S3440P Substitution in C-Terminal Region of Human Reelin Dramatically Impairs Secretion of Reelin from HEK 293T cells

Elika Esmaeilzadeh-Gharehdaghi,Amirreza Bitaraf,Mahdi Mahmoudi,Ehsan Razmara,Masoud Garshasbi

doi:10.14715/cmb/2019.65.6.3

Elika Esmaeilzadeh-Gharehdaghi, Amirreza Bitaraf + Show 3 more

Open Access

https://doi.org/10.14715/cmb/2019.65.6.3

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Abstract

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. In this study, we performed experimental analyses on a sub-region of Human Reelin containing 5th and 6th repeats (R5-R6), a part of 8th repeat and the CTR which were amplified from cDNA of K562 and HEPG2(HepatocellularG2) 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. In-vitro study demonstrated that the CTR alteration (S3440P) leads to impairment of Reelin secretion even after overexpression. Our results indicate that S3440P substitution is the highly conserved structure of the CTR has an important effect on Reelin secretion.

Highlights

Reelin is a large extracellular glycoprotein (420-450 KD) which is secreted by Cajal–Retzius cells in the cerebral cortex of the brain [1]
Our results indicate that S3440P substitution is the highly conserved structure of the C-Terminal Region (CTR) has an important effect on Reelin secretion
We showed that the secretion of Reelin could be altered by S3440P substitution in highly conserved residues of CTR

Summary

Introduction

Reelin is a large extracellular glycoprotein (420-450 KD) which is secreted by Cajal–Retzius cells in the cerebral cortex of the brain [1]. RELN gene encodes Reelin protein which has a key function in neuronal migration during the complex processes of brain development [2]. Reelin plays a pivotal role in different processes in synaptic plasticity, learning, and memory in the adult brain through interaction with NMDA(N-Methyl-DAspartate ) receptor [3]. Reelin is composed of three distinctive subdomains: the N-terminal containing Fspondin-like domain and signal peptide, eight Reelin Repeats (RRs), and a short highly basic C-Terminal Region (CTR) [4]. Each repeat of Reelin is subdivided by Epidermal-like Growth Factor (EGF) domain into two distinctive subdomains A and B [5]. The EGF-like domains have important roles in protein-protein interactions, protein folding, and receptor binding [6]. Mutations in EGF-like domains have a potential effect on protein secretion [7]

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