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S3440P Substitution in C-Terminal Region of Human Reelin Dramatically Impairs Secretion of Reelin from HEK 293T Cells
: Received: 30 June 2019 / Approved: 1 July 2019 / Online: 1 July 2019 (17:22:14 CEST)
A peer-reviewed article of this Preprint also exists.
Journal reference: Cellular and Molecular Biology 2019
Background: Reelin is a large extracellular glycoprotein secreted by Cajal–Retzius cells and has a main role during brain development, especially in neuronal migration. Reelin is comprised of N-terminal F-spondin like domain, eight tandem repeats, and a highly conserved basic C-terminal region (CTR). The CTR main role in the secretion of Reelin has been investigated by advertently inducing deletion in whole or a part of this region; however, the role of CTR point mutations on the secretion of Reelin is shrouded in mystery. Materials and Methods: In this study, we performed experimental analyses on a subregion of Human Reelin containing 5th and 6th repeats (R5-R6), a part of 8 th repeat and the CTR which were amplified from cDNA of K562 and HEPG2 cells and cloned into a mammalian expressional plasmid (pVP22/myc-His). Bioinformatics investigation was performed on the CTR at both level of nucleotide and amino acid as well as mutant type. Random mutagenesis by error-prone PCR method was utilized to induce mutation in the CTR. The secretion efficiency of recombinant wild-type and mutant Reelin constructs compared in cell lysate and supernatant isolated from the transiently transfected HEK 293T cells using 6XHistag ELISA method. Results: In-vitro study demonstrated that the CTR alteration(S3440P) leads to impairment of Reelin secretion even after overexpression. Conclusions: Our results indicate that S3440P substitution in highly conserved structure of the CTR has an important effect on Reelin secretion.
reelin; CTR; point mutation; secretion; RELN
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