoform of human T-cadherin (isoform 4) (NM_001220490.2) was 68% for the whole sequence and 95% for the mutual regions. Comparative analysis of the truncated and full-length mouse T-cadherin revealed the presence of a prodomain in the full-length T-cadherin protein that was absent in the truncated form of the protein lacking the 3rd exon. Among the five predicted structures for the truncated isoform, the structure with the highest cumulative pLDDT score was selected. The predicted structure visually displayed five cadherin domains. Conclusion: Surface charge analysis of the truncated isoform indicated the preservation of charges in the cadherin domain regions with a redistribution of surface charges in the region lacking the 3rd exon. This suggests the retention of cadherin domain functionality. Further studies are warranted to reveal the mechanism underlying the reported physiological effects of truncated T-cadherin in Cdh13∆Exon3 mice including the interactions of T-cadherin with its well-known ligands, high molecular weight adiponectin and low density lipoproteins (https://doi.org/10.1016/j.ejcb.2021.151183). В нетранкированной форме присутствует ранее предсказанный при помощи InterPro прокадгериновый домен, отсутствующий в транкированной. Прокадгериновый домен является вторым в структуре Т-кадгерина (относительно первой N-концевой альфа-спирали) и является внеклеточным доменом вместе с 5 кадгериновыми доменами, тогда как по результатам InterPro Phobius трансмембранный и внутриклеточный домены располагаются на противоположном С-конце Т-кадгерина. Grant References: The study was supported by the Russian Science Foundation, grant No. 23-11-00205. This research was performed under the State Assignment # 03р-23/110-03 of Lomonosov Moscow State University.