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J. Biol. Chem., Vol. 279, Issue 49, 51331-51337, December 3, 2004

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The Gly-Gly Linker Region of the Insect Cytokine Growth-blocking Peptide Is Essential for Activity^*

Masanobu Yoshida, Tomoyasu Aizawa, Takashi Nakamura, Kunio Shitara, Yoichi Hayakawa¶, Kimiaki Matsubara, Kazunori Miura, Takahide Kouno, Kevin D. Clark||, Michael R. Strand||, Mineyuki Mizuguchi, Makoto Demura, Katsutoshi Nitta, and Keiichi Kawano**

From the Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan, the Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan, the ^¶Department of Agriculture, Saga University, Saga 840-8502, Japan, and the ^||Department of Entomology, University of Georgia, Athens, Georgia 30602

Growth-blocking peptide (GBP) is a 25-amino acid cytokine isolated from the lepidopteran insect Pseudaletia separata. GBP exhibits various biological activities such as regulation of larval growth of insects, proliferation of a few kinds of cultured cells, and stimulation of a class of insect immune cells called plasmatocytes. The tertiary structure of GBP consists of a well structured core domain and disordered N and C termini. Our previous studies revealed that, in addition to the structured core, specific residues in the unstructured N-terminal region (Glu¹ and Phe³) are also essential for the plasmatocyte-stimulating activity. In this study, a number of deletion, insertion, and site-directed mutants targeting the unstructured N-terminal residues of GBP were constructed to gain more detailed insight into the mode of interaction between the N-terminal region and GBP receptor. Alteration of the backbone length of the linker region between the core structure and N-terminal domain reduced plasmatocyte-stimulating activity. The substitutions of Gly⁵ or Gly⁶ in this linker region with more bulky residues, such as Phe and Pro, also remarkably reduced this activity. We conclude that the interaction of GBP with its receptor depends on the relative position of the N-terminal domain to the core structure, and therefore the backbone flexibility of Gly residues in the linker region is necessary for adoption of a proper conformation suited to receptor binding. Additionally, antagonistic experiments using deletion mutants confirmed that not only the core domain but also the N-terminal region of GBP are required for "receptor-binding," and furthermore Phe³ is a binding determinant of the N-terminal domain.

Received for publication, August 16, 2004 , and in revised form, September 17, 2004.

^* This work was supported by the Program for the Promotion of Basic Research Activities for Innovation Biosciences, Japan, and the Ministry of Education, Culture, Sports, Science and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed. Tel.: 81-11-706-2770; Fax: 81-11-706-4993; E-mail: kawano{at}sci.hokudai.ac.jp.

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