Presentation Information
[P04-486]Development of the Hyaluronic Acid-Binding Protein with Angiogenic Activity
○Jingyuan Zhao1, Natsuko F Inagaki1, Momoko Kamedani1, Masashi Okawa1, Kohei Tsumoto1, Taichi Ito1 (1. Graduate School of Engineering, The University of Tokyo (Japan))
Keywords:
Link module,Tumor necrosis factor-stimulated gene-6,Angiogenesis,HA-binding capacity
[Purpose]
Hyaluronan (HA) is one of the main components of extracellular matrix. Tumor necrosis factor-stimulated gene-6 (TSG-6) can bind to HA via its Link module. Recently, the functional peptide has been fused to C-terminal of TSG-6–derived Link module to obtain the engineered Link module with specific functions. Vascular endothelial growth factor (VEGF) is a potent signaling protein interacting with receptor tyrosine kinases on endothelial cells to promote angiogenesis. However, it has disadvantages such as low stability and high production costs. QK peptide (KLTWQELYQLKYKGI) is a synthetic VEGF mimetic effectively mimicking the receptor-binding domain of VEGF. Herein, in this study, we designed and developed a novel TSG-6–derived Link module by fusing the sequence of QK into the C-terminal of Link module, obtaining Link-QK with both HA-binding ability and VEGF-mimicking capacity.
[Method]
A 6HisSUMO-tag was introduced at the N-terminal of Link module, and a QK-tag was fused into the C-terminal of Link module. Link-QK was expressed using SHuffle® T7 Competent E. coli (C3029J) and further purified. The secondary structure of Link-QK was measured via Circular dichroism assay. The thermal stability of Link-QK was measured via Differential scanning calorimetry assay. The thermodynamic parameters of the interaction between Link-QK and HA8 were determined using a VP-ITC instrument. Further, the angiogenic ability of Link-QK was studied via tube formation assay and aortic ring assay. In tube formation assay, Human Umbilical Vein Endothelial Cells (HUVECs) were treated with different groups. At 8 h, microscopy was adopted to observe the tubes formed. In aortic ring assay, the aorta taken from Sprague-Dawley rats were cut into 2-mm pieces and further cultured under different treatments. On Day 7, the cell sprouting of aortic rings were observed via microscopy and the sprouting area among different groups were compared. Finally, mouse pressure ulcer model was adopted to evaluate the ability of Link-QK to promote wound healing. The body weight and the wound area among different groups were recorded for comparison.
[Results]
Physicochemical properties of Link-QK were characterized. Link-QK had the obvious α-helix structure and the peak appeared at the wavelength of 222 nm. At pH 6.0, Tm of Link-QK was 47.0 ± 0.4 °C while at pH 7.4, Tm was 53.5 ± 1.0 °C. Link-QK was more stable at pH 7.4 than pH 6.0. Representative curve of calorimetric titration showed that the HA-binding property of Link-QK with HA8 was evident at pH 6.0 and weak at pH 7.4. At 8 h, Link-QK group displayed better ability to form tubes than Negative control as for number of branches and total branches length, comparable to QK peptide, VEGF, and Positive control. On Day 7, microvessels can be observed to grow out from the aortic rings in Link-QK group and the sprouting area in Link-QK group was larger than that in Negative control. In the mouse pressure ulcer model, Link-QK started to display the promoting ability for wound healing from Day 3.
[Conclusion]
Link-QK was successfully expressed and characterized. Link-QK displayed both pH-dependent HA-binding ability and fair ability to promote angiogenesis of HUVECs and cell sprouting of aortic rings. Link-QK displayed fair ability to promote wound healing using mouse pressure ulcer model. Link-QK is capable of specifically binding to HA and has demonstrated promising potential in pro-angiogenic applications, indicating broad prospects for translational research.
Hyaluronan (HA) is one of the main components of extracellular matrix. Tumor necrosis factor-stimulated gene-6 (TSG-6) can bind to HA via its Link module. Recently, the functional peptide has been fused to C-terminal of TSG-6–derived Link module to obtain the engineered Link module with specific functions. Vascular endothelial growth factor (VEGF) is a potent signaling protein interacting with receptor tyrosine kinases on endothelial cells to promote angiogenesis. However, it has disadvantages such as low stability and high production costs. QK peptide (KLTWQELYQLKYKGI) is a synthetic VEGF mimetic effectively mimicking the receptor-binding domain of VEGF. Herein, in this study, we designed and developed a novel TSG-6–derived Link module by fusing the sequence of QK into the C-terminal of Link module, obtaining Link-QK with both HA-binding ability and VEGF-mimicking capacity.
[Method]
A 6HisSUMO-tag was introduced at the N-terminal of Link module, and a QK-tag was fused into the C-terminal of Link module. Link-QK was expressed using SHuffle® T7 Competent E. coli (C3029J) and further purified. The secondary structure of Link-QK was measured via Circular dichroism assay. The thermal stability of Link-QK was measured via Differential scanning calorimetry assay. The thermodynamic parameters of the interaction between Link-QK and HA8 were determined using a VP-ITC instrument. Further, the angiogenic ability of Link-QK was studied via tube formation assay and aortic ring assay. In tube formation assay, Human Umbilical Vein Endothelial Cells (HUVECs) were treated with different groups. At 8 h, microscopy was adopted to observe the tubes formed. In aortic ring assay, the aorta taken from Sprague-Dawley rats were cut into 2-mm pieces and further cultured under different treatments. On Day 7, the cell sprouting of aortic rings were observed via microscopy and the sprouting area among different groups were compared. Finally, mouse pressure ulcer model was adopted to evaluate the ability of Link-QK to promote wound healing. The body weight and the wound area among different groups were recorded for comparison.
[Results]
Physicochemical properties of Link-QK were characterized. Link-QK had the obvious α-helix structure and the peak appeared at the wavelength of 222 nm. At pH 6.0, Tm of Link-QK was 47.0 ± 0.4 °C while at pH 7.4, Tm was 53.5 ± 1.0 °C. Link-QK was more stable at pH 7.4 than pH 6.0. Representative curve of calorimetric titration showed that the HA-binding property of Link-QK with HA8 was evident at pH 6.0 and weak at pH 7.4. At 8 h, Link-QK group displayed better ability to form tubes than Negative control as for number of branches and total branches length, comparable to QK peptide, VEGF, and Positive control. On Day 7, microvessels can be observed to grow out from the aortic rings in Link-QK group and the sprouting area in Link-QK group was larger than that in Negative control. In the mouse pressure ulcer model, Link-QK started to display the promoting ability for wound healing from Day 3.
[Conclusion]
Link-QK was successfully expressed and characterized. Link-QK displayed both pH-dependent HA-binding ability and fair ability to promote angiogenesis of HUVECs and cell sprouting of aortic rings. Link-QK displayed fair ability to promote wound healing using mouse pressure ulcer model. Link-QK is capable of specifically binding to HA and has demonstrated promising potential in pro-angiogenic applications, indicating broad prospects for translational research.
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