Human interleukin-3 (hIL-3) (
)is a multilineage
hematopoietic cytokine acting in the bone marrow to promote the growth
of most lineages of blood cell precursors(1) . Recently,
exogenously administered hIL-3 has shown promise for the clinical
relief of neutropenia and thrombocytopenia induced by cancer
chemotherapy(2, 3) . Sequence homology comparisons of
hIL-3 with other proteins indicate that it is a member of the
hematopoietic cytokine family (4, 5, 6) and
that it adopts a four-
-helix bundle topology (7, 8, 9, 10) . The protein binds to
a receptor comprising at least two nonidentical
subunits(11, 12) . Although the precise nature of
interaction between hIL-3 and its receptor is not known, studies using
site-specific mutants have shed some light on which portions of the
protein are important for
function(8, 13, 14, 15, 16, 17) .
In particular, mutagenesis of the adjacent helices A and D indicate
that these regions are important for interaction with the receptor.
This is similar to the findings for human interleukin-5 and human
granulocyte-macrophage colony stimulating factor, whose receptors share
a common
subunit with the hIL-3
receptor(11, 18, 19) . Other members of the
hematopoietic cytokine family also have important residues in helices A
and D (19, 20, 21, 22, 23) and in
helix C(20, 22, 24, 25) .
In this
paper we have undertaken an extensive mutagenesis of hIL-3 in order to
discover mutants with enhanced proliferative activity and to define
residues necessary for activity. Although alanine scanning mutagenesis
has been successfully used to derive structure-activity
information(20, 26, 27, 28, 29, 30, 31, 32) ,
we chose to perform a more extensive mutagenesis, permitting the
incorporation of any of the possible 19 substitutions(33) .
MATERIALS AND METHODS
Production of hIL-3 and Variants in the Escherichia
coli Cytoplasm
General techniques for manipulation of DNA are
described elsewhere(34) . The hIL-3 gene (35) was
obtained from British Biotechnology (Cambridge, United Kingdom) and
expressed in the cytoplasm of E. coli using pMON5847 (ATCC
68912), which contains the recA promoter and g10-L
ribosome binding site(36) . Human IL-3 and hIL-3 variants were
expressed with an initiator methionine followed by an alanine at the N
terminus of the genes. The initiator methionine was removed in E.
coli by methionine aminopeptidase (data not shown). Production of
hIL-3 in cultures of JM101 cells ((37) ; ATCC 33876) harboring
hIL-3 expression plasmids and examination of total cell protein on
polyacrylamide gels was performed as described previously(34) .
For measurements of the solubility of hIL-3 variants, cell pellets from
induced cultures were lysed by sonication(38) , and the
proteins were fractionated by centrifugation.
Production of hIL-3
Variants
in the Periplasm of E. coli
hIL-3
variants were produced in the periplasm of E. coli JM101
using a secretion vector(39) . These plasmids contained the E. coli araBAD promoter (40) governing expression of
fusions of the LamB signal peptide to hIL-3 variants. Culture growth
was performed as described previously (36) except that the
medium contained 0.2% glycerol in place of glucose. Arabinose was added
to cultures to a final concentration of 0.05% to induce expression from p
, and cultures were grown at 30 °C for a
further 3-4 h.
Osmotic Shock Release of Secreted
hIL-3
from E. coli
The osmotic
shock method used was similar to that of Neu and Heppel(41) . A
1-ml aliquot of cells was harvested by centrifugation for 5 min at
5,400
g. The pellet was resuspended gently by
pipetting in 500 µl of a room temperature sucrose solution (20%
sucrose (w/v), 30 mM Tris-HCl, pH 7.5, 1 mM EDTA).
Following a 10-min incubation at room temperature, the cells were spun
for 10 min at 8,400
g. The sucrose fraction was
carefully removed from the cell pellet and saved. The cells in the
pellets were then resuspended gently by pipetting in 500 µl of
ice-cold water. Following a 10-min incubation on ice, the cells were
spun for 10 min at 14,200
g. The supernatant water
fraction was carefully removed and saved. Equal volumes of the sucrose
and water fractions were pooled and used in the hIL-3 ELISA and AML
cell proliferation assay. Some cultures and fractions were analyzed by
immunoblots (34) using a rabbit polyclonal antibody raised
against an hIL-3
peptide. The level of loading on
the gel was normalized to the densities of the cultures, and protein
corresponding to 4 Klett units was loaded in each lane.
Two-step Site-directed PCR Mutagenesis
Libraries
of all 19 possible single-site variants were constructed at each
position from residues 17-123 in the hIL-3
molecule, using two PCR mutagenesis steps. This two-step approach
facilitated the rapid identification of single amino acid substitutions
using differential DNA hybridization. Libraries at positions
94-105 were created from an intermediate plasmid that contained a
12-base replacement. This approach for the creation of a library at
residue 105 is illustrated (see Fig. 2a). In the first
mutagenesis step, one of the PCR primers replaced codons 102-105
in the hIL-3
gene with 12 bases encoding two
translation stop codons and six additional bases (5`-TAATAAGTCGAC-3`).
The reverse PCR primer was complementary to the 5`-end of the
hIL-3
gene. Plasmid DNA containing the
hIL-3
gene in a secretion expression vector
served as the template in the PCR reaction. PCR mutagenesis (42) was performed using reagent kits, and thermal cycler from
Perkin-Elmer Cetus. Primer extension was carried out using 20 pmol of
each oligonucleotide and 0.5 pmol of template plasmid DNA for 35 cycles
(94 °C for 1 min, 50 °C for 2 min and 72 °C for 3 min). The
PCR-generated DNA product was extracted with phenol/chloroform and
precipitated with ethanol. The DNA was digested with NcoI and EcoRI and ligated into the corresponding restriction sites in
the hIL-3
secretion plasmid. The resulting
``intermediate plasmid'' served as the template in the second
mutagenesis step. The 12-base segment described above was restored with
a PCR primer encoding the parental DNA sequence at codons 102-104
and a 32-fold degeneracy at codon 105. The degenerate oligonucleotides
were synthesized to have G, A, T, or C (in equal proportions) in the
first and second positions and G or C in the third position of the
codon. The reverse PCR primer was complementary to the 5`-end of the
hIL-3
gene. The PCR reaction mixture was
purified as above, digested with NcoI and EcoRI, and
ligated into the corresponding sites of the intermediate plasmid.
Single colonies were selected at random and grown in liquid culture in
a 96-well plate format. Plasmid DNA was prepared from the resulting
cultures in a 96-well format and DNA dot-blot hybridization was used to
distinguish mutants from parental plasmids using a probe specific for
the parental plasmid. This process was repeated in a similar manner to
create libraries of mutants at positions 94-104. A limited number
of positive clones from the hybridization screen were sequenced to
confirm the quality of the libraries.
Figure 2:
Construction of libraries of single-site
mutants using two-step PCR mutagenesis. Using the replacement
mutagenesis approach (a), stop codons were first
inserted in the hIL-3
coding region, and the
coding region was then restored in a second step, using a degenerate
PCR primer. For insertion mutagenesis (b), a segment of
hIL-3
was first deleted, and then restored using
a degenerate PCR primer.
Libraries at positions
17-93 and 106-123 were created using intermediate plasmids
that contained an 18-base deletion. This approach for the creation of a
library at a single position, residue 71, is illustrated (see Fig. 2b). Again, a two-step PCR mutagenesis approach
was used, but in this case, the first mutagnesis step led to an
intermediate plasmid with a portion of the coding region deleted. In
the second step, the mutagenic PCR primer encoded the parental DNA
sequence at codons 72-76 and a 32-fold degeneracy at residue 71.
Similarly, the same intermediate plasmid was used for creation of
libraries at residues 72-76. Other intermediate plasmids carrying
6-codon deletions throughout the remainder of the
hIL-3
(residues 17-22, 23-28,
29-34, 35-40, 41-46, 47-52, 53-58,
59-64, 65-70, 71-76, 77-82, 83-88,
88-93, 106-111, 112-117, and 118-123) were
constructed and used as templates for the remaining libraries.
Mutants from each library were selected at random and identified by
DNA sequencing. In addition, a small number of selected single-site
mutants were constructed by substitution of a portion of the gene with
appropriate synthetic oligonucleotides.
hIL-3 ELISA
Concentrations of hIL-3 variants in
osmotic shock fractions were determined using a sandwich ELISA.
Dynatech Immulon II microtiter plates were coated with
affinity-purified polyclonal goat-anti-hIL-3 (see below) at 1 µg/ml
in 100 mM NaHCO
, pH 8.2. Plates were incubated
overnight at room temperature in a humidified chamber. The plates were
blocked with Dulbecco's phosphate-buffered saline (D-PBS)
containing 3% bovine serum albumin and 0.05% Tween 20, pH 7.4, for 1 h
at 37 °C in a humidified chamber. Plates were then washed 4 times
with 150 mM NaCl containing 0.05% Tween 20. Unknowns were
serially diluted in assay buffer (Dulbecco's phosphate-buffered
saline containing 0.1% bovine serum albumin, 0.01% Tween 20, pH 7.4). A
standard curve of purified recombinant hIL-3 diluted in assay buffer
ranged from 0.125 to 5 ng/ml. Plates were incubated for 2.5 h at 37
°C in a humidified chamber. After four washes, goat anti-hIL-3
conjugated to horseradish peroxidase was added to each plate. Plates
were incubated 1.5 h at 37 °C in a humidified chamber. Plates were
washed 4 times and ABTS peroxidase substrate solution (Kirkegaard and
Perry Labs, Gaithersburg, MD) was added. The plates were read at a test
wavelength of 410 nm and a reference wavelength of 570 nm on a Dynatech
microtiter plate reader (Chantilly, VA). Concentrations of hIL-3 in
unknown samples were calculated from the standard curve using software
supplied with the plate reader. To raise polyclonal antibodies to
recombinant hIL-3, goats were initially immunized with 3 mg of purified
recombinant hIL-3 in complete Freund's adjuvant. Each received
two monthly boosts of 1 mg in incomplete Freund's adjuvant.
Thereafter, each goat was given monthly boosts with 0.25 mg of hIL-3 in
incomplete Freund's adjuvant. Blood was collected 10 days after
each boost and allowed to coagulate at room temperature. Serum was
separated from whole blood by centrifugation, sterile filtered, and
stored at -70 °C. Antibody titers were verified prior to
purification using Ouchterlony immunodiffusion. Goat polyclonal
antibodies to hIL-3 were purified from serum using an affinity
chromatography column of 10 mg hIL-3 coupled to 1 ml Affi-Gel 10
agarose beads (Bio-Rad). Purified antibody was conjugated to
horseradish peroxidase for use in the ELISA assay.
AML193.1.3 Cell Proliferation Assay for
hIL-3
AML193 cells (43) were obtained from the ATCC.
The cells were adapted for long term growth in hIL-3 by starving for
growth factor (granulocyte-macrophage colony stimulating factor) for 24
h and replating in media containing hIL-3 (100 units/ml, Amgen,
Thousand Oaks, CA). After 2 months, the cells could grow rapidly in
hIL-3. These cells were designated AML193.1.3 and were maintained in
Iscove's modified Dulbecco's medium (Life Technologies,
Inc.) supplemented with 5% fetal bovine serum, 50 µM 2-mercaptoethanol and 4 ng/ml hIL-3. AML193.1.3 proliferation in
response to hIL-3 or hIL-3 variants was determined by
[
H]thymidine incorporation. Cells were seeded at
2.5
10
/well in 96-well microtiter plates (Costar,
Cambridge, MA). Human IL-3 samples were added in Iscove's
modified Dulbecco's medium supplemented with 500 µg/ml bovine
albumin, 100 µg/ml human transferrin, 50 µg/ml soy bean lipids
(all from Boehringer Mannheim) and 50 µM
2-mercaptoethanol. After 72 h, 0.5 µCi of
[
H]thymidine was added to each well. After
16-24 h, the cells were harvested onto a glass fiber filter mat
(Pharmacia Biotech Inc.) and counted in an LKB 1205 Betaplate
scintillation counter (Bromma, Sweden). Osmotic shock fractions with
hIL-3
accumulation levels greater than 1
µg/ml were tested in the AML193.1.3 bioassay, and the concentration
of hIL-3
variants that gave 50% of maximal
proliferation was determined. Each assay included secreted
hIL-3
as an internal standard and was performed
1-7 times. All activity values were expressed relative to this
control. Purified protein variants were assayed at least 3 times, and
EC
values were calculated by fitting a four-parameter
logistic model to the data.
Purification of hIL-3
Inclusion bodies from E.
coli cell pellets were isolated by sonication in 10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl, and 0.1
mM phenylmethylsulfonyl fluoride followed by
centrifugation(38) . One gram of inclusion body pellet was
extracted for 15-30 s with 5 ml of 6 M guanidine HCl, 50
mM CHES, pH 9.5, 20 mM dithiothreitol, using a
Bio-Homogenizer. The solution was gently rocked for 2 h at 5 °C and
dialyzed (1,000 molecular weight cut-off) overnight at 5 °C against
100 volumes of 4 M guanidine HCl, 50 mM CHES, pH 8.0.
Dialysis was repeated against 2 M guanidine HCl, 50
mM CHES, pH 8.0. The protein solution was acidified by the
addition of an equal volume of 40% acetonitrile (CH
CN),
0.2% trifluoroacetic acid. After clarification by centrifugation
(16,000
g for 5 min), the supernatant was loaded onto
a Vydac C-18 reversed phase column (10
250 mm), equilibrated in
20% CH
CN, 0.1% trifluoroacetic acid. The column was eluted
at 3 ml/min with a linear gradient (0.2% CH
CN/minute)
between 40 and 50% CH
CN, 0.1% trifluoroacetic acid.
Fractions were analyzed by SDS-polyacrylamide gel electrophoresis, and
the appropriate fractions were pooled and dried by lyophilization or in
a Speed Vac concentrator. The dry powder was reconstituted with 10
mM ammonium bicarbonate, pH 7.5, and clarified by
centrifugation (16,000
g for 5 min). The amino acid
composition and concentration of purified proteins were determined from
samples that had been subjected to acid hydrolysis (6 M HCl,
evacuated sealed tubes, 90 min, 150 °C). All analyses were
performed after postcolumn derivatization of the hydrolysates using
ninhydrin as modified from (44) . A Beckman model 6300
Autoanalyzer was employed for the actual determinations. All proteins
were recovered at a purity of >90%, as determined by densitometric
scanning of Coomassie-stained SDS-polyacrylamide gels.
RESULTS
N- and C-terminal Deletions of hIL-3 Define a Core
Required for Full Activity
Deletions of the N and C termini of
hIL-3 were constructed in order to determine the minimal protein
required for full activity. The variants were expressed in the
cytoplasm of E. coli, refolded in vitro,purified, and tested in the AML193.1.3 cell proliferation
bioassay. As shown in Table 1, residues 15-118 were
required for full activity, while C-terminal deletions extending beyond
residue 118 retained less than 1% activity. Our data on the minimal
size of a bioactive hIL-3 molecule differ somewhat from those of
Dorssers et al.(14) who found that
hIL-3
retained approximately 10% bioactivity.
Interestingly, we routinely found that variants lacking residues
119-133 had a 2-3-fold increase in activity.
Human IL-3
produced in the cytoplasm of E. coli accumulated as insoluble
inclusion bodies and was recovered in the cell pellet (see Table 1). During the course of our deletion analysis of hIL-3, it
was found that the deletion of the last eight amino acids (TTLSLAIF)
resulted in approximately 50% of the hIL-3 variant fractionating in the
cell supernatant rather than the cell pellet. The reason for this
apparent change in solubility is unclear, but it might be due to the
removal of a number of hydrophobic amino acids.
Secretion of hIL-3
in E.
coli
Although refolding and purification of cytoplasmically
produced hIL-3 variants was suitable for testing a small number of
proteins, it was too cumbersome for an extensive mutagenesis study. We
have previously demonstrated the utility of secretion in E. coli as a route for generating soluble, correctly folded recombinant
bovine somatotropin(45) , and therefore tested whether hIL-3
could expressed as a secreted protein when fused to the E. coli LamB signal peptide. The truncated protein,
hIL-3
, could be expressed using this vector (Fig. 1a). Efficient signal processing of the LamB
signal peptide was observed, releasing hIL-3
,
which was soluble and could be extracted from the periplasmic space by
osmotic shock. Accurate processing of the signal peptide was confirmed
by N-terminal sequence analysis. As shown in Fig. 1, b and c, the secreted hIL-3
had
equivalent activity to cytoplasmically-produced
hIL-3
, which had been refolded, purified, and
accurately quantified. Hence, secretion provided a simple route for the
production of correctly folded hIL-3
and
provided a critical tool for the extensive mutagenesis of hIL-3.
Figure 1:
Secretion of
bioactive hIL-3
. a, immunoblot analysis
of secretion of hIL-3
from E. coli. Cultures were induced and proteins fractionated as described under
``Materials and Methods.'' Loading in lanes1-6 was normalized to culture density prior to
sample preparation (4 Klett units loaded). Lane 1, negative
control, JM101 cells 3 h post-induction; lane2,
whole cell pellet of JM101 expressing secreted
hIL-3
, prior to induction; lane3, whole cell pellet of JM101 expressing secreted
hIL-3
, 3 h post-induction; lane4, osmotic shock water fraction of secreted
hIL-3
, 3 h post-induction; lane5, sucrose fraction; lane6, remaining
cell pellet after osmotic shock; lane7, 50 ng
purified hIL-3
standard, denoted by the arrow. b, AML193.1.3 proliferation assay of purified
hIL-3
and purified hIL-3
.
Purified proteins were quantified by amino acid composition. c, AML proliferation assay of crude, secreted
hIL-3
. Proteins were released from E. coli by osmotic shock and assayed for bioactivity. hIL-3 immunoreactive
material was quantified by ELISA.
Construction and Screening of Libraries of Single-site
Mutants of hIL-3
An extensive
site-directed mutagenesis of the essential portion of hIL-3 was
undertaken. At the outset of this study, we wished to avoid any
preconceived ideas about which residues would be important for
biological activity and therefore constructed a series of 105 libraries
of single-site mutants at most positions in the hIL-3
molecule (Fig. 2). Approximately eight mutants were
selected at random from each library, and expression of the mutants in E. coli was induced by the addition of arabinose. After
extracting the proteins by osmotic shock, the level of hIL-3 variant
was measured by ELISA. Typically, secreted hIL-3
could be recovered from osmotic shock fractions of E. coli at a level of about 5-10 µg/ml. As illustrated in Fig. 1c, the secreted hIL-3
showed a biphasic dose-response relationship, with reduced
proliferative activity at concentrations above approximately 100
pM. This effect was due to the presence of inhibitory material
in the osmotic shock fraction, which was manifested at low sample
dilutions (data not shown). Consequently, the bioassay screen was not
used to measure potency when the concentration of hIL-3 variant was
<1 µg/ml. Approximately 20% of the mutants were recovered at
this level. The results of the 770 mutants characterized in this study
are presented in Table 2. Substitutions were divided into three
activity classes: full (
20% specific activity of native
hIL-3
), moderate (5-19% specific activity)
and low (<5%).
Most Residues in hIL-31
Were
Tolerant of Substitution
A representation of the
structure-function relationship for hIL-3
is
shown in Fig. 3. As a rule of thumb, residues were considered
tolerant of substitution if at least three substitutions were tested
and found to have
5% bioactivity of the parental
hIL-3
. 71% of residues tested fell into this
category. Residues considered to be intolerant of substitution had no
more than one mutant with
5%, but at least two mutants with <5%
activity. 16% of residues tested fell into this category. The
assignment of 14 residues in hIL-3
is uncertain,
either because mutants were not recovered or because the accumulation
level of the variants was low. It is likely that residues 36-40
are not necessary for biological activity, because a hybrid protein
containing the corresponding murine amino acids had substantial
activity.
Residue Asn-70 is not likely to be important for
receptor binding since it can be N-glycosylated in active
hIL-3 produced in mammalian cells.
Figure 3:
Relationship between predicted secondary
structure of hIL-3
and bioactivity of
single-site mutants. Predicted helical regions (7, 47, 48) are indicated. !, intolerant of
substitution; %, partially tolerant of substitution; ., not necessary
for activity; ?, insufficient data; +, site of mutant(s) with
increased activity; c, residue predicted to be on the
hydrophobic side of an
-helix; h, predicted
-helical
region. The predicted helical regions are similar to those recently
identified from the NMR structure of a highly-substituted hIL-3
variant(60) .
Mutants with Increased Activity
Approximately
1-2% of the single-site mutant proteins appeared to have elevated
activity in the screening assay relative to that of
hIL-3
(data not shown). In order to validate
these results with purified proteins, 16 mutants were selected (see Table 3and Fig. 3). The mutant hIL-3
genes were transferred to a cytoplasmic vector, permitting the
proteins to be produced at high levels in inclusion bodies. The
variants were then refolded in vitro, purified, and
quantified by amino acid analysis. This approach eliminated the
possible artifacts that might have arisen out of the screening
approach. All 16 purified proteins showed at least 5-fold increased
activity. Remarkably, K116W was 26-fold more active than native hIL-3.
DISCUSSION
We have measured the effect of 770 amino acid substitutions
on the cell proliferative activity of hIL-3
.
Most surprising was that single-amino acid substitutions at 12
positions in hIL-3
resulted in at least 5-fold
increased cell proliferative activity (see Table 3and Fig. 3). It seems unlikely that these 12 residues make essential
hIL-3 receptor contacts, since all of the positions were highly
tolerant of substitution or, in the case of Gln-122, could be deleted
without loss of cell proliferative activity (cf. Table 1). Furthermore, two different substitutions with increased
activity were found at positions 34, 42, 45, and 116. It is interesting
to note that two critical residues, Glu-43 and Asp-44, are flanked by
residues where substitutions with increased activity were observed
(positions 42, 45, and 46). Perhaps mutations with increased activity
function by providing an improved context for critical residues.
Increased potency could result by bringing essential contact residues
into more favorable alignment with the receptor by reducing steric
hindrance through moving unfavorable residues away from the receptor or
by having an effect on the flexibility of the ligand. For most of the
variant proteins tested, increased bioactivity correlated with
increased affinity for the hIL-3 receptor. (
)
Only 16
residues of hIL-3 were identified that were critical for activity (see Fig. 3). Five of these critical amino acids, Glu-22, Glu-43,
Asp-44, Arg-94, and Lys-110 are charged residues that are candidates
for contact with the receptor. Our interpretation of the role of these
amino acids is consistent with previous reports (8, 14, 15, 17) and supports the
model that electrostatic interactions have been proposed to be
important for ligand-receptor interactions of the hematopoietic
cytokines(4, 49) . Most of the other residues
interpreted to be critical for activity of hIL-3 are nonpolar (Leu-27,
Leu-48, Leu-53, Leu-58, Phe-61, Ala-64, Leu-81, and Leu-115).
``Helical wheel'' (7, 50) projections
predict that all but residue Leu-81 are on the hydrophobic faces of
amphipathic
-helices. It is likely that these nonpolar residues
are in the interior of the protein and do not interact directly with
the receptor. Substitution of these residues could result in disruption
of the structure of hIL-3 and hence render the protein
inactive(21, 51, 52) . Other nonpolar
residues, Ile-20, Ile-23, Ile-24, Ile-47, Met-49, Leu-68, Ile-97, and
Ile-99 were found to be partially tolerant of substitution. At some
positions, 23, 24, 97, and 99, only other nonpolar residues were
identified in active hIL-3
variants.
Interpretation of the effect of substitutions at residues Phe-107,
Leu-111, Tyr-114, and Leu-118 is problematic since few mutants were
expressed at sufficient levels for assay. However, all four residues
are located on the predicted hydrophobic face of helix D and therefore
seem unlikely to be involved directly in interaction with the receptor.
It should be noted that atoms in the protein backbone can also interact
with the receptor(24, 53) .
We have not determined
the nature of the expression defect seen for approximately 20% of the
mutants. However, it is tempting to speculate that several of these
single-site mutants might cause a disruption in protein structure that
could result either in aggregation, increased susceptibility to host
proteases and/or faulty recognition by the host secretion
machinery(54) . Examination of Table 2and Fig. 3suggests that substitution of residues predicted to lie in
the hydrophobic core of the protein can often lead to a reduction in
accumulation. This would be consistent with a disruption in protein
folding, as has been observed for other
proteins(21, 51, 52, 55) . 12
hIL-3
mutants with levels of recovery slightly
below 1 µg/ml (indicated by in Table 2) were assayed for
proliferative activity. In all cases, the mutants had low activity,
supporting the notion that the phenotype of low expression can provide
useful biological information. We have not undertaken physical studies
(such as CD spectroscopy) of poorly expressed mutants to determine
whether substitutions do indeed disrupt protein folding or thermal
stability. Tryptophan, lysine, and cysteine substitutions were strongly
overrepresented in the low expression category (
analysis not shown). In the case of tryptophan, it is possible
that the presence of the bulky side chain could result in a disruption
of protein structure. The reason for overrepresentation of lysine is
unknown. The overrepresentation of cysteine in this class of mutants
might be a consequence of aberrant disulfide bond formation and/or
protein dimerization(21) . Cysteine substitution did not always
disrupt the protein, since we observed several examples of cysteine
substitutions retaining considerable activity (at positions 41, 42, 46,
56, 104, and 122).
Our choice of mutagenesis was more extensive than
two other commonly used approaches, alanine
scanning(20, 26, 27, 28, 29, 30, 31, 32) and
charge
reversal(14, 19, 22, 56, 57) .
In general, the 46 alanine substitutions recovered in our random
mutagenesis support the value of alanine scanning mutagenesis. Seven
alanine substitutions gave low accumulation levels, so bioactivity
could not be determined. At 36 positions, the conclusions from alanine
substitutions we recovered agree with the interpretation from the
substitutions of other amino acids at those positions. However, at
residues Ser-67, Leu-115, and Asn-120, alanine substitution did not
have the same effect as substitution of other amino acids. Hence,
alanine scanning would have led to false interpretation about the
importance of these residues. Moreover, it should be stressed that
alanine scanning would not have identified most of the mutants with
enhanced proliferative activity. An alternative mutagenic approach is
charge
reversal(14, 19, 22, 56, 57) ,
in which charged residues are substituted with those with the opposite
charge to test the effect on activity. The assumption is that
electrostatic interactions may be an important element in
ligand-receptor interaction (4, 49, 58) . In
our study, results from three charge reversal substitutions D21K, R54D,
and E119K would have led to a conclusion that was in disagreement with
that from our larger data base. Consequently, applying a charge
reversal scanning mutagenesis strategy to hIL-3 would have led to a
limited and potentially misleading interpretation.
Our mutagenesis
work led to the identification of 770 amino acid substitution mutations
in hIL-3
. The catalogue of tolerated amino acid
substitutions generated in this study permits the construction of
highly-mutated hIL-3 variants(59) . The solution NMR structure
for one highly substituted variant of hIL-3 (SC-65369) has recently
been solved(60) . This structure should provide an excellent
tool for refining the interpretation of the mutant data base presented
in this paper. The random mutagenesis approach we have employed is an
effective way to discover variants of a protein hormone with increased
activity, particularly in cases where little structural information is
available. The relatively large number of mutations of
hIL-3
with elevated cell proliferative activity
was unexpected, considering that the potency of hIL-3 is already high
(EC
= 10-100 pM). Perhaps the
optimal in vivo properties of hIL-3 do not include the maximal
affinity for its receptor. We anticipate that mutants of other protein
hormones may also have considerably increased activity. If so, this may
be important for the development of clinically superior molecules.