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

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.


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-D-Aspartate ) 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).
N-Terminal Region (NTR) is necessary for dimerization of Reelin during binding to specific receptors (8).
The central fragment of Reelin involves third to sixth RRs which are sufficient for attaching to very-low-density lipoprotein receptor (VLDLR) and Apolipoprotein receptor 2 (ApoER2) and leading to phosphorylation of adaptor protein Disabled-1 (Dab1) (9). C-Terminal Re-gion (CTR) of Reelin composes less than 1% of entire Reelin 32 AA(amino acids) of 3460 AA (10).
RELN gene consists of 65 exons and most part of CTR (from Thr 3431 to the end ) is encoded by exon 65 (11). Exon 64 consists of 6 nucleotides and called as a micro-exon, this sequence is expressed in neuron cells but skipped by alternative splicing in other tissues (12). The main role of micro-exons has been revealed in various processes such as protein-protein interaction, generation of additional sites for post-translational modifications, and association with various neurological diseases (13). Alternative polyadenylation creates a short form of Reelin protein without the last 33 amino acids which are involving 10-25% of reelin mRNA in the mouse brain (12). Overexpression studies in COS-7 have shown that this truncated form of Reelin will be secreted much lower than full-length type, Reelin Wildtype (WT) (12). The primary sequence of CTR is highly conserved among vertebrates ( Figure 1A) which could be justified by its essential physiological function (14).
There is controversial evidence on the involvement of CTR in Reelin secretion; Although some studies have noted that the presence of CTR is essential for secretion, (15) others have shown that is not required. However, some studies have elucidated that CTR is only necessary for efficient transduction of downstream signaling (14,16). Previously it has been shown that a mutant form of Reelin without 20 C-terminal residues of the CTR could be secreted as efficiently as wild-type Reelin. Additional it has been shown that replacing the CTR with eight Arginine, or Glutamate residues have little impression on the secretion efficiency, but replacing with eight Alanine or Histidine residues dramatically impairs Reelin secretion (4). Effect(s) of CTR point mutations in the secretion efficiency of Reelin protein has not been revealed.
Production and secretion of the mouse recombinant reelin protein involving receptor binding domains (R5-R6) and a part of repeat 8 (R8B) with or without (CTR) have been detected in the supernatants and cell lysates of HEK-293T (Human Embryonic Kidney 293T)cells transfected by related constructs (16).
Expression of RELN gene has been detected in the liver as a blood source of Reelin protein (17). RELN gene is expressed in K562 cells and has a negative effect on erythroid cells differentiation (18).
Here, we used cDNA derived from HEPG2 (human liver carcinoma cells) cells and random mutagenesis by error-prone PCR method for inducing mutation in the CTR. Eventually, a random mutation, c.10318T>C, p. S3440P was detected in this highly conserved region and two vectors, containing the R5-R6 domains and a part of the R8B domain of Human Reelin with either Wild-Type or Mutant forms of CTR (designated as R5-6/ CWT and R5-6/CMUT respectively) were constructed meticulously.
The S3440P substitution in the CTR dramatically impaired secretion of the recombinant Reelin from HEK 293T cells.

Cells and Reagents
This study was approved by the ethics committee of Tarbiat Modares University, Tehran, Iran. In this study, we utilized HEK 293T, K562 cells, and PRSVβgal plasmid (kindly gifted from Dr. Mehdi Banan, Uni-

Cell culture and Construction of Expression Plasmids
HEPG2 cells were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin in a standard condition as in 5% Co 2 atmosphere and 37 • C. Total RNA of the cells was isolated using RNAX-plus. The CTR fragment including coding region of the amino acids 3365-3460, harboring the 32 highly conserved amino acids of the Reelin CTR (3430-3460) was amplified from cDNA derived HEPG2 cells by following primers; CTR-Forward 5'-aaagaattcGTCAACAACG-GGATCAC-3', CTR-Reverse 5'-aaatctagacgTGGG-TATCGCCTAAGT-3'using a low fidelity enzyme and high concentration of Mgcl2 (3mM in total volume 25 µL). The mammalian expression plasmid, pVP22/myc-His2, was utilized for constructing the recombinant Human Reelin involving (R5-R6), a part of R8B and CTR. VP22 gene is related to one of the structural proteins of herpes simplex virus type 1 (HSV-1) and this gene was cut and replaced by CTR region by means of EcoRI/XbaI restriction enzymes. After affirming by Sanger sequencing, 5th and 6th repeats of Human Reelin protein were sub-cloned into constructs and ligated to CTR (wild-type and mutant) by using HindIII/EcoRI and appropriate ligating enzymes. This long fragment was comprised amino acids 1917-2673 of Reelin protein and amplified using PfuUltra High-Fidelity DNA Polymerase from cDNA derived from K562 cells by following primers; R5/R6-Forward-5-aaaaaagcttacc atgGCCCAAACCAATGCTACA-3́ and R5/R6-Reverse-5'-aaaagaattcGGTGTCCAGCATAACGGTC -3. The gene-specific sequences in the primers are bold and the remaining added as restriction or transcription initiation sites.

Enzyme-Linked Immunosorbent Assay (ELISA)
We utilized the GST 6XHis tag ELISA kit and 6XHis tag antibody, for evaluating the recombinant Reelin secretion and its intracellular level which was applied for 24 and 48 hours after transfection. All the reagents were applied based on standard protocols and a stan- The R5-6/CWT levels in the supernatants were 7.36 ± 1.2 ng mL -1 per 2×10 6 cells after 24 h and 26.53± 1.6 ng mL -1 after 48h. In contrast, nothing was detected in the R5-6/C MUT supernatants 24h after transfection dard curve was constructed by applying a serial dilution of the standard recombinant GST-6XHistag. ELISA assays were obtained from three independent experiments which were performed in duplicates of all samples and standards. The supernatants and cell lysates of nontransfected cells were used as a negative control for this test.

Statistical analysis
Statistical analysis was applied by means of SPSS Version 25.0 (SPSS Inc., Chicago, IL, USA) and Graphpad Prism v.6.0 (Graph Pad Software, San Diego, CA, USA). Continuous variables were presented as Mean value ± SD for n =3 experiments.

Results
Reelin's CTR in both level of nucleotide sequences and amino acid residues is highly conserved ( Figure 1A and B). The primary sequence of the CTR is completely conserved among all mammals, Figure 1A indicates this fact that 90 of 99 (90.9%) nucleotide sequences encoding CTR are conserved among the species listed. CTR has highly positive charged peptides especially Arginine-rich domains. 12 out of 32 amino acids (38%) are basic (green highlighted in Figure 1B), while none of them are acidic. Reelin's 8th repeat ended at Val 3429 that leaves the last 32 amino acid residues for CTR (Figure 1B). All of the 32 comprising amino acid residues are conserved, except the first one in the crocodile, due to lack of micro-exon containing 6 nucleotides coding Val 3429 and Ser 3430 . This evolutionary observation could suggest that the CTR has an important physiological function in vertebrates.
Given the controversial evidence about the role of CTR in the secretion of Reelin protein, in this study, we constructed two expressional plasmids (R5-6/CWT) and (R5-6/C MUT). To do so, the CTR fragment amplified from cDNA derived from HEPG2 cells and cloned. Several plasmids were sequenced, and clones containing wild-type sequence of CTR and a transition (c.10318T>C, p. S3440P) in the CTR (Figure 2) were identified. The long fragments harboring repeat 5 and 6 -RR6 and RR5-of the Human Reelin were ligated to the upstream of CTR in both plasmids and Sanger sequencing did not show any other mutations in ORF of the two constructs.
In-silico prediction using I-Mutant2.0 (Predictor of Protein Stability Changes upon Mutations) showed that the S3440P substitution has a free energy change value (DDG: DeltaG new protein-DeltaG wild-type in Kcal/ mol) of -0.69 which predicts that this change reduces protein stability ( Figure 1C); therefore, it was hypothesized that the secretion of Reelin protein could be altered by this substitution.
RAMPAGE online tool indicated almost 97% of residues in the most favored regions, around 2.1% of residues in allowed regions, and only 0.4% of residues in the outlier regions, which suggested that the modeled structures of RR8 and CTR were acceptable ( Figure  1C).
The secretion efficiency of the wild-type and mutant recombinant Reelin were compared in HEK 293T cells. Cell lysates and supernatants were collected at different  and its amount after 48h was 0.33 ± 0.57 ng mL -1 . Difference between the supernatant concentration of the R5-6/CWT and the R5-6C/CMUT was significantly obvious after 48h ( Figure 2C). All data are abstracted in Table 1.

Discussion
In this report, we showed that the secretion of Reelin could be altered by S3440P substitution in highly conserved residues of CTR. Learning about the mechanism of Reelin secretion is important not only for the elucidation of its function in brain development but also for understanding the exact role(s) of Reelin in synaptic plasticity in the adult brain (19).
Reelin protein is released by Cajal-Retzus in the marginal zone (MZ) of the brain and is essential for developing neocortex by regulating neuronal cells migration (1,20). It has been shown that the intact CTR is pivotal for the formation of the MZ and development of dendritic cells (21). Homozygous mutations in the RELN gene lead to similar reeler mice phenotype in human including; lissencephaly with cerebellar hypoplasia, ataxia, and cognitive dysfunction (22). Deletion(s) in a part of the eighth RR (RR8) and CTR resulting from retroviral insertion is responsible for reeler phenotype in Orleans strain which is not able to secret Reelin (15,23). The prevention of Reelin secretion in transfected COS-7 cells has been reported by D'Arcangelo et al, as a result of an inserted stop codon in the middle of repeat eight of Reelin (RR8) leading to lack of 133 c-terminal residues (4). In contrast, in 1999, Lambert de Rouvroit et al. have shown that a Reelin protein without CTR (32 amino acids) resulting from alternative polyadenylation, could be secreted if overexpressed in COS-7 cells (12). From these results, it could be concluded that Reelin secretion probably requires 100 amino acids which are located between 3328 to 3428 AA.
In the present study, releasing a subregion of Human Reelin containing apart of eighth Reelin repeat (3365 to3429) from HEK 293T cells after 24 and 48 hours from transfection bolster this idea that the presence of the predicted 37 residues (3328 to 3365) is not critical for the secretion.
In this study, the ELISA data from cell lysates revealed the higher intracellular concentration of the mutant Reelin (including Proline instead of Serine at position 3440) compare to wild-type Reelin, especially 48 hours after transfection ( Figure 2C). Serine is a polar, hydrophilic amino acid, while Proline is hydrophobic, which is mostly detected buried inside the protein. Also, Proline is found in loop regions and gives rigidity to the polypeptide chain due to imposing certain torsion angles on the protein structure (24,25). It seems a hydrophilic structure in the CTR is required for efficient secretion of Reelin.
As indicated in Fig 2C, the sum of Reelin mutant in both conditioned medium and cell lysate of R5-6/ CMUT were less than the sum of R5-6/CWT after 24h and 48 h. As the cloned fragments are expressed under the control of a strong promoter (CMV: Cytomegalovirus), modification in translational or post-translational steps and not transcription are expected. No posttranslational modifications like glycosylation have been reported for the CTR, therefore, effects on folding or reducing the stability of the folded Reelin protein which also was predicted by an in-silico study using I-Mu-tant2.0 could be considered to be responsible for these differences. Endoplasmic reticulum (ER) has a main role in packaging and exporting the correctly folded proteins. The ER system is a quality control apparatus that solves intracellular retention of misfolded proteins through degradation of them (26).
Several heterozygous mutations or polymorphisms in the RELN gene have been reported in association with some neurological disorders such as schizophrenia, autism spectrum disorder (ASD), bipolar disorder, and major depression. Additionally, reduced Reelin protein in the brain and blood of these patients have been observed, previously (27-29). Furthermore, a heterozygous mutation, R3441Q, in the CTR has been recognized juxtaposed to our finding substitution in ASD patients (30).
Nutshell, although similar publications have reported the presence of CTR is not essential for the secretion of Reelin, our results illustrated that the substitution S3440P in this highly conserved residue of CTR has great effects on Reelin secretion. ND: Not Detectable; * it was out of the detection range of ELISA Kit. FC: Fold Change Table 1. Comparison between R5-6 C/ WT and R5-6 C/MUT by using the HEK 293T cells which are transfected by appropriate vectors after 24 and 48 hours follow up.