Limits...
Mutation of arginine residues to avoid non-specific cellular uptakes for hepatitis B virus core particles.

Bin Mohamed Suffian IF, Nishimura Y, Morita K, Nakamura-Tsuruta S, Al-Jamal KT, Ishii J, Ogino C, Kondo A - J Nanobiotechnology (2015)

Bottom Line: However, since the arginine-rich domain that is located in the C-terminal region of the HBc monomer binds to the heparan sulphate proteoglycan on the cell surface due to its positive charge, HBc particles are introduced non-specifically into a wide range of cells.The mutated HBc particles in which R154 was replaced with glycine (G) residue (R154G) showed a drastic decrease in the ability to bind to the heparan sulphate proteoglycan and to avoid non-specific cellular uptake by several types of cancer cells.Because this mutant particle retains most of its C-terminal arginine-rich residues, it would be useful in the targeting of specificity-altered HBc particles in the delivery of nucleic acids.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan. izzat_fahimuddin.bin_mohamed_suffian@kcl.ac.uk.

ABSTRACT

Background: The hepatitis B virus core (HBc) particle is known as a promising new carrier for the delivery of drugs and nucleic acids. However, since the arginine-rich domain that is located in the C-terminal region of the HBc monomer binds to the heparan sulphate proteoglycan on the cell surface due to its positive charge, HBc particles are introduced non-specifically into a wide range of cells. To avoid non-specific cellular uptake with the intent to control the ability of cell targeting, we individually replaced the respective arginine (R) residues of the arginine-rich domain located in amino acid positions 150-159 in glycine (G) residues.

Results: The mutated HBc particles in which R154 was replaced with glycine (G) residue (R154G) showed a drastic decrease in the ability to bind to the heparan sulphate proteoglycan and to avoid non-specific cellular uptake by several types of cancer cells.

Conclusions: Because this mutant particle retains most of its C-terminal arginine-rich residues, it would be useful in the targeting of specificity-altered HBc particles in the delivery of nucleic acids.

No MeSH data available.


Related in: MedlinePlus

Analyses of purified HBc particles. (A) Atomic force microscope images of HBc-WT particle (left) and HBc-R154G particle (right). Scale bar: 50 nm. (B) Scanning electron microscope images of HBc-WT particle (left) and HBc-R154G particle (right). Scale bar: 100 nm. (C) Size distribution using DLS analysis. The average size of the HBc-R154G particle was 28.7 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4334417&req=5

Fig1: Analyses of purified HBc particles. (A) Atomic force microscope images of HBc-WT particle (left) and HBc-R154G particle (right). Scale bar: 50 nm. (B) Scanning electron microscope images of HBc-WT particle (left) and HBc-R154G particle (right). Scale bar: 100 nm. (C) Size distribution using DLS analysis. The average size of the HBc-R154G particle was 28.7 nm.

Mentions: Wild-type and singly mutated (respectively replacing R with G among aa 150–159 in the arginine-rich domain) HBc monomers (Additional file 1) were expressed in E. coli, and the proteins were extracted with lysis buffer as well as with dissociation buffer. HBc dimers were then purified by affinity chromatography. It has been proved that the C-terminal histidine-tag on HBc monomer had no significant adverse effect on the particle formation and the cell binding [11]. The expression of each HBc monomer (21 kDa) was confirmed by western blot analysis using anti-His6 antibody (data not shown). The particle formation was confirmed by atomic force microscopy (AFM), scanning electron microscope (SEM) and dynamic light scattering (DLS) (Figure 1). These results indicated that point-mutations replacing R with G in the arginine-rich domain (150–159 aa) did not affect the self-assembly capacity of the HBc dimers.Figure 1


Mutation of arginine residues to avoid non-specific cellular uptakes for hepatitis B virus core particles.

Bin Mohamed Suffian IF, Nishimura Y, Morita K, Nakamura-Tsuruta S, Al-Jamal KT, Ishii J, Ogino C, Kondo A - J Nanobiotechnology (2015)

Analyses of purified HBc particles. (A) Atomic force microscope images of HBc-WT particle (left) and HBc-R154G particle (right). Scale bar: 50 nm. (B) Scanning electron microscope images of HBc-WT particle (left) and HBc-R154G particle (right). Scale bar: 100 nm. (C) Size distribution using DLS analysis. The average size of the HBc-R154G particle was 28.7 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4334417&req=5

Fig1: Analyses of purified HBc particles. (A) Atomic force microscope images of HBc-WT particle (left) and HBc-R154G particle (right). Scale bar: 50 nm. (B) Scanning electron microscope images of HBc-WT particle (left) and HBc-R154G particle (right). Scale bar: 100 nm. (C) Size distribution using DLS analysis. The average size of the HBc-R154G particle was 28.7 nm.
Mentions: Wild-type and singly mutated (respectively replacing R with G among aa 150–159 in the arginine-rich domain) HBc monomers (Additional file 1) were expressed in E. coli, and the proteins were extracted with lysis buffer as well as with dissociation buffer. HBc dimers were then purified by affinity chromatography. It has been proved that the C-terminal histidine-tag on HBc monomer had no significant adverse effect on the particle formation and the cell binding [11]. The expression of each HBc monomer (21 kDa) was confirmed by western blot analysis using anti-His6 antibody (data not shown). The particle formation was confirmed by atomic force microscopy (AFM), scanning electron microscope (SEM) and dynamic light scattering (DLS) (Figure 1). These results indicated that point-mutations replacing R with G in the arginine-rich domain (150–159 aa) did not affect the self-assembly capacity of the HBc dimers.Figure 1

Bottom Line: However, since the arginine-rich domain that is located in the C-terminal region of the HBc monomer binds to the heparan sulphate proteoglycan on the cell surface due to its positive charge, HBc particles are introduced non-specifically into a wide range of cells.The mutated HBc particles in which R154 was replaced with glycine (G) residue (R154G) showed a drastic decrease in the ability to bind to the heparan sulphate proteoglycan and to avoid non-specific cellular uptake by several types of cancer cells.Because this mutant particle retains most of its C-terminal arginine-rich residues, it would be useful in the targeting of specificity-altered HBc particles in the delivery of nucleic acids.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan. izzat_fahimuddin.bin_mohamed_suffian@kcl.ac.uk.

ABSTRACT

Background: The hepatitis B virus core (HBc) particle is known as a promising new carrier for the delivery of drugs and nucleic acids. However, since the arginine-rich domain that is located in the C-terminal region of the HBc monomer binds to the heparan sulphate proteoglycan on the cell surface due to its positive charge, HBc particles are introduced non-specifically into a wide range of cells. To avoid non-specific cellular uptake with the intent to control the ability of cell targeting, we individually replaced the respective arginine (R) residues of the arginine-rich domain located in amino acid positions 150-159 in glycine (G) residues.

Results: The mutated HBc particles in which R154 was replaced with glycine (G) residue (R154G) showed a drastic decrease in the ability to bind to the heparan sulphate proteoglycan and to avoid non-specific cellular uptake by several types of cancer cells.

Conclusions: Because this mutant particle retains most of its C-terminal arginine-rich residues, it would be useful in the targeting of specificity-altered HBc particles in the delivery of nucleic acids.

No MeSH data available.


Related in: MedlinePlus