First published online September 9, 2005
Journal of Experimental Biology 208, 3609-3622 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01795
Cloning, characterization and expression of escapin, a broadly antimicrobial FAD-containing L-amino acid oxidase from ink of the sea hare Aplysia californica
Hsiuchin Yang1,*,
Paul Micah Johnson1,2,4,*,
Ko-Chun Ko1,
Michiya Kamio1,2,
Markus W. Germann3,
Charles D. Derby1,2,
and
Phang C. Tai1
1 Department of Biology, Georgia State University, Atlanta, GA 30302-4010,
USA
2 Center for Behavioral Neuroscience, Georgia State University, Atlanta, GA
30302-4010, USA
3 Department of Chemistry, Georgia State University, Atlanta, GA 30302-4010,
USA
4 Department of Zoology, University of Washington, Seattle, WA 98195-1800,
USA
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Fig. 1. (A) The ink of Aplysia californica containing escapin, a 60 kDa
protein. (B) SDS-PAGE of raw purple ink (left lane) and ink purified as
described in the Materials and methods to yield escapin, a 60 kDa protein
(right lane). Molecular mass standards are also shown. (C) Analytical gel
filtration shows that escapin elutes as a single peak. Absorbance is expressed
on a relative scale; mAu, milliabsorbance units. Arrow and arrowhead indicate
elution times for escapin and BSA, respectively. The inset shows elution
volumes of molecular mass standards: BSA, 67 kDa; ovalbumen, 43 kDa;
chymotrypsinogen A, 25 kDa, demonstrating that native (non-denatured) escapin
has molecular mass of ca. 60 kDa, similar to that of the denatured escapin, as
shown in B. The photograph in A is courtesy of Genevieve Anderson.
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Fig. 2. Amino acid sequence alignment of escapin and related proteins. Solid and
broken underlines indicate DMB and GG motifs, respectively (see text for
details). *Predicted signal sequence cleavage site at A 18 and D
19. **Predicted glycosylation site at Thr 463. Boxed areas indicate
regions of homology.
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Fig. 3. The yellow pigment associated with escapin is FAD. (A) Aromatic region of
1H NMR spectrum for FAD standard and the yellow pigment from
escapin, showing identical signals. (B) Positive ion ESI-TOF mass spectrum of
the yellow pigment from escapin. See text for explanation of these
signals.
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Fig. 4. LAAO enzyme activity of escapin, and its substrate specificity. 0.6 µg
escapin in 100 µl was incubated at 22°C for 1 min in 2 mmol
l-1 of each L-amino acid and taurine. LAAO activity was
measured by absorbance at 436 nm and normalized to the value for arginine.
Values are mean ± S.E.M.,
N=2. Inset shows a Lineweaver-Burk plot of data for LAAO activity at
different concentrations for lysine and arginine. Values are means ±
S.E.M., N=2. Km
and Vmax values calculated from this experiment are
shown.
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Fig. 5. Escapin has a very long antimicrobial shelf-life at room temperature.
E. coli cells in stationary phase were grown as a lawn on solid
medium and tested in a plate assay for ability of escapin to inhibit growth.
Escapin was tested at concentrations of 3-125 µg ml-1, under two
conditions: `Fresh': freshly isolated; `5 months': after storage for 5 months
at room temperature.
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Fig. 6. Escapin preferentially kills E. coli cells in their growing but
not in their resting state. 2 x108 cells ml-1 in
early-log growth or stationary phase were centrifuged, and the pellet was
resuspended in fresh LB medium. The absorbance reading was adjusted to that of
early-log phase cells (=0.5). Cells were then coincubated in escapin at 0-50
µg ml-1 at 37°C for 2 h. Cultures were then placed on LB
agar plates and incubated at 37°C for 18 h, at which time the number of
viable colonies was counted. Values are means ±
S.E.M., N=3.
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Fig. 7. Escapin can be bacteriostatic or bactericidal. (A) Inhibition of growth.
E. coli cells were incubated in LB medium at 37°C with 10 µg
ml-1 escapin (Esc) or without escapin (Control: buffer added
instead), and with or without 0.4-1.6 mg catalase (Cat). Absorbance at 600 nm
was measured at the indicated incubation times to determine cell density. This
experiment was repeated twice more with similar effects. (B) Bactericidal
effect. E. coli cells were grown in LB medium containing 3
x108 cells ml-1, then incubated in escapin (10
µg ml-1) in LB medium at 37°C (closed squares) or 0°C
(open squares). Values are means ±
S.E.M., N=3.
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Fig. 8. Escapin's bactericidal effect was greatest in the enriched growth medium
and L-lysine. (A,B) E. coli and (C) Staphylococcus
aureus cells in early-log growth phase were cultured in M9-glucose medium
(for E. coli) or LB (for S. aureus), and 4 samples of equal
cell density were resuspended in different growth media in the presence of
escapin (Esc: 50 µg ml-1) or (C) buffer control. Cells were then
incubated at 37°C for 60 min. Ye, 1% yeast extract; Try, 1% Tryptone
Peptone; Lys, 50 mmol l-1 L-lysine; Arg, 50 mmol
l-1 L-arginine; His, 50 mmol l-1
L-histidine; Val, 50 mmol l-1 L-valine; aa,
amino acid mixture containing 20 L-amino acids each at 50 µmol
l-1. In a follow-up experiment, a 10 times higher concentration of
Ye or aa was used, and similar results were observed. Values are means
± S.E.M., N=3. An asterisk
indicates a significant reduction in the number of viable clones
(P<0.05, t-test).
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Fig. 9. Concentration dependence of the effects of amino acids on escapin's
antimicrobial activity. (A) Plate assay of growth inhibition. E. coli
cells were grown at 37°C on a plate in minimal medium, (M9+glucose) in the
presence of amino acids and escapin, or in hydrogen peroxide alone (without
escapin) at concentrations from 3 to 50 mmol l-1. (B) Bactericidal
assay. E. coli cells were grown in M9+glucose containing 3
x108 cells ml-1, then incubated at 37°C with
escapin (60 µg ml-1) in M9+glucose with L-lysine,
L-arginine, or L-tyrosine at 3-50 mmol l-1.
Values are means ± S.E.M.,
N=3.
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Fig. 10. Escapin's bactericidal effect does not require protein synthesis. E.
coli at 2 x108 cells ml-1 were incubated with
or without 50 µg ml-1 chloramphenicol (Cam), an inhibitor of
protein synthesis, in the presence of 50 µg ml-1 escapin (Esc)
or buffer control (C) in LB medium at 37°C. Values are means ±
S.E.M., N=3. An asterisk indicates a
significant reduction in the number of viable clones (P<0.05,
t-test).
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Fig. 11. Recombinant escapin and its antibacterial activity. (A) Western blots
demonstrating successful expression of recombinant escapin. (B) Plate assay of
inhibition of growth of E. coli by wild-type escapin and recombinant
escapin. E. coli cells in stationary phase were grown at 37°C as
a lawn on LB solid medium and tested against wild-type escapin and recombinant
escapin at 7-60 µg ml-1. Concentrations of wild-type and
recombinant escapin were estimated from western blots. WEsc, wild-type escapin
purified from ink; r-proEsc, recombinant escapin, with 18 amino acid signal
peptide, in E. coli cell lysate; rEsc, recombinant in E.
coli cell lysate, escapin without signal sequence (i.e. lacking 18 amino
acids at N terminus); M, molecular mass markers.
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© The Company of Biologists Ltd 2005
