Structural and functional characterization of a recombinant sticholysin I (rSt I) from the sea anemone Stichodactyla helianthus.
Sticholysins I and II (Sts I and II) are two potent cytolysins from the sea anemone Stichodactyla helianthus. These isoforms present 13 substitutions, with three non-conservative located at the N-terminus. St II is considerably more hemolytic than St I in human red blood cells, a result explained by the smaller number of negatively charged groups present at St II's N-terminus. In the present work, we have obtained a recombinant St I (rSt I), differing from the wild type in a single amino acid residue (E16Q). This pseudo-wild type is structurally similar to St I and shows a similar capacity to interact with and form pores in model membranes. This was assessed by the intrinsic fluorescence increase in the presence of liposomes, their adsorption to bilayers (measured by SPR), their concentration at the air-water interface, their interaction with lipid monolayers and their capacity to promote the release of carboxyfluorescein entrapped in liposomes. In spite of these similarities, rSt I presents a larger hemolytic activity in human red blood cells than St I, being intermediate in activity between Sts I and II. The results obtained in the present work emphasize that even the change of one single E by Q at the N-terminal segment may modify the toxin HA and show that this functional property is the most sensitive to subtle changes in the protein primary structure.
Pazos
F
F
Center for Protein Studies, Faculty of Biology, University of Havana, Habana, Cuba.
Valle
A
A
Martínez
D
D
Ramírez
A
A
Calderón
L
L
Pupo
A
A
Tejuca
M
M
Morera
V
V
Campos
J
J
Fando
R
R
Dyszy
F
F
Schreier
S
S
Horjales
E
E
Alvarez
C
C
Lanio
M E
ME
Lissi
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D004912:Erythrocytes / Q000187:drug effects; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009930:Organic Chemicals / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010539:Permeability / Q000187:drug effects; D052899:Pore Forming Cytotoxic Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012615:Sea Anemones / Q000737:chemistry*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D020349:Surface Plasmon Resonance; D013500:Surface Tension / Q000187:drug effects
D008051:Lipid Bilayers; D008081:Liposomes; D009930:Organic Chemicals; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins; C121348:stycholysin I