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The C-terminal Extension Peptide of Non-photoconvertible Water-Soluble Chlorophyll-Binding Proteins (Class II WSCPs) Affects Their Solubility and Stability: Comparative Analyses of the Biochemical and Chlorophyll-Binding Properties of Recombinant Brassica, Raphanus and Lepidium WSCPs with or Without Their C-terminal Extension Peptides

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Abstract

Numerous members of the Brassicaceae possess non-photoconvertible water-soluble chlorophyll (Chl)-binding proteins (Class II WSCPs), which function as Chl scavengers during cell disruption caused by wounding, pest/pathogen attacks, and/or environmental stress. Class II WSCPs have two extension peptides, one at the N-terminus and one at the C-terminus. The N-terminal peptide acts as a signal peptide, targeting the protein to the endoplasmic reticulum body, a unique defensive organelle found only in the Brassicaceae. However, the physiological and biochemical functions of the C-terminal extension peptide had not been characterized previously. To investigate the function of the C-terminal extension peptide, we produced expression constructs of recombinant WSCPs with or without the C-terminal extension peptide. The WSCPs used were of Brussels sprouts (Brassica oleracea), Japanese wild radish (Raphanus sativus) and Virginia pepperweed (Lepidium virginicum). The solubility of all of the WSCPs with the C-terminal extension peptide was drastically lower than that of the recombinant WSCPs without the C-terminal extension peptide. In addition, the stability of the reconstituted WSCPs complexes with the C-terminal extension peptide was altered compared with that of the proteins without the C-terminal extension peptide. These finding indicate that the C-terminal extension peptide affects not only the solubility, but also the stability of Class II WSCP. Furthermore, we characterized the Chl-binding properties of the recombinant WSCP from Japanese wild radish (RshWSCP-His) in a 40 % methanol solution. An electrophoretic mobility shift assay revealed that RshWSCP-His required a half-molar ratio of Chls to form a tetramer.

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Abbreviations

CD:

Circular dichroism

Chl:

Chlorophyll

C-terminal:

Carboxy-terminal

EMSA:

Electrophoretic mobility shift assay

ER:

Endplasmic reticulum

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

MALDI-TOF-MS:

Matrix assisted laser desorption ionization time of flight mass spectrometry

N-terminal:

Amino-terminal

WSCP:

Water soluble chlorophyll-binding protein

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Acknowledgments

We thank Dr. Shunsuke Kuwahara for his kind help in the measurements of CD spectra.

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Authors and Affiliations

  1. Department of Biomolecular Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan

    Shigekazu Takahashi, Akira Uchida, Katsumi Nakayama & Hiroyuki Satoh

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  1. Shigekazu Takahashi
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  2. Akira Uchida
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  3. Katsumi Nakayama
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  4. Hiroyuki Satoh
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Correspondence to Hiroyuki Satoh.

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Takahashi, S., Uchida, A., Nakayama, K. et al. The C-terminal Extension Peptide of Non-photoconvertible Water-Soluble Chlorophyll-Binding Proteins (Class II WSCPs) Affects Their Solubility and Stability: Comparative Analyses of the Biochemical and Chlorophyll-Binding Properties of Recombinant Brassica, Raphanus and Lepidium WSCPs with or Without Their C-terminal Extension Peptides. Protein J 33, 75–84 (2014). https://doi.org/10.1007/s10930-013-9539-5

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