Longistatin in tick saliva blocks advanced glycation end-product receptor activation
Anisuzzaman, … , Kozo Fujisaki, Naotoshi Tsuji
Anisuzzaman, … , Kozo Fujisaki, Naotoshi Tsuji
Published September 2, 2014
Citation Information: J Clin Invest. 2014;124(10):4429-4444. https://doi.org/10.1172/JCI74917.
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Research Article Infectious disease

Longistatin in tick saliva blocks advanced glycation end-product receptor activation

Abstract

Ticks are notorious hematophagous ectoparasites and vectors of many deadly pathogens. As an effective vector, ticks must break the strong barrier provided by the skin of their host during feeding, and their saliva contains a complex mixture of bioactive molecules that paralyze host defenses. The receptor for advanced glycation end products (RAGE) mediates immune cell activation at inflammatory sites and is constitutively and highly expressed in skin. Here, we demonstrate that longistatin secreted with saliva of the tick Haemaphysalis longicornis binds RAGE and modulates the host immune response. Similar to other RAGE ligands, longistatin specifically bound the RAGE V domain, and stimulated cultured HUVECs adhered to a longistatin-coated surface; this binding was dramatically inhibited by soluble RAGE or RAGE siRNA. Treatment of HUVECs with longistatin prior to stimulation substantially attenuated cellular oxidative stress and prevented NF-κB translocation, thereby reducing adhesion molecule and cytokine production. Recombinant longistatin inhibited RAGE-mediated migration of mouse peritoneal resident cells (mPRCs) and ameliorated inflammation in mouse footpad edema and pneumonia models. Importantly, tick bite upregulated RAGE ligands in skin, and endogenous longistatin attenuated RAGE-mediated inflammation during tick feeding. Our results suggest that longistatin is a RAGE antagonist that suppresses tick bite–associated inflammation, allowing successful blood-meal acquisition from hosts.

Authors

Anisuzzaman, Takeshi Hatta, Takeharu Miyoshi, Makoto Matsubayashi, M. Khyrul Islam, M. Abdul Alim, M. Abu Anas, M. Mehedi Hasan, Yasunobu Matsumoto, Yasuhiko Yamamoto, Hiroshi Yamamoto, Kozo Fujisaki, Naotoshi Tsuji

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Figure 1

Longistatin binds with RAGE.

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Longistatin binds with RAGE. (A) Longistatin binds with RAGE in a concen...
(A) Longistatin binds with RAGE in a concentration-dependent manner. RAGE (5 μg/ml) was coated and blocked. Longistatin or BSA (0–384 nM) was added and incubated in buffer A for 1 hour at RT and treated with anti-longistatin (1:1000) or anti-BSA (1:1000) and detected with HRP-conjugated IgG. Amounts of bound longistatin were determined, and KD value was calculated. (B) RAGE-dependent binding. Longistatin-coated (4 μg/ml) wells were reacted with RAGE/TLR4 (0–5 μg/ml) in 50 μl of buffer A and treated with anti-RAGE or anti-TLR4. (C) Competitive binding of longistatin with the V domain. RAGE (4 μg/ml) was coated, blocked, and treated with longistatin alone or with a mixture of longistatin (1 μg/ml) and each of the other RAGE ligands (5 μg/ml) as indicated, and bound longistatin was detected. (D) Longistatin binds with recombinant V domain. V, C1, or C2 domain (5 μg/ml) was coated and interacted with longistatin, and bound longistatin was detected. (E) Computational docking of longistatin to the V domain of RAGE was performed using ClusPro 2.0. (F) Ca2+-dependent RAGE binding. RAGE-coated (5 μg/ml) wells were treated with longistatin or metal-free longistatin (6 μg/ml), and binding was detected. n = 3. *P < 0.05; **P < 0.01.