| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Protein Engineering: The Amino Acid Residues at Positions 86 and 90 Are Important for Antiproliferative Activity1
*
Division of Therapeutic Proteins, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892; and
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
is a family of structurally related proteins that
exhibit a wide range of antiproliferative activities. To understand the
structural basis for these different antiproliferative activities,
eight recombinant human IFN- hybrids (HY) of 21a/2c (HY-4,
HY-5) and mutants (site-directed mutagenesis (SDM)-1, 2 and cassette
mutagenesis (CM)-1, 2, 3, and 4) have been expressed, purified, and
characterized. The data showed that the amino acid region 81–95 is
important for antiproliferative activity. Site-directed mutagenesis and
cassette mutagenesis studies showed that if serine (S) 86 and
asparagine (N) 90 were replaced by tyrosine (Y), the antiproliferative
activity was increased. We have also observed that if Y86 was replaced
by isoleucine (I), the antiproliferative activity was comparable.
However, if Y86 was replaced by aspartic acid (D), lysine (K), or
alanine (A), the antiproliferative activity was substantially
decreased. Our results indicate that Y and/or I at position 86 and Y at
position 90 are very important in antiproliferative activity of human
IFN-. Circular dichroism spectra showed that the amino acid
replacements at position 86 did not change the secondary structure.
Thus the biological activity changes among those mutants do not appear
to be due to conformational changes. The results also suggest that
hydrophobic residue(s) at position 86 may be important for the
interaction of the molecule with its receptor. The competitive
binding data correlated with the antiproliferative activity. The
N-terminal region of the molecule and the hydrophobic residues
(including Y and I) on the C-helix region at positions 86 and/or 90 are
important for binding and antiproliferative activities of human
IFN-s. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, IFN-
, and IFN-
(type 1 IFN) constitute one of the most
complex families of proteins exhibiting high homology in primary,
secondary, and tertiary structure. These proteins are related members
of the cytokine family and are composed of five helices (1, 2). In general, the effects of these IFNs are believed to be
mediated through interaction with a common IFN receptor comprised of at
least two proteins, IFNAR1 (3) and IFNAR2
(4, 5, 6), and signal transduction to the nucleus through the
Janus kinase 1/STAT1 and 2 pathways. However, these IFNs have small
differences in the primary, secondary, and tertiary structures that may
be responsible for a significant variety of biological actions
(7).
There are a large number of reports on the structure-function
relationship of these IFNs (7, 8, 9, 10, 11, 12, 13, 14). To elucidate the
mechanism by which IFNs bind to and activate their receptor, detailed
structure-activity studies are required. Currently, the structures of
several IFNs have been solved, such as murine IFN-
(15), human IFN-2 (16, 17), human IFN-
(18), ovine IFN-
(19), and human IFN-
consensus (20). Numerous mutational studies have revealed
important functional residues on IFNs (12). However, many
of these studies predate the determination of the crystal structures of
these molecules. Hence, it was not possible to design mutations based
on any knowledge of the location of the mutated residue in the
three-dimensional structure of the IFN molecule, or of its involvement
in intramolecular interactions that are required for maintaining
structural integrity (13).
Understanding the structure and function of human IFN-s and their
receptors is the primary focus of our studies. Previously we have
purified and characterized 22 IFN- components from the human
lymphoblastoid cell line, Namalwa, induced with Sendai virus
(21). One of them, component o, was found to be noteworthy
for its high antiproliferative activity and its poor ability to compete
for the IFN-2b binding site (22). Based on our partial
amino acid sequence data, component o was indistinguishable from
IFN-21a. Therefore, IFN-21a was cloned and expressed (23, 24). To determine the specific region(s) of IFN- responsible
for antiviral and antiproliferative activity, three IFN-2c and
IFN-21a chimeras, hybrid
(HY)3-1
(IFN-21a(1–75)/2c(76–165)), HY-2
(IFN-21a(1–95)/2c(96–165)), and HY-3
(IFN-2c(1–95)/21a(96–166)), were constructed based on the
three-dimensional structures of human IFN- and -, and the
biological activities of these human IFN-s were determined
(23). HY-3 exhibited antiproliferative activity greater
than either of the parent IFN-s.
The goal of this study was to identify the amino acid residues of
IFN- hybrids that are important for antiproliferative activity. HY-4
(IFN-21a(1–75)/IFN-2c(76–81)/IFN-21a(82–95)/IFN-2c(96–166))
and HY-5 (IFN-21a(1–81)/IFN-2c(82–166)) were constructed using
PCR. In addition, two mutant IFN-s, site-directed mutagenesis
(SDM)-1 (HY-4(86S
Y)) and SDM-2 (HY-4(90NY)), were prepared by
SDM. Four additional mutants were constructed using cassette
mutagenesis (CM): CM-1 (SDM-1(86YD)), CM-2 (SDM-1(86YI)), CM-3
(SDM-1(86YK)), and CM-4 (SDM-1(86YA)). The antiviral,
antiproliferative receptor binding properties of IFN- hybrids and
mutants were analyzed and compared. We have found that the region
81–95 and specifically amino acid residues at positions 86 and 90 are
important in the antiproliferative activity and receptor binding of
human IFN-. In addition, the amino-terminal amino acid sequence is
also important for interaction with the IFN- receptor proteins.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Recombinant human IFN-2b was obtained from Schering-Plough
(Kenilworth, NJ) and has an antiviral specific activity of 2 x
108 IU/mg protein. IFN-2c cDNA was
obtained from Stratagene (La Jolla, CA). HY-2 (21a (1–95)/2c
(96–166)) with a antiviral sp. act. of 3 x
108 IU/mg protein were expressed and purified
from Escherichia coli strain JM109 as previously described
(23). IFN-2b and IFN-21a were radiolabeled with
125I-labeled Bolton-Hunter reagent
(Amersham, Arlington Heights, IL) as previously described (22, 23).
Cells and cell culture
Human Daudi cells were obtained from Dr. P. Grimley (Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD). Cells were grown in suspension using RPMI 1640 (Life Technologies, Gaithersburg, MD) containing 10% FBS, 2 mM glutamine, and 0.2% gentamicin. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2. All cultures were determined to be free of mycoplasma.
Construction of IFN- hybrids and mutants
Fig. 1
shows the construction scheme of IFN-
hybrids and mutants. HY-4
(IFN-21a(1–75)/IFN-2c(76–81)/IFN-21a(82–95)/IFN-2c(96–166))
and HY-5 (IFN-21a(1–81)/IFN-2c(82–166)) cDNA were constructed
using PCR (23, 25) as shown in Fig. 1. PCR primers for the
construction of HY-4 and HY-5 include 1) 5'-GXT GCT TGG GAT GAG ACC CTC
CTA-3', 2) 5'-TAG GAG GGT CTC ATC CCA AGC AGC-3', 3) 5'-CTA GAC AAA TTC
TAC ACT GAA CTC TAC CAG-3', 4) 5'-CTG GTA GAG TTC AGT GTA GAA TTT GTC
TAG-3', 5) 5'-TCC GGA TCC TGT GAT CTG CCT CAG ACC-3', and 6)
5'-GAG CTC GCA TGC TCA TCA TTC CTT ACT TCT TAA
ACT-3'.
|
Site-directed mutagenesis
SDM-1 (HY-4(86SY)) and SDM-2 (HY-4(90NY)) were generated
using SDM (26). Plasmid DNA pQE30/HY-4 was used as the
template for mutants SDM-1 and SDM-2 construction by PCR. The outside
primers are primers 5 and 6, and the two inside mutant primers for
SDM-1 are primer 7 (5'-GAA AAA TTT TAC ACT GAA CTT-3',
sense) and primer 8 (5'-AG TTC AGT GTA AAA TTT TTC-3',
antisense). The other two inside primers for SDM-2 are primer 9
(5'-CT GAA CTT TAC CAG CAG CTG-3', sense) and primer 10
(5'-CAG CTG CTG GTA AAG TTC AGT-3', antisense). The
underlined bases are modified bases that yield the mutants. The
amplified cDNA constructs were digested with BamHI and
SphI and ligated into pQE30 expression vector, then the
plasmids were transformed into E. coli strain JM109 cells.
All constructs were confirmed by DNA sequencing with an automated DNA
sequencing system.
Cassette mutagenesis
We also used a modification of CM (semisaturation mutation)
(25, 27, 28) to generate multiple mutations of codons at
amino acid position 86 in the SDM-1 for CM-1 (SDM-1(86YD)), CM-2
(SDM-1(86YI)), CM-3 (SDM-1(86YK)), and CM-4 (SDM-1(86YA)). The
two outside primers for all CM mutants are primers 5 and 6. The
degenerate inside primers for codon 86 of the SDM-1 mutants are primer
11, 5'-GAA AAA TTT (A/G/T) (C/T/A)(T/A) ACT GAA CTT AAC-3' (sense), and
primer 12, 5'-GTT AAG TTC AGT (T/A)(A/G/T)(C/T/A) AAA TTT TTC-3'
(antisense). Plasmid DNA pQE30/SDM-1 was used as template. The
amplified modified cDNA constructs were digested with BamHI
and SphI and ligated into the expression vector pQE30.
Mutants were identified by DNA sequencing with an automated DNA
sequencing system.
Expression and purification
All plasmid DNAs of IFN- hybrids and mutants were
individually transformed into E. coli strain JM109. Bacteria
were grown in super broth (Biofluids, Rockville, MD) containing 100
µg/ml ampicillin in a 37°C shaker incubator overnight. The cultures
were diluted 1/50 and incubated at 37°C with shaking. Protein
expression was induced by 1 mM isopropyl -D-thiogalactoside. The
bacteria were then incubated at 30°C for 4 h, after which
cells were harvested by centrifugation and sonicated on ice (1-min
bursts/1-min cooling/250 W) (23). IFN purification was
performed by Ni-nitrilotriacetic acid agarose resin (Qiagen)
(23, 29) and NK2 (a mAb against IFN-) mAb affinity
chromatography (21) as previously described
(23).
Protein determination
Purified recombinant IFN protein concentrations were determined using the Coomassie Plus Protein Assay (Pierce, Rockford, IL). Purity of IFNs was assessed by SDS-PAGE (23) (data not shown).
Antiviral and antiproliferative assays
Antiviral and antiproliferative assays were performed as
previously described respectively (21, 22). MDBK cells
(American Type Culture Collection (ATCC), Manassas, VA) and Wish cells
(ATCC) were used for the antiviral assay(s). All IFN units are
expressed with reference to the National Institutes of Health Human
Recombinant IFN-2a standard Gxa01-901-535. Human Daudi cells were
used for the antiproliferative assay.
Receptor binding assays
IFN- receptor binding assays were performed as previously
described on human Daudi cells (22).
Circular dichroism (CD) spectral analysis
CD spectra were measured at room temperature in a Jasco-J-715 spectropolarimeter at 25°C, using 1-mm-pathlength quartz cuvettes. Protein concentrations were 100 µg/ml in 5 mM sodium acetate buffer (pH 5.5), containing 0.1 M sodium perchlorate. Four scans were made between 260 and 185 nm, speed 50 nm/min, time constant equal to 1 s. Measured ellipticities (millidegrees) were converted into mean residue ellipticity. Secondary structure was determined from averaged spectral data using the CONTIN program (30).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
hybrids and mutants
Eight recombinant human IFN- hybrids and mutants have been
expressed and purified. To facilitate the purification process, an
affinity tag consisting of six histidine residues was attached to the
amino terminus of the recombinant IFN-s. These His tags do not
appear to interfere with the structure or function of the recombinant
proteins (31).
Human HY-4 and HY-5, and mutants SDM-1, SDM-2, CM-1, CM-2, CM-3, and
CM-4 were expressed in E. coli using a pQE30 expression
system. Sequencing of the IFN- hybrid and mutant cDNAs demonstrated
that the mutated residues and positions agreed with the designed
structure (Fig. 1
). The expression levels
of IFN hybrids and mutants were 0.5 mg/L (HY-4 and 5, and SDM-1 and 2)
and 1 mg/L (CM-1, 2, 3, and 4). Initial purification of the IFNs on a
Ni-nitrilotriacetic acid agarose column yielded partially purified
protein, with antiviral specific activities ranging from 3 to 4.5
x 106 IU/mg protein on MDBK cells. These IFNs
were further purified by NK2 mAb affinity chromatography
(21). After the two purification steps, each IFN appeared
as a single band with an apparent m.w. of 20,000 on reducing SDS-PAGE.
The antiviral specific activities of the purified IFN-s ranged from
2.0 to 4.6 x 108 IU/mg protein on MDBK
cells. The antiviral specific activities of the hybrids and mutants
were similar to each other on MDBK cells.
The ability of the IFN- hybrids and mutants to inhibit the growth of
Daudi cells was also examined. Some inhibition curves are shown in Fig. 2. The concentrations of these IFN-s
that inhibited Daudi cell growth by 50% are shown in Table I. Overall, HY-1 and 5, and mutants
SDM-1, 2, and CM-2 have higher antiproliferative activities than HY-2,
4, and CM-1, 3, and 4. HY-3 exhibited the highest antiproliferative
activity compared with the other hybrids and mutants on Daudi cells. In
contrast, HY-2 has the lowest antiproliferative activity. SDM-1
displayed 300-fold higher antiproliferative activity compared with HY-2
on Daudi cells. Our earlier report (23) indicated that
HY-1(IFN-21a(1–75)/2c(76–166)) had a 40-fold greater
antiproliferative specific activity than HY-2
(IFN-21a(1–95)/2c(96–166)) on Daudi cells (23).
Comparing the sequence of HY-1 and HY-2 there are seven amino acid
differences in the 75–95 region. Based on this data, we constructed
HY-4 (IFN-21a(1–75)/2c(76–81)/21a(82–95)/2c(96–166))
and HY-5 (IFN-21a(1–81)/2c(82–166)). HY-4 has the 76–81 region
of 2c and the same antiproliferative activity as HY-2 on Daudi
cells. However, HY-5, which has the 82–95 region of 2c, has a
30-fold greater antiproliferative activity than HY-4 and 60-fold
greater activity than HY-2 on Daudi cells. These data show that the
amino acid region 81–95 is important for antiproliferative activity.
Further comparing IFN-21a and IFN-2c there are only three amino
acid differences in the 81–95 region (see Fig. 3). IFN-2c has D, Y, and Y at
positions 83, 86, and 90, respectively, and IFN-21a has E, S, and N
at these respective positions. Because the change from D to E is
conservative, i.e., acidic amino acid residues are negatively charged,
we constructed two mutants of HY-4 at positions 86 and 90, SDM-1
(HY-4(86SY)) and SDM-2 (HY-4(90NY)) by SDM. Fig. 2 and Table I
show that if 86S was replaced by Y (SDM-1) or 90N was replaced by Y
(SDM-2) the antiproliferative activity of HY-4 was increased similar to
the level of HY-5. These data suggest that the tyrosines at positions
86 and 90 are important for antiproliferative activity. To determine
whether only tyrosine at position 86 has this effect or whether
other amino acid residues could substitute for tyrosine, amino acids
that were either negatively or positively charged or that possessed
long or short side chains were substituted using CM (32).
Four mutants CM-1 (SDM-1(86YD), CM-2 (SDM-1(86YI)), CM-3
(SDM-1(86YK)), and CM-4 (SDM-1(86YA)) were selected. Tyrosine at
position 86 was replaced with D, I, K, or A. We observed that I at
position 86 (CM-2) had the same level of antiproliferative activity as
SDM-1 (Table I). In contrast, replacement with D (CM-1), K (CM-3), or A
(CM-4) decreased antiproliferative activity by 60-fold (CM-1), 100-fold
(CM-3), and 80-fold (CM-4). These results suggest that tyrosine or
isoleucine at position 86 is very important for the antiproliferative
activity of IFN-.
|
|
|
Spectroscopic analysis was conducted on IFN- mutants SDM-1,
CM-2, and CM-4 to compare their secondary structure. Fig. 4 shows the far UV spectra of SDM-1,
CM-2, and CM-4. These mutants all have very similar structures.
Estimates of secondary structure were: 58–64% helix, 9–40%
sheet for SDM-1; 63–71% helix, 5–35% sheet for CM-2; and
58–65% helix, 9–42% sheet for CM-4. These values were in
the range of that described previously; human IFN- has 66%
helix, 15% sheet (14). Of interest, these results
showed that the amino acid replacements at position 86 did not
significantly change the secondary structure. Thus, the biological
activity changes among those mutants do not appear to be due to major
conformation changes.
|
To examine the cell surface binding properties of IFN- hybrids
and mutants, competitive binding analysis was performed
using 125I-labeled IFN-2b
and 125I-labeled IFN-21a. Figs. 5 and 6 show examples of the ability of
IFN-21a, 2c, hybrids, and mutants to compete for IFN-2b and
IFN-21a binding sites on Daudi cells. The concentrations of the
IFN-s, which inhibit binding by 50% on Daudi cells, are shown in
Table I. IFN-2c inhibited the binding of
125I-labeled IFN-2b to Daudi cells very
effectively. The concentration of IFN-2c, which inhibits binding by
50%, is 5.0 ± 2 ng/ml. In contrast, hybrids (HY-4 and 5) and
mutants (SDM-1, 2, and CM-1, 2, 3, and 4) like IFN-21a, which have
the N-terminal part of IFN-21a, poorly inhibited
125I-labeled IFN-2b binding to Daudi cells
(Fig. 5 and Table I). The concentration of IFN-21a, which inhibits
binding of 125I-labeled IFN-2b by 50%, is
2000 ± 208 ng/ml on Daudi cells.
|
|
shows the ability of IFN-2c, 21a, hybrids, and mutants to
compete for 125I-labeled IFN-21a binding sites
on Daudi cells. HY-5 and mutants SDM-1, 2, and CM-2 compete very well
and similarly with IFN-21a for the
125I-labeled IFN-21a binding site. The
concentration of IFN-21a, which inhibits binding by 50%, is
60.0 ± 15 ng/ml. But HY-4, CM-1, 3, and 4 competed poorly for the
125I-labeled IFN-21a binding site on Daudi
cells. CM-1 exhibits the lowest affinity for the IFN-21a binding
site (900 ± 100 ng/ml inhibits binding by 50%). Each of the IFN
hybrids and mutants that competes well with
125I-labeled IFN-21a had tyrosine or
isoleucine either at position 86 or 90 and had the N-terminal part of
IFN-21a or the original sequence of 21a in this region (86S,
90N). These binding results correlated with antiproliferative activity.
Higher antiproliferative activity was observed for SDM-1 and 2, CM-2,
and HY-5, whereas lower antiproliferative activity was seen for HY-4,
CM-1, 3, and 4. Thus, these data suggest that the N-terminal region of
the IFN- molecule and amino acid residues at position 86 and 90 are
important for the binding and antiproliferative activities of
IFN-. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and - (7, 9, 31) show that the
surface structures of these IFNs are likely to be involved in
interaction with a specific cellular receptor, a step required to
elicit a biological response in target cells. Several reports
have proposed that two conservative amino acid regions 30–41 and
120–145 appear to constitute the basic framework of receptor binding
site (14). The site in the N-terminal region would
determine the binding to high-affinity receptors, and the site in the
C-terminal region would influence low-affinity binding to cells
(33, 34). Extensive evidence suggests that this receptor
system is complex, possibly consisting of either multiple receptors or
a multi-subunit receptor (22, 35). The amino acids in the
80–92 region (helix C) may be easily accessible to react with their
appropriate receptors and thus may be important for biological
activity. Dimarco et al. (8) conducted SDM in the C helix
of the IFN molecule. They have proposed a model of IFN-receptor
interaction in which these residues define a potential binding site.
They have reported that mutated amino acids (at position 84, 86, 87,
and 90) of helix C of IFN- faces toward one of the subunits of the
receptor and are part of one of two binding site of IFN-. Korn et
al. (20) and Fish (34) have proposed that
there may be sufficient complexity in the type 1 IFN receptor (IFN-
and - receptor) to account for the differential sensitivities
between IFN-s and IFN- that may be associated with a residue
difference in region 78–95. The IFN sequence 78–95 has been
implicated as influencing species specificity among the murine and
human IFN-s and affecting the differential specificity of action
between human IFN-s and . It is intriguing to speculate that
these residue positions may constitute specific recognition sites
associated with an accessory component of the IFN- receptor. Earlier
studies reported that the replacement of Ser86 of
IFN-4 with Cys increased the antiviral activity on murine cells by
10-fold, but did not affect the antiviral activity on human and bovine
cells (36). Recent studies indicate that the high affinity
binding site for human IFN- is IFNAR2 (2, 13, 37). The binding face is formed by regions A, AB, and E of the
IFNs. Based on the tertiary structure of IFN-, it appears
the helix C, which includes residues 86 and 90, is on the opposite face
of the molecule and is more likely to bind to IFNAR1.
One prominent effect of the IFN-s is their ability to inhibit cell
growth, which has also been suggested to be of major importance in
determining antitumor action. In our earlier studies the
antiproliferative activity of several purified native and hybrid
IFN-s were compared (23). The data showed that HY-1
(IFN-21a (1–75)/2c(76–166)) has a higher antiproliferative
activity than HY-2 (IFN-21a(1–95)/2c(96–166)) on Daudi and Wish
cells, and that these two hybrids differ structurally by only 7 aa in
the 75–95 region. To understand the structural basis for these
different activities we used SDM and CM to create individual specific
amino acid substitutions in this region. CM allows the manipulation of
the IFN- at position 86 with all possible amino acid substitutions
to create several different mutants simultaneously. Our present studies
show that HY-4 has the same antiproliferative activity as HY-2, but
HY-5 like HY-1 has a higher antiproliferative activity than HY-4 and
HY-2 (Fig. 2). The concentration of IFN that inhibits Daudi cell growth
by 50% is 1.5 ng/ml for HY-4, and 0.05 ng/ml for HY-5 (Table I); HY-5
is 30-fold more active then HY-4. These data suggest that amino acid
region 81–95 is important for antiproliferative activity. Further
studies by SDM and CM show that tyrosines at positions 86 and 90 are
important in the antiproliferative activity of human IFN-. Likewise,
substitution of 86Y with a hydrophobic amino acid residue, i.e.,
isoleucine, results in a similar level of antiproliferative activity
and thus can replace Y at this position.
The members of the human IFN- family, due to local
differences in structure, sometimes show distinct properties. Even
minute changes or differences in the primary sequences could be
responsible for a significant variety of biological actions. Our
results indicated that if the Y at position 86 or 90 is replaced, the
biological activity also changes. The Y at position 86 seems to act as
a spacer. The substitution of this residue with positively charged
residue (K) like CM-3, negatively charged residue (D) like CM-1, or
noncharged short chain (A) like CM-4, decreased the antiproliferative
activity by 60-fold (CM-1), 100-fold (CM-3), and 80-fold (CM-4)
compared with the original Y. However, substitution with isoleucine
results in comparable antiproliferative activity to the tyrosine. The
CD spectral analysis shows that the amino acid replacements at position
86 did not appear to change the secondary structure. The biological
activity changes among those mutants cannot be attributed to
conformation changes alone. It is possible that the amino acid residue
at position 86 is involved in the interaction between IFN- and its
receptor. Our competitive binding data using
125I-labeled IFN-2b and
125I-labeled IFN-21a also provide evidence
that demonstrated that the N-terminal region and residues at positions
86 and 90 are involved in IFN- and receptor interaction. Table II shows the binding properties of
IFN- hybrids and mutants. If IFN hybrids and mutants have N-terminal
part from IFN-21a and Y (or I) at either position 86 or 90
(HY-1, HY-5, SDM-1, SDM-2, and CM-2), they compete best for the
125I-IFN-21a binding site on Daudi cells.
However, HY-1, SDM-1, SDM-2, and CM-2 do not have the N-terminal of
2c, but they have Y or I at position 86 or 90, so they compete less
for the 125I-labeled IFN-2b binding site. HY-2
and 4, CM-1, 3, and 4 have no Y at position 86 or 90 and have no
N-terminal part from 2c; they compete least. It is possible that the
differences in the competitive binding results obtained with the
hybrids and mutants can be explained on the basis of the dual binding
face human IFN- and - and their interaction with the IFNAR1 and
IFNAR2 subunits. The fact that human IFN-2c and IFN-21a are poor
competitors for one another might indicate that they possess discrete
docking regions of interaction on the surface of IFNAR2. This is
supported by our results in Figs. 5B and 6B. The
SDM-1- and SDM-2-derived hybrids from HY-4 have the N-terminal 1–75 aa
from 21a, but the majority of the remainder amino acid is 2c
related in structure. These mutants compete strongly for 21a but not
2b. Thus, the A and AB domains appear to account for most of the
binding specificity of these IFNs. In addition, SDM-1 and SDM-2 cause a
significantly increasing affinity shift in the ability of HY-4 to
compete with 21a binding (see Fig. 6B). These changes of
Ser (SDM-1) and Asn (SDM-2) to Tyr make these molecules more like
IFN-2c. These data also suggest that this region may be involved in
binding to IFNAR1 and that these mutants may be able to adopt a more
favorable conformation.
|
s with
the IFN-2c domain at the N terminus compete well with the
125I-labeled IFN-2b binding sites, and all
hybrids and mutants with the IFN-21a domain at the N terminus show
reduced ability to compete with 125I-labeled
IFN-2b binding sites. In contrast, the IFN-s with 21a domain
at N-terminal and that had Y at position 86 or 90 (SDM-1, 2, CM-2, and
HY-5) compete well with 125I-IFN-21a binding
sites. These data suggest that the N-terminal and the residues at
positions 86 and 90 are involved in IFN- receptor binding
interaction. Cutrone et al. have recently defined a IFN2 ligand
binding site on IFNAR1. They reported that five alanine
substitution of hydrophobic residues (132W, 139F, 141Y, 160Y, and 253W)
on the receptor component, IFNAR1, could each decrease ligand binding
by a minimum of 80%. These data suggest that the hydrophobic residues
of the receptor are critical for their ligand interactions
(38). The recent article of Piehler et al.
(37) indicated that the IFNAR2 binding site overlaps the
largest continuous hydrophobic patch on IFN-2. Thus, hydrophobic
interactions may play a significant role stabilizing this interaction
with the charged residues contributing to the rapid association of the
complex. In conclusion, our data demonstrate that amino acid region 81–95 is important for antiproliferative activity, and that the tyrosine or the hydrophobic amino acid, isoleucine, at positions 86 and 90 are important for antiproliferative activity. Finally, positions 86 and/or 90 may be involved in the interaction of the IFN molecule with its receptor.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Renqiu Hu, Division of Therapeutic Proteins, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD 20892. E-mail address: hur{at}cber.fda.gov
3 Abbreviations used in this paper: HY, hybrid; SDM, site-directed mutagenesis; CM, cassette mutagenesis.
Received for publication February 13, 2001. Accepted for publication May 21, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
receptor into mouse cells: cloning and expression of its cDNA. Cell 60:225.[Medline]
/ receptor: characterization and molecular cloning. Cell 77:391.[Medline]
. Biochem. Biophys. Res. Commun. 202:1445.[Medline]
: mapping of a conformational epitope by homologue scanning. J. Immunol. 152:705.[Abstract]
hybrids. Pharmacol. Ther. 66:507.[Medline]
. Pharmacol. Ther. 58:93.[Medline]
-1a that are important for receptor binding and functional activity. Biochemistry 39:2538.[Medline]
. EMBO J. 11:3193.[Medline]
2b revealed by x-ray crystallography. Structure 4:1453.[Medline]
2a determined by heteronuclear NMR spectroscopy in solution. J. Mol. Biol. 274:661.[Medline]
at 2.2A resolution. Proc. Natl. Acad. Sci. USA 94:11813. at 2.1A resolution. J. Mol. Biol. 286:151.
consensus sequence. J. Interferon Res. 12:1.
. J. Biol. Chem. 267:15210.. J. Biol. Chem. 268:12591.21a. J. Interferon Res. 14:s98.
subtypes IFN-conl, IFN-8 and IFN-1 derived from the crystal structure of IFN-2b. Semin. Oncol. 24:S52.
. J. Interferon Res. 12:257.[Medline]
2c and their role in binding to cells. Mol. Immunol. 28:1289.[Medline]
receptor. Pharmacol. Ther. 52:227.[Medline]
enhanced activity on human and murine cells. Antiviral Res. 15:27.[Medline]
This article has been cited by other articles:
|
|
 |
|
  T. Matsumiya, S. M. Prescott, and D. M. Stafforini IFN-{epsilon} Mediates TNF-{alpha}-Induced STAT1 Phosphorylation and Induction of Retinoic Acid-Inducible Gene-I in Human Cervical Cancer Cells J. Immunol., October 1, 2007; 179(7): 4542 - 4549. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. A. Jaitin, L. C. Roisman, E. Jaks, M. Gavutis, J. Piehler, J. Van der Heyden, G. Uze, and G. Schreiber Inquiring into the Differential Action of Interferons (IFNs): an IFN-{alpha}2 Mutant with Enhanced Affinity to IFNAR1 Is Functionally Similar to IFN-{beta}. Mol. Cell. Biol., March 1, 2006; 26(5): 1888 - 1897. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Grace, S. Lee, S. Bradshaw, J. Chapman, J. Spond, S. Cox, M. DeLorenzo, D. Brassard, D. Wylie, S. Cannon-Carlson, et al. Site of Pegylation and Polyethylene Glycol Molecule Size Attenuate Interferon-{alpha} Antiviral and Antiproliferative Activities through the JAK/STAT Signaling Pathway J. Biol. Chem., February 25, 2005; 280(8): 6327 - 6336. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and IFN-
) are used as controls. Results are
expressed as a percentage of growth relative to a control cell culture.
A, CM group. B, SDM group.
C, HY group.
, CM-4.
