Structural factors governing binding of curvature-sensing peptides to bacterial extracellular vesicles covered with hydrophilic polysaccharide chains

Kawano, Kenichi; Kamasaka, Kouhei; Yokoyama, Fumiaki; Kawamoto, Jun; Ogawa, Teiichiro; Kurihara, Tatsuo

doi:10.1016/j.bpc.2023.107039

Cited by 5 publications

(4 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The procedure has been described previously. 12 For single-molecule observation by TIRFM, the Nterminus of NIC4 (1 equiv) was labeled with 5-TAMRA Nsuccinimidyl ester (1.2 equiv) (Tokyo Chemical Industry, Tokyo, Japan) in the presence of DIEA (6 equiv) on Rink amide resin for 38 h at 25 °C. After deprotection and cleavage of the TMR-NIC4 from the resin, the peptide was purified by RP-HPLC.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…The NBD fluorescence intensity was measured using EnVision (PerkinElmer, Yokohama, Japan) 10 min after peptides mixed liposomes. The K d values were determined using the following equations: B c = B max 1 + K d c B c B max = F − F 0 F t − F 0 × 100 = 1 1 + K d c where B c , B max , and c indicate the binding level of the peptides to the vesicles, the maximum level of binding, and the given lipid concentration of the vesicles, respectively. The ratio of ( F – F 0 )/( F t – F 0 ) was calculated and plotted as a function of c , where F 0 and F are the fluorescence intensities of NBD-peptides at each point in the absence and presence of vesicles, respectively, and F t indicates the fluorescence intensities of NBD-nFAAV5 at [L] = 300 μM at pH 6.…”

Section: Methodsmentioning

confidence: 99%

“…For EV detection, we have previously created a curvature-sensing peptide, nFAAV5, , based on the sequence of sorting nexin protein 1 of the Bin/Amphiphysin/Rvs protein family. nFAAV5 selectively binds to bacterial EVs with high sensitivity, even in the presence of EV-secretory cells in cultured media.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Extracellular-Vesicle Catch-and-Release Isolation System Using a Net-Charge Invertible Curvature-Sensing Peptide

Kawano,

Kuzuma,

Yoshio

et al. 2024

Anal. Chem.

Self Cite

View full text Add to dashboard Cite

Extracellular vesicles (EVs) carry various informative components, including signaling proteins, transcriptional regulators, lipids, and nucleic acids. These components are utilized for cell−cell communication between donor and recipient cells. EVs have shown great promise as pharmaceutical-targeting vesicles and have attracted the attention of researchers in the fields of biological and medical science because of their importance as diagnostic and prognostic markers. However, the isolation and purification of EVs from cell-cultured media remain challenging. Ultracentrifugation is the most widely used method, but it requires specialized and expensive equipment. In the present study, we proposed a novel methodology to isolate EVs using a simple and convenient method, i.e., an EV catch-and-release isolation system (EV-CaRiS) using a net-charge invertible curvature-sensing peptide (NIC). Curvature-sensing peptides recognize vesicles by binding to lipid-packing defects on highly curved membranes regardless of the expression levels of biomarkers. NIC was newly designed to reversibly capture and release EVs in a pH-dependent manner. NIC allowed us to achieve reproducible EV isolation from three human cell lines on resin using a batch method and single-particle imaging of EVs containing the ubiquitous exosome markers CD63 and CD81 by total internal reflection fluorescence microscopy (TIRFM). EV-CaRiS was demonstrated as a simple and convenient methodology for EV isolation, and NIC is promising for applications in the single-particle analysis of EVs.

show abstract

Section: ■ Materials and Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Extracellular-Vesicle Catch-and-Release Isolation System Using a Net-Charge Invertible Curvature-Sensing Peptide

Kawano,

Kuzuma,

Yoshio

et al. 2024

Anal. Chem.

Self Cite

View full text Add to dashboard Cite

show abstract

“…In-depth proteomic and biochemical analyses have revealed that bEVs, like their parent bacteria, transport a variety of cargo, including immunostimulatory ligands such as membrane-bound and periplasmic pathogen-associated molecular patterns (PAMPs) [ 54 ], enzymes, toxins [ 55 ], polysaccharides [ 56 ], nucleic acids (DNA and RNA) [ 57 ], LPS, lipoteichoic acid (LTA) [ 58 ], and peptidoglycan [ 59 ]. Both commensal and pathogenic bEVs have been shown to modulate the host immune system via PAMPs that are recognized by host pattern recognition receptors (PRRs) such as TLRs and nucleotide-binding oligomerization domain-containing protein 1 (NOD1) [ 11 ].…”

Section: Microbiome Revelations Spur Interest In Bugs-as-drugs For Ca...mentioning

confidence: 99%

Harnessing Bacterial Extracellular Vesicle Immune Effects for Cancer Therapy

Karaman,

Pathak,

Bayik

et al. 2024

PAI

View full text Add to dashboard Cite

There are a growing number of studies linking the composition of the human microbiome to disease states and treatment responses, especially in the context of cancer. This has raised significant interest in developing microbes and microbial products as cancer immunotherapeutics that mimic or recapitulate the beneficial effects of host-microbe interactions. Bacterial extracellular vesicles (bEVs) are nano-sized, membrane-bound particles secreted by essentially all bacteria species and contain a diverse bioactive cargo of the producing cell. They have a fundamental role in facilitating interactions among cells of the same species, different microbial species, and even with multicellular host organisms in the context of colonization (microbiome) and infection. The interaction of bEVs with the immune system has been studied extensively in the context of infection and suggests that bEV effects depend largely on the producing species. They thus provide functional diversity, while also being nonreplicative, having inherent cell-targeting qualities, and potentially overcoming natural barriers. These characteristics make them highly appealing for development as cancer immunotherapeutics. Both natively secreted and engineered bEVs are now being investigated for their application as immunotherapeutics, vaccines, drug delivery vehicles, and combinations of the above, with promising early results. This suggests that both the intrinsic immunomodulatory properties of bEVs and their ability to be modified could be harnessed for the development of next-generation microbe-inspired therapies. Nonetheless, there remain major outstanding questions regarding how the observed preclinical effectiveness will translate from murine models to primates, and humans in particular. Moreover, research into the pharmacology, toxicology, and mass manufacturing of this potential novel therapeutic platform is still at early stages. In this review, we highlight the breadth of bEV interactions with host cells, focusing on immunologic effects as the main mechanism of action of bEVs currently in preclinical development. We review the literature on ongoing efforts to develop natively secreted and engineered bEVs from a variety of bacterial species for cancer therapy and finally discuss efforts to overcome outstanding challenges that remain for clinical translation.

show abstract

Addressing Heterogeneity in Direct Analysis of Extracellular Vesicles and Their Analogs by Membrane Sensing Peptides as Pan‐Vesicular Affinity Probes

Gori,

Frigerio,

Gagni

et al. 2024

Advanced Science

View full text Add to dashboard Cite

Extracellular vesicles (EVs), crucial mediators of cell‐to‐cell communication, hold significant diagnostic potential due to their ability to concentrate protein biomarkers in bodily fluids. However, challenges in isolating EVs from biological specimens hinder their widespread use. The preferred strategy involves direct analysis, integrating isolation and analysis solutions, with immunoaffinity methods currently dominating. Yet, the heterogeneous nature of EVs poses challenges, as proposed markers may not be as universally present as thought, raising concerns about biomarker screening reliability. This issue extends to EV‐mimics, where conventional methods may lack applicability. Addressing these challenges, the study reports on Membrane Sensing Peptides (MSP) as pan‐vesicular affinity ligands for both EVs and their non‐canonical analogs, streamlining capture and phenotyping through Single Molecule Array (SiMoA). MSP ligands enable direct analysis of circulating EVs, eliminating the need for prior isolation. Demonstrating clinical translation, MSP technology detects an EV‐associated epitope signature in serum and plasma, distinguishing myocardial infarction from stable angina. Additionally, MSP allow analysis of tetraspanin‐lacking Red Blood Cell‐derived EVs, overcoming limitations associated with antibody‐based methods. Overall, the work underlines the value of MSP as complementary tools to antibodies, advancing EV analysis for clinical diagnostics and beyond, and marking the first‐ever peptide‐based application in SiMoA technology.

show abstract

Structural factors governing binding of curvature-sensing peptides to bacterial extracellular vesicles covered with hydrophilic polysaccharide chains

Cited by 5 publications

References 45 publications

Extracellular-Vesicle Catch-and-Release Isolation System Using a Net-Charge Invertible Curvature-Sensing Peptide

Extracellular-Vesicle Catch-and-Release Isolation System Using a Net-Charge Invertible Curvature-Sensing Peptide

Harnessing Bacterial Extracellular Vesicle Immune Effects for Cancer Therapy

Addressing Heterogeneity in Direct Analysis of Extracellular Vesicles and Their Analogs by Membrane Sensing Peptides as Pan‐Vesicular Affinity Probes

Contact Info

Product

Resources

About