Basic Fibroblast Growth Factor Fused with Tandem Collagen-Binding Domains from Clostridium histolyticum Collagenase ColG Increases Bone Formation

Hiroyuki Sekiguchi, Kentaro Uchida, Osamu Matsushita, Gen Inoue, Nozomu Nishi, Ryo Masuda, Nana Hamamoto, Takaki Koide, Shintaro Shoji, Masashi Takaso

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Basic fibroblast growth factor 2 (bFGF) accelerates bone formation during fracture healing. Because the efficacy of bFGF decreases rapidly following its diffusion from fracture sites, however, repeated dosing is required to ensure a sustained therapeutic effect. We previously developed a fusion protein comprising bFGF, a polycystic kidney disease domain (PKD; s2b), and collagen-binding domain (CBD; s3) sourced from the Clostridium histolyticum class II collagenase, ColH, and reported that the combination of this fusion protein with a collagen-like peptide, poly(Pro-Hyp-Gly)10, induced mesenchymal cell proliferation and callus formation at fracture sites. In addition, C. histolyticum produces class I collagenase (ColG) with tandem CBDs (s3a and s3b) at the C-terminus. We therefore hypothesized that a bFGF fusion protein containing ColG-derived tandem CBDs (s3a and s3b) would show enhanced collagen-binding activity, leading to improved bone formation. Here, we examined the binding affinity of four collagen anchors derived from the two clostridial collagenases to H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly)12-NH2, a collagenous peptide, by surface plasmon resonance and found that tandem CBDs (s3a-s3b) have the highest affinity for the collagenous peptide. We also constructed four fusion proteins consisting of bFGF and s3 (bFGF-s3), s2b-s3b (bFGF-s2b-s3), s3b (bFGF-s3b), and s3a-s3b (bFGF-s3a-s3b) and compared their biological activities to those of a previous fusion construct (bFGF-s2b-s3) using a cell proliferation assay in vitro and a mouse femoral fracture model in vivo. Among these CB-bFGFs, bFGF-s3a-s3b showed the highest capacity to induce mesenchymal cell proliferation and callus formation in the mice fracture model. The poly(Pro-Hyp-Gly)10/bFGF-s3a-s3b construct may therefore have the potential to promote bone formation in clinical settings.

Original languageEnglish
Article number8393194
JournalBioMed Research International
Volume2018
DOIs
Publication statusPublished - Jan 1 2018

Fingerprint

Microbial Collagenase
Collagenases
Fibroblast Growth Factor 2
Osteogenesis
Bone
Collagen
Fusion reactions
Clostridium histolyticum
Cell proliferation
Cell Proliferation
Bony Callus
Peptides
Proteins

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Cite this

Basic Fibroblast Growth Factor Fused with Tandem Collagen-Binding Domains from Clostridium histolyticum Collagenase ColG Increases Bone Formation. / Sekiguchi, Hiroyuki; Uchida, Kentaro; Matsushita, Osamu; Inoue, Gen; Nishi, Nozomu; Masuda, Ryo; Hamamoto, Nana; Koide, Takaki; Shoji, Shintaro; Takaso, Masashi.

In: BioMed Research International, Vol. 2018, 8393194, 01.01.2018.

Research output: Contribution to journalArticle

Sekiguchi, Hiroyuki ; Uchida, Kentaro ; Matsushita, Osamu ; Inoue, Gen ; Nishi, Nozomu ; Masuda, Ryo ; Hamamoto, Nana ; Koide, Takaki ; Shoji, Shintaro ; Takaso, Masashi. / Basic Fibroblast Growth Factor Fused with Tandem Collagen-Binding Domains from Clostridium histolyticum Collagenase ColG Increases Bone Formation. In: BioMed Research International. 2018 ; Vol. 2018.
@article{7689ca7937ce430a8b55863bb6759a65,
title = "Basic Fibroblast Growth Factor Fused with Tandem Collagen-Binding Domains from Clostridium histolyticum Collagenase ColG Increases Bone Formation",
abstract = "Basic fibroblast growth factor 2 (bFGF) accelerates bone formation during fracture healing. Because the efficacy of bFGF decreases rapidly following its diffusion from fracture sites, however, repeated dosing is required to ensure a sustained therapeutic effect. We previously developed a fusion protein comprising bFGF, a polycystic kidney disease domain (PKD; s2b), and collagen-binding domain (CBD; s3) sourced from the Clostridium histolyticum class II collagenase, ColH, and reported that the combination of this fusion protein with a collagen-like peptide, poly(Pro-Hyp-Gly)10, induced mesenchymal cell proliferation and callus formation at fracture sites. In addition, C. histolyticum produces class I collagenase (ColG) with tandem CBDs (s3a and s3b) at the C-terminus. We therefore hypothesized that a bFGF fusion protein containing ColG-derived tandem CBDs (s3a and s3b) would show enhanced collagen-binding activity, leading to improved bone formation. Here, we examined the binding affinity of four collagen anchors derived from the two clostridial collagenases to H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly)12-NH2, a collagenous peptide, by surface plasmon resonance and found that tandem CBDs (s3a-s3b) have the highest affinity for the collagenous peptide. We also constructed four fusion proteins consisting of bFGF and s3 (bFGF-s3), s2b-s3b (bFGF-s2b-s3), s3b (bFGF-s3b), and s3a-s3b (bFGF-s3a-s3b) and compared their biological activities to those of a previous fusion construct (bFGF-s2b-s3) using a cell proliferation assay in vitro and a mouse femoral fracture model in vivo. Among these CB-bFGFs, bFGF-s3a-s3b showed the highest capacity to induce mesenchymal cell proliferation and callus formation in the mice fracture model. The poly(Pro-Hyp-Gly)10/bFGF-s3a-s3b construct may therefore have the potential to promote bone formation in clinical settings.",
author = "Hiroyuki Sekiguchi and Kentaro Uchida and Osamu Matsushita and Gen Inoue and Nozomu Nishi and Ryo Masuda and Nana Hamamoto and Takaki Koide and Shintaro Shoji and Masashi Takaso",
year = "2018",
month = "1",
day = "1",
doi = "10.1155/2018/8393194",
language = "English",
volume = "2018",
journal = "BioMed Research International",
issn = "2314-6133",
publisher = "Hindawi Publishing Corporation",

}

TY - JOUR

T1 - Basic Fibroblast Growth Factor Fused with Tandem Collagen-Binding Domains from Clostridium histolyticum Collagenase ColG Increases Bone Formation

AU - Sekiguchi, Hiroyuki

AU - Uchida, Kentaro

AU - Matsushita, Osamu

AU - Inoue, Gen

AU - Nishi, Nozomu

AU - Masuda, Ryo

AU - Hamamoto, Nana

AU - Koide, Takaki

AU - Shoji, Shintaro

AU - Takaso, Masashi

PY - 2018/1/1

Y1 - 2018/1/1

N2 - Basic fibroblast growth factor 2 (bFGF) accelerates bone formation during fracture healing. Because the efficacy of bFGF decreases rapidly following its diffusion from fracture sites, however, repeated dosing is required to ensure a sustained therapeutic effect. We previously developed a fusion protein comprising bFGF, a polycystic kidney disease domain (PKD; s2b), and collagen-binding domain (CBD; s3) sourced from the Clostridium histolyticum class II collagenase, ColH, and reported that the combination of this fusion protein with a collagen-like peptide, poly(Pro-Hyp-Gly)10, induced mesenchymal cell proliferation and callus formation at fracture sites. In addition, C. histolyticum produces class I collagenase (ColG) with tandem CBDs (s3a and s3b) at the C-terminus. We therefore hypothesized that a bFGF fusion protein containing ColG-derived tandem CBDs (s3a and s3b) would show enhanced collagen-binding activity, leading to improved bone formation. Here, we examined the binding affinity of four collagen anchors derived from the two clostridial collagenases to H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly)12-NH2, a collagenous peptide, by surface plasmon resonance and found that tandem CBDs (s3a-s3b) have the highest affinity for the collagenous peptide. We also constructed four fusion proteins consisting of bFGF and s3 (bFGF-s3), s2b-s3b (bFGF-s2b-s3), s3b (bFGF-s3b), and s3a-s3b (bFGF-s3a-s3b) and compared their biological activities to those of a previous fusion construct (bFGF-s2b-s3) using a cell proliferation assay in vitro and a mouse femoral fracture model in vivo. Among these CB-bFGFs, bFGF-s3a-s3b showed the highest capacity to induce mesenchymal cell proliferation and callus formation in the mice fracture model. The poly(Pro-Hyp-Gly)10/bFGF-s3a-s3b construct may therefore have the potential to promote bone formation in clinical settings.

AB - Basic fibroblast growth factor 2 (bFGF) accelerates bone formation during fracture healing. Because the efficacy of bFGF decreases rapidly following its diffusion from fracture sites, however, repeated dosing is required to ensure a sustained therapeutic effect. We previously developed a fusion protein comprising bFGF, a polycystic kidney disease domain (PKD; s2b), and collagen-binding domain (CBD; s3) sourced from the Clostridium histolyticum class II collagenase, ColH, and reported that the combination of this fusion protein with a collagen-like peptide, poly(Pro-Hyp-Gly)10, induced mesenchymal cell proliferation and callus formation at fracture sites. In addition, C. histolyticum produces class I collagenase (ColG) with tandem CBDs (s3a and s3b) at the C-terminus. We therefore hypothesized that a bFGF fusion protein containing ColG-derived tandem CBDs (s3a and s3b) would show enhanced collagen-binding activity, leading to improved bone formation. Here, we examined the binding affinity of four collagen anchors derived from the two clostridial collagenases to H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly)12-NH2, a collagenous peptide, by surface plasmon resonance and found that tandem CBDs (s3a-s3b) have the highest affinity for the collagenous peptide. We also constructed four fusion proteins consisting of bFGF and s3 (bFGF-s3), s2b-s3b (bFGF-s2b-s3), s3b (bFGF-s3b), and s3a-s3b (bFGF-s3a-s3b) and compared their biological activities to those of a previous fusion construct (bFGF-s2b-s3) using a cell proliferation assay in vitro and a mouse femoral fracture model in vivo. Among these CB-bFGFs, bFGF-s3a-s3b showed the highest capacity to induce mesenchymal cell proliferation and callus formation in the mice fracture model. The poly(Pro-Hyp-Gly)10/bFGF-s3a-s3b construct may therefore have the potential to promote bone formation in clinical settings.

UR - http://www.scopus.com/inward/record.url?scp=85045466702&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85045466702&partnerID=8YFLogxK

U2 - 10.1155/2018/8393194

DO - 10.1155/2018/8393194

M3 - Article

VL - 2018

JO - BioMed Research International

JF - BioMed Research International

SN - 2314-6133

M1 - 8393194

ER -