Identification and recombinant analysis of botrocetin-2, a snake venom cofactor for von willebrand factor-induced platelet agglutination+

Yukiyo Yamamoto-Suzuki, Yoshihiko Sakurai, Yoshihiro Fujimura, Masanori Matsumoto, Jiharu Hamako, Tetsuro Kokubo, Hitoshi Kitagawa, Sarkar M.A. Kawsar, Yuki Fujii, Yasuhiro Ozeki, Fumio Matsushita, Taei Matsui

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Botrocetin is a heterodimer snake venom protein that induces von Willebrand factor (VWF)- and platelet glycoprotein Ib (GPIb)-dependent platelet agglutination in vitro. We have cloned cDNAs for a botrocetin-2 from a cDNA library of the venom gland of Bothrops jararaca having a high similarity with botrocetin subunits. Recombinant botrocetin-2, expressed in 293T cells, showed cofactor activity comparable to natural botrocetin. In a single subunit expression experiment, a dimer of the β subunit was obtained, and it showed reduced, but apparent, platelet agglutination activity. Ala scanning mutagenesis showed that substitutions at Asp62, Asp70, Arg115, or Lys117 in the β subunit reduced platelet agglutination activity. The 3D homology modeling of botrocetin-2 complexed with the VWF A1 domain and GPIbα indicated that Asp62, Arg115, and Lys117 of the β subunit are located near Arg218 and Asp222 of GPIbα, respectively, and that Aspβ70 is in proximity to Gln1391 of the A1 domain. Our results indicate that these charged amino acid residues in the β subunit have a preferential role in the activity of botrocetin-2. Since it has been time-consuming and difficult to obtain homogeneous botrocetin from natural venom, recombinant botrocetin-2 has potential benefits for clinical and basic investigations into hemostasis and thrombosis as a standard reagent.

Original languageEnglish
Pages (from-to)5329-5338
Number of pages10
JournalBiochemistry
Volume51
Issue number26
DOIs
Publication statusPublished - 03-07-2012

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Snake Venoms
Agglutination
von Willebrand Factor
Platelets
Blood Platelets
Platelet Glycoprotein GPIb-IX Complex
Venoms
Bothrops
Platelet Membrane Glycoproteins
botrocetin
Mutagenesis
HEK293 Cells
Hemostasis
Gene Library
Dimers
Thrombosis
Substitution reactions
Complementary DNA
Proteins
Scanning

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Yamamoto-Suzuki, Y., Sakurai, Y., Fujimura, Y., Matsumoto, M., Hamako, J., Kokubo, T., ... Matsui, T. (2012). Identification and recombinant analysis of botrocetin-2, a snake venom cofactor for von willebrand factor-induced platelet agglutination+. Biochemistry, 51(26), 5329-5338. https://doi.org/10.1021/bi300442c
Yamamoto-Suzuki, Yukiyo ; Sakurai, Yoshihiko ; Fujimura, Yoshihiro ; Matsumoto, Masanori ; Hamako, Jiharu ; Kokubo, Tetsuro ; Kitagawa, Hitoshi ; Kawsar, Sarkar M.A. ; Fujii, Yuki ; Ozeki, Yasuhiro ; Matsushita, Fumio ; Matsui, Taei. / Identification and recombinant analysis of botrocetin-2, a snake venom cofactor for von willebrand factor-induced platelet agglutination+. In: Biochemistry. 2012 ; Vol. 51, No. 26. pp. 5329-5338.
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abstract = "Botrocetin is a heterodimer snake venom protein that induces von Willebrand factor (VWF)- and platelet glycoprotein Ib (GPIb)-dependent platelet agglutination in vitro. We have cloned cDNAs for a botrocetin-2 from a cDNA library of the venom gland of Bothrops jararaca having a high similarity with botrocetin subunits. Recombinant botrocetin-2, expressed in 293T cells, showed cofactor activity comparable to natural botrocetin. In a single subunit expression experiment, a dimer of the β subunit was obtained, and it showed reduced, but apparent, platelet agglutination activity. Ala scanning mutagenesis showed that substitutions at Asp62, Asp70, Arg115, or Lys117 in the β subunit reduced platelet agglutination activity. The 3D homology modeling of botrocetin-2 complexed with the VWF A1 domain and GPIbα indicated that Asp62, Arg115, and Lys117 of the β subunit are located near Arg218 and Asp222 of GPIbα, respectively, and that Aspβ70 is in proximity to Gln1391 of the A1 domain. Our results indicate that these charged amino acid residues in the β subunit have a preferential role in the activity of botrocetin-2. Since it has been time-consuming and difficult to obtain homogeneous botrocetin from natural venom, recombinant botrocetin-2 has potential benefits for clinical and basic investigations into hemostasis and thrombosis as a standard reagent.",
author = "Yukiyo Yamamoto-Suzuki and Yoshihiko Sakurai and Yoshihiro Fujimura and Masanori Matsumoto and Jiharu Hamako and Tetsuro Kokubo and Hitoshi Kitagawa and Kawsar, {Sarkar M.A.} and Yuki Fujii and Yasuhiro Ozeki and Fumio Matsushita and Taei Matsui",
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Yamamoto-Suzuki, Y, Sakurai, Y, Fujimura, Y, Matsumoto, M, Hamako, J, Kokubo, T, Kitagawa, H, Kawsar, SMA, Fujii, Y, Ozeki, Y, Matsushita, F & Matsui, T 2012, 'Identification and recombinant analysis of botrocetin-2, a snake venom cofactor for von willebrand factor-induced platelet agglutination+', Biochemistry, vol. 51, no. 26, pp. 5329-5338. https://doi.org/10.1021/bi300442c

Identification and recombinant analysis of botrocetin-2, a snake venom cofactor for von willebrand factor-induced platelet agglutination+. / Yamamoto-Suzuki, Yukiyo; Sakurai, Yoshihiko; Fujimura, Yoshihiro; Matsumoto, Masanori; Hamako, Jiharu; Kokubo, Tetsuro; Kitagawa, Hitoshi; Kawsar, Sarkar M.A.; Fujii, Yuki; Ozeki, Yasuhiro; Matsushita, Fumio; Matsui, Taei.

In: Biochemistry, Vol. 51, No. 26, 03.07.2012, p. 5329-5338.

Research output: Contribution to journalArticle

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T1 - Identification and recombinant analysis of botrocetin-2, a snake venom cofactor for von willebrand factor-induced platelet agglutination+

AU - Yamamoto-Suzuki, Yukiyo

AU - Sakurai, Yoshihiko

AU - Fujimura, Yoshihiro

AU - Matsumoto, Masanori

AU - Hamako, Jiharu

AU - Kokubo, Tetsuro

AU - Kitagawa, Hitoshi

AU - Kawsar, Sarkar M.A.

AU - Fujii, Yuki

AU - Ozeki, Yasuhiro

AU - Matsushita, Fumio

AU - Matsui, Taei

PY - 2012/7/3

Y1 - 2012/7/3

N2 - Botrocetin is a heterodimer snake venom protein that induces von Willebrand factor (VWF)- and platelet glycoprotein Ib (GPIb)-dependent platelet agglutination in vitro. We have cloned cDNAs for a botrocetin-2 from a cDNA library of the venom gland of Bothrops jararaca having a high similarity with botrocetin subunits. Recombinant botrocetin-2, expressed in 293T cells, showed cofactor activity comparable to natural botrocetin. In a single subunit expression experiment, a dimer of the β subunit was obtained, and it showed reduced, but apparent, platelet agglutination activity. Ala scanning mutagenesis showed that substitutions at Asp62, Asp70, Arg115, or Lys117 in the β subunit reduced platelet agglutination activity. The 3D homology modeling of botrocetin-2 complexed with the VWF A1 domain and GPIbα indicated that Asp62, Arg115, and Lys117 of the β subunit are located near Arg218 and Asp222 of GPIbα, respectively, and that Aspβ70 is in proximity to Gln1391 of the A1 domain. Our results indicate that these charged amino acid residues in the β subunit have a preferential role in the activity of botrocetin-2. Since it has been time-consuming and difficult to obtain homogeneous botrocetin from natural venom, recombinant botrocetin-2 has potential benefits for clinical and basic investigations into hemostasis and thrombosis as a standard reagent.

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