Form 1

2018 Report Form for Collaboration withResearch Center for Biomedical Engineering

2019/3/11

Year/month/dateP-69

NumberDate:

Date /Month/Year
11/March/2019

To Chairman, Board of Directors, Research Center for Biomedical Engineering

ApplicantAffiliation : Jahangirnagar University
Title: Assistant Professor
Name: Dr. Md. Zahidul Islam
Address: Department of Biotechnology and Genetic Engineering,
Jahangirnagar University, Savar, Dhaka-1342, Bangladesh.
Phone
Fax
E-mail: +88-01787-086965: zahidul_ku@yahoo.com; zahidul.islam@bgeju.edu.bd
Report Form for Collaboration Research

Effect of membrane potential on entry of cell-penetrating peptide Transportan10

Research Theme

Research Area

Research Period

into single vesicles.
1. Biomaterials
2. Bioengineering3. Functional molecules
4. Chemistry/Electrical Engineering/Mechanical Engineering/Materials Science

From:Date/month/Year
14/05/2018

To: Date/month/Year
31/03/2019

Applicant Organization
Name
Md. Zahidul Islam

Department
Department of Biotechnology and Genetic
Engineering, Jahangirnagar University,
Bangladesh

Title
Assistant
Professor

Role
Experiments andanalysis

Md. Mizanur Rahman
MoghalIntegrated Bioscience Section, Graduate
School of Science and Technology, Shizuoka
Univ.

Ph. D
studentExperiments

Masahito Yamazaki

Research Institute of Electronics, Shizuoka
Univ.

Professor

Collaboration Partners
Research CenterintheMasahito Yamazaki

courseAnalysis

Research Results (Including Purpose, Results, Figures, etc.)

1. Purpose of the research
Cell-penetrating peptide (CPPs) such as TAT peptide and transportan 10 (TP10) can penetrate plasma
membrane of cells but the mechanism of its entry is not clearly understood. To elucidate the mechanism of thetranslocation of CPPs across plasma membrane and to reveal its elementary processes, we have developed thesingle giant unilamellar vesicle (GUV) method for CPPs, and succeeded in observing the elementary processes ofthe entry of fluorescent probe-labeled CPPs into single GUVs [1-3]. To reveal the mechanisms of entering of
TP10 into a GUV lumen, in the present study we investigated the effects of the membrane potential across lipid
bilayers on the entering of CF-TP10 using the single GUV method.
2. Results and discussion
First, we developed the method to prepare GUVs with various negative membrane potentials and examined
the membrane potential by measuring the fluorescence intensity of the GUV membranes (i.e., the rim intensity)
due to a potential-sensitive dye using confocal laser scanning microscopy. Then, we analyzed the membranepotential dependence of the rim intensity, indicating the application of membrane potential across the membranes.
Then, we examined the effect of membrane potential on the entry of CF-TP10 into GUVs comprised ofdioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (2/8, molar ratio) using single
GUVs containing small GUVs inside the GUV lumen [1] or single GUVs containing LUVs inside the GUV lumen[2]. We found that CF-TP10 entered the GUV without pore formation and the rate of entry of CF-TP10 into theGUV lumen increased with an increase in negative potential. These results indicate that the rate of translocation ofCF-TP10 across the membrane increased with negative membrane potential. We discussed the mechanism of thiseffect of membrane potential based on the prepore model of the translocation of CF-TP10 across the membrane[3].
3. References[1] M.Z. Islam, H. Ariyama, M.J. Alam, M. Yamazaki, Biochemistry, 53, 386-396 (2014).
[2] M. M. R. Moghal, M. Z. Islam, S. Sharmin, V. Levadnyy, M. Moniruzzaman, M. Yamazaki, Chem. Phys.
Lipids. 21, 120-129 (2018). [3] M. Z. Islam, S. Sharmin, M. Moniruzzaman, M. Yamazaki, Appl. Microbiol.
Biotechnol. 102, 3879-3892 (2018).
List of Publications Related to the Collaboration Research(1) Md. Zahidul Islam, Sabrina Sharmin, Md. Moniruzzaman, and Masahito Yamazaki
Elementary Processes for the Entry of Cell-Penetrating Peptides into Lipid Bilayer Vesicles and BacterialCells, Appl. Microbiol. Biotechnol. 102, 3879-3892 (2018).
List of Presentations ( Conference, Meeting, etc)
(1) Moynul Hasan, Md. Zahidul Islam, and Masahito Yamazaki, “Single GUV Studies on Mode of Action of
Antimicrobial Peptides and Cell-Penetrating Peptides”, 2018 Asian Biophysics Association Symposium, , at
RMIT University City Campus, Melbourne, Australia, 2-6 Dec., 2018 (Invited Talk)(2) M. M. R. Moghal, M. Z. Islam, S. Sharmin, and M. Yamazaki, “Continuous detection of entry ofcell-penetrating peptide transportan 10 into single vesicles”, The 3rd International Symposium on BiomedicalEngineering, Satake Memorial Hall, Hiroshima Univ., Higashi-hiroshima, 8-9 Nov., 2018.
(3) M. M. R. Moghal, M. Z. Islam, S. Sharmin, and M. Yamazaki, “Continuous detection of entry ofcell-penetrating peptide transportan 10 into single vesicles”, 20th Takayanagi International Symposium,Sanarukaikan, Hamamatsu, 27-29 Nov., 2018.
(4) Md. Mizanur Rahman Moghal, Md. Zahidul Islam, Sabrina Sharmin, and Masahito Yamazaki, “Continuousdetection of entry of cell-penetrating peptide transportan 10 into single vesicle lumen”, The 56th Annual
Meeting of Biophysical Society of Japan, 1517 Sept., 2018, Okayama Univ., Okayama
List of Awards

Registration of research-theme continuation for next year
Prior consent from the collaboration partner in the Research Center is necessary.

Yes

Research plan for the next year (from April 1, 2019 to March 31, 2020), if the collaboration research is continued.
As described above, this year we found that CF-TP10 entered the GUV without pore formation and the rate ofentry of CF-TP10 into the GUV lumen increased with an increase in negative potential. These results indicate thatthe rate of translocation of CF-TP10 across the membrane increased with negative membrane potential. We haveproposed a hypothesis on the mechanism of effect of membrane potential on entry of CF-TP10 into single GUVs.
Next year we continue to perform this project to obtain several experimental results to support of this hypothesis.
Next, to generalize the concept of the effect of the membrane potential on the entry of CPPs, we investigate theeffect of membrane potential on the entry of other CPPs such as Rh-LfcinB (4-9) (1) and its elementary processes.
Generally, it is considered that the labeling of fluorescent probe to peptides affects their behavior. To overcome
this problem, we will develop a new method to detect the entry of non-labeled peptide to single vesicles. After wedevelop the method, we examine the effect of membrane potential on the entry of non-labeled TP10 into single
vesicles.
The results of the effect of membrane potential on the entry of CPPs and the mechanism of the translocation ofCPPs across the membranes are very helpful to develop novel vectors for the delivery of drugs, chemicals,oligonucleotides, and proteins into cells for curing various diseases.
<References>(1) Moniruzzaman M, Islam MZ, Sharmin S, Dohra H, Yamazaki M (2017) Entry of a Six-Residue Antimicrobial
Peptide Derived from Lactoferricin B into Single Vesicles and Escherichia coli Cells without Damaging theirMembranes. Biochemistry 56:4419-4431.