A new cytolysin from the sea anemone, Heteractis magnifica: isolation, cDNA cloning and functional expression.
We purified a new cytolysin (HMgIII) from the sea anemone, Heteractis magnifica. HMgIII, which has a molecular mass of approximately 19 kDa, functions as both a cytolysin and a hemolysin. The full-length HMg III cDNA was obtained by reverse transcriptase-polymerase chain reaction, using primers designed from its N-terminal amino acid sequence and an internal conserved region of two other sea anemone cytolysins: equinatoxin II (EqT II) and cytolysin III. The cDNA contained an open reading frame of 633 bp, which encodes a protein of 211 amino acids. The nascent HMg III protein contained a prepropeptide of 34 amino acids, which includes a signal peptide of 19 amino acids. The mature HMg III has a predicted molecular mass of 19 kDa and a pI of 9.1, and shares 91%, 89%, 65% and 63% amino acid sequence similarity with cytolysin III, cytolysin ST I, tenebrosin-C and equinatoxin (EqT II), respectively. The predicted secondary structure of the mature HMg III comprises 16% alpha-helix, 23% extended strand and 60% random coils. The characteristic amphiphilic alpha-helix of cytolysins is located at the N-terminus of the processed HMg III. Recombinant HMg III (rHMg III) was expressed in Escherichia coli as a fusion protein containing a 6xHisTag at the N-terminus. The hemolytic and cytotoxic activities of the purified rHMg III were comparable to those of the native HMg III. The hemolytic activities of both proteins were similarly potentiated with 8-anilino-1-naphthalenesulfonate (ANS). Increasing the length of the peptide tag on the N-terminal of rHMg III correlated with decreasing hemolytic activity, thus confirming the importance of the N-terminal amphiphilic alpha-helix for its cytolytic activity.
Wang
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Department of Biochemistry, Faculty of Medicine, National University of Singapore, 10 Kent Ridge Crescent, Singapore, Singapore.
Chua
K L
KL
Khoo
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HE
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003064:Cnidarian Venoms / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003603:Cytotoxins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006460:Hemolysin Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012615:Sea Anemones / Q000235:genetics / Q000378:metabolism*
D003064:Cnidarian Venoms; D003603:Cytotoxins; D018076:DNA, Complementary; D006460:Hemolysin Proteins; D010455:Peptides; C407990:magnificalysin III