Form 1

2019 Report Form for Collaboration with
Research Center for Biomedical Engineering

Year/month/date
Numberdate:

Date /Month/Year
6/March/2020

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

ApplicantAffiliation ; Jahangirnagar University
Title: Associate 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
14/05/2019

To: Date/month/Year
31/03/2020

Applicant Organization
Name
Md. Zahidul Islam

Department
Department of Biotechnology and Genetic
Engineering, Jahangirnagar University, Ban
gladeshTitle
Associate
Professor

Role
Experiments andanalysis

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

Ph. D
student

courseExperiments

Ph. D
student

courseExperiments

Masahito Yamazaki

Research Institute of Electronics, Shizuoka
Univ.

Professor

Collaboration Partners
Research CenterinFarzana Hossain

theMasahito Yamazaki

Analysis

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

1. Purpose of the research
In the report last year, we described the effect of membrane potential, ∆, on entry of a cell-penetrating
peptide (CPP), transportan 10 (TP10) into the lumen of single giant unilamellar vesicles (GUVs). The rate of entryof TP10 into single GUVs increased with negative membrane potential, ∆ , without pore formation in the GUVmembrane [1]. To elucidate the effect of ∆ on the interactions of peptides with GUVs in more detail, here we
examined the effect of ∆ on antimicrobial peptide (AMP)-induced damage of plasma membrane of bacterial cells
and lipid bilayers [2]. As AMP, we selected lactoferricin B (LfcinB), because it is reported that LfcinB-induced
damage of the bacterial plasma membrane is a key factor in its antimicrobial activity [3].
2. Results and discussion
First, we investigated the interaction of LfcinB with live single E. coli cells loaded with fluorescent dye,calcein, in the cytoplasm using confocal laser scanning microscopy (CLSM). LfcinB induced rapid calceinleakage from single E. coli cells, indicating that LfcinB induced damage or pore formation in the plasma
membrane. To examine direct interaction of LfcinB with the plasma membrane, we investigated its interaction
with single spheroplasts (loaded with calcein) derived from E. coli cells. LfcinB induced rapid leakage of calcein
from spheroplasts. The presence of a proton ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP)
suppressed the LfcinB-induced leakage of calcein from single E. coli and spheroplasts. It is considered that CCCP
dissipates ∆ of cells, and thus, this result indicates the importance of ∆ in the LfcinB-induced leakage fromsingle E. coli and spheroplasts.
Next, we examined the interactions of LfcinB with GUVs of E. coli polar extract lipids (E. coli-lipid)
containing fluorescent dye, AF647, in their lumens using the single GUV method. LfcinB induced rapid leakageof AF647 stochastically with some damage in the GUV structure, indicating that LfcinB induced local rupture in
the GUV membranes. We obtained the rate constant of local rupture from its quantitative analysis. However,higher LfcinB concentrations were required to induce a significant rate constant of local rupture compared with E.
coli cells and spheroplasts. To identify this reason, we applied ∆ to the GUVs using K+ concentration gradient,and examined the effect of ∆ on LfcinB-induced local rupture. We found that the rate constant of LfcinB-induced
local rupture in GUVs increased greatly with ∆ .
The results in this study show that the ∆ greatly affects the LfcinB-induced rapid leakage from E. coli cells,
their spheroplasts, and E. coli lipid-GUVs, indicating that ∆ plays an important role in the LfcinB-induced local
rupture or pore formation in the lipid bilayers and E. coli plasma membrane. On the basis of these results, wediscuss the mode of action of LfcinB’s antimicrobial activity.
3. References[1] M. M. R. Moghal, M. Z. Islam, F. Hossain, S. K. Saha, M. Yamazaki, Biophys. J. 118, 57-69 (2020)[2] F. Hossain, M.M.R. Moghal, M.Z. Islam, M. Moniruzzaman, M. Yamazaki, J. Biol. Chem. 294,10449-10462 (2019).
[3] M. Moniruzzaman, M.J. Alam, H. Dohra, M. Yamazaki, Biochemistry, 54, 5802-5814 (2015).

List of Publications Related to the Collaboration Research(1) Md. Mizanur Rahman Moghal, Md. Zahidul Islam, Farzana Hossain, Samiron Kumar Saha, and Masahito
Yamazaki, Role of Membrane Potential on Entry of Cell-Penetrating Peptide Transportan 10 into SingleVesicles, Biophys. J. 118, 57-69, 2020
(2) Farzana Hossain, Md. Mizanur Rahman Moghal, Md. Zahidul Islam, Md. Moniruzzaman, and Masahito
Yamazaki, Membrane potential is vital for rapid permeabilization of plasma membranes and lipid bilayers by
the antimicrobial peptide lactoferricin B, J. Biol. Chem., 294, 10449-10462, 2019.

List of Presentations ( Conference, Meeting, etc)
(1) Farzana Hossain, Md. Mizanur Rahman Moghal, Md. Zahidul Islam, Md. Moniruzzaman, MasahitoYamazaki, “Membrane potential is vital for rapid permeabilization of plasma membranes and lipid bilayers by
the antimicrobial peptide lactoferricin B”, American Biophysical Society 64th Annual Meeting, San Diego
Convention Center, San Diego, USA, 15-19, Feb., 2020.
(2) Md. Mizanur Rahman Moghal, Md. Zahidul Islam, Samiron Kumar Saha, Masahito Yamazaki, “Effect ofmembrane potential on entry of cell-penetrating peptide transportan10 into single vesicles”, American
Biophysical Society 64th Annual Meeting, San Diego Convention Center, San Diego, USA, 15-19, Feb., 2020.
(3) Farzana Hossain, Md. Mizanur Rahman Moghal, Md. Zahidul Islam, Md. Moniruzzaman, Masahito
Yamazaki, “Membrane potential is vital for rapid permeabilization of plasma membranes and lipid bilayers by
the antimicrobial peptide lactoferricin B”, The 4th International Symposium on Biomedical Engineering, Act
City Hamamatsu Congress Center, Hamamatsu, 14-15 Nov., 2019.
(4) Farzana Hossain, Md. Mizanur Rahman Moghal, Md. Zahidul Islam, Md. Moniruzzaman, Masahito
Yamazaki, “Membrane potential is vital for rapid permeabilization of plasma membranes and lipid bilayers bythe antimicrobial peptide lactoferricin B”, The 57th Annual Meeting of Biophysical Society of Japan, 2426Sept., 2019, Miyazaki Seagaia Convention center, Miyazaki.
(5) Md. Mizanur Rahman Moghal, Md. Zahidul Islam, Samiron Kumar Saha, Masahito Yamazaki, “Effect ofmembrane potential on the entry of cell-penetrating peptide transportan10 into the lumen of single vesicles
and its mechanism”, The 57th Annual Meeting of Biophysical Society of Japan, 2426 Sept., 2019, Miyazaki
Seagaia Convention center, Miyazaki.

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, 2020 to March 31, 2021), if the collaboration research is continued.
We want to continue and expand this project. As the research theme, we want to use a new title “Effects ofmembrane potential on action of antimicrobial peptides and cell-penetrating peptides”.
Under this project, we have found so far the effects of membrane potential on actions of an AMP, LfcinB and aCPP, CF-TP10 and revealed clearly the parts of their elementary processes using the single GUV method. Thesestudies have been just started. If we use the advantage of the single GUV method, we will get more informationon the elementary processes of actions of AMPs and CPPs on membrane potential and their mechanisms. In theproject at new fiscal year, we will investigate the effect of membrane potential on the action of an AMP, magainin
2 (mainly pore formation in the membrane), and the action of a CPP, LfcinB (4-9) (mainly the entry into cells andvesicles) from various aspects of the interactions of these peptides with single GUVs. We will also examine thephysical properties of GUVs under membrane potential using various methods. The results of the effect ofmembrane potential on the action of these AMPs and CPPs are very helpful to develop new peptides with abactericidal activity and novel vectors for the delivery of drugs, chemicals, oligonucleotides, and proteins intocells for curing various diseases.