|
|
|
|||||||
|
|||||||||
(Received for publication, January 27, 1995; and in revised form, July 31, 1995) From the
Both diphtheria toxin and Pseudomonas exotoxin A
inhibit eukaryotic protein synthesis by ADP-ribosylating diphthamide, a
posttranslationally modified histidine residue present in the
elongation factor 2 (EF-2) protein. Elongation factor 2 cannot be
ADP-ribosylated by the toxins unless this histidine is modified. In
this report we identify three new point mutations in toxin-resistant
alleles of the Chinese hamster ovary cell elongation factor 2 gene. The
mutations resulted in amino acid substitutions at positions 584 (serine
to glycine), 714 (isoleucine to asparagine), and 719 (glycine to
aspartic acid). All three amino acid substitutions prevented the
biosynthesis of diphthamide. The amount by which the toxins reduced
protein synthesis in each of these mutant cell strains suggested that
all three mutations also either impaired the function of EF-2 or
reduced its steady state level in the cytoplasm. Western blot analysis
showed that equal amounts of EF-2 were present in each of the cell
strains, indicating that the mutations impaired the catalytic function
of EF-2.
Elongation factor 2 (EF-2) ( Both diphtheria toxin (DT) and Pseudomonas exotoxin A (PEA)
catalyze transfer of the ADP-ribose moiety of NAD In the present study we screened toxin-resistant mutant strains
derived from CHO-K1 cells to identify EF-2 gene mutations. We used
restriction fragment length polymorphism (RFLP) analysis and DNA
sequencing to characterize point mutations in the EF-2 gene that are
responsible for toxin resistance. We determined if these mutations
disrupted the biosynthesis of diphthamide. Expression of cloned mutant
and wild-type EF-2 in wild-type CHO-K1 cells confirmed that each point
mutation was sufficient to account for the toxin-resistant phenotype.
RFLP analysis
for an MboII-site indicative of a mutation in codon 717.
Genomic DNA was harvested from 4
Figure 1:
Plasmids used in this study. Cloning
strategy showing restriction maps of PCR products and plasmids. The
plasmids were constructed as described under ``Experimental
Procedures.''
Figure 2:
Intact cell assay for inhibition of
protein synthesis by DT. Incorporation of
Dose-response curves were generated for each mutant cell
strain using an intact cell assay(8) . With this assay, in
which incorporation of radiolabeled amino acids into protein is
measured after exposing cells to a range of concentrations of DT or
PEA, it is possible to classify toxin-resistant mutant strains into
three general categories: (i) class I mutants are defective in some
aspect of the entry of toxins into the cytosol, and protein synthesis
is completely inhibited by high concentrations of toxin; (ii) class IIa
mutants either lack enzymes needed to modify histidine 715 to
diphthamide (MOD
Each class IIb mutant was screened for the presence of
an MboII restriction endonuclease recognition sequence (GAAGA)
indicative of a G to A transition in codon 717 of the EF-2 gene (GAGGA
Figure 3:
MboII restriction digest of exon
11 PCR products. A 316-base pair region, which included exon 11 of the
EF-2 gene, was amplified from genomic DNA harvested from wild-type and
mutant strains of CHO-K1 cells as described under ``Experimental
Procedures.'' The PCR products were digested with MboII,
separated on a 5% nondenaturing polyacrylamide gel, and stained with
ethidium bromide.
After determining that DT-resistant
cell strains RPE.11a, R1.41, and K1W1.P17 did not have a G to A
transition in codon 717, we identified the mutations in their EF-2
alleles responsible for resistance to DT. Because the histidine
precursor of diphthamide is encoded by exon 11 of the EF-2 gene, we
first sequenced exons 11-13 from each of the mutants. PCR
amplification of this region of genomic DNA using primers which
hybridize to the previously published cDNA sequence (25) proved
problematic. The PCR product produced by such primers always produced a
product that was shorter in length than would be predicted for the
gene, but identical in length to the product predicted for cDNA.
Pretreatment of the template genomic DNA with RNase did not eliminate
this artifact. The shorter product is most likely derived from an EF-2
pseudogene, based on partial sequencing of the artifact band (data not
shown). Use of primers complementary to sequences in intron 10 and exon
13 enabled us to successfully amplify the correct region of the EF-2
gene. The intron 10-exon 13 PCR products from RPE.11a, R1.41, and
K1W1.P17 were ligated into M13mp18 and M13mp19 and single-stranded DNA
from several clones of each was harvested and sequenced. Roughly half
of the R1.41 clones contained a T to A transversion in the second
position of codon 714, resulting in a change from isoleucine to
asparagine in the predicted protein, the other half were derived from
the wild-type allele. Roughly half of the clones derived from K1W1.P17
contained a G to A transition in the second position of codon 719,
resulting in a glycine to aspartic acid substitution in the predicted
protein. No mutation was found in the intron 10-exon 13 clones derived
from RPE.11a. We then cloned full-length cDNA from RPE.11a, in order to
avoid sequencing introns. RPE.11a cDNA contained an A to G transition
in the first position of codon 584, resulting in a serine to glycine
substitution in the predicted protein. Having demonstrated that
RPE.11a, R1.41, and K1W1.P17 each possessed a unique mutation resulting
in an amino acid change in EF-2, we analyzed the EF-2 protein from each
cell strain to determine the effect of these mutations on the
posttranslational biosynthesis of diphthamide. The posttranslational
modification of histidine 715 to diphthamide proceeds in several steps
and four intermediate forms have been identified. The intermediates are
2-(3-carboxy-3-aminopropyl)histidine and the unamidated 1-, 2-, and
3-methylammonio forms of diphthamide. Each form can be resolved
chromatographically, all eluting before histidine with the buffer
system used(7, 16) . Amino acid analysis of EF-2
tryptic peptides, that contained the histidine 715 residue, was carried
out for each mutant. Peptides from ADP-ribosylated and nonribosylated
EF-2 from CHO-K1 cells were included as controls. Because each mutant
cell strain is heterozygous at the EF-2 locus and produces both
toxin-sensitive and toxin-resistant EF-2, the EF-2 in cellular extracts
was first ADP-ribosylated to completion with an excess of
NAD
Figure 4:
Amino acid analysis of
His-715/diphthamide. Elution profiles of
[
Because the tryptic peptide contains one other
histidine residue in addition to histidine 715, and the procedure for
preparing the tryptic peptide does not eliminate all other
histidine-containing peptides, a large histidine peak was always
detected. Neither the size of the ribosyl-diphthamide peak, nor the
ratio of histidine to ribosyl-diphthamide can be used to calculate the
amount of EF-2 present in cell extracts. Thus, these analyses did not
prove that the mutant EF-2 contained only an unmodified histidine 715.
The possibility remained that the mutant EF-2 contained diphthamide
which had been ADP-ribosylated, but was still able to function in
protein synthesis. Analysis of the amount of
[
Figure 5:
ADP-ribosylatable EF-2. Cytosolic extracts
prepared from CHO-K1 cells and mutant strains were ADP-ribosylated with
[
In cell strains RPE.11a, R1.41, and K1W1.P17,
protein synthesis was reduced to 20.8, 27.3, and 24% of control,
respectively, by high concentrations of diphtheria toxin (Table 1). In order to address the issue of whether the mutant,
toxin-resistant EF-2 produced from the mutant allele in cell strains
RPE.11a, R1.41, and K1W1.P17 was functionally impaired, or was less
stable, we performed quantitative Western blots to assess the total
amount of EF-2 present in each of these strains (Fig. 6). There
was no significant difference in the total amount of EF-2 detected in
these cell strains, compared with CHO-K1 and RE1.22, indicating that
the mutant EF-2 is not degraded more rapidly or produced in lower
quantity than the wild-type EF-2. We therefore conclude that these
mutations reduce the catalytic efficiency of the EF-2 protein.
Figure 6:
Immunodetection of EF-2 by Western
blotting. Cytosolic cell extracts were prepared from cells, and the
protein concentration in each extract was determined. Five, 10, 15, and
20 µg of protein from each extract was assayed. Extract from CHO-K1 (lane 1,
In
order to prove that the point mutations we identified were responsible
for the DTX-resistant phenotype of these cell strains, we constructed
EF-2 cDNA expression vectors which differed from each other only by
these single point mutations by domain swapping, as described under
``Experimental Procedures'' (Fig. 1). We first
sequenced a clone of RE1.22c to ascertain that no reverse transcription
or PCR misincorporations were present. The sequence of the clone
derived from cell strain RE1.22c was identical to the sequence with
GenBank(TM) accession number M13708 (25) with two
exceptions: (i) position 1400 in M13708 is A, whereas it is C in
RE1.22c and also C in the genomic clone with accession number J03200.
The authors of J03200 (26) found a C at this position in the
genomic DNA sequence and speculated that the cDNA sequence in M13708
might have had an error at this position. Our sequence data indicate
that the C is correct, and the A in M13708 is an error. (ii) In the
first position of codon 717, we found a G to A transition, predicted by
the presence of an MboII site in exon 11. CHO-K1 cells were
transfected with wild-type and toxin-resistant EF-2 cDNA in the pCEP4
expression vector. The expression constructs were constructed by
replacing the 3` end of the fully sequenced RE1.22c cDNA clone with the
3` end from wild-type or mutant clones which had been sequenced (Fig. 1). The region swapped into each construct was then
sequenced again. Twenty-four hours after transfection, 600 units/ml of
hygromycin B was added. Within 7 days untransfected control cells
exposed to hygromycin had died, and transfected cells had formed
colonies of 20-60 cells. Response to DT was then measured in an
intact cell assay. Cells transfected with mutant EF-2 showed
significant levels of incorporation of labeled amino acids into protein
after exposure to high concentrations of DT, while those transfected
with wild-type EF-2 were not significantly different from untransfected
controls (Table 2). The results of these assays appear to agree
very well with results previously published for mouse L cells
transiently transfected with mutant EF-2 cDNA in a plasmid utilizing
the SV40 early promoter(12, 13) . However, it is not
possible to make a direct comparison, because toxin-free controls were
not included in the previous studies.
Although our results were in
agreement with those previously published for expression of cloned EF-2
cDNA in mammalian cells, the level of protein synthesis obtained in
high concentrations of toxin was lower than expected. In an attempt to
achieve higher levels of expression than previously
published(12, 13) , we used pCEP4, an expression
vector with the cytomegalovirus enhancer/promoter, which has been
reported to be severalfold more active than the SV40 promoter/enhancer (27) and we selected for hygromycin resistance, rather than
assaying the whole population of transiently transfected cells. We had
hoped that the recombinant EF-2 would be stably expressed at high
levels. Our results indicate that in a population of cells transformed
to hygromycin resistance, the level of EF-2 protein produced from the
plasmid is less than 10% of that produced from the two functional EF-2
alleles in the CHO-K1 genome (Table 2). Transfections with the
pMSG vector constructs, containing the murine mammary tumor virus
promoter produced similar results (data not shown). In order to
select cells which stably expressed high levels of toxin-resistant
EF-2, we exposed transfected populations, which had grown in
hygromycin-containing medium for a period of 2 weeks, to PEA at a
concentration of 100 ng/ml for 48 h. This exposure to PEA resulted in
cessation of cell division or cell death in roughly 80-90% of the
cells. The remaining cells grew into discrete colonies of varying
sizes. One week after exposure to toxin, several individual colonies
were picked and grown in toxin-free medium for 2 months. Intact cell
assays were performed on these clonal lines, and their resistance to
toxin was highly variable (Table 3). The resistance correlated
with the size of the colonies from which each clone was picked.
We have characterized 13 mutant strains derived from CHO-K1
cells that are resistant to diphtheria toxin and Pseudomonas exotoxin A due to mutations in the elongation factor 2 gene. Ten
of the strains have a G to A transition in the first position of codon
717 of the EF-2 gene, resulting in a glycine to arginine substitution
in the protein. Three others have unique mutations: (i) a G to A
transition in the second position of codon 719, resulting in a glycine
to aspartic acid substitution in the predicted protein; (ii) a T to A
transversion in the second position of codon 714, resulting in an
isoleucine to asparagine change in the protein; (iii) an A to G
transition in the first position of codon 584, resulting in a serine to
glycine change in the protein. The mutant strains contained one-half as
much ADP-ribosylatable EF-2 as parental cells and equal amounts of
total EF-2. Only ribosyldiphthamide, but not free diphthamide or
intermediate forms of diphthamide, was detected following
ADP-ribosylation of the EF-2 in vitro. All four mutations
prevent the addition of the backbone of methionine from S-adenosyl-methionine to the imidazole ring of histidine 715
in the posttranslational synthesis of
diphthamide(2, 3) . Although it is tempting to
label codon 717 of the EF-2 gene as a mutational ``hot spot'' (13) due to the high proportion of mutations to toxin
resistance which occurred in this codon, our results indicate that this
may be an artifact of the method of selection. While mutations at sites
other than the first position of codon 717 result in EF-2 that is
resistant to ADP-ribosylation, they also may result in a rate of
protein synthesis in the presence of toxin that is well below 50% of
control. These cell strains grow much more slowly, if at all, in the
presence of DT or PEA. Previous studies, using somatic cell fusions
between wild-type and class IIb mutants, showed that the resulting
hybrids could not survive in high levels of toxin, although they
synthesized roughly 25% as much protein in the presence of toxin as
they did in toxin-free medium(14) . Thus, these mutant cell
strains may be overlooked when picking clones of toxin-resistant cells
selected in toxin. Other investigators, using site-directed mutagenesis
of the Saccharomyces cerevisiae EF-2 gene, have also recently
identified new toxin-resistance mutations, all of which result in an
increased doubling time, compared with cells expressing only wild-type
EF-2(10, 28) . In these studies Kimata and Kohno (28) recovered nine amino acid substitutions at codon 699
(equivalent to codon 715 in CHO-K1 EF-2) that were not recovered by
Phan et al.(10) and postulated that the difference in
growth rates led to this differential recovery(28) . A scan
of the GenBank(TM) nucleotide sequence data base release 84.0 (31) shows that the EF-2 or EF-G gene or cDNA has been
sequenced from 27 species, including that from eukaryotic mitochondria
and chloroplast organelles. A multiple sequence alignment of the
protein translations of these sequences allowed us to identify many
regions that are conserved in all 27 sequences (data available in
computer-readable format upon request). Fig. 7is a multiple
sequence alignment of the two regions of EF-2/EF-G in which we found
mutations. We chose sequences from five eukaryotes, two archaebacteria,
and two eubacteria as representative. The mutation in codon 584,
identified in cell strain RPE.11a, lies at the carboxyl-terminal end of
one of the conserved regions of the protein (Fig. 7B).
This mutation is of particular interest, because it is located far from
the modified histidine. The three-dimensional structure of EF-G from Thermus thermophilus has recently been determined by
crystallography(29, 30) . The structure shows that
amino acid 584 is located close to amino acid 715, on the surface of
domain 4 of the protein. Thus, in the biosynthesis of diphthamide one
or both of the enzymes (16) required for the addition of the
backbone of methionine to the imidazole ring of histidine 715 in EF-2
may interact directly with amino acid 584. Alternatively, alteration of
amino acid 584 could possibly disrupt the native conformation of domain
4 and prevent the enzymes(s) from modifying histidine 715.
Figure 7:
Multiple sequence alignment of EF-2/EF-G.
EF-2 and EF-G protein sequences were obtained from GenBank(TM) and
aligned using the University of Wisconsin Genetics Computer Group
PILEUP program. Five eukaryotic sequences (Cricetulus griseus,
U17362; Homo sapiens, Z11692; Drosophila
melanogaster, X15805; Saccharomyces cerevisiae, M59369)
are aligned with two archaebacterial (Methanococcus vannielii,
X12384; Thermoplasma acidophilum, X56840) and two eubacterial (Micrococcus luteus, M17788; Escherichia coli,
X00415) sequences. Fig. 6A shows the regions in which
we report mutations. Amino acids which are conserved between all nine
species are highlighted in black, and amino acids
which are conserved between members of two of the three different
``domains'' (Eucarya, Archaea, Bacteria) (37) are highlighted in gray. Bold amino acids above
the alignment indicate the mutations discussed in this study. Bold amino acids below the alignment indicate the mutations discussed
in this study. Bold amino acids below the alignment indicate
the E. coli EF-G mutations described in(32) . The
histidine residue which is posttranslationally modified in eukaryotes
and archaebacteria is indicated with an asterisk and highlighted in bold. Fig. 6B is a
schematic representation of domains conserved between all nine of the
proteins. The University of Wisconsin Genetics Computer Group
PLOTSIMILARITY program was used with the Dayhoff table (38) to
score similar as well as identical amino acids. Regions with a
similarity score above 1.0 are indicated with thick boxes. Gray
boxes indicate domains conserved among other GTP-binding
proteins.
The
mutation in codon 584 results in EF-2, which apparently functions less
well in protein synthesis than wild-type EF-2, as indicated by levels
of protein synthesis that are 20% of control, while expressing equal
amounts of wild-type and total EF-2 ( Fig. 5and Fig. 6),
when cell strain RPE.11a is grown in the presence of DT ( Fig. 2and Table 1). Two mutations that result in kanamycin
resistance have been identified in the E. coli fusA gene that
encodes EF-G(32) . Both of the fusA mutations result in
colonies that exhibit very slow growth at 42 °C. These mutations
are both located within eight amino acids of the mutation we identified
in cell strain RPE.11a (Fig. 7). The similar effects of
mutations in this region of both EF-2 and EF-G suggest a common
conserved functional role for this domain of the protein. Given that
the mutations we have studied interfere with the biosynthesis of
diphthamide, the location of mutations near histidine 715 is less
surprising than the mutation in codon 584. A combination of multiple
sequence alignments with the recently published three-dimensional x-ray
crystallographic structure of EF-G(29, 30) shows that
the diphthamide residue occurs in EF-2 at a position equivalent to the
tip of domain four of the EF-G protein. Amino acids in this domain
which are conserved between eukaryotic EF-2 and prokaryotic EF-G are
primarily located on the surface of the three-dimensional structure.
The multiple sequence alignments show that diphthamide occurs within a
region of 100 amino acids near the carboxyl-terminal end of EF-2 that,
like the GTP-binding domain near the amino-terminal end of the protein,
is highly conserved (Fig. 7B). The conservation of
amino acid sequences in the region surrounding the site of diphthamide
indicates that this region is important to the function of EF-2. The
results in Table 3demonstrate that CHO-K1 cells can survive
exposure to toxin even when protein synthesis is inhibited to less than
10% of control. Previous studies on cell-cell hybrids showed that cells
could not survive in saturating doses (0.5 µg/ml) of DT if their
level of protein synthesis was less than 25% of control(14) .
These results have important implications regarding strategies for
recovering new mutations in the EF-2 gene. A brief exposure to toxin,
followed by selecting clones that grow at different rates, may allow
recovery of a wider variety of mutations than is possible by selecting
clones which grow when continuously exposed to toxin. With continuous
exposure to toxin, colonies of class IIa mutants and class IIb mutants
with mutations in codon 717 may overgrow the plate before other mutants
can form colonies large enough to pick. Many of the clones of stable
transfectants expressing EF-2 with a Gly to Arg mutation in codon 717
synthesized protein in the presence of toxin at a rate near 50% of
toxin-free control (Table 3). It is unlikely that this is due to
allelic replacement of one of the two chromosomal copies of the EF-2
gene via homologous recombination, because homologous recombination has
previously been shown to be an extremely rare
event(33, 34) . Modified forms of DT and PEA are
being tested for use in cancer therapy. These toxins are used because
of their extreme toxicity to mammalian cells, and the ability to link
the toxic domain to different ligand-binding domains, such as
immunoglobulins specific for a certain cell type, either by chemical
cross-linking or by genetic engineering (35) . As with any
chemotherapy, a major concern is the development of a toxin-resistant
subset of target cells that escape the chemotherapy. The frequency of
occurrence of class II toxin-resistant mutants varies widely from one
cell line to another. Class II toxin-resistant cells arise at a
frequency of less than 5.6 Despite extensive study, the role of
diphthamide in eukaryotes is unknown. Also unknown is the extent to
which EF-2 participates in the regulation of protein synthesis and the
role that regulation of protein synthesis plays in growth, development,
and cell cycle regulation. In this report we identify several new
mutations which help map critical regions of the EF-2 protein and
provide insight into modifications of selection procedures which will
help to identify additional mutations that can be used to address these
important issues. The mapping of bacterial EF-G mutations and
eukaryotic EF-2 mutations to homologous regions of the protein sequence
provides evidence that studies of the mechanism of ribosomal
translocation in either system will be applicable to the other. The
role of diphthamide, however, must be studied in archaebacterial or
eukaryotic cells. Our expression studies demonstrate that transfected
cell strains expressing different levels of toxin-resistant EF-2 can be
produced. The strains should prove valuable in studies to determine the
role EF-2 plays in regulating protein synthesis.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
U17362[GenBank].
Volume 270,
Number 39,
Issue of September 29, pp. 23218-23225, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
GENETIC AND BIOCHEMICAL ANALYSES (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)is responsible for the
GTP hydrolysis-dependent translocation of eukaryotic and
archaebacterial ribosomes during polypeptide synthesis(1) . Its
counterpart in eubacteria, mitochondria, and chloroplasts is elongation
factor G (EF-G). The primary distinction between EF-2 and EF-G is the
presence in EF-2 of a unique posttranslationally modified histidine
residue
2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine which
has been characterized in detail and given the trivial name
diphthamide(2, 3) . Posttranslational modification of
histidine to diphthamide requires the participation of at least six
gene products in yeast(4) . Despite the evolutionary
conservation of this complex enzymatic pathway in all eukaryotes and
archaebacteria, the role of diphthamide in the cell remains enigmatic. to
N1 of the histidine imidazole ring of diphthamide(5) .
Toxin-resistant mutants lacking enzyme activities required for
diphthamide synthesis have been isolated and studied in detail in both
yeast and mammalian cells (6, 7, 8, 9) and show no phenotypic
alterations other than resistance to DT and PEA. Several mutations in
the EF-2 structural gene can also result in the production of
toxin-resistant
EF-2(10, 11, 12, 13) . Mutant cell
lines with mutations in the EF-2 gene can be distinguished from those
with mutations in genes encoding enzymes required for diphthamide
synthesis by somatic cell dominance hybridization and complementation
analyses, dose-response assays with DT and PEA, and in vitro ADP-ribosylation of EF-2 by
DT(6, 8, 14, 15, 16) .
Cells, Media, and Culture Conditions
CHO-K1
(Pro) Chinese hamster ovary cells were obtained from
the American Type Culture Collection. A hypoxanthine
phosphoribosyltransferase negative strain, P1R2, was selected from
CHO-K1. The procedure for isolation of toxin-resistant mutant strains
has been described(8) . The mutant strains used in these
studies were selected by exposure of mutagenized cells to DT, with the
exception of RPE.10, RPE.11a, and K1W1.P17, which were selected by
exposure to PEA. Cell strains RE1.22 which has one wild-type and one
toxin-resistant EF-2 allele, and RE1.22c (derived from RE1.22) in which
both EF-2 alleles are toxin-resistant have been previously
characterized in detail(11, 16) . Cells were
maintained in Dulbecco's modified Eagle's
medium/Ham's nutrient mixture F-12 (Sigma) supplemented with 5%
fetal bovine serum, hereafter referred to as growth medium, at 37
°C in an atmosphere of 5% CO
in air.
10
cells. A
polymerase chain reaction (PCR) was used to amplify exon 11. Primers
were 11F = 5`-cctgggtgttccttctc-3` (bases 4812-4828;
numbered as in GenBank(TM) (31) entry J03200(26) )
and 11R = 5`-gggtgaatgggcgccacacaggcagg-3` (complimentary to
bases 5128-5103), generating a 316-base pair product. The PCR
reactions contained 0.75 µM concentration of each primer,
PCR mix (200 µM of each dNTP, 3 mM
MgCl, 50 mM KCl, 10 mM Tris-HCl, pH 8.3,
100 µg/ml bovine serum albumin, 0.05% Triton X-100), 0.1 µg of
genomic DNA template and 1 unit of Taq polymerase (Life
Technologies, Inc.) in a 100-µl reaction volume, overlaid with 50
µl of sterile mineral oil. Reaction mixtures were cycled between 94
°C for 30 s, and 72 °C for 90 s, for a total of 25 cycles,
followed by a 7-min incubation at 72 °C and storage at 4 °C.
The PCR product generated from a wild-type allele contains no MboII recognition sequence. The product derived from an allele
containing a G to A transition in codon 717 contains one MboII
site which results in cleavage into two fragments of 180 and 136 base
pairs. Ten µl of each PCR mixture were digested with MboII
and resolved on a 5% polyacrylamide gel, stained with ethidium bromide,
and photographed under UV light. The validity of this method was
confirmed by Southern blots of MboII-digested genomic DNA
performed as described in(11) .Intact Cell Assay for Inhibition of Protein Synthesis by
Toxins
This assay, which involves the exposure of intact cells
to various concentrations of DT or PEA for 24 h followed by pulse
labeling with medium containing radiolabeled amino acids is derived
from a previously published protocol (8) except that 10
µCi/ml TranS-label was used in place of 0.4 µCi/ml
C-labeled amino acid mixture, in order to increase the
sensitivity of the assay at low rates of protein synthesis.In Vitro ADP-ribosylation of EF-2
EF-2 was
prepared and ADP-ribosylated in vitro with
[C]NAD as described (8) except that cells were lysed with a rapid freeze-thaw
cycle, rather than by Dounce homogenization.
Labeling and Enzymatic Digestion of the Tryptic Peptide
Containing the His-715 Residue
Metabolic labeling of EF-2 with
[
H]histidine was done as described (EF-2 was
purified by SDS-polyacrylamide gel electrophoresis and digested with
trypsin as described(7, 16) . Tryptic peptides were
purified using a Pharmacia fast protein liquid chromatography system,
PepRPC HR5/5 column, using a 0-30% gradient of 0.1%
trifluoroacetic acid in acetonitrile against 0.1% trifluoroacetic acid
in water. The fractions containing the tryptic peptide, including
histidine 715 or its modified form(s), were pooled, dried, and
resuspended in 15 mM MgCl and enzymatically
digested as described(3) . As discussed in (3) , the
enzymatic digestion of the tryptic peptide included incubation with
snake venom phosphodiesterase, which removes the ADP moiety from
ADP-ribosylated diphthamide. Thus detection of ribosyl-diphthamide in
amino acid analysis of the digests is indicative of the presence of
ADP-ribosyldiphthamide prior to digestion.Amino Acid Analysis
Amino acids derived from the
enzymatically digested peptide were then analyzed on a Waters Maxima
820 chromatography workstation, using a buffer gradient (Pierce
Buffelute(TM) high performance liquid chromatography buffers for
amino acid analysis) that expanded the amino acid profile in the region
of histidine. A gradient profile from 100% buffer C, pH 9.94, to 100%
buffer A, pH 3.17, over 120 min was used. Under these conditions,
analysis of amino acid standards showed that phenylalanine eluted at 65
min, histidine at 87 min, and arginine at 102 min. One-minute fractions
were collected from the amino acid analyzer outflow and counted in a
scintillation spectrometer following addition of scintillation mixture. Western Blots
The same cellular extracts used to
determine concentrations of ADP-ribosylatable EF-2 were used for
quantitative immunoblotting. Protein concentrations in each of the
extracts were determined both by a Bio-Rad assay kit (Bio-Rad) and by
Amido Black binding(17) . Five, 10, 15, and 20 µg of
cytosolic extract protein from each cell strain were electrophoresed on
7.5% polyacrylamide gels and electroeluted onto nylon membranes
(Amersham Corp.). The blot was then incubated with anti-EF-2 rabbit
serum, kindly provided by Dr. A. Nairn(18) . Rabbit antibody
binding to the blot was detected with I-labeled Staphylococcus protein A and quantified in a Bio-Rad GS-250
molecular imager. The blots were also autoradiographed with DuPont
X-Omat AR film.
Cloning EF-2 cDNA by Reverse Transcription PCR
RNA
was harvested from 4.0 10
log phase cells by the
method of Peppel and Baglioni(19) . Polyadenylated messenger
RNA (poly(A) mRNA) was prepared from total RNA using an oligo(dT)
column (Life Technologies, Inc.). The first strand of cDNA was produced
from 50 ng of poly(A) mRNA using SuperScript RNase H reverse transcriptase (Life Technologies, Inc.) and either an
oligo(dT) primer or a primer specific for the 3` end of the EF-2 mRNA
with equal success. Following reverse transcription, the cDNA was
ethanol-precipitated and used as template for PCR. PCR was performed
under the following conditions: 75 µM of each primer, 1F
= 5`-catggatccgaatccgtcgccatccgccactatgg-3` (bases 395-420
of J03200, plus a BamHI site at the 5` end) and 13R =
5`-catgaattcgggcactgtgcagtgtgtggc-3` (complementary to bases 5717 to
5742 of J03200 plus an EcoRI site), PCR mix (detailed above),
and 1 unit of Taq polymerase in a 100-µl reaction volume,
overlaid with 50 µl of sterile mineral oil. Forty cycles (30 s, 95
°C; 30 s, 65 °C; and 3 min, 74 °C) were performed. The
reaction mixture was extracted twice with chloroform to remove the
mineral oil and precipitated with ethanol. The 2.64-kilobase pair PCR
product, containing the complete coding region of the EF-2 cDNA, was
purified by agarose gel electrophoresis, digested with BamHI
and EcoRI and ligated into pBlueScript KS+ (Fig. 1). The plasmid was not dephosphorylated. Several
independent reverse transcription PCR reactions and ligations were
performed on RNA from each cell strain, so that if misincorporation(s)
occurred during either reverse transcription or PCR, the same ones
would not be found in clones isolated from different reactions.
Sequencing
All sequencing was done using either
single-stranded DNA from M13 clones or double-stranded plasmid DNA as
template and Sequenase-modified T7 DNA polymerase (U. S. Biochemical
Corp.) following the manufacturer's standard protocol. Template
plasmid DNA was prepared by alkaline lysis, followed by a diatomaceous
earth-based clean-up protocol as described in(20) . The
sequence of the entire BamHI to EcoRI insert of clone
pBS-22c-EF2-A2 was determined on both strands. The sequences of the
region, including the BglII to EcoRI sites were
determined on both strands for clones derived from both wild-type and
mutant cDNAs from cell strains R1.41 and K1W1.P17. The ApaI to ApaI region was sequenced on both strands for clones derived
from RPE.11a.Construction of Domain-swapped Plasmids
Having
determined by sequencing that the plasmid pBS-22c-EF2-A2 had a single
point mutation in codon 717, this clone was then used for domain
swapping. The BglII-EcoRI fragment containing the
point mutation was excised and replaced with the 671-base pair BglII-EcoRI fragments derived from the wild-type and
mutant cDNAs from heterozygous cell strains R1.41 and K1W1.P17 (Fig. 2). For RPE.11a a similar strategy was employed except
that the swapped fragment was bounded by two ApaI sites, one
in exon 7 of the EF-2 cDNA, and one from the multiple cloning site of
pBlueScriptKS+. The entire EF-2 cDNA insert was then excised from
pBlueScriptKS+ with XbaI and XhoI and ligated
into the multiple cloning site of the mammalian cell expression vectors
pCEP4 (Invitrogen) and pMSG (Pharmacia Biotech Inc.), which had been
digested with NheI and XhoI. Plasmid pCEP4 contains a
hygromycin phosphotransferase gene conferring resistance to hygromycin
as a selectable marker and the cytomegalovirus immediate early
enhancer/promoter as a promoter for expression of the cDNA insert.
Plasmid pMSG carries the Escherichia coligpt gene
conferring resistance to hypoxanthine-aminopterin-thymidine medium when
transfected into hypoxanthine phosphoribosyltransferase negative cell
strains(21) , and the mouse mammary tumor virus long terminal
repeat as a promoter for expression of the cDNA insert.
S-labeled amino
acids into trichloroacetic acid-precipitable protein during a 1-h pulse
after 24-h incubation in toxin is plotted as percentage of toxin-free
control.
, CHO-K1;
, RE1.22;
, RE1.22c; 
,
RPE.10; 
, R1.41; , K1W1.P17;
, RPE.11a. Each data
point represents the average of three separate vials of cells. The
values for S incorporation in the toxin-free controls
ranged from 22,775 ± 247 cpm for RE1.22 to 19,484 ± 422
cpm for RPE.11a in this assay.
Transfections
5.0 10
cells
were plated in 60-mm tissue culture dishes and incubated 18 to 24 h
prior to transfection. Cells were transfected with Transfectam
(Promega) according to the manufacturer's recommended protocol
for use with medium without serum. Trial transfections with varying
ratios of Transfectam to DNA proved that 7 µg of plasmid DNA used
with 7 µl of Transfectam solution gave the best results. After
transfection with pCEP4 or pMSG plasmid constructs the cells were
allowed to recover for 24 h and then selected in growth medium
containing 600 units of hygromycin B (Calbiochem)/ml or
hypoxanthine-aminopterin-thymidine medium, respectively.
) or have mutations in both copies of
the elongation factor 2 gene, and protein synthesis is unaffected by
any concentration of toxin; (iii) class IIb mutants have mutations in
one of the two copies of the elongation factor 2 gene, and protein
synthesis is typically reduced to 47-56% of control, by high
concentrations of toxin(8, 14, 22) . In this
study, 12 mutant cell strains (Table 1) produced typical class
IIb dose-response curves as illustrated in Fig. 2. Incorporation
of amino acids into protein in the presence of high doses of DT was
reduced to between 47.5 and 56% of toxin-free controls. In the case of
the mutant cell strains RPE.11a, R1.41, and K1W1.P17, protein synthesis
was reduced well below 50%, but was never reduced to less than 15% ( Table 1and Fig. 2). Wild-type CHO-K1, and class IIa
RE1.22c cell strains, were included as controls. Thus, the mutant
strains which are the focus of this study were confirmed to be class
IIb mutants.
GAAGA)(11, 13) . This mutation, which was
originally reported in four out of four independent isolates of
mutagenized CHO-K1 cells selected for toxin resistance(13) ,
prevents the first step in the biosynthesis of
diphthamide(11) . Fig. 3presents results from a
representative PCR RFLP analysis. Ten of the 13 class IIb mutant cell
strains analyzed possessed an MboII site indicative of a G to
A transition in codon 717, and three did not. Wild-type CHO-K1,
heterozygous RE1.22 and homozygous RE1.22c (11) were included
as controls. The three strains which did not possess an MboII
site, were the same three that showed significantly less than 50%
protein synthesis in the intact cell assay. This suggested either that
their toxin-resistant EF-2 was functionally impaired or that it was
less stable than wild-type EF-2.
and DT to ADP-ribosylate all of the wild-type
EF-2. This enabled us to determine by chromatographic analysis if the
product of the mutant allele possessed diphthamide that could not be
ADP-ribosylated by toxin. Amino acid analysis revealed that only
ribosyl-diphthamide and histidine were recovered from the EF-2
extracted from each mutant. No intermediate forms in the diphthamide
biosynthetic pathway were detected (Fig. 4). A small but
significant amount of ADP-ribosylated diphthamide was detected in
extracts of CHO-K1 cells that had not been exposed to toxin. This is in
agreement with other studies, which have shown that a small percentage
of EF-2 in normal cells is ADP-ribosylated by an endogenous
enzyme(23) , apparently in a cell cycle-dependent
manner(24) .
H]histidine (A-E)- or
[C]NAD (F)-labeled
enzymatic digests of EF-2 tryptic peptide. A, CHO-K1. B, CHO-K1 ADP-ribosylated with DT adn NAD
. C, R1.41 ADP-ribosylated with DT and NAD
. D, RPE.11a ADP-ribosylated with DT and NAD+. E,
K1W1.P17 ADP-ribosylated with DT and NAD
. F,
CHO-K1 ADP-ribosylated with DT and
[
C]NAD. The ADP group was
removed from ADP-ribosylated diphthamide by phosphodiesterase during
the enzymatic digestion of the tryptic peptide, leaving
ribosyl-diphthamide.
C]ADP-ribose incorporated into EF-2 by DT in
cell extracts from each of the mutants eliminated this possibility.
Mutant cell strains RPE.11a, K1W1.P17, and R1.41 each possessed half as
much ADP-ribosylatable EF-2 per mg of cellular protein as did CHO-K1 (Fig. 5).
C]NAD and DT as described
under ``Experimental Procedures.'' Results are plotted as a
percentage of the ADP-ribosylated EF-2 in CHO-K1 cells (114 pmol/mg of
extract protein). Each bar represents the average of three
separate determinations, the error bars indicate the maximum
value obtained.
) was compared with extracts from RE1.22 (lane 2,
), RPE.11a (lane 3, ), R1.41 (lane 4,
) and K1W1.P17 (lane 5,
).
The radioactivity in each band was quantified using a Bio-Rad GS-250
molecular imager and is expressed in pixel density units per band after
a 12-h exposure to the phosphor screen. Data points for the 15
µg/lane sample are the average of two separate gel
loadings.
10 in recently
cloned CHO-K1 cells(14) , but occur at a very high frequency
(approximately 1
10) in Vero monkey kidney
cells. (
)Within the class II mutants, the ratio of EF-2 gene
mutations to mutations in genes responsible for the posttranslational
modification of His-715 to diphthamide is also variable. In CHO-K1
cells, both MOD and EF-2 mutants have been isolated.
In S. cerevisiae only MOD
mutants have been
described, but this is due to the selection criteria used(36) ,
not an indication of a low rate of mutation in the EF-2 genes. In most
cultured mammalian cell lines, only class IIb mutants have been
isolated.
Cancer cells are often found to be defective in
one or more DNA damage and repair pathways, and this leads to the
possibility that the frequency of occurrence of DT and PEA-resistant
cells within a population of tumor cells will be highly variable from
tumor to tumor. More studies with human tumor cell lines will be
necessary to confirm this.
))
We thank Dr. Douglas Johnson for helpful comments on
the manuscript. We also thank Dr. Angus Nairn for providing rabbit
anti-EF-2 antiserum.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. Nobukuni, K. Kohno, and K. Miyagawa Gene Trap Mutagenesis-based Forward Genetic Approach Reveals That the Tumor Suppressor OVCA1 Is a Component of the Biosynthetic Pathway of Diphthamide on Elongation Factor 2 J. Biol. Chem., March 18, 2005; 280(11): 10572 - 10577. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Shulga-Morskoy and B.E. Rich Bioactive IL7-diphtheria fusion toxin secreted by mammalian cells Protein Eng. Des. Sel., January 1, 2005; 18(1): 25 - 31. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Jorgensen, S. P. Yates, D. J. Teal, J. Nilsson, G. A. Prentice, A. R. Merrill, and G. R. Andersen Crystal Structure of ADP-ribosylated Ribosomal Translocase from Saccharomyces cerevisiae J. Biol. Chem., October 29, 2004; 279(44): 45919 - 45925. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Di Paolo, J. Willuda, S. Kubetzko, I. Lauffer, D. Tschudi, R. Waibel, A. Pluckthun, R. A. Stahel, and U. Zangemeister-Wittke A Recombinant Immunotoxin Derived from a Humanized Epithelial Cell Adhesion Molecule-specific Single-Chain Antibody Fragment Has Potent and Selective Antitumor Activity Clin. Cancer Res., July 1, 2003; 9(7): 2837 - 2848. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Qiao and M. Caruso PG13 Packaging Cells Produce Recombinant Retroviruses Carrying a Diphtheria Toxin Mutant Which Kills Cancer Cells J. Virol., June 14, 2002; 76(14): 7343 - 7348. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. K. Ichikawa, A. Norris, M. G. Bangera, G. K. Geiss, A. B. van 't Wout, R. E. Bumgarner, and S. Lory Interaction of Pseudomonas aeruginosa with epithelial cells: Identification of differentially regulated genes by expression microarray analysis of human cDNAs PNAS, August 6, 2000; (2000) 160140297. [Abstract] [Full Text]
|
|
|
|
|
|
M. V. Rodnina and W. Wintermeyer Form follows function: Structure of an elongation factor G-ribosome complex PNAS, June 23, 1998; 95(13): 7237 - 7239. [Full Text] [PDF]
|
|
|
|
|
|
J. K. Ichikawa, A. Norris, M. G. Bangera, G. K. Geiss, A. B. van 't Wout, R. E. Bumgarner, and S. Lory Interaction of Pseudomonas aeruginosa with epithelial cells: Identification of differentially regulated genes by expression microarray analysis of human cDNAs PNAS, August 15, 2000; 97(17): 9659 - 9664. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |