Translocation of the nonlabeled antimicrobial peptide PGLa across lipid bilayers and its entry into vesicle lumens without pore formation

Md Hazrat Ali; Madhabi Lata Shuma; Hideo Dohra; Masahito Yamazaki

doi:10.1016/j.bbamem.2021.183680

Translocation of the nonlabeled antimicrobial peptide PGLa across lipid bilayers and its entry into vesicle lumens without pore formation

Biochim Biophys Acta Biomembr. 2021 Oct 1;1863(10):183680. doi: 10.1016/j.bbamem.2021.183680. Epub 2021 Jun 18.

Authors

Md Hazrat Ali¹, Madhabi Lata Shuma¹, Hideo Dohra², Masahito Yamazaki³

Affiliations

¹ Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan.
² Instrumental Research Support Office, Research Institute of Green Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan; Department of Science, Graduate School of Integrated Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan.
³ Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan; Department of Science, Graduate School of Integrated Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan; Nanomaterials Research Division, Research Institute of Electronics, Shizuoka University, Shizuoka 422-8529, Japan; Department of Physics, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan. Electronic address: yamazaki.masahito@shizuoka.ac.jp.

PMID: 34153295
DOI: 10.1016/j.bbamem.2021.183680

Abstract

Fluorescent-probe-labeled peptides are used to study the interactions of peptides with cells and lipid vesicles but labeling peptides with fluorescent probes can significantly change these interactions. We recently developed a new method to detect the entry of nonlabeled peptides into the lumen of single giant unilamellar vesicles (GUVs). Here we applied this method to examine the interaction of the antimicrobial peptide PGLa with single GUVs to elucidate whether PGLa can enter the GUV lumen without pore formation. First, we examined the interaction of nonlabeled PGLa with single GUVs comprising dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (4/6) whose lumens contain the fluorescent probe AF647 and DOPG/DOPC (8/2)-large unilamellar vesicles encapsulating a high concentration of calcein. After a large lag period from starting the interaction with PGLa, the fluorescence intensity of the GUV lumen due to calcein (I_calcein) increased gradually without leakage of AF647, indicating that PGLa enters the GUV lumen without pore formation in the GUV membrane. The fraction of entry of PGLa increased with increasing PGLa concentration. Simultaneous measurement of the fractional area change of the GUV membrane (δ) and PGLa-induced increase in I_calcein showed that the entry of PGLa occurs only during the second increase in δ, indicating that PGLa enters the lumen during its translocation from the outer leaflet to the inner leaflet. The fraction of entry of PGLa without pore formation increased with increasing membrane tension. Based on these results, we discuss the elementary processes and the mechanism of the entry of PGLa into the GUV lumen.

Keywords: Antimicrobial peptides; Entry; Giant unilamellar vesicle; PGLa; Pore formation; Translocation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Fluorescent Dyes
Lipid Bilayers*
Pore Forming Cytotoxic Proteins / metabolism*
Protein Precursors / metabolism*
Protein Transport
Unilamellar Liposomes

Substances

Fluorescent Dyes
Lipid Bilayers
PGLa prepropeptide
Pore Forming Cytotoxic Proteins
Protein Precursors
Unilamellar Liposomes