Download significance of the putative upstream polybasic nuclear localisation

SIGNIFICANCE OF THE PUTATIVE UPSTREAM POLYBASIC NUCLEAR
LOCALISATION SEQUENCE FOR THE BIOLOGICAL ACTIVITY OF
HUMAN INTERFERON-GAMMA
Stefan Petrov*, Maya Boyanova*, Alfredo Berzal-Herranz+, Andrey Karshikoff#, Genoveva
Nacheva* and Ivan Ivanov*
* Institute of Molecular Biology, Bulgarian Academy of Sciences, “Acad. G. Bonchev” Str., 21,
1113 Sofia, Bulgaria; + Instituto de Parasitologia y Biomedicina "Lopez-Neyra", CSIC, Avda. del
Conocimiento s/n Armilla, 18100 Granada, Spain; # Department of Biosciences at Novum,
Karolinska Institutet, Huddinge, S-14157 Huddinge, Sweden
Correspondence to: Stefan Petrov, e-mail: [email protected]
ABSTRACT
Interferon-gamma (IFNγ) accomplishes its multiple biological effects by activating the STAT transcription
factors, which are translocated to the nucleus through a specific nuclear localization sequence(s) (NLS)
located in the IFNγ molecule. Two putative NLS have been pointed out in the human interferon gamma
(hIFN): an upstream located in helix E (residues 83-89) and a downstream located at the C-terminal
unstructured region (residues 124-132). To investigate the significance of the putative upstream NLS for the
biological activity of hIFN we have introduced a point mutation in the hIFN gene to disturb the polybasic
sequence typical for the conventional NLS. In the new gene a gln codon was substituted for the Lys88 codon
and the mutated gene was cloned and expressed in E. coli LE392. This mutation led to a 1000-fold decrease
in both hIFN antiviral and antiproliferative activities. When co-incubated with the wild-type hIFN (standard),
the mutant hIFN competed for the cellular receptors that led to a 30% inhibition of the standard activity. This
indicates that the mutation does not interfere with the interaction of the protein to the receptor but probably
affects the intracellular signal transduction pathway. To avoid any putative compensatory effect in the function
of the upstream NLS caused by the downstream C-terminal NLS, 21 C-terminal codons were deleted from the
mutant hIFN gene. The latter resulted in only 10-fold additional decrease in biological activity and 50%
inhibition of the standard activity in the competition assay. Our data indicate that the upstream NLS is
endowed with a greater functional significance for the hIFNmediated signal transduction rather than the
downstream NLS.
Keywords: human interferon gamma; nuclear localization sequence; biological activity
Introduction
Human interferon gamma (hIFNγ) is a
cytokine secreted in minute amounts by the T
lymphocytes upon induction with viruses,
bacteria, fungal antigens, immunotoxins,
mitogens, etc. It is endowed with multiple
biological activities and plays a key role in the
modulation of the immune response (21).
hIFN is a 17 kDa single chain protein
consisting of 143 amino acids (16). The active
form of hIFN is a non-covalent homodimer
1
organised in 6 -helixes linked by short
unstructured regions (6). It activates the target
cells via interaction with the extracellular
domain of the hIFNγ receptor complex (5)
followed by activation of the receptor
associated JAK kinases (12). The JAK mediated
phosphorylation of tyrosine residues at specific
receptor sites provides a docking motif for
STAT, which is itself also phosphorylated (2,
15). The activated STAT dissociates from the
receptor, dimmerizes and is then translocated to
the nucleus through a Ran/importin transport
system (17). The active nuclear import of the
protein complex is directed by a short amino
acid sequence called nuclear localization
sequence (NLS). The NLS generally consists of
one or two clusters of basic amino acids
separated by a spacer of variable length (8).
Several groups have attempted to locate the
elusive NLS in STATs trough mutagenesis of
arginine/lysine rich motifs in their molecules (9,
17, 18). The mutational analyses, however,
failed to reveal any conventional NLS in
STATs, which is in accordance with a concept
that the necessary basic NLS is provided by the
STAT ligands. A similar examination of hIFNγ
revealed two putative NLS: a downstream,
located in the unstructured C-terminus of the
protein between amino acid residues 124-132
(20) and an upstream (resembling the NLSs of
the SV40 large tumour antigen, polyoma virus
and the steroid hormone receptors) identified
between the amino acid residues 83-89 (23).
Larkin and co-workers (11) found that the
upstream NLS is much less efficient than the Cterminal one, which was consistent with the
observed loss of more than 90% of biological
activity in the protein lacking the downstream
NLS. The finding that the deletion of the last 23
C-terminal amino acids resulted in inactivation
of the hIFNγ was explained by the absence of
C-terminal NLS (19). Nacheva et al., (14)
demonstrated that the removal of the entire
unstructured (21 amino acids) C-terminal part
from the molecule of hIFNγ led to a 10-fold
decrease in its biological activity only. It was
postulated that the absence of the downstream
NLS in hIFNγ can be compensated (at least
partly) by its upstream analogue. It was
assumed also that the unstructured C-terminal
domain in hIFNγ plays modulating but not
crucial role for the biological activity of this
cytokine.
The present study aims to investigate the
significance of the putative upstream NLS in
hIFNγ for its biological activity. To this end
two new variants of the hIFNγ gene are
constructed. The first one (Lys/Gln88) contains
a gln substitution for the Lys88 in order to
disturb the corresponding polybasic NLS and
the second one (Lys88/Gln/ΔC) carries both the
same substitution plus a deletion of the last 21
3’-terminal codons (these amino acid residues
carry the entire downstream NLS). The two
hIFN derivative proteins are studied for the
basic hIFNγ biological activities (antiviral and
antiproliferative) as well as for their affinity to
the hIFNγ receptor.
Materials and methods
Chemicals and Bacterial Strains
Restriction endonucleases and other DNA
modifying enzymes were purchased from
Gibco-BRL
(Life
Technologies,
Inc.,
Gaithersburg, MD, USA). All other reagents for
electrophoresis, DNA manipulations, antiviral
and kynurenine assays were products of Merck
(Darmstadt, Germany) and Sigma (USA). E.coli
LE 392 cells were used for expression of the
hIFNγ genes.
2
DNA Manipulations
The oligonucleotides necessary for the
modification of the hIFNγ gene were
synthesized on a Millipore Cyclon Plus DNA
synthesizer (MilliGene, Division of Millipore,
Burlington, MA, USA) by the phosphoramidite
method and purified by electrophoresis on a
polyacrylamide (PAA) urea gel. Their
nucleotide sequences are as follows:
Forward primer:
5’-CCC AAG CTT ATG CAG GAC C - 3’
Reverse primer:
5’-GCTT TTC GAA GTC ATC ACG TTG CT
T TTT GTT G -3’
The forward primer carries a HindIII
cloning site (shown in italic) and an ATG codon
followed by a nucleotide sequence identical to
that of the IFN gene.
The reverse primer carries an AsuII
cloning site (shown in italic) and introduces a
point mutation (in bold) designed to substitute a
gln codon (CAA) for the Lys88 codon AAA in
the hIFNγ gene.
To obtain the mutant hIFNγ genes, the
genes encoding for the full size (143 amino
acids) and the 3’-end truncated (122 amino
acids) hIFNγ (14) were amplified by a two step
PCR including 5 cycles of annealing at 500C for
1 min and 35 cycles at 600C for 1 min. The
PCR products were digested with HindIII and
AsuII and cloned in a pBR322-based expression
vector (Fig.1). The primary structure of the
derivative hIFNγ genes was verified by DNA
sequencing on a DNA Cycle Sequencing
System (Gibco–BRL).
Bacterial Transformation
E.coli LE 392 cells were transformed with
the expression plasmids by the CaCl2 method.
PAA-SDS gel electrophoresis
PAA-SDS gels (15%) were prepared
according to Laemmly (10) and run at 20 mA
for 1.5 h. After electrophoresis, the gels were
stained by Coomassie blue R250.
Protein yield determination
Bacterial cells (6 OD595) from overnight
cultures of transformed E.coli LE 392 cells
were harvested by centrifugation, resuspended
in 1 ml 0.14 M NaCl, 10 mM Tris-HCl, 0.1 mM
PMSF and disrupted by sonication. After
centrifugation (15 min, 14,000g) the
supernatants (clear lysates) were collected. The
protein concentration was determined by the
Bradford method (4). Cell lysates were diluted
with phosphate-buffered saline (pH 7.2-7.4) to a
final protein concentration of 27 μg/ml.
Samples of 50 μl were applied on
polyvinylchloride 96-well microplates (Costar
Ltd., USA) by overnight incubation at 40C and
the content of hIFNγ was determined by ELISA
with sequence specific monoclonal antibodies.
Bioassays
Clear cell lysates were sterilized by
filtration and subjected to serial dilutions
appropriate for testing of both hIFNγ antiviral
and antiproliferative activities. Antiviral activity
was determined by measuring the protective
effect of the protein on WISH cells against the
cytopathic action of the vesicular stomatitis
virus (VSV) (7). Antiproliferative activity was
determined by a modified kynurenine bioassay
on WISH cells as described earlier (3).
Competition assay
3
The affinity of the hIFNγ derivative
proteins to the cell receptor was analyzed by
measuring their capacity to compete with the
wild-type protein. Appropriate serial dilutions
of clear cell lysates and purified (99.5% purity)
recombinant hIFNγ (specific antiviral activity
5x107) were prepared. The mutant proteins (in
cell lysates) were mixed in equimollar amounts
with pure wild-type protein and the
antiproliferative activity of the obtained
mixtures was determined in a standard
kynurenine bioassay. The results were
compared with that of the pure wild-type
hIFNγ.
Results and Discussion
1. Design of hIFNγ analogs
It is a routine approach to study the
structure-function relationship by mutating the
protein molecule by routine recombinant DNA
techniques. As mentioned above, to investigate
the functional significance of the putative
upstream NLS in hIFNγ, two derivative proteins
(Lys/Gln88 and Lys88/Gln/ΔC) were created
(Fig. 2).
The introduced mutations had to carry
minimum or no effect on the secondary
structure of hIFNγ and on its ability to bind the
cell receptor. Thus hIFNγ derivative proteins
were designed on the basis of a model
describing the electrostatic interactions in the
homodimer (the active form) of hIFNγ as free
molecule and hIFNγ bound to the extracellular
part of the receptor (1).
One of the most general characteristics
of the behavior of proteins in solution is the set
of pK values of their titratable (ionizing)
groups. Because the electrostatic interactions
cannot be experimentally measured in the
protein molecule, their theoretical prediction is
the main tool for their analysis. In the above
paper (1) the electrostatic interactions in hIFN
are calculated for two different states of the
molecule - alone (in the form of homodimer)
and hIFNγ bound to the receptor. The study was
based on the tree-dimensional structures of
hIFNγ (6, 22) and clearly demonstrated that a
number of titratable groups (mainly basic) had a
remarkable shifts in their pK values after
binding to the receptor. It is worth mentioning
that the groups having high desolvation energy
in both states (free and in a complex with the
receptor) usually corresponded to highly
conserved amino acids. Four of them, Tyr53,
Lys61, Lys88, and Glu112 were conserved in
all IFNγ sequences (6, 13), which is an
indication for their functional significance.
Calculating the ionisation equilibrium of
titratable groups in the area of the putative
upstream NLS for the two states of hIFNγ we
found that most of the basic groups were
drastically desolvated. Analysis of the free
state of the molecule showed that the
protonation states and the solvent accessibility
of Lys86 and Lys87 in the putative upstream
NLS essentially differ in the two subunits
(monomers) of the homodimer and also that the
Lys88 was inaccessible to the solvent in both
subunits. In the hIFNγ/receptor complex Lys86
and Lys87 had high solvent accessibility in both
subunits, while Lys88 was still inaccessible
(Table 1). These data indicated that the Lys88
was located at a distance from the receptor
binding site and therefore its mutation would
not affect the affinity of the protein to the
receptor. In order to minimize possible changes
in the solubility of the protein due to change of
the local polarity it was chosen to substitute
4
Lys88 with glutamine (Fig. 2). Furthermore, to
evaluate the potential compensatory effect of
the downstream NLS on the function of the
upstream one, we constructed a second mutant
hIFNγ in witch the last 21 C-terminal amino
acids (together with the entire downstream
NLS) were deleted (Fig.2).
The mutated hIFNγ genes were prepared
and expressed in E. coli LE392 as described in
Materials and Methods. The expression of the
resulted proteins (Fig. 3) is presented in Fig. 4.
2. Biological activity of the hIFNγ analogs
In order to avoid artifacts related to the
incorrect folding of the recombinant proteins
during their purification, the biological activity
of the hIFNγ derivatives was measured directly
in clear cell lysates. Thus the insoluble fraction
(inclusion bodies) was removed and the activity
of the proteins was determined using the soluble
(cytosolic fraction) only. Two biological
activities, antiviral and antiproliferative, which
are typical for the hIFNγ, were measured. The
first activity was determined on the basis of the
protective effect of hIFNγ on WISH cells
against the cytopathic action of the VSV and
the second one was measured by the so called
“kynurenine bioassay” using the same cell line.
To determine the specific activity of the tested
proteins, their content in the clear lysates was
measured by ELISA using anti-hIFNγ
monoclonal antibodies and pure recombinant
hIFN as a standard.
As seen in Table 2, the single amino acid
substitution in the construct Lys/Gln88 had a
1000-fold lower hIFN activity and the activity
of the truncated construct (Lys88/Gln/ΔC) was
10-fold lower than that of the Lys/Gln88
mutant. To shed light on the reason for the
lower biological activity of the new hIFNγ
constructs we have analyzed their affinity to the
hIFNγ receptor by measuring their capacity to
compete with the wild-type protein. The
experiments were performed as described in
Material and Methods on WISH cells (known to
be enriched in hIFN receptors. The results
presented in Table 2 indicate that the coincubation of the mutant Lys/Gln88 with the
wild-type hIFN led to a 30% decrease in its
antiproliferative activity. We assume that
inhibition higher than 30% could not be
achieved because of the residual biological
activity of the hIFN mutant (see Table 2). The
mutant hIFNγ with fully truncated C-terminus
(Lys88/Gln/ΔC) showed approximately 50 %
inhibition of the standard activity in the
competition assay, confirming our previous data
that the lack of the C-terminus stabilizes the
hIFN/receptor complex (1). These data
indicate that the mutant proteins are capable of
interacting with the hIFN receptor but are
inefficient in triggering the intracellular signal
transduction pathway.
Conclusions
The role of the putative upstream NLS of
hIFN was investigated by site directed
mutagenesis at the area of both upstream and
downstream NLSs. Two derivative proteins
were thus obtained containing either a single
gln substitution for the Lys88 (Lys/Gln88) or
both the single substitution and a 21 aminoacid
C-terminal deletion (Lys88/Gln/ΔC). The first
one demonstrated a 1000-fold lower biological
activity in comparison with the wild-type hIFNγ
and the second one was only 10- times less
active than the Lys/Gln88 mutant. In a
competition bioassay both constructs behaved
as efficient competitors of the wild-type hIFN
for the cellular receptor. The latter means that
5
the putative upstream NLS is crucial for the
biological activity of hIFN but it is dispensable
for its interaction of the protein with the cellular
receptor. Based on these data we are tempted to
speculate that the upstream NLS is of a greater
functional
importance
for
the
signal
transduction as compared to the downstream
NLS.
REFERENCES
1. Altobelli G., Nacheva G., Todorova K.,
Ivanov I. and Karshikoff A. (2001)
PROTEINS: Structure, Function and Genetics,
43, 125-133
Acknowledgments
This work is supported by National Science
Fund, grant K-1405
11. Larkin J. P., Subramaniam B. A., Torres
Johnson, H. M. (2001) J. Interferon Cytokine
Res. 21, 341-348
12. Leaman D. W., Leung S., Li,X., and Stark
G.R. (1996) FASEB J. 10, 1578-1588
2. Bach E. A., Aguet M., and Schreiber R. D.
(1997) Annu. Rev. Immunol. 5, 563-591.
13. Lundell D.J., Narula
Pharmacol. Ther. 64, 1-21
3. Boyanova M., Tsanev R., Ivanov I. (2002)
Annal. Biochem. 308, 178-181
14. Nacheva G., Boyanova, M., Todorova, K.,
Karshikoff, A., Ivanov, I. (2003) ABB
4. Bradford M. M. (1976) Anal. Biochem. 72,
248-254
15. Pestka S., Kotenko S. V., Muthukumaran
G., Izotova L. S., Cook J. R., and Garotta G.
(1997) Cytokine Growth Factor Rev. 8, 189206.
5. Darnell J.E. Jr., Kerr I. M., Stark G. M.
(1994) Science 264, 1415-1421
6. Ealick S.E., Cook W.J., Vijay-Kumar S.,
Carson M., Nagabhushan T.L., Trotta P.P.,
Bugg C.E. (1991) Science 252, 698-702
7. Forti R. L., Schuffman S.S., Davies H.A.,
Mitchell W.M. (1986) Methods Enzymol. 119,
533-540
8. Gorlich D., and Mattaj I. W. (1996) Science
271, 1513-1518
9. Herrington J., Rui L., Luo G., Yu-Lee L.,
and Carter-Su C. (1999) J. Biol. Chem. 274,
5138–5145
S.K.
(1994)
16. Rinderknecht E., O”Connor B.H.,
Rodriguez H. (1984) J.Biol.Chem. 259, 67906797
17. Sekimoto T., Imamoto N., Nakajima K.,
Hirano T., and Yoneda Y. (1997) EMBO J. 16,
7067-7077
18. Strehlow I. and Schindler C. (1998) J.
Biol. Chem. 273, 28049–28056
19. Subramaniam P. S., Larkin, J., III,
Mujtaba, M. G., Walter, M.R., and Johnson,
H., M. (2000) J.Cell. Sci. 113, 2771-2781
10. Laemmly U. (1970) Nature 227, 680-685
6
20. Subramaniam P. S., Mujtaba M. G.,
Paddy M.R., and Johnson H.M. (1999) J. Biol.
Chem. 274, 403-407
21. Tsanev R. and Ivanov I. (2002) CRC
Press
22. Walter M. R., Windsor W.T.,
Nagabhushan T.L., Lundell D.J., Lunn C.A.,
Zauondy P. J., and Narula S.K. (1995) Nature
376, 230-235
23. Zu. X. W., Jay F. (1991) J. Biol. Chem.
266, 6023-6026
7
Figure legends
Fig. 1 Schematic structure of the vector pP1SD-IFNγ for expression of hIFNγ gene in E.coli. P1 –
strong synthetic constitutive promoter, R9 – ribosome binding site SD.
Fig. 2 Mutations introduced in the hIFN-gene. The putative NLSs are marked in orange and yellow
and the polybasic sequences are underlined. The amino acid substitution is indicated by arrow. The
scissors indicate the truncation of the unstructured C-terminus.
Fig. 3 Amino acid sequence of hIFNγ and its mutant derivatives. The C-terminal amino acids are
shown in green.
Fig. 4 SDS-PAAGE of crude lysates of E. coli LE392 cells expressing hIFNg derivative genes. 1:
E.coli LE 392 host (nontransformed) cells; 2-4: bacterial cells expressing the wild type hIFNγ,
Lys/Gln88 and Lys88/Gln/ΔC respectively. The arrow indicates the position of the hIFNγ.
Table 1 Charge values and solvent accessibility of selected amino acids in the putative upstream
NLS in a free state and in a complex with the cellular receptor.
Table 2 Specific biological activities of the mutant derivatives and competitive inhibition of hIFNγ
by its analogs.
8
Fig.1
1
84
143
94
125
N
134
C
Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys
Gln
AAA
(Lys88)
Lys Thr Gly Lys Arg Lys Arg Ser Gln Met
CAA
(gln)
Lys88/Gln/ΔC
Lys88/Gln
Fig.2
hIFN
Gln1…..Lys86 Lys87 Lys88…..Leu 120 Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser
Gln Met Leu Phe Arg Gly Arg Arg140 Ala Ser Gln 143
Lys88/Gln
Gln1…..Lys86 Lys87 Gln88…..Leu 120 Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser
Gln Met Leu Phe Arg Gly Arg Arg140 Ala Ser Gln 143
Lys88/Gln/C
Gln1…..Lys86 Lys87 Gln88…..Leu 120 Ser Pro122
Fig.3
9
1
2
3
4
Fig. 4
10
Table 1
Amino
acids
Lys 80
Lys 86
Lys 87
Lys 88
Arg 89
Asp 90
Asp 91
Charge
A
0.40
0.85
0.99
0.38
1
-1
-1
Charge
B
0.99
0
0.09
0.02
1
-1
0
Free state
Accessibility
A (Å2)
62.91
134.18
122.13
9.02
77.72
56.06
7.52
Accessibility
B (Å2)
40.97
69.30
87.71
47.98
83.01
61.65
6.49
Charge
A
0.92
1
1
1
1
-1
-0.99
In a complex with the receptor
Charge Accessibility Accessibility
B
A (Å2)
B (Å2)
0.28
23.92
37.08
1
128.66
127.53
1
143.96
140.35
1
17.81
23.15
1
63.41
63.34
-1
60.45
63.34
0.91
22.35
19.29
Table 2
Protein
Antiviral activity
(IU/mg)
Antiproliferative Inhibition
activity (IU/mg)
(%)
hIFNγ
6.1 х 107
1.1 х 108
0
Lys88/Gln
5.7 x 104
1.7 x 105
32(±4%)
Lys88/Gln/ΔC
1.2 x 103
6.7 x 103
50(±5%)
11