Comparative analysis of the metal-dependent structural and functional properties of mouse and human SMP30.

Roshan Kumar Dutta,Parmanand Pandey,Nidhi Dhama,Md Summon Hossain,Fauzia Parween,Rinkoo Devi Gupta,Eugene A Permyakov

doi:10.1371/journal.pone.0218629

Roshan Kumar Dutta, Parmanand Pandey + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0218629

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Journal: PloS one	Publication Date: Jun 20, 2019
Citations: 8	License type: CC BY 4.0

Affiliation: South Asian University

Abstract

Senescence Marker Protein (SMP30) is a metalloenzyme that shows lactonase activity in the ascorbic acid (AA) biosynthesis pathway in non-primate mammals such as a mouse. However, AA biosynthesis does not occur in the primates including humans. Several studies have shown the role of SMP30 in maintaining calcium homeostasis in mammals. In addition, it is also reported to have promiscuous enzyme activity with an organophosphate (OP) substrate. Hence, this study aims to recombinantly express and purify the SMP30 proteins from both mouse and human, and to study their structural alterations and functional deviations in the presence of different divalent metals. For this, mouse SMP30 (MoSMP30) as well as human SMP30 (HuSMP30) were cloned in the bacterial expression vector. Proteins were overexpressed and purified from soluble fractions as well as from inclusion bodies as these proteins were expressed largely in insoluble fractions. The purified proteins were used to study the folding conformations in the presence of different divalent cations (Ca2+, Co2+, Mg2+, and Zn2+) with the help of circular dichroism (CD) spectroscopy. It was observed that both MoSMP30 and HuSMP30 acquired native folding conformations. To study the metal-binding affinity, dissociation constant (Kd values) were calculated from UV-VIS titration curve, which showed the highest affinity of MoSMP30 with Zn2+. However, HuSMP30 showed the highest affinity with Ca2+, suggesting the importance of HuSMP30 in maintaining calcium homeostasis. Enzyme kinetics were performed with γ-Thiobutyrolactone and Demeton-S in the presence of different divalent cations. Interestingly, both the proteins showed lactonase activity in the presence of Ca2+. In addition, MoSMP30 and HuSMP30 also showed lactonase activity in the presence of Co2+ and Zn2+ respectively. Moreover, both the proteins showed OP hydrolase activities in the presence of Ca2+ as well as Zn2+, suggesting the metal-dependent promiscuous nature of SMP30.

Highlights

SMP30 is known to possess calcium binding property [1] and is involved in cellular Ca2+ homeostasis [2]
The mouse SMP30 (MoSMP30) gene amplified from the cDNA prepared using mouse kidney and codon optimized human SMP30 (HuSMP30) gene were cloned in pET28a with 6xHis tag at N-terminus (S1 and S2 Figs)
SDS-PAGE analysis showed that both MoSMP30 and HuSMP30 proteins were significantly expressed after IPTG induction

Summary

Objectives

This study aims to recombinantly express and purify the SMP30 proteins from both mouse and human, and to study their structural alterations and functional deviations in the presence of different divalent metals. We aim to clone human and mouse SMP30, express and purify the recombinant proteins of both human and mouse SMP30, and to study the structural conformation and functional deviations in the presence of different divalent metals

Methods

Results

Discussion

Conclusion