(Received for publication, October 10, 1995; and in revised form, February 7, 1996 )
From the
The cytotoxic and antibacterial polypeptide NK-lysin has a molecular mass of approximately 9 kDa and contains three disulfide bonds. The activity was highly dependent on intact disulfides, because the bactericidal effect on Escherichia coli and the cytolytic effect on human 3B6 lymphocytes was inhibited when NK-lysin was treated with dithiothreitol prior to incubation with the cells. NK-lysin was a direct substrate for human or calf thymus thioredoxin reductase and preincubation of the peptide with mammalian thioredoxin reductase, and NADPH abolished its antibacterial and cytolytic activities. The addition of human thioredoxin further enhanced the inhibitory effect of thioredoxin reductase and NADPH. In contrast, E. coli thioredoxin reductase showed no direct disulfide reductase activity with NK-lysin in agreement with previous data showing large differences in structure and substrate specificity between the mammalian and E. coli enzymes. NK-lysin is the first identified macromolecular disulfide substrate for human thioredoxin reductase apart from human thioredoxin. When 3B6 cells were incubated with NADPH, thioredoxin, and thioredoxin reductase prior to addition of NK-lysin, cytotoxicity was markedly reduced. These data suggest that thioredoxin reductase inactivates NK-lysin and provides a mechanism by which the cytotoxic activity of NK-lysin is regulated.
Peptide antibiotics are considered to be an important part of
the innate immunity of animals(1, 2, 3) . A
large number of antimicrobial peptides have been identified that are
used under different physiological conditions. Many animals have
circulating phagocytic cells containing antibiotic peptides stored in
granula that participate in the destruction of engulfed
bacteria(4) . The mucosal surfaces of the intestine, lung, and
tongue have epithelial cells that produce antibiotic peptides (5, 6, 7, 8, 9) that are
likely to act extracellularly and locally protect the host from
microbial invasion. In insects, antibiotic peptides are released in the
hemolymph and are potent host defense effector molecules. Animal
peptides that have evolved to kill engulfed microbes inside phagocytic
vacuoles may be cytotoxic also to the host as exemplified by mammalian
defensin and bactenecin(10, 11) . On the other hand
peptide antibiotics that are delivered to the circulating system like
insect cecropins (12) and insect defensins (13) are not
harmful to the producing organism. Although most of these peptides were
discovered on the basis of the antimicrobial potency, other properties
such as promotion of wound healing(14, 15) ,
stimulation of monocyte chemotaxis(16) , and effects on
intracellular signal transduction pathways (17, 18) have been reported. Recently, a novel
antimicrobial peptide, NK-lysin, was isolated and shown to be present
in CD2, CD4
, and CD8
cells, suggesting a function as effector peptide in
T-lymphocytes(19) . NK-lysin is a cyst(e)ine-containing peptide
that in addition to its antimicrobial activity is also cytotoxic to
certain tumor cells but does not lyse red blood cells. The six
cysteines in NK-lysin form three disulfide bonds that are well
characterized(19) .
Thioredoxin is a small protein (12 kDa)
present in all prokaryotic and eukaryotic cells(20) . In its
oxidized form (Trx-S), (
)it contains a redox
active disulfide with the sequence Cys-Gly-Pro-Cys localized in a
protrusion of the known three-dimensional
structure(21, 22) . Trx-S
is reduced by
the flavoenzyme thioredoxin reductase (20) and NADPH (the
thioredoxin system), and Trx-(SH)
operates as a general
protein disulfide reductase ( and ):
The flow of electrons in catalysis by thioredoxin reductase is
from NADPH to FAD and then to the active site disulfide, which forms a
dithiol that reduces the disulfide of oxidized
thioredoxin(23) . Escherichia coli TR has a molecular
mass of 70 000, with two identical subunits and known three-dimensional
structure(24) . Mammalian thioredoxin reductases are larger
proteins (M 116,000) also with two identical
subunits but a wider substrate specificity as compared with the E.
coli enzyme(25, 26) .
The thioredoxin system
has been implicated in a large variety of physiological
processes(20) . It serves, for example, as a hydrogen donor for
ribonucleotide reductase (27) and a regulator of enzymes by
thiol redox control(28) . Trx also modulates the activity of
transcription factors such as NF-B(29) ,
AP-1(30) , and steroid receptors(31, 32) .
More recently several cytokines or cytokine-like factors such as adult
T cell leukemia-derived factor(33) ,
3B6-interleukin-1(34) , T-hybridoma-derived (MP-6) B cell
stimulatory factor(35) , and early pregnancy factor (36) have been reported to be identical to thioredoxin. Trx is
thus secreted by activated human lymphocytes(37) . These
results show that Trx has cytokine-like extracellular activities.
In contrast to thioredoxin, which can act as a general disulfide oxidoreductase, thioredoxin reductase has only few known substrates. Beside small molecules like vitamin K, alloxan, and selenite(26, 38, 39) , only Trx and proteins that contain thioredoxin domains (protein disulfide isomerase, CaBP1, and CaBP2) are known substrates for thioredoxin reductase(40, 41) . In addition to being located in the cytosol, thioredoxin reductase has been reported to be present on the plasma membranes as well as on the surface of some cancer cells and could be responsible for the reductive activation of extracellular Trx (42) . Thus the Trx system may play an important role in modulating the activity of extracellularly circulating peptides and proteins. The Trx system has previously been shown to prevent toxic effects of venom neurotoxin (43) and cytolysis by tumor necrosis factor(44) , but the mechanism of protection is not clear.
In this report, we show that the antibacterial and cytotoxic activities of NK-lysin are dependent on intact disulfide bonds. NK-lysin kills human 3B6 lymphocytes, and this lytic effect may be inhibited by pretreatment of the peptide with thioredoxin reductase or the complete Trx system.
Recombinant human thioredoxin was prepared as described by
Ren et al.(45) ; E. coli thioredoxin and E. coli TR were prepared as described(20) . Mammalian
thioredoxin reductase was prepared from human placenta or calf thymus
essentially as described by Luthman and Holmgren(26) . Yeast
glutaredoxin reductase was from Sigma. RPMI medium was from Flow
Laboratories (Ayshire, UK), and fetal calf serum, glutamine, and
antibiotics were from Life Technologies, Inc. The cyto96 cytotoxic kit
was from Promega. The columns used for HPLC analyses were: C18 column
(Vydac 4.6 250 mm, The Separation Group, Hesperia, CA) and C8
column (Sephasil 2.1
10 mm, Pharmacia Biotech Inc.). All other
chemicals were from Sigma.
Figure 1: Inhibition of NK-lysin anti-E. coli activity by DTT. NKL (20 µM) was incubated with different concentrations of DTT for 20 min at 37 °C prior to incubation with bacteria. The results are shown as percentages of the bactericidal effect obtained with NKL incubated under the same conditions but in the absence of DTT.
Figure 2:
Inhibition of NK-lysin anti-E. coli activity by TR and the Trx system. A, NKL (20
µM) was incubated with different concentrations of TR and
0.25 mM NADPH for 20 min prior to incubation with bacteria.
, E. coli TR;
, human placenta TR;
, calf
thymus TR. B, NKL (20 µM) was incubated with
different concentrations of human Trx in the presence of 50 nME. coli (
) or human placenta TR (
) and 0.25
mM NADPH prior to incubation with bacteria. The results are
shown as percentages of the bactericidal effect obtained with
nontreated NKL.
In contrast to the mammalian enzyme, E. coli TR does not inactivate NK-lysin. Even higher concentrations of E. coli TR (up to 0.5 µM) did not show any effect. When NK-lysin was incubated with reduced human Trx or E. coli Trx alone at concentrations up to 5 µM, 100% of its anti-E. coli activity was preserved.
We then tested if the complete thioredoxin system was a more efficient inactivating system. The concentrations of the HP-TR and the E. coli TR were kept constant at 50 nM while increasing concentrations of the homologous thioredoxins were used. As shown in Fig. 2B, human Trx dose-dependently enhanced the inactivation of NK-lysin anti-E. coli activity. E. coli Trx also inactivated NK-lysin when combined with E. coli TR but not as efficiently as the mammalian homologue. NK-lysin incubation with the Trx system was also performed in the presence of 0.1 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, and 1 µM pepstatin with no change in the results (data not shown), demonstrating that the reduction in activity is not caused by enzymatic degradation.
Figure 3: Time and concentration dependence of NK-lysin cell cytotoxicity. 3B6 cells (20 000/well) in 115 µl of RPMI 1640 medium plus 2% fetal calf serum were incubated with different concentrations of NKL, and the percentage of lysis was determined by the lactate dehydrogenase release method.
When NK-lysin was preincubated with 2.5 mM DTT or 220 nM HP-TR or with the complete thioredoxin system (2.8 µM human Trx/60 nM HP-TR) and 0.25 mM NADPH prior to addition to 3B6 cells, more than 95% of the lytic effect was blocked (Fig. 4). If NK-lysin was incubated with 5 µM human Trx alone, there was no inactivating effect.
Figure 4: Inhibition of NK-lysin cytotoxicity against 3B6 cells. NKL (20 µM) was incubated with 2.5 mM DTT, 5 µM human Trx, 220 nM HP-TR, or 60 nM HP-TR/2.8 µM human Trx and 0.25 mM NADPH for 20 min, and aliquots were added to 3B6 cells (20,000/well) to give a final concentration of 23 µg/ml NKL. The incubation was stopped after 4 h, and the percentage of lysis was determined by the lactate dehydrogenase release method.
Likewise, if 3B6 cells were incubated for 15 min with a fixed concentration of TR (25 nM) and increasing concentrations of human Trx prior to the addition of NK-lysin, there was a dose-dependent inhibition of the cytolytic activity (Fig. 5). This protective effect was not seen when the cells were incubated with human Trx alone. At present, the limited amount of TR available did not permit experiments with preincubation of cells with higher concentrations of TR only.
Figure 5: Pretreatment of 3B6 cells with Trx system reduces NK-lysin cytotoxicity. 3B6 cells (20,000/well) were preincubated with human Trx or human Trx/HP-TR (25 nM) and 0.25 mM NADPH for 15 min prior to the addition of NKL (final concentration, 23 µg/ml). The incubation was stopped after 4 h, and the percentage of lysis was determined by the lactate dehydrogenase release method. The results are given as percentages of maximal lysis in the absence of Trx and TR.
Figure 6: Schematic drawing of NK-lysin and the resulting chromatogram of TR-reduced and 4-vinyl pyridine-treated NK-lysin. A, schematic drawing of the S-S bonds in NK-lysin. B, NK-lysin was reduced by 100 nM TR and 0.25 mM NADPH, and the SH group of the cysteines was reacted with 4-vinyl pyridine and analyzed by reverse-phase HPLC on a C18 column.
This analysis showed that in peak a all cysteines were
alkylated, and in peak b all Cys and Cys
were
alkylated, whereas no alkylation was found in Cys
and
Cys
. This indicates that the
Cys
-Cys
disulfide bond is more amenable
to reduction by TR than Cys
-Cys
or
Cys
-Cys
. NK-lysin not treated with TR
and incubated in parallel with 4-vinyl pyridine did not change its
elution mobility and gave no change in absorption at 280 nm, suggesting
no interference of 4-vinyl pyridine with the disulfide bonds.
Many effector molecules contribute to host immunity, and one group that may play an important role is the group of antibiotic peptides. At present, a large number of peptide sequences (or cDNA sequences coding for peptides) with antibiotic activity are known(3) . On a chemical basis, these peptides may be divided into groups based on structural similarities, e.g. (i) linear peptides devoid of cysteines, often forming amphipatic helices, (ii) linear peptides with a high proportion of certain residues like proline and arginine, (iii) loop-forming peptides with one disulfide bond, and (iv) peptides with one or more internal disulfide bonds.
A
78-residue peptide, NK-lysin, belonging to group iv was recently
purified from pig intestine and shown to have a disulfide pattern
different from other members in this group like the - and
-defensins(19) . Immunostaining with a polyclonal antibody
against NKL co-stained with monoclonal markers for
CD2
, CD4
, or CD8
,
suggesting its presence in cytolytic T-lymphocytes (often localized in
granules)(19) . Most, but not all, peptide antibiotics are
believed to interact with the lipid bilayer and express their activity
through membrane destruction. Defensins are postulated to form
-sheet structures that dimerize (50) and interact with
the lipid bilayer that may eventually form multimeric
pores(51) . The structure and mode of action is not known for
NKL, but the structural similarities of NKL to the saposin-like family
of peptides suggest that membrane interaction is likely to occur (52) . Cysteine-rich peptides like snake toxins and amoebapore
A are usually stable on exposure to acid or high temperature but loose
all activity upon disulfide reduction(49, 53) . On the
other hand reduction of disulfide bonds in guinea pig defensin does not
reduce its biological activities(54) . NK-lysin also resists
100 °C for 10 min but is inactivated by DTT. Thus it was of
interest to investigate if the thioredoxin system could modulate the
activity of NK-lysin because NK-lysin is potentially an effector
peptide of pig cytotoxic T lymphocytes. Trx has been shown to have many
extracellular activities, some playing a role as mediators in the
immune system or as regulators of cell growth (35, 48, 55) in an autocrine manner. Trx may
also prevent venom neurotoxin (43) and tumor necrosis factor (44) cytotoxic effects. In this report we show that thioredoxin
reductase alone or the complete Trx system can inhibit the
antibacterial and cytotoxic effects of NK-lysin.
The Trx system has
a broad substrate specificity and can reduce disulfides in a number of
proteins and peptides (56) and also inhibits the anti-E.
coli activity of NK-lysin, most probably by reduction of
disulfides. Thioredoxin reductase has a narrower substrate specificity
than Trx, and so far only a limited number of substrates, all proteins
containing thioredoxin domains, are
identified(40, 41) . Interestingly, NK-lysin is
inactivated by mammalian TR in the absence of Trx and represents a new
substrate for TR. E. coli TR is more substrate-specific than
mammalian TR (56) and does not affect the activity of NKL (this
might be a positive factor for the anti-E. coli activity of
NKL). Another protein with thiol-disulfide reductase activity present
in mammalian cells is glutaredoxin, which is reduced by glutathione and
glutathione reductase, the glutaredoxin system(57) . Both human
and E. coli glutaredoxin efficiently inactivate NK-lysin when
combined with yeast glutaredoxin reductase, glutathione, and NADPH. ()The complete system was needed, and no single component
showed any effect.
NK-lysin is cytolytic for the mouse tumor cell
line YAC-1 (19) and also kills virus-infected B-lymphocytes
(3B6) as well as other cells (1G8 and U937) at similar half-maximal
concentrations (2-5 µM). In contrast with
defensin(58) , the lysis proceeds rapidly and is clearly
detected within 10 min compared with a lag time of approximately 4 h
for defensin. Also, NKL lytic activity is not efficiently quenched by
serum protein ( (19) and this paper) as is the case with
defensin(58) . At present, red blood cells are the only example
of mammalian cells not lysed by NKL. The reason for this is not clear.
TR appears not to specifically reduce a particular disulfide bond in
NKL because all cysteines could be modified by 4-vinyl pyridine.
However Cys-Cys
was preferentially
reduced because no Cys
-Cys
bond could
be found in the partially reduced NK-lysin.
Thioredoxin reductase
has been reported to be associated with plasma membranes and in some
cells to be present on the surface. Thus, the amount of thioredoxin
reductase in different cell types may influence the sensitivity of
certain cells to NK-lysin. Thioredoxin reductase as well as thioredoxin
vary during the cell cycle, reaching a maximum at the S phase and are
decreased when cells enter G/M. (
)These
variations during the cell cycle, and the heterogeneity of the cell
population may explain why NKL lysis proceeds with a fast initial rate (Fig. 3). Immunohistochemical localization showed that TR and
Trx and colocalized in most cells. Nevertheless, there are cells, e.g. spermatocytes and Sertoli cells, that are positive for
thioredoxin reductase but negative for thioredoxin(59) . These
observations suggest that thioredoxin reductase has more functions than
reducing thioredoxin.
Thus, we describe a novel mechanism by which the cytotoxic effect of NK-lysin can be inhibited. The direct action of thioredoxin reductase alone or of the thioredoxin system can inactivate NK-lysin and could play a role in modulating NK-lysin cytotoxicity. However, other mechanisms such as dilution or quenching by protein absorption may also reduce the toxic effect of NK-lysin.