From the Institut de Génétique Moléculaire, CNRS,
F-34293 Montpellier, Cedex 5, France, Biogen Inc.,
Cambridge, Massachusetts 02142, § Department of Pathology,
University of Tennessee Health Science Center, Memphis, Tennessee
38163, and ¶ Institut Pasteur, INSERM U 276, Paris 75724, Cedex 15, France
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ABSTRACT |
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Type I interferon (IFN) subtypes and
share a common multicomponent, cell surface receptor and elicit a
similar range of biological responses, including antiviral,
antiproliferative, and immunomodulatory activities. However,
and
IFNs exhibit key differences in several biological properties. For
example, IFN-
, but not IFN-
, induces the association of
tyrosine-phosphorylated receptor components ifnar1 and ifnar2, and has
activity in cells lacking the IFN receptor-associated, Janus kinase
tyk2. To define the structural basis for these functional differences
we produced human IFN-
with point mutations and compared them to
wild-type IFN-
in assays that distinguish
and
IFN subtypes.
IFN-
mutants with charged residues (N86K, N86E, or Y92D) introduced
at two positions in the C helix lost the ability to induce the
association of tyrosine-phosphorylated receptor chains and had reduced
activity on tyk2-deficient cells. The combination of negatively charged residues N86E and Y92D (homologous with IFN-
8) increased the cross-species activity of the mutant IFN-
s on bovine cells to a
level comparable to that of human IFN-
s. In contrast, point mutations in the AB loop and D helix had no significant effect on these
subtype-specific activities. A subset of these latter mutations did,
however, reduce activity in a manner analogous to IFN-
mutations.
The effects of these mutations on IFN-
activity are discussed in the
context of a family of related ligands acting through a common
receptor and signaling pathway.
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INTRODUCTION |
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The mammalian type I
IFNs,1 produced in response
to viral infection and other inducers, are divided into and
subtypes on the basis of their reactivity with antisera raised against
IFNs derived, respectively, from leukocytes and fibroblasts (1). The
human IFN-
s are encoded by a family of at least 15 different genes,
while IFN-
is the unique member of its subtype (2). Primary sequence
comparison between the
and
subtypes reveal an approximately
50% amino acid homology, while the amino acid homologies between the
IFN-
subtypes are approximately 80% (2, 3), reinforcing the
division between IFN-
and -
subtypes.
As the pleiotropic nature of these cytokines became apparent, with both
subtypes eliciting a similar range of biological activities (3),
differences between subtypes, and between IFN-
and -
s, in
potency and cell type specific activities were noted (4). In
particular, IFN-
elicits a markedly higher antiproliferation response in some cell types such as (5), embryonal carcinoma, melanoma
and melanocytes than do IFN-
s (6, 7, and references therein). Higher
potency of IFN-
in treatment of multiple sclerosis and certain
cancers has been observed (7).
The entire class of type I IFNs elicit their biological activities
through engagement of a common cell surface receptor (8-10). Two
chains of the receptor, ifnar1 and ifnar2, both members of the type two
cytokine receptor family, have been identified (11-15). Both
components are necessary for function and in the absence of either
there is neither high affinity binding nor biological effect (14, 16,
17). The intracellular portions of the receptor subunits are bound by
tyrosine kinases, jak1 (12, 18) and tyk2 (19, 20), members of the Janus
kinase family. Upon ligand binding these kinases are activated and
phosphorylate members of the STAT family of transcription factors (21),
as well as ifnar1 and 2. A further property that distinguishes these
IFN subtypes is that IFN-, but not IFN-
, induces association of tyrosine phosphorylated ifnar1 and 2, detectable by precipitation with
anti-ifnar1 antibodies (22-25). In addition, tyk2-deficient cells
retain partial responsiveness to IFN-
, but are completely unresponsive to IFN-
s (26). Complementation of the tyk2 deficiency by expression of a kinase-inactive tyk2 partially restores IFN-
8 binding and activity, but has no effect on the IFN-
binding to these
cells although it augments IFN-
-induced signaling (27). Thus,
potency and specificity differences between IFN-
s and -
s may
reflect differences in receptor interaction.
The type I IFNs are closely related members of the helical cytokine
family (28). Resolution of the three-dimensional structures for
crystals of murine IFN- (29, 30) and human IFN-
2b (31) revealed
that their overall structure is very similar, and that these IFNs are
composed of 5 helices joined by loops of various lengths. Inspection of
the crystal structures in light of previous extensive IFN-
mutational analyses (reviewed in Refs. 15 and 32) allows identification
of putative domains likely to be involved in receptor interactions.
Examination of structural models in regions of mutational hotspots,
such as the AB loop, D helix, DE loop (32), and the C helix (33, 34),
directed us to exposed residues available for receptor binding.
In this study we describe the functional consequences of site-directed
mutations of human IFN-. Our results reveal the importance of C
helix residues in conferring subtype specific activities on IFN-
as
judged from activity and biochemical assays. Mutation of AB loop
(Arg27 and Arg35) and D helix
(Lys123, but not Arg124) residues reduces
activity in all assays and are qualitatively similar to effects seen
for homologous IFN-
mutations. However, the AB loop and D helix
IFN-
mutants retain IFN-
-specific activities in assays that
distinguish between IFN-
and -
subtypes.
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EXPERIMENTAL PROCEDURES |
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Notation for IFN Amino Acid Sequences--
We use the single
letter code for amino acids. Substitutions are given as XnY,
where X is the amino acid replaced, n, its number
in the sequence and Y, the replacement. To facilitate
discussion, we number the IFN- amino acid sequences from the
NH2 terminus according to the convention that counts the
deletion at position 44 of IFN-
2 (29, 30, 32). Human IFN-
is
numbered from the NH2 terminus without deletion. This
alignment leaves all
positions, n, with homologous
positions at n + 2, up to the COOH terminus of IFN-
. The
five
helices of IFN are labeled from the NH2 terminus:
ABCDE, the loops are labeled with the letters of the helices at their
NH2-terminal and COOH-terminal ends.
Construction, Production, and Evaluation of Mutant
IFNs--
Substitutions introduced into human IFN- are given in
Fig. 1. The mutations were introduced into the human IFN-
gene
carried on plasmid pJC017, a COS cell expression vector (Biogen, Inc. Cambridge, MA), using the transformer site-directed mutagenesis kit
from CLONTECH Laboratories, Inc. (Palo Alto, CA).
COS 7 cells were transiently transfected, and the supernatants from
cultures (3 days posttransfection) were screened for the presence of
immunoreactive IFN-
and then for biological activity on human HL116
cells, which carry a luciferase reporter under control of an
IFN-
-
-inducible promoter (33). Quantitation of IFN expression
levels was performed by enzyme-linked immunosorbent assay utilizing
rabbit polyclonal anti-IFN-
1a serum to coat plates, biotinylated
rabbit polyclonal antibodies as a secondary reagent, followed by
streptavidin-coupled horseradish peroxidase (Jackson Immunochemical,
West Grove, PA). Enzyme-linked immunosorbent assay values were
confirmed by Western blot analyses using a second rabbit anti-IFN-
1a
polyclonal serum. Western blotting revealed two interferon bands
corresponding to unglycosylated and glycosylated forms of the molecule
which were present in roughly equal proportions. Molarities of
interferons based on biological activity referenced to fully
glycosylated, purified IFN-
1a were always less than those estimated
by enzyme-linked immunosorbent assay. Experiments to investigate the
effect of glycosylation on IFN-
activity suggest a 2-fold lower
specific activity of unglycosylated versus glycosylated
forms (53). All the results presented in this report compare mutant and
wild-type preparations that had similar proportions of glycosylated
interferon. Supernatants were assayed for activity on cells with
luciferase as an IFN-inducible reporter. Each production batch included
a transfection with cDNA for unmutated wild-type IFN-
and a mock transfection control. Preliminary studies had shown that IFN-
could
be routinely produced with supernatants yielding in a range around 1.0 nM and that such supernatants, stored at 4 °C, retained their activity over several months.
Immunoprecipitations--
Receptor chain coimmunoprecipitations
were performed essentially as described previously (24). For each
experimental point 2 × 108 human Daudi B cells (ATCC
no. CCL 213) were treated with wild-type or mutant IFN- (2,500 units/mL, 10 min, 37 °C). Receptor proteins were immunoprecipitated
from extracts using either anti-ifnar1 monoclonal antibody (AA3, Biogen
Inc.), anti-ifnar2 monoclonal antibody (AB.B7.2, Biogen Inc.), or
control mouse sera. Immune complexes were analyzed by Western blotting
of SDS-polyacrylamide gel electrophoresis, 7.5% gels, probed with
anti-phosphotyrosine monoclonal AB-2 antibodies (Oncogene Research
Products, Cambridge, MA) followed by anti-mouse IgG coupled with
horseradish peroxidase (Amersham Corp.). Blots were developed using
enhanced chemiluminescence (ECL, Amersham). The blots were stripped and
reblotted with anti-ifnar1 monoclonal antibody to verify that equal
amounts of protein were loaded in each lane.
Cells and IFN Assays--
The recombinant IFNs, human IFN-
was from Biogen Inc.; human IFN-
2 (2c) was a gift from Dr G. Adolf,
Ernst Boehringer Institute, Vienna, Austria; human IFN-
8 and -
1
and hybrids of these were a gift from Ciba-Geigy, Basel, Switzerland.
Concentrations of the IFNs were estimated against IFN reference
preparations, pure recombinant IFNs, IFN-
at 5.0 nM, and
IFN-
at 250 nM of active monomer. The IFN-
reference
at 5 nM corresponds to 20,000 IU (MRC 69/19). Cell
sensitivity to IFN was estimated in terms of the mean concentration
required to obtain a 50% response in any given assay (see Table
I).
Modeling and Assessment of Side Chain Accessibility--
Models
of IFN- and IFN-
were generated by homology modeling with the
Modeler, Protein Design, and Protein Health functions available with
the Quanta package (MSI Inc., San Diego CA), using the
carbon
coordinates of murine IFN-
(Brookhaven accession no. 2RMI) as
template. Solvent accessibility of residues and their side chains and
were obtained with the default settings of the Protein Design function
of Quanta. Inspection of the recently published figures for IFN-
2b
(31) and comparison with the unpublished coordinates for human IFN-
(37) suggest that these models were useful for the
helices and for
the AB loop, COOH-terminal to cysteine 29 (cysteine 31 in human
);
less so for the positioning of residues in the other loops.
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RESULTS |
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Design of IFN- Mutants--
The putative distribution of
helices (A through E) and loops (identified by their bounding helices)
are shown in the primary sequence comparison shown in Fig.
1. The mutations in the C helix of
IFN-
were confined to residues 86 and 92 (homologous with 84 and 90 in the IFN-
s) and were concerned with substituting the noncharged
side chains with residues that would be charged at neutral pH. Previous
work had demonstrated that these residues participate to confer subtype
8-specific activities onto a hybrid IFN-
1 (33), implicating them
as important residues for defining the character of receptor mediated,
subtype-specific activities. Single mutations N86E, N86K, and Y92D, as
well as doubly substituted mutants at these positions, N86E,Y92D and
N86K,Y92D, were investigated in this study.
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Substitutions in the C Helix--
Substitution of the charged
residues at IFN- positions 86 and 92, either individually (N86E,
N86K, and Y92D) or together (N86E,Y92D and N86K,Y92D), did not alter
the specific activity as measured by luciferase induction on human
HL116 cells, antiviral potency on equine NBL6 cells (Table II), or
antiproliferative activity on human Daudi B cells (data not shown).
However, in several assays where IFN-
and -
properties are
distinctly different, the double substitutions were shown to result in
biological activities different from wild-type IFN-
.
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AB Loop and D Helix Mutations--
The substitutions R27A, Y30R,
and R35T in the AB loop were made to mimic analogous residues of the
equine IFN- (Fig. 1). None of the mutant IFN-
s showed higher
activity on equine cells than wild-type IFN-
(Table II), as would
have been expected if these residues were an important site for equine
receptor recognition. Substitution Y30R increased by nearly 3-fold the
IFN-
activity on tyk2-deficient cells, but had neither effect on
activity in bovine antiviral assays nor on the IFN-
-induced
association of receptor chains (Table II). Mutations at two positions
in the AB loop, R27A and R35T, caused a diminution in activity in
assays on bovine and human cells. Residue Arg27 is not
conserved between IFN-
and -
subtypes, while Arg35 is
conserved in all human IFN-
s and -
s (2, 3). The IFN-
homologue
Arg33 is particularly sensitive to mutational change, where
even the charge conserved mutation R33K produces more than 100-fold
loss in activity (33). The mutation R35T in IFN-
produced a modest 10-30-fold loss in antiviral potency in all assay systems, except on
equine cells where it assayed as wild-type activity.
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DISCUSSION |
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The aim of this study was to characterize the functional
consequences of point mutations of IFN-, which IFN-
mutational analyses and sequence comparisons had implicated as important for
receptor interactions. We examined the biological activity of these
mutant IFN-
s in assays that distinguish between IFN-
and -
subtypes, and in further assays that detect overall losses in activity
(i.e. HL116 and WISH cells, which are equally sensitive to
INF-
and -
subtypes). We primarily targeted solvent exposed residues of the AB loop, C helix and D helix, which are regions of the
molecule shown to be important for IFN-
binding and function (32-34). We found that mutations in the NH2-terminal AB
loop and D helix had no effect on subtype-specific activities.
Substitutions of residues highly conserved between IFN-
and -
s,
R35T in the AB loop, and K123A in the D helix moderately reduced
activity on human cells. By comparison with analogous mutations in
IFN-
(32), these residues are relatively insensitive to mutation in
IFN-
, suggesting quantitatively different contributions of these
residues to IFN-
-receptor interactions. We cannot exclude the
possibility, that since much of the structure/function studies were
performed on bacterially produced IFN-
s, those mutants may have been
misfolded or less quantitatively assayed due to technological limitations (38-44). The eukaryotic cos cell expressed IFN-
mutants described in this study were soluble, highly glycosylated, and retained
full activity over many months in conditioned medium. The importance of
glycosylation for IFN-
stability and solubility have been recently
described (53).
Heterologous systems have been used to define important domains of
IFN- necessary for cross-species reactivities, presumably by
creating hybrid IFNs that interact better than parental forms with
receptor components (42-44). We sought to extend these studies for
human IFN-
by substituting AB loop residues, Arg27,
Tyr30, and Arg35, for equine IFN-
residues,
since these residues are not well conserved between equine and human
IFN-
s (Fig. 1). Neither substitutions of the AB loop nor C helix
mutants showed increased activity on equine cells. This result
implicates other regions of the molecule, possibly in the A helix or
proximal D helix (homology comparisons shown in Fig. 1), as important
determinants for equine receptor binding.
The introduction of charged residues at two positions in the C helix
(N86E, N86K, Y92D, N86E,Y92D, and N86K,Y92D) resulted in IFN-
mutants with altered activities in several assays that distinguish
between
and
subtypes. The tandem substitutions (N86E,Y92D and
N86K,Y92D) had the most striking effect on subtype-specific activities,
eliminating the IFN-
-induced association of phosphorylated ifnar1
and ifnar2 receptor chains in human Daudi Burkitt's lymphoma cells,
increasing antiviral activity on bovine MDBK cells, and lowering
activity on tyk2-deficient human cells. While these changes represent a
loss or decrease of specifically IFN-
characteristics and a shift
toward the properties of IFN-
, it is not clear that they represent
an acquisition of
-like properties. In particular, recent results
show that the 11.1, tyk2-deficient cells from which the A27 strain was
derived, express low levels of ifnar1 (45), and the C helix mutation
may represent simply a reduced functionality in a specifically
IFN-
-type interaction.
Considering that the two chains of the IFN receptor provide binding sites for different jak kinases, (12, 18-20) and STAT transcription factors, STAT1, STAT2 (46-48), and STAT3 (49), whose activities are induced by type I IFN binding, it is interesting to consider how alternative geometries of ligand-receptor complexes may achieve distinct signals through a common receptor.
STAT proteins bind to distinct receptor cytoplasmic domains and
considerable overlap exists in their activation profiles in response to
a wide spectrum of cytokines (46). Specificity of cytokine action may
be achieved through finely tuned activation events mediated through
specific receptor associations with Janus kinases, interdependent STAT
binding and phosphorylation events, and differential assembly of homo-
and heteromeric STAT complexes (21, 47, 48, 50), which distinguish
promoter elements on the basis of their distinctive binding properties
(21, 47). The potential for IFN-s and -
s to differentially
activate different STAT complexes, or to induce other signaling events
(51, 52), could result in distinctive gene activation events. Further
studies to delineate putative distinctive signaling events and
differentially inducible genes will be necessary to test these
possibilities.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. M. Karpusas of Biogen
Inc. for checking our models of IFN- against his coordinates for the
crystal structure of human IFN-
. We thank Prof. Ph. Jeanteur for his support. We are grateful to Paula Hochman, Joe Rosa, and Michael Karpusas for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by grants from ARC, DRET: 94/2513A, Ligue National contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Institut de
Génétique Moléculaire, CNRS, 1919 Route de Mende,
F-34293 Montpellier, Cedex 5, France. Tel.: (0)4 67 61 36 76; Fax: (04)
67 04 02 45/31.
1 The abbreviations used are: IFN, interferon; HAT, hypoxanthine, aminopterin, and thymidine; 6TG, 6-thioguanine; MDBK, Madin-Darby bovine kidney; tyk, tyrosine kinase; STAT, signal transducers and activators of transcription.
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REFERENCES |
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