Channel-forming activity of the perforin N-terminus and a putative alpha-helical region homologous with complement C9.
Cytolytic lymphocytes are endowed with a pore-forming protein called perforin. Recently, a cytolytic domain was located in the first 34 residues of the perforin N-terminus. It has been proposed that the first 19 residues are composed of a 3-domain structure including a putative amphipathic beta-sheet and that the 19 residues are sufficient for cytolytic activity. This model has now been tested by synthesizing peptides covering different portions of the N-terminus, and testing their ability to lyse lipid vesicles or increase the conductance of lipid bilayers or plasma membranes. It was found that the putative beta-sheet is indispensable for lytic activity and that the first 19 residues of the N-terminus are required for optimal lytic activity but that shorter peptides, containing only 16 residues, can form pores in lipid bilayers and cell membranes. A putative amphipathic alpha-helix from the central portion of perforin, homologous to complement C9, is nonlytic to lipid vesicles, but it can form pores in lipid bilayers. Taken together, these results support the model that the perforin N-terminus is important in initial pore formation and that the putative alpha-helical domain may be involved in subsequent perforin polymerization into large pores.
Persechini
P M
PM
Rockefeller University, New York, New York 10021.
Ojcius
D M
DM
Adeodato
S C
SC
Notaroberto
P C
PC
Daniel
C B
CB
Young
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D003186:Complement C9 / Q000737:chemistry*; D006801:Humans; D008051:Lipid Bilayers; D008562:Membrane Glycoproteins; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D013602:T-Lymphocytes, Cytotoxic / Q000502:physiology*; D014407:Tumor Cells, Cultured
D003186:Complement C9; D008051:Lipid Bilayers; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin