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

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

doi:10.14715/cmb/2019.65.6.3

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Vol. 65 No. 6: Issue 6

Issue Published : August 5, 2019

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

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

Elika Esmaeilzadeh-Gharehdaghi

Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

Ehsan Razmara

Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

http://orcid.org/0000-0001-9926-3975

Amirreza Bitaraf

Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University

Mahdi Mahmoudi

Rheumatology Research Center, Tehran University of Medical Sciences, Tehran, Iran

Masoud Garshasbi

Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

Corresponding Author(s) : Masoud Garshasbi

masoud.garshasbi@gmail.com

Cellular and Molecular Biology, Vol. 65 No. 6: Issue 6
Article Published : July 31, 2019

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.

Keywords

Reelin CTR Point mutation Secretion RELN.

Esmaeilzadeh-Gharehdaghi, E., Razmara, E., Bitaraf, A., Mahmoudi, M., & Garshasbi, M. (2019). S3440P Substitution in C-Terminal Region of Human Reelin Dramatically Impairs Secretion of Reelin from HEK 293T cells. Cellular and Molecular Biology, 65(6), 12–16. https://doi.org/10.14715/cmb/2019.65.6.3

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References

References
D'arcangelo G, Miao GG, Chen S-C, Scares HD, Morgan JI, Curran T. A protein related to extracellular matrix proteins deleted in the mouse mutant reeler. Nature. 1995;374(6524):719.
Verbrugghe P, Bouwer S, Wiltshire S, Carter K, Chandler D, Cooper M, et al. Impact of the reelin signaling cascade (ligands–receptors–adaptor complex) on cognition in schizophrenia. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics. 2012;159(4):392-404.
Chen Y, Beffert U, Ertunc M, Tang T-S, Kavalali ET, Bezprozvanny I, et al. Reelin modulates NMDA receptor activity in cortical neurons. Journal of Neuroscience. 2005;25(36):8209-16.
Fatemi SH. Reelin Glycoprotein: Structure, Biology and Roles in Health and Disease: Springer; 2008.
De Bergeyck V, Naerhuyzen B, Goffinet A, de Rouvroit CL. A panel of monoclonal antibodies against reelin, the extracellular matrix protein defective in reeler mutant mice. Journal of neuroscience methods. 1998;82(1):17-24.
He X, Semenov M, Tamai K, Zeng X. LDL receptor-related proteins 5 and 6 in Wnt/Î²-catenin signaling: arrows point the way. Development. 2004;131(8):1663-77.
Schrijver I, Liu W, Brenn T, Furthmayr H, Francke U. Cysteine substitutions in epidermal growth factor–like domains of fibrillin-1: distinct effects on biochemical and clinical phenotypes. The American Journal of Human Genetics. 1999;65(4):1007-20.
Utsunomiya-Tate N, Kubo K-i, Tate S-i, Kainosho M, Katayama E, Nakajima K, et al. Reelin molecules assemble together to form a large protein complex, which is inhibited by the function-blocking CR-50 antibody. Proceedings of the National Academy of Sciences. 2000;97(17):9729-34.
Jossin Y, Ignatova N, Hiesberger T, Herz J, de Rouvroit CL, Goffinet AM. The central fragment of Reelin, generated by proteolytic processing in vivo, is critical to its function during cortical plate development. Journal of Neuroscience. 2004;24(2):514-21.
Abdolmaleky HM, Cheng Kh, Russo A, Smith CL, Faraone SV, Wilcox M, et al. Hypermethylation of the reelin (RELN) promoter in the brain of schizophrenic patients: a preliminary report. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics. 2005;134(1):60-6.
Royaux I, de Rouvroit CL, D'arcangelo G, Demirov D, Goffinet AM. Genomic Organization of the MouseReelinGene. Genomics. 1997;46(2):240-50.
de Rouvroit CL, Bernier B, Royaux I, de Bergeyck V, Goffinet AM. Evolutionarily conserved, alternative splicing of reelin during brain development. Experimental neurology. 1999;156(2):229-38.
Curry-Hyde A, Chen BJ, Mills JD, Janitz M. Microexons: novel regulators of the transcriptome. Journal of Human Transcriptome. 2018;2(1):1-6.
Nakano Y, Kohno T, Hibi T, Kohno S, Baba A, Mikoshiba K, et al. The extremely conserved C-terminal region of Reelin is not necessary for secretion but is required for efficient activation of downstream signaling. Journal of Biological Chemistry. 2007;282(28):20544-52.
De Bergeyck V, Nakajima K, de Rouvroit CL, Naerhuyzen B, Goffinet A, Miyata T, et al. A truncated Reelin protein is produced but not secreted in the ‘Orleans' reeler mutation (Relnrl-Orl). Molecular brain research. 1997;50(1-2):85-90.
Okoro EU, Zhang H, Guo Z, Yang F, Smith Jr C, Yang H. A subregion of reelin suppresses lipoprotein-induced cholesterol accumulation in macrophages. PloS one. 2015;10(8):e0136895.
Lammert DB, Middleton FA, Pan J, Olson EC, Howell BW. The de novo autism spectrum disorder RELN R2290C mutation reduces Reelin secretion and increases protein disulfide isomerase expression. Journal of neurochemistry. 2017;142(1):89-102.
Beffert U, Weeber EJ, Durudas A, Qiu S, Masiulis I, Sweatt JD, et al. Modulation of synaptic plasticity and memory by Reelin involves differential splicing of the lipoprotein receptor Apoer2. Neuron. 2005;47(4):567-79.
Ogawa M, Miyata T, Nakajimat K, Yagyu K, Seike M, Ikenaka K, et al. The reeler gene-associated antigen on Cajal-Retzius neurons is a crucial molecule for laminar organization of cortical neurons. Neuron. 1995;14(5):899-912.
Kohno T, Honda T, Kubo K-i, Nakano Y, Tsuchiya A, Murakami T, et al. Importance of Reelin C-terminal region in the development and maintenance of the postnatal cerebral cortex and its regulation by specific proteolysis. Journal of Neuroscience. 2015;35(11):4776-87.
Hong SE, Shugart YY, Huang DT, Al Shahwan S, Grant PE, Hourihane JOB, et al. Autosomal recessive lissencephaly with cerebellar hypoplasia is associated with human RELN mutations. Nature genetics. 2000;26(1):93.
Derer P, Derer M, Goffinet A. Axonal secretion of reelin by cajal"retzius cells: Evidence from comparison of normal and RelnOrl mutant mice. Journal of Comparative Neurology. 2001;440(2):136-43.
Krieger F, Möglich A, Kiefhaber T. Effect of proline and glycine residues on dynamics and barriers of loop formation in polypeptide chains. Journal of the American Chemical Society. 2005;127(10):3346-52.
Karadaghi SA. The 20 Amino Acids and Their Role in Protein Structures. Basics of Protein Structure. 2015.
Guerriero CJ, Brodsky JL. The delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology. Physiological reviews. 2012;92(2):537-76.
Botella-López A, Burgaya F, Gavín R, García-Ayllón MS, Gómez-Tortosa E, Peña-Casanova J, et al. Reelin expression and glycosylation patterns are altered in Alzheimer's disease. Proceedings of the National Academy of Sciences. 2006;103(14):5573-8.
Folsom TD, Fatemi SH. The involvement of Reelin in neurodevelopmental disorders. Neuropharmacology. 2013;68:122-35.
Habl G, Schmitt A, Zink M, Von Wilmsdorff M, Yeganeh-Doost P, Jatzko A, et al. Decreased reelin expression in the left prefrontal cortex (BA9) in chronic schizophrenia patients. Neuropsychobiology. 2012;66(1):57-62.
Lammert DB, Howell BW. RELN mutations in autism spectrum disorder. Frontiers in cellular neuroscience. 2016;10:84.

References

D'arcangelo G, Miao GG, Chen S-C, Scares HD, Morgan JI, Curran T. A protein related to extracellular matrix proteins deleted in the mouse mutant reeler. Nature. 1995;374(6524):719.

Verbrugghe P, Bouwer S, Wiltshire S, Carter K, Chandler D, Cooper M, et al. Impact of the reelin signaling cascade (ligands–receptors–adaptor complex) on cognition in schizophrenia. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics. 2012;159(4):392-404.

Chen Y, Beffert U, Ertunc M, Tang T-S, Kavalali ET, Bezprozvanny I, et al. Reelin modulates NMDA receptor activity in cortical neurons. Journal of Neuroscience. 2005;25(36):8209-16.

Fatemi SH. Reelin Glycoprotein: Structure, Biology and Roles in Health and Disease: Springer; 2008.

De Bergeyck V, Naerhuyzen B, Goffinet A, de Rouvroit CL. A panel of monoclonal antibodies against reelin, the extracellular matrix protein defective in reeler mutant mice. Journal of neuroscience methods. 1998;82(1):17-24.

He X, Semenov M, Tamai K, Zeng X. LDL receptor-related proteins 5 and 6 in Wnt/Î²-catenin signaling: arrows point the way. Development. 2004;131(8):1663-77.

Schrijver I, Liu W, Brenn T, Furthmayr H, Francke U. Cysteine substitutions in epidermal growth factor–like domains of fibrillin-1: distinct effects on biochemical and clinical phenotypes. The American Journal of Human Genetics. 1999;65(4):1007-20.

Utsunomiya-Tate N, Kubo K-i, Tate S-i, Kainosho M, Katayama E, Nakajima K, et al. Reelin molecules assemble together to form a large protein complex, which is inhibited by the function-blocking CR-50 antibody. Proceedings of the National Academy of Sciences. 2000;97(17):9729-34.

Jossin Y, Ignatova N, Hiesberger T, Herz J, de Rouvroit CL, Goffinet AM. The central fragment of Reelin, generated by proteolytic processing in vivo, is critical to its function during cortical plate development. Journal of Neuroscience. 2004;24(2):514-21.

Abdolmaleky HM, Cheng Kh, Russo A, Smith CL, Faraone SV, Wilcox M, et al. Hypermethylation of the reelin (RELN) promoter in the brain of schizophrenic patients: a preliminary report. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics. 2005;134(1):60-6.

Royaux I, de Rouvroit CL, D'arcangelo G, Demirov D, Goffinet AM. Genomic Organization of the MouseReelinGene. Genomics. 1997;46(2):240-50.

de Rouvroit CL, Bernier B, Royaux I, de Bergeyck V, Goffinet AM. Evolutionarily conserved, alternative splicing of reelin during brain development. Experimental neurology. 1999;156(2):229-38.

Curry-Hyde A, Chen BJ, Mills JD, Janitz M. Microexons: novel regulators of the transcriptome. Journal of Human Transcriptome. 2018;2(1):1-6.

Nakano Y, Kohno T, Hibi T, Kohno S, Baba A, Mikoshiba K, et al. The extremely conserved C-terminal region of Reelin is not necessary for secretion but is required for efficient activation of downstream signaling. Journal of Biological Chemistry. 2007;282(28):20544-52.

De Bergeyck V, Nakajima K, de Rouvroit CL, Naerhuyzen B, Goffinet A, Miyata T, et al. A truncated Reelin protein is produced but not secreted in the ‘Orleans' reeler mutation (Relnrl-Orl). Molecular brain research. 1997;50(1-2):85-90.

Okoro EU, Zhang H, Guo Z, Yang F, Smith Jr C, Yang H. A subregion of reelin suppresses lipoprotein-induced cholesterol accumulation in macrophages. PloS one. 2015;10(8):e0136895.

Lammert DB, Middleton FA, Pan J, Olson EC, Howell BW. The de novo autism spectrum disorder RELN R2290C mutation reduces Reelin secretion and increases protein disulfide isomerase expression. Journal of neurochemistry. 2017;142(1):89-102.

Beffert U, Weeber EJ, Durudas A, Qiu S, Masiulis I, Sweatt JD, et al. Modulation of synaptic plasticity and memory by Reelin involves differential splicing of the lipoprotein receptor Apoer2. Neuron. 2005;47(4):567-79.

Ogawa M, Miyata T, Nakajimat K, Yagyu K, Seike M, Ikenaka K, et al. The reeler gene-associated antigen on Cajal-Retzius neurons is a crucial molecule for laminar organization of cortical neurons. Neuron. 1995;14(5):899-912.

Kohno T, Honda T, Kubo K-i, Nakano Y, Tsuchiya A, Murakami T, et al. Importance of Reelin C-terminal region in the development and maintenance of the postnatal cerebral cortex and its regulation by specific proteolysis. Journal of Neuroscience. 2015;35(11):4776-87.

Hong SE, Shugart YY, Huang DT, Al Shahwan S, Grant PE, Hourihane JOB, et al. Autosomal recessive lissencephaly with cerebellar hypoplasia is associated with human RELN mutations. Nature genetics. 2000;26(1):93.

Derer P, Derer M, Goffinet A. Axonal secretion of reelin by cajal"retzius cells: Evidence from comparison of normal and RelnOrl mutant mice. Journal of Comparative Neurology. 2001;440(2):136-43.

Krieger F, Möglich A, Kiefhaber T. Effect of proline and glycine residues on dynamics and barriers of loop formation in polypeptide chains. Journal of the American Chemical Society. 2005;127(10):3346-52.

Karadaghi SA. The 20 Amino Acids and Their Role in Protein Structures. Basics of Protein Structure. 2015.

Guerriero CJ, Brodsky JL. The delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology. Physiological reviews. 2012;92(2):537-76.

Botella-López A, Burgaya F, Gavín R, García-Ayllón MS, Gómez-Tortosa E, Peña-Casanova J, et al. Reelin expression and glycosylation patterns are altered in Alzheimer's disease. Proceedings of the National Academy of Sciences. 2006;103(14):5573-8.

Folsom TD, Fatemi SH. The involvement of Reelin in neurodevelopmental disorders. Neuropharmacology. 2013;68:122-35.

Habl G, Schmitt A, Zink M, Von Wilmsdorff M, Yeganeh-Doost P, Jatzko A, et al. Decreased reelin expression in the left prefrontal cortex (BA9) in chronic schizophrenia patients. Neuropsychobiology. 2012;66(1):57-62.

Lammert DB, Howell BW. RELN mutations in autism spectrum disorder. Frontiers in cellular neuroscience. 2016;10:84.

Author biographies is not available.