JBC, Vol. 251, Issue 18, 5580-5588, Sep, 1976
Effect of single and double peptide bond scission by trypsin on the structure and activity of staphylococcal enterotoxin C
L. Spero, B. Y. Griffin, J. L. Middlebrook and J. F. Metzger
Two peptide bonds of staphylococcal enterotoxin C, were hydrolyzed
concurrently at quite different rates during limited digestion with
trypsin. A Lys-Val at about position 92 in the disulfide loop was the first
bond cleaved, followed by a Lys-Asx at about position 57 on the
NH2-terminal side of the loop. Preparations of singly cleaved material
(enterotoxin C1-T1) contained about 93% of the cleaved protein and 7%
unreacted enterotoxin. Preparations of the doubly cleaved material
(enterotoxin C1-T2) consisted of 98% enterotoxin C1-T2 and 2% enterotoxin
C1-T1. In the absence of denaturant, enterotoxin C1-T2 behaved as a single
particle. It gave a single peak on Sephadex G-75 with a sedimentation
coefficient of 2.85 S and a molecular weight of 29,100 by sedimentation
equilibrium. Circular dichroic spectra indicated only minor conformational
differences between enterotoxins C1-T2 and C1. However conformational
stability was significantly affected with the unfolding of enterotoxin
C1-T2 in 4 M guanidine hydrochloride proceeding at about twice the rate of
native enterotoxin. Enterotoxin C1-T2 was separated into 6,500 and 22,000
molecular weight polypeptides by gel filtration on Sepharose 6B in 6 M
guanidine hydrochloride. Complementation (as measured by CD spectra,
serologic activity and mitogenicity) of the two polypeptides was readily
achieved from solution in 6 M guanidine hydrochloride by dialysis against
phosphate buffer. The 22,000 molecular weight polypeptide was further
separated into two peptides (Mr = 4,000 and 19,000 after alkylation of the
reduced disulfide bridge. Summation of the amino acid composition of the
constituent peptides of enterotoxin C1-T2 agreed well with the composition
of enterotoxin C1. A comparison of the 6,500 and 4,000 molecular weight
polypeptides from enterotoxin C1-T2 with structurally equivalent segments
of enterotoxin B suggested structural homology between the two antigenic
variants. Enterotoxins C1, C1-T1, and C1-T2 gave reactions of complete
identity in Ouchterlony immunodiffusion and were indistinguishable in the
quantitative precipitin reaction. Enterotoxins C1-T1 and C1-T2 were highly
mitogenic but were slightly less potent than the native enterotoxin.
Enterotoxin C1-T2 had equivalent emetic activity to enterotoxin C1 in
rhesus monkeys. It is suggested that the exceptional lability to limited
enzymic hydrolysis exemplified by enterotoxin C1 is associated with beta
turn structures at protein surfaces.
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