Disrupting the entry barrier and attacking brain tumors: the role of the Neisseria H.8 epitope and the Laz protein.
Azurin is a periplasmic 128 amino acid protein in Pseudomonas aeruginosa, termed Paz, which has been shown to enter preferentially and induce apoptosis in cancer cells such as human melanoma or breast cancer. Its effectiveness against brain tumors such as glioblastomas has not been studied. The meningitis-causing bacterium Neisseria meningitidis also harbors an azurin-like protein. Unlike all other known azurins, Neisserial azurin, termed Laz, is surface-exposed and has in its N-terminal region a 39 amino acid epitope called H.8. Upstream of this H.8 moiety is a lipobox that results in the truncation of the protein at the N-terminal cysteine residue with modification by a lipid group. No function of Laz is known. We demonstrate that while Paz is deficient in entering glioblastoma cells and exhibits low cytotoxicity, Laz is much more proficient in entering glioblastoma cells and shows a higher level of cytotoxicity. When the Neisserial H.8 moiety containing the lipobox is fused in frame with Paz either in its N-terminal (H.8-Paz) or in its C-terminal (Paz-H.8), both had high cytotoxicity for glioblastoma cells and a higher level of internalization. When expressed in E. coli, H.8-Paz was much more exposed on the surface than Paz-H.8. The replacement of the Laz N-terminal cysteine residue involved in acylation with an alanine residue abolished the surface display, but had no effect on cytotoxicity or entry in glioblastoma cells, suggesting a role of the H.8 moiety, but not its lipidation, in disrupting the entry barrier in brain tumor cells.
Hong
Chang Soo
CS
Department of Microbiology, University of Illinois College of Medicine, Chicago, Illinois 60612, USA.
Yamada
Tohru
T
Hashimoto
Wataru
W
Fialho
Arsenio M
AM
Das Gupta
Tapas K
TK
Chakrabarty
Ananda M
AM
D001400:Azurin / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001932:Brain Neoplasms / Q000473:pathology*; D016923:Cell Death; D003001:Cloning, Molecular; D000939:Epitopes / Q000276:immunology*; D004926:Escherichia coli; D015870:Gene Expression; D005909:Glioblastoma / Q000473:pathology; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D009345:Neisseria meningitidis / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D033903:Periplasmic Proteins / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes; C055700:H.8 protein (Neisseria); D008055:Lipids; D008565:Membrane Proteins; D050505:Mutant Proteins; D010455:Peptides; D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins; D001400:Azurin