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Research Communications |
a Cell Engineering Facility GBB, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands; and
b N.V. Organon, P.O. Box 20, 5340 BH Oss, The Netherlands
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Key Words: heterologous protein expression • follicle-stimulating hormone • infertility
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The gonadotropins human choriogonadotropin
(hCG),2
luteinizing hormone (LH), and
follicle-stimulating hormone (FSH) regulate the cellular and endocrine
function of the reproductive organs (4)
. These glycoproteins consist of
two subunits (
and ß) that are associated by hydrogen bonds and
hydrophobic contacts. The -subunit of the gonadotropins is identical
for a given species, whereas the ß-subunits are different and are
responsible for the receptor specificity. The glycosylation
(N-linked; for hCG, also O-linked) is important
for the folding, secretion, and stability (5)
of these proteins and
also influences their in vivo behavior. Since the
biosynthesis of the gonadotropins is a very complex process that also
involves so-called cystine knot formation (6)
and
attachment/modification of oligosaccharide side chains (7)
, only
eukaryotic cells can be used to express these glycoproteins. Thus, the
recombinant gonadotropins used clinically (rec FSH, rec LH) have been
expressed in Chinese hamster ovary (CHO) cells (3)
.
Despite the efforts of many research groups over two decades, we estimate that fewer than 500 gonadotropin mutants have been reported in the scientific literature. Although these site-directed mutagenesis studies have led to the identification of structural determinants for receptor binding and bioactivity, there are still many issues to be addressed. Perhaps even more important, there is an ongoing clinical need for novel gonadotropin drugs like LH antagonists (to treat ovarian hyperstimulation syndrome) for which there are no candidates (8) . It may be anticipated that random mutagenesis approaches in combination with high throughput screening may lead to the generation of such compounds.
However, due to the limited number of mutants that can be generated, mammalian cells such as CHO cells are not suitable for random mutagenesis of protein domains. In addition, the use of CHO cells is expensive and labor intensive. The soil amoeba Dictyostelium discoideum is an organism that provides an attractive alternative for heterologous expression of the human glycoprotein hormones 9, 10) . Though it can be grown and transformed with the same ease as the yeast Saccharomyces, it has some complex features that resemble mammalian cells, such as glycosylation and chemotaxis. Furthermore, it has been shown that Dictyostelium provides a useful system for random mutagenesis approaches (11) . Several heterologous proteins have been successfully expressed in Dictyostelium, such as the human muscarinic receptor (12) , the rat GLUT 1 glucose transporter (13) , and human antithrombin III, which could be recovered from growth medium (14) . Very recently, we have demonstrated that Dictyostelium is also able to express a single-chain variant of hCG (15) , which shows bioactivity albeit somewhat less than CHO-produced material (16) .
In this paper we explain that heterodimeric hCG and FSH can be
expressed by Dictyostelium cells and that these secreted
hormones display bioactivities of the same order as the corresponding
CHO-generated gonadotropins, suggesting that the - and ß-subunits
are folded and assembled correctly. We selected two amino acid
positions of hCG (ß94, ß95) that are part of an established hot
spot (the so-called `determinant loop') 17, 18)
of hCG and generated
a sample of 87 random mutants. We studied the immuno- and bioactivity
of these mutants and found mutants with widely varying properties that
provide new insights into the structure-function relationships. Since a
complete generation testing cycle of several thousands of mutants can
be completed within 2 months, this approach may allow the
identification of variant forms of hCG that are of therapeutic
interest.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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- and ß-subunits of the gonadotropins is
essentially identical to that of MB12n, which has been described
previously (16)
. In short, each subunit was amplified from cDNA
containing plasmids by using the appropriate 5' and 3' primers to
generate a DNA fragment that is cloned in a Dictyostelium
extra-chromosomal expression vector (16)
. To facilitate expression of
two independent plasmids in Dictyostelium, we required two
plasmids with different selection markers. For this purpose, we
replaced the blasticidin resistance cassette in MB12n with the 2.4 kb
KpnI-XbaI fragment from p155d1 (19)
containing a
neomycin cassette, creating MB12neo. For construction of the expression
vector for the -subunit, its natural cDNA sequence, including the
mammalian secretion signal sequence, was produced by polymerase chain
reaction (PCR) by using primers that introduce a BglII
restriction site both at the 5' and 3' end of the fragment, so that it
could be cloned in MB12neo. For construction of the expression vector
for the ß-subunit of hCG, MB12n was modified to contain another
unique restriction site (SphI) 3' of the BglII
cloning site (16)
. About 40% of the codons in the ß-subunit of CG
are infrequently used in Dictyostelium (20)
. To limit
possible problems in mRNA translation at the start of the open reading
frame, the first 30 bases of the leader sequence of the ß-subunit
were altered conform to Dictyostelium preferred codon usage
(16)
. The hCG ß-subunit cDNA was amplified using a 5' primer (16)
,
resulting in alteration of the first 30 bases of the coding sequence
conform the Dictyostelium preferred codon usage. The primers
also introduced appropriate restriction sites at both the 5'
(BglII) and 3' (SphI) end of the fragment to
facilitate directional cloning. To construct the expression vector for the ß-subunit of FSH, the cDNA was amplified using a 5' primer resulting in the alteration of the first 27 bases of the coding sequence conform the Dictyostelium preferred codon usage [CGACTC ACTATAGGGC AGATCTCACC ATG AAG ACT CTC CAA TTT TTC TTC CTC TTC TGT TGC TGG], and cloning was performed as described for the ß-subunit of hCG. All DNA sequences were confirmed by using an automated sequencer.
PCR mutagenesis and mutant library construction
Specific base substitutions were introduced by site-specific
mutagenesis and combining PCR fragments that overlap in sequences, as
described, by using standard PCR conditions (21)
. The primers were
designed to alter amino acids 94 and 95 of the ß-subunit of hCG. The
first two nucleotides of both codons were altered fully randomly (A,C,T
or G), whereas the third base was restricted to G or T to minimize the
introduction of stop codons. After PCR and subcloning in the
BglII and SphI sites of pCR 2.1 (Invitrogen,
Leek, The Netherlands), the pool of constructs was transformed to
Escherichia coli. Subsequently, DNA was isolated from a pool
of 400 transformants and, after restriction digestion, the
BglII/SphI mutated fragments were subcloned in
MB12n containing the BgIII and SphI site (see above).
Expression of recombinant hormones
After plating E. coli transformants of MB12 plasmids
containing the random mutant fragments, 400 colonies were pooled, DNA
was prepared, and Dictyostelium was transformed by
electroporation as described (16)
. Selection with blasticidin (10
µg/ml) was introduced 5 h after electroporation. The next day,
cells were clonally diluted in 96-well plates using fourfold dilutions
and neomycin selection (10 µg/ml) was added. In total, sixteen
96-well plates were used. Medium was replaced every 3–4 days,
maintaining selective conditions. Positive wells were identified 11–14
days after electroporation, and the transformation efficiency was
estimated from the dilution series. Typically, ~500 transformants
were obtained by electroporation of 107 cells
with 1 µg of both the hCG and hCG ß vectors.
Transformants from single wells were then selected for further
experiments. A single well contains 200 µl of medium. Larger amounts
of media for dose-response in vitro analysis were harvested
from 10 cm culture dishes. Concentrations of wild-type and mutant hCG
were measured using a DELFIA hLH assay (Wallac Oy, Turku, Finland),
which has a 100% cross-reactivity with hCG, as described by the
manufacturer. FSH was quantified by a sandwich immunoassay as described
previously (22)
. Both assays are based on the enzyme-linked immunoassay
principle, with a solid-phase anti-ß chain antibody and a soluble
anti- chain antibody. In the DELFIA assay, 15,000 units is
equivalent to ~1 mg hCG protein; in the FSH assay, 10,000 units is
equivalent to ~1 mg FSH protein.
In vitro bioassay
In vitro bioactivity was determined on either the
human FSH receptor or the human LH/CG receptor, as described previously
(5)
. In short, CHO cells expressing human HL/CG or FSH receptors also
contain a reporter construct with luciferase driven by a cAMP
responsive element. On exposure of the cells to the hormone, the
reporter gene is activated through the receptor-mediated signal
transduction pathway and luciferase activity is measured (5)
. Highly
purified, recombinant gonadotropins produced by CHO cells were used as
standards.
Sequence analysis of the hCG mutants
Total DNA of several selected Dictyostelium clones
was isolated and used to transform E. coli. For each
Dictyostelium clone, a number of transformants was analyzed
by colony PCR for the presence of the -subunit plasmid or the
ß-subunit plasmid. Subsequently, DNA was isolated from several
transformants containing the ß-subunit plasmid and sequence analysis
was performed on the mutated region by using an automated sequencer
(Pharmacia, Piscataway, N.J.).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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showsan overview of the different steps involved in selection of
gonadotropin analogues by random mutagenesis in conjunction with a
screen for immuno- and biological activity. The first step in a random
mutagenesis approach is the selection of a protein domain for analysis.
Based on the X-ray structure of hCG 23, 24)
combined with
site-directed mutagenesis studies, we selected part of the so-called
`determinant loop' for construction of the mutant library. Since this
region of hCG has been demonstrated to be involved in receptor binding
and signal transduction (18)
, we expected to generate a set of
gonadotropin analogues with altered biological profiles. The X-ray
structure of hCG is shown at the top of Fig. 1
(Protein Data bank code
1hcn) in which the selected amino acids ß94 and ß95 are shown in
red.
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Gonadotropin expression in Dictyostelium
A major obstacle to overcome was the functional expression
of gonadotropins in Dictyostelium. For the expression of the
- and ß-subunit of hCG, two expression plasmids were generated.
The first construct contained the -subunit of hCG together with the
neomycin gene as a selectable marker, whereas the second construct
contained the hCG ß-subunit with the blasticidin gene as a selectable
marker. The two wild-type expression plasmids were transformed
simultaneously to Dictyostelium. After transformation, cells
were plated and clonal transformants that express both selection
markers were identified and further grown for analysis. The amount of
hCG secreted by Dictyostelium was determined by a sandwich
immunoassay that indicated that heterodimeric, immunologically active
hCG was produced by Dictyostelium (200-400 mU/ml), which is
equivalent to ~13-26 ng/ml. This amount is produced in 4-5 days in a
petri dish with ~106 cells per ml of medium.
For comparison, the expression level of wt hCG in CHO cells is
800mU/106 cells per 24 h (22)
.
We also studied the production of FSH by Dictyostelium. In line with the strategy for hCG (see Materials and Methods), two expression plasmids were generated and transformed simultaneously. An FSH specific sandwich immunoassay demonstrated that immunologically active FSH was produced by Dictyostelium (200–400 mU/ml, ~20-40 ng/ml). We expressed each subunit by using their own signal peptide, confirming our earlier observation that mammalian signal sequences are able to facilitate secretion of these proteins in Dictyostelium (16) .
Biological activities of gonadotropins produced by
Dictyostelium
Although the presence of heterodimeric hCG was demonstrated in the
medium by means of epitope detection, additional experiments were
necessary to establish whether hCG produced by Dictyostelium
is biologically active. The bioactivity of heterodimeric hCG from
Dictyostelium was analyzed by examination of its ability to
activate the human LH/CG receptor in a luciferase reporter assay. The
results demonstrate that the heterodimeric hCG produced by
Dictyostelium is able to activate the human LH/CG receptor
(Fig. 2
A). Moreover, its bioactivity is comparable
(IC50 value approximately twofold higher) to the
bioactivity of wild-type hCG produced by CHO cells. This suggests that
the overall conformation of heterodimeric hCG produced by
Dictyostelium is comparable to the material produced in CHO
cells. Heterodimeric FSH produced by Dictyostelium is also
able to activate the human FSH receptor (Fig. 2B
).
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The gonadotropins probably belong to the most complex glycoproteins produced by mammalian cells. It is axiomatic that gonadotropin subunit assembly is vital for the biological activity of the hormones. Furthermore, correct pairing of the intrachain disulfide bonds is essential for the conformation of the subunits and assembly. Finally, both hCG and FSH contain four N-linked sugars that are essential for intracellular folding and transport, as well as for their biological activity. Thus, we conclude that Dictyostelium contains an extensive set of proteins involved in assisted folding and glycosylation. The fact that Dictyostelium is able to perform all posttranslational modifications necessary for the proper folding and bioactivity of gonadotropins suggests that this organism is probably also capable of folding a variety of other complex mammalian glycoproteins.
Random mutagenesis of a selected region of hCG
Since Dictyostelium was found to be capable of
producing biologically active gonadotropins, the construction of the
mutant library became feasible. Using site-directed mutagenesis, six
random base substitutions were introduced in the codons for amino acids
94 and 95 in the expression vector for the hCG ß-subunit.
Simultaneously with this pool of altered expression vectors for the
ß-subunit of hCG, the expression vector for the -subunit was
transformed to Dictyostelium (see Fig. 1
for schematic
overview). After transformation and serial dilution in 96-well plates,
clonal transformants were selected and analyzed.
Initial screen for bioactivity
To identify interesting clones, we screened supernatants of the
Dictyostelium clones for the presence of immuno- and
bioactivity (see lower part of Fig. 1
). As controls, wild-type hCG
producing Dictyostelium clones and cells transformed with
control plasmids were present on the 96-well plates (9 wells in total).
The amount of hCG produced by different Dictyostelium clones
varied considerably. The relatively high variation in production levels
is most likely due to differences in growth rate and/or production
levels between the individual clones. However, by calculating the
biological activity per amount of immunological active protein, it is
possible to eliminate the influence of this variation in the assay.
Nevertheless, since each activity in this random mutagenesis screen is
determined by single-point measurement, some variation and scatter in
the data can be expected, as demonstrated by the ratio of biological
and immunological activity (B/I) of three individual wild-type
hCG-producing clones (Fig. 3
, open bars). As expected, all Dictyostelium clones that were
transfected with a control plasmid showed no immuno- or biological
activity (not shown).
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The results of a typical mutant screen are displayed in Fig. 3
. The
in vitro bioactivity of individual clones is depicted as a
function of their immunological activity. Of the 87 mutants analyzed,
12 clones do not show any hCG production (these clones are not
presented in Fig. 3
). This is probably due to interference of the
altered amino acids with appropriate folding of the mutated
ß-polypeptide and/or association with the -subunit. The value of
the three wild-type hCG-producing clones is in the same range
(0.42-0.79). About half of the remaining 75 mutants show B/I ratios
that are not significantly different from wild-type hCG produced by
Dictyostelium. One of the clones (#78, Fig. 3
) shows an
increased B/I ratio of ~1.9. Remarkably, ~40% of the mutants show
significantly decreased B/I ratio's. This indicates that a significant
number of mutant proteins show decreased biological activity compared
with wild-type hCG, suggesting that amino acids 94 and/or 95 of the
ß-subunit are indeed involved in receptor binding and/or signal
transduction. From the 75 hCG-producing clones, we selected 18
individual clones with varying B/I ratios for detailed analysis.
Bioactivity and sequence analysis of selected mutants
The 18 selected clones were further grown on 10 cm plates until
confluence and cell culture supernatants were collected after 4
additional days for detailed analysis of the mutants (see bottom part
of Fig. 1
). Of the 18 clones, 4 did not produce any detectable hCG,
both in an immuno- and an in vitro bioassay. This is in
agreement with the results obtained in the initial screen. Among the
other clones, the amount of hCG produced varied considerably (116-2118
mU/ml). Since all clones were grown until confluence, the difference in
their production levels can only be explained by differences in
assembly and/or secretion of the mutants. This is not unlikely, as the
determinant loop is also known to be involved in assembly of the -
and ß-subunit of hCG (25)
.
To identify the mutations that are responsible for biological
activities of the selected mutants, we isolated total DNA from selected
Dictyostelium clones and transformed it to E.
coli. First, E. coli transformants were analyzed by PCR
for the presence of an - or a ß-subunit containing expression
vector. Subsequently, sequence analysis of ß-subunit containing
plasmids was performed on multiple E. coli colonies (on
average, 5.2 E. coli clones per Dictyostelium DNA
isolation). In 15 of 18 Dictyostelium DNAs tested, a single
sequence was detected in all E. coli colonies. These
findings indicate that in the majority of cases the gonadotropin
expressed by the Dictyostelium clones is encoded by a unique
mutant sequence in the hCGß expression vector. In addition, the
sequence data show no obvious sequence bias indicating that the
mutagenesis has been random.
The cell culture supernatants were analyzed for the presence of
in vitro biological activity of hCG at a range of
concentrations based on their immunological activities. The
dose/response curves for nine of the mutants are shown in Fig. 4
. Most mutants display a decreased biological activity compared with
wild-type hCG. This result could be anticipated, since amino acid ß94
and ß95 are involved in receptor binding and/or signal transduction.
However, to our surprise, the mutant with a Pro-Ser sequence showed the
same bioactivity as wild-type hCG, illustrating that the effect of
different mutations on the biological activity of the hCG analogies
cannot be predicted beforehand on the basis of structure/function
relationships. Another interesting observation is that one of the
mutants with a large charge alteration (Arg-Arg to Gly-Asp) showed an
unexpected high bioactivity as compared with wild-type (Fig. 4)
(26)
.
It also illustrates that popular techniques like Ala scanning of
regions in order to determine the `hot' amino acids may lead to too
simple views on which amino acids are essential for receptor binding
and/or bioactivity.
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In a random mutagenesis screen, it is important to check that the first
high throughput assay is robust and representative for most samples.
The Arg-Leu mutant (#78, Fig. 3
), which showed an increased B/I ratio
in the initial screen, now displayed wild-type activity. Probably one
or both activities of this mutant were not measured accurately in the
initial screen. On the other hand, all mutants that showed B/I ratios
in the range of wild-type hCG in the initial screen also displayed a
wild-type in vitro biological activity when analyzed in more
detail. Furthermore, all mutants with decreased B/I ratios in the
initial screen indeed show a clear increase in
IC50 in the more extensive biological activity
assay. Their B/I ratios varied from a 2- to >100-fold lower than
wild-type hCG produced by Dictyostelium. Thus, the initial
screen is sufficient to identify the clones that display decreased B/I
ratios, as the majority of clones give rise to comparable results when
analyzed in more detail. As discussed earlier, some variation and
scatter cannot be excluded, since in the initial screen both
immunological activity and in vitro biological activity are
determined by single-point analyses. A successful screening approach
should therefore aim to identify activities that sufficiently deviate
from wild-type activity, such as the significant decreased B/I ratios
in this study, which can then be studied in more detail.
We conclude that screening of thousands of mutants for a specific activity or property is now feasible, because only a small portion of the gonadotropin analogs will display the desired biological properties and need further analyses. The current random mutagenesis and screening approach is applicable in different ways. First, although several structural determinants for receptor binding and bioactivity of gonadotropins have been elucidated 4, 23) , the function of the majority of the domains in these large glycoproteins is more or less unknown. The large number of mutants that can be screened for facilitates exhaustive screening of different domains of these molecules without any bias whatsoever. A much more complete structure/function analysis may be the starting point for the design of low molecular weight lead compounds. Several recent studies have shown how, by a combination of random mutagenesis approaches with structural analyses and screening, protein-protein interactions could be effected by relatively small oligopeptides 27, 28) . Second, regions of interest can be analyzed in detail, facilitating the optimization of desired biological activities without the limitations of site-directed mutagenesis.
From the results described in this paper, we conclude that Dictyostelium discoideum is an attractive alternative host cell for the expression of complex mammalian glycoproteins. The use of Dictyostelium discoideum for random mutagenesis of gonadotropins in combination with screening now opens the way to the generation of new fertility drugs. We anticipate that this method also may be generally useful for engineering potential new therapeutic analogies of other clinically important glycoproteins.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Present address: McKinsey & Company, Amstel 344, 1017 AS
Amsterdam, The Netherlands.
2 Abbreviations: CHO, Chinese hamster ovary; FSH,
follicle-stimulating hormone; hCG, human choriogonadotropin; LH,
luteinizing hormone; PCR, polymerase chain reaction; rec,
recombinant.
Received for publication September 9, 1998.
Revision received November 23, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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subunit. Glycobiology 5,3-10This article has been cited by other articles:
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