Molecular Dissection of Mitogillin Reveals That the Fungal
Ribotoxins Are a Family of Natural Genetically Engineered
Ribonucleases*
Richard
Kao
and
Julian
Davies
From the Department of Microbiology and Immunology, The University
of British Columbia, 6174 University Blvd.,
Vancouver, British Columbia V6T 1Z3, Canada
 |
ABSTRACT |
Mitogillin and the related fungal ribotoxins are
highly specific ribonucleases which inactivate the ribosome
enzymatically by cleaving the 23-28 S RNA of the large ribosomal
subunit at a single phosphodiester bond. The site of cleavage occurs
between G4325 and A4326 (rat ribosome
numbering) which are present in one of the most conserved sequences
(the
-sarcin loop) among the large subunit ribosomal RNAs of all
living species. Amino acid sequence comparison of ribotoxins and
guanyl/purine ribonucleases have identified domains or residues likely
involved in ribonucleolytic activity or cleavage specificity. Fifteen
deletion mutants (each 4 to 8 amino acid deletions) in motifs of
mitogillin showing little amino acid sequence homology with
guanyl/purine ribonucleases were constructed by site-directed
mutagenesis. Analyses of the purified mutant proteins identified those
regions in fungal ribotoxins contributing to ribosome targeting and
modulating the catalytic activity of the toxin; some of the identified
motifs are homologous to sequences in ribosomal proteins and elongation
factors. This mutational study of mitogillin together with the recently
published x-ray structure of restrictocin (a close relative of
mitogillin) supports the hypothesis that the specific cleavage
properties of ribotoxins are the result of natural genetic engineering
in which the ribosomal targeting elements of ribosome-associated
proteins were inserted into nonessential regions of T1-like ribonucleases.
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INTRODUCTION |
Mitogillin and the related Aspergillus fungal
ribotoxins restrictocin and
-sarcin, are small basic proteins of
~17 kDa (kilodaltons). Mitogillin differs from restrictocin by only 1 amino acid and has 86% amino acid sequence identity with
-sarcin
(1-4). The fungal ribotoxins are a family of highly specific
ribonucleases which inactivate the ribosome by cleavage of the 23-28 S
RNA of the large ribosomal subunit (5) at a single phosphodiester bond.
The site of cleavage occurs between G4325 and
A4326 (rat ribosome numbering) in a 14-base sequence (the
-sarcin loop) found in the large subunit ribosomal RNAs
(rRNAs)1 of all living
species. This single cleavage completely abolishes the capability of
ribosomes to carry out protein synthesis (6-8) by inhibiting
elongation factor 1-dependent binding of aminoacyl-tRNA and
GTP-dependent binding of elongation factor 2 to ribosomes. Mitogillin-like ribotoxins are among the most potent inhibitors of
translation so far identified and studies have been carried out to
investigate their potential as anti-tumor agents or components of
immunotoxins (9-14).
The recognition elements of the structure of the
-sarcin loop in 28 S rRNA have been studied extensively (15, 16) and G4319 was
proposed to be the identity element for
-sarcin (17). However, very
little is known about the ribosome-targeting elements of the protein
toxins. Mitogillin and related ribotoxins are known to share amino acid
sequence similarity with T1-like ribonucleases (8) and
-sarcin has
been shown to behave as a cyclizing ribonuclease like many other
ribonucleases (18), but their property to interact specifically with
the ribosome and cause a single ribonucleolytic cleavage in the large
subunit rRNA is unique. Previous studies indicated that the
similarities and differences detected in amino acid sequence comparison
of ribotoxins and a large family of other guanyl/purine ribonucleases
may represent domains or residues key to ribonucleolytic activity and
specificity (19, 20). The presence of these "extra" protein domains
(some of which are similar to sequences in ribosome-associated
proteins) in fungal ribotoxins led to the hypothesis that fungal
ribotoxins are a family of naturally engineered toxins with ribosomal
targeting elements acquired from different ribosome-associated proteins (19). The prediction that the fungal ribotoxins are T1-like ribonucleases with additional protein domains extended from the catalytic core of the RNase is confirmed by the crystal structure of
restrictocin determined by x-ray analysis (21) and the
three-dimensional structure of
-sarcin in solution determined by NMR
(22). These structural analyses also support the proposal that the
ribosomal protein-like region Lys106-Lys113 is
the major ribosomal recognition element in mitogillin (19). In the
present study we report the characterization and properties of 15 deletion mutants (4 to 8 amino acid deletions) in motifs of mitogillin
having little amino acid sequence homology with guanyl/purine
ribonucleases and the identification of elements in mitogillin (and
related ribotoxins) contributing to its specific cleavage of ribosomes.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains and Plasmids--
An Escherichia
coli host (derivative of W3110) and the plasmid pING3522 were used
for the production of mitogillin and mutant mitogillin proteins.
pING3522 is an inducible secretion vector with the expression of the
inserted gene under the control of the Salmonella typhimurium
araB promoter; the secretion of recombinant proteins through the
cytoplasmic membrane is directed by the Erwinia carotovora
pelB leader sequence (14).
Design and Construction of Deletion Mutants--
Polymerase
chain reaction (PCR)-mediated site-directed mutagenesis was employed to
construct deletion mutants in three regions (Gln8-Thr43,
Asp56-Lys88, and
Lys106-Lys113) of mitogillin (Fig.
1A). Nucleotide sequences of
oligonucleotides used to construct mutants are shown in Table
I. PCR-mediated site-specific mutagenesis
is described in Fig. 1B. RK-01 (forward "universal"
primer) was mixed with a reverse mutagenic oligo and RK-02 (reverse
universal primer) was mixed with a forward mutagenic oligo; PCR was
carried out directly on single E. coli colonies (containing
pING3522 plasmid) in two separate 100-µl reactions (50 mM
KCl, 10 mM Tris-Cl, pH 8.3, 1.5 mM
MgCl2, 100 pmol of each primer oligonucleotide, and 200 µM of each dNTP). The tubes were heated at 94 °C for 5 min before the addition of 2.5 units of Taq polymerase,
overlaid with paraffin oil and 30 cycles of PCR carried out (94 °C
for 1 min, 55 °C for 2 min, and 72 °C for 3 min). The PCR
products were electrophoresed on a 1% agarose gel and purified using
QIAquick gel extraction kit (QIAGEN). Purified PCR products were mixed
and the volume was brought to 100 µl with 50 mM KCl, 10 mM Tris-Cl, pH 8.3, 1.5 mM MgCl2,
200 µM of each dNTP, and 2.5 units of Taq
polymerase, and incubated at 94 °C for 1 min, 55 °C for 2 min,
and 72 °C for 10 min to achieve the 3' extension of the mixed PCR
products. PCR amplification of the mutagenized mitogillin gene was then
accomplished by adding 100 pmol of each of oligomers RK-01 and RK-02,
and 2.5 units of Taq polymerase, overlaid with paraffin oil
and reincubated at 94 °C for 1 min, 55 °C for 2 min, and 72 °C
for 3 min, for 30 cycles. The final PCR product was purified by
electrophoresis followed by QIAquick extraction. The purified fragment
was ligated to PstI-HindIII-digested pING3522 and
transformed into E. coli. The nucleotide sequence of each
mutant was verified. Production of mutant mitogillin proteins was
detected by SDS-PAGE (0.1% SDS, 15% polyacrylamide) of the induced
culture supernatants followed by Western blotting using rabbit antisera
raised against mitogillin.

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Fig. 1.
Mutagenesis of mitogillin. A,
sites of deletion are indicated. Shown also are the secondary
structures of mitogillin: 1, -sheet 1; L1, loop 1; 2,
-sheet 2; H1, -helix 1; L2, loop 2; 3, -sheet 3; L3, loop
3; H2, -helix 2; 4, -sheet 4; L4, loop 4; 5, -sheet 5;
L5, loop 5; 6, -sheet 6; L6, loop 6; 7, -sheet 7. B, schematic representation of the PCR-mediated
site-specific deletion of the mitogillin gene. Shown are the
PstI/HindIII restriction enzyme sites and the
relative positions of various PCR primers. Site of deletion is
indicated by an *.
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Table I
Oligonucleotides used for site-directed mutagenesis of the mitogillin
gene
Oligonucleotides RK-01 and RK-02 were "universal" primers used for
site-directed mutagenesis and DNA sequencing. RK-01 binds upstream of
the mitogillin gene near the araC gene, and RK-02 binds near
the tet gene in the vector region of pING3522. For detailed
information on DNA sequences of plasmid pING3522 and the mitogillin
gene, see Better et al. (14).
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Production and Purification of Mutant Mitogillins--
E.
coli harboring pING3522-derived plasmids containing the
mutagenized mitogillin gene were grown at 37 °C to
A600 = 0.4 in Tryptone broth (containing 10 µg
of tetracycline/ml), induced by the addition of 0.1%
L-arabinose (Sigma) and grown for 18 h. Secreted
proteins were purified by cation exchange chromatography followed by
size-exclusion chromatography as described previously (20) except that
50 mM sodium phosphate buffer, pH 7.2, was used for cation
exchange chromatography.
Assay for Nonspecific Ribonucleolytic
Activity--
Ribonucleolytic activity of ribonuclease T1, mitogillin,
and mutant mitogillins against poly(I) homopolymer (Sigma) was examined by methods described previously (20). 200 µM poly(I)
homopolymer substrate was used in all cleavage assays. In initial rate
experiments, 50 nM of each protein was used and the extent
of poly(I) homopolymers hydrolysis was less than 10% of the total and
MicroCon-100 concentrators (molecular cut-off = 300 nucleotides
for single-stranded RNA; purchased from Amicon) were used to separate
cleavage products from poly(I) homopolymers. Ribonucleolytic activity
against MS-2 phage RNA was determined by incubating RNA (400 ng) with
300 nM purified protein in 10 µl of reaction buffer (15 mM Tris-HCl, pH 7.6, 15 mM NaCl, 50 mM KCl, 2.5 mM EDTA). The reaction was incubated at 37 °C for 30 min, stopped by the addition of 1.0 µl
of 5% SDS, and separated by electrophoresis (1.2% agarose) as
described previously (19); the gel was stained with ethidium bromide to
visualize the degradation of the RNA substrate. An activity staining
assay (zymogram electrophoresis) described by Blank et al.
(23) was employed to monitor the presence of contaminating ribonucleases. Briefly, SDS-PAGE of 150 ng of each of the mutant mitogillins was performed under nonreducing condition on a 0.1% SDS,
15% polyacrylamide gel containing 0.3 mg of poly(I) substrate. The gel
was treated as described by Blank et al. (23) and incubated in 100 mM Tris-HCl, pH 7.4, at 37 °C for 1 h,
stained with 0.2% toluidine blue for 10 min at room temperature and
washed extensively with distilled water for 1 h. RNase activity
was indicated by the appearance of a clear zone on a dark blue background.
Specific Cleavage of Rabbit Ribosomes and Synthetic
-Sarcin
Loop RNA--
Specific cleavage of eukaryotic ribosomes was done in
reaction mixtures with 10 µl of untreated rabbit reticulocyte lysate (Promega), 600 nM toxin protein, 15 mM
Tris-HCl, pH 7.6, 15 mM NaCl, 50 mM KCl, and
2.5 mM EDTA, incubated at 37 °C for 15 min. Extracted
RNA was separated by electrophoresis (1.5% agarose) and RNA species
visualized by staining with ethidium bromide. The specific
ribonucleolytic activity of the toxin derivatives was followed by the
appearance of the
-fragment, a ~400 nucleotide RNA species on the
gel. A RNA oligonucleotide (a 35-mer) that mimics the
-sarcin domain
of the eukaryotic large subunit rRNA was prepared with synthetic DNA
templates, T7 RNA polymerase, and the four nucleotide triphosphates and
purified using denaturing polyacrylamide gel electrophoresis (24, 25).
1.0 µM synthetic RNA 35-mer was combined with 600 nM mitogillin or mutant mitogillins for a total volume of
6.0 µl in 10 mM Tris-HCl, pH 7.4, and incubated at
37 °C for 15 min. The reaction was stopped by the addition of 4.0 µl of deionized formamide and heating at 95 °C for 3 min. Cleavage
products were separated by denaturing polyacrylamide gel
electrophoresis and the gel stained with SYBR-GOLD nucleic acid stain
(Molecular Probes).
 |
RESULTS |
Mutant Construction and Protein Purification--
Fifteen deletion
mutants of mitogillin in regions predicted to be functional elements
inserted into a T1 ribonuclease core structure were constructed. The
mutant genes were completely sequenced to confirm the nature of the
deletions; no other alterations were detected. Production of mutant
proteins on growth in liquid culture was detected from 10 clones (Table
II) by Western blotting with a rabbit
antiserum (data not shown). SDS-PAGE followed by silver staining of the
purified proteins confirmed their purity (Fig. 4A).
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Table II
Production of mutant mitogillin proteins
Production was detected by SDS-PAGE of induced culture supernatants
followed by Western blotting using rabbit anti-mitogillin antisera.
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Nonspecific Ribonucleolytic Activity--
RNase activity of
mitogillin and all 10 mutant proteins was detected when poly(I)
homopolymers (Fig. 2) or MS-2 phage RNA (Fig. 3) were used as substrates. The
comparison between the ribonuclease activities of mutant and wild-type
mitogillin using poly(I) homopolymer is shown in Fig. 2; initial rates
of reaction are tabulated in Table III.
The data indicates that deleting Lys13-Lys16,
Lys16-Asp19, or
Lys20-Leu23 increased the RNase activity;
Lys28-Ser31,
Arg77-Gln83, or
Lys106-Lys113 decreased the RNase activity,
while the deletion of Asp56-Lys60,
Gly59-Ile62,
Lys63-Ile68, or
Asn84-Lys88 had little effect as judged by the
initial rates of ribonucleolytic reaction. Results obtained from the
zymogram electrophoresis assay (Fig.
4B) eliminate the possibility
of the presence of contaminating ribonucleases in the preparations.

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Fig. 2.
Nonspecific ribonucleolytic activity of
mitogillin and its mutants on poly(I) substrate. Results were
plotted as percent of poly(I) degradation versus time when 3 µM mitogillin or a mutant protein was used.
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Fig. 3.
MS-2 phage RNA degradation assay. The
ribonucleolytic activity of the mutant mitogillin proteins was assayed
by examining the extent of degradation of MS-2 phage RNA. Lane
1, mitogillin; lane 2, no toxin; lane 3,
Lys13-Lys16 mutant; lane 4,
Lys16-Asp19 mutant; lane 5,
Lys20-Leu23 mutant; lane 6,
Lys28-Ser31 mutant; lane 7,
Asn56-Lys60 mutant; lane 8,
Gly59-Ile62 mutant; lane 9,
Lys63-Ile68 mutant; lane 10,
Arg77-Gln83 mutant; lane 11,
Asn84-Lys88 mutant; and lane 12,
Lys106-Lys113 mutant.
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Table III
Comparison of the ribonucleolytic activity (initial rate of cleavage)
of mitogillin, mutant mitogillins, and ribonuclease T1 on poly(I)
homopolymer
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Fig. 4.
A, SDS-PAGE of mutant mitogillins. 1 µg of each mutant protein was run on a 0.1% SDS-15% polyacrylamide
gel under nonreducing conditions. Presence of protein was detected by
silver staining procedures. B, zymogram electrophoresis.
SDS-PAGE of 150 ng of each of the mutant mitogillin proteins was
performed under nonreducing condition on a 0.1% SDS-15%
polyacrylamide gel containing 0.3 mg of poly(I) substrate (Sigma).
RNase activity was indicated by the appearance of clearing band on the
poly(I) containing polyacrylamide gel stained with toluidine blue.
Lane 1, mitogillin; lane 2,
Lys13-Lys16 mutant; lane 3,
Lys16-Asp19 mutant; lane 4,
Lys20-Leu23 mutant; lane 5,
Lys28-Ser31 mutant; lane 6,
Asp56-Lys60 mutant; lane 7,
Gly59-Ile62 mutant; lane 8,
Lys63-Ile68 mutant; lane 9,
Arg77-Gln83 mutant; lane 10,
Asn84-Lys88 mutant; and lane 11,
Lys106-Lys113 mutant. Shown also are the
positions of molecular markers in kilodaltons.
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Specific Ribonucleolytic Activity--
On treatment of rabbit
reticulocyte lysates with purified mitogillin or the deletion variants,
a distinctive
-fragment band was detected in all samples except the
Lys106-Lys113 deletion mutant (Fig.
5A). Extensive degradation of
28 S RNA in ribosomes by Lys13-Leu23 deletion
mutants suggests that this region in the fungal ribotoxin is involved
in modulating the activity and specific recognition of the cleavage
site in the ribosome. Results obtained from assays using synthetic
-sarcin loop (a 35-mer) as substrate showed that deletions in
regions Lys13-Lys16,
Lys16-Asp19, or
Lys20-Leu23 gave rise to mitogillin variants
with elevated ribonucleolytic activity while deletion of
Lys106-Lys113 created a mutant which fails to
recognize and cleave the
-sarcin loop (Fig. 5B).

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Fig. 5.
A, specific ribonucleolytic activity
(in vitro -fragment release) of mitogillin and its
variants. Positions of 28 S rRNA, 18 S rRNA, and -fragment are
indicated. B, synthetic -sarcin loop cleavage assay.
Positions of 35-mer, 21-mer, and 14-mer are also indicated. Details of
the assay conditions are described under "Experimental Procedures."
Lane 1, mitogillin; lane 2, no toxin; lane
3, Lys13-Lys16 mutant; lane
4, Lys16-Asp19 mutant; lane
5, Lys20-Leu23 mutant; lane
6, Lys28-Ser31 mutant; lane
7, Asp56-Lys60 mutant; lane
8, Gly59-Ile62 mutant; lane
9, Lys63-Ile68 mutant; lane
10, Arg77-Gln83 mutant; lane
11, Asn84-Lys88 mutant; and lane
12, Lys106-Lys113 mutant.
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 |
DISCUSSION |
Structural comparison between the three-dimensional structures of
restrictocin (99% identity with mitogillin) and T1 ribonuclease reveals strong structural similarities with certain domains in the
fungal ribotoxins absent from the T1 ribonucleases; the most obvious
differences are the
1-loop 1-
2 region, the loop 3 region, and the
loop 4 region of restrictocin (Fig. 6).
It has been postulated that these domains are "inserted" elements
contributing to the specific targeted, cytotoxicity of this class of
proteins; preliminary studies on the loop 4 region indicated that this
region indeed is the major ribosome-targeting elements of mitogillin
(19). We have generated deletions (4-8 amino acid residues) in those regions of mitogillin proposed to be inserted elements. Deletions were
preferred over amino acid substitutions for this analysis in order to
identify domains responsible for targeting mitogillin to the ribosome
and examine the influence of these inserted regions on the general
properties of the fungal ribotoxin. Ten of the 15 deletion mutants
produce biologically active proteins using the E. coli
expression system. It is interesting to note that the five
non-expressible mutant mitogillin proteins have deletions located near
or extending into elements such as disulfide bridge-forming cysteine
residues, proline residues, or helixes, which are likely to be
susceptible to host protease activity. The majority of the deletions
obtained retain the catalytic activity of the ribotoxin which supports
the proposal that these domains have roles in ribotoxin function
distinct from the RNase activity.

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Fig. 6.
Structural comparison of restrictocin and
ribonuclease T1. Secondary structures of the proteins are labeled:
1, -sheet 1; L1, loop 1; 2, -sheet 2; H1, -helix 1; L2,
loop 2; 3, -sheet 3; L3, loop 3; H2, -helix 2; 4, -sheet
4; L4, loop 4; 5, -sheet 5; L5, loop 5; 6, -sheet 6; L6,
loop 6; 7, -sheet 7. Positions of the catalytically important
residues His49, Glu95, Arg120, and
His136 of restrictocin and the corresponding residues
His40, Glu58, Arg77, and
His92 of ribonuclease T1 are also indicated. Noted is the
absence of 1-loop 1- 2, loop 3, and loop 4 domains of mitogillin
in ribonuclease T1. The coordinates of restrictocin and ribonuclease T1
are taken from Protein data bank files 1AQZ (21) and 1RNT (28),
respectively.
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Deletions in loop 4 and in
1-loop 1-
2 regions generate mutants
with interesting properties in terms of their ribonucleolytic activities. We have shown that the octapeptide Lys106 to
Lys113 in the loop 4 region has sequence similarity to
various ribosome-associated proteins (19) and that the heptapeptide
Thr14 to Lys20 in the
1-loop 1-
2 region
is strongly related to motifs in various elongation factors (Table
IV). Although the significance of these relationships has yet to be examined, it is noteworthy that the
Lys106-Lys113 mutant (previously termed
2
mutant in Ref. 19) has lost the ability to recognize and cleave the
-sarcin loop (19) (Fig. 5, A and B, this
study), and that the ribonucleolytic activity of the deletion mutants
in the
1-loop 1-
2 region is greatly elevated (20-30-fold higher
than that of wild-type mitogillin, Table III). The
-sarcin
loop/restrictocin docking model of Yang and Moffat (21) suggests that
the loop 4 region of mitogillin is in close proximity to the 28 S rRNA
identity element (G4319) of the fungal ribotoxin; our
results support the conclusion that elements in the loop 4 domain
interact with the "bulged" G4319 in the
-sarcin loop
to promote specific recognition and cleavage of the substrate.
However, the
1-loop 1-
2 region of mitogillin is relatively
distant from the catalytic center of the nuclease; the role of this
domain in attenuating the catalytic activity of the toxin is
intriguing. We propose that this is the result of hydrogen bond
formation between amino acid residues in
1 and
2, and residues in
6 in which the catalytic residue His136 is situated
(Fig. 7). It is possible that the
1-loop 1-
2 domain attenuates the catalytic activity of the toxin
by keeping the catalytic residue His136 in a configuration
suboptimal for nucleolytic activity through hydrogen bonding with
6.
Upon binding to the ribosome, the interactions between loop 4 and the
-sarcin loop trigger a conformational change in the protein which
disrupts the interactions between the
1-loop 1-
2 domain and
6,
so positioning the catalytic residue His136 in a optimal
environment for cleavage of phosphodiester bond of the RNA substrate.
Evidence in favor of this interpretation comes from the analysis of a
N7A mutant of mitogillin (which presumably eliminates the hydrogen
bonding between Asn7 and His136) which shows
greatly elevated ribonuclease activity (Table III). The detailed
biochemical properties of the N7A mutant are currently under
investigation. Our interpretation also agrees with the findings of
Takeda et al. (27) which suggest that the fungal ribotoxins bind to two sites in the RNA substrate; one through the bulged G4319 and the other around the GAGA tetraloop cleavage
site. An RNA oligomer substrate with the deleted bulged G is
unsusceptible to
-sarcin cleavage but becomes a strong
noncompetitive inhibitor of the wild-type RNA molecule (with the bulged
G), which suggests that the association between the RNA recognition
element (bulged G4319) and the first substrate-binding site
in mitogillin (elements in loop 4 of the protein) may trigger a
conformational change in the active site of the ribotoxin which permits
the efficient cleavage of the GAGA tetraloop. Shapiro (26) demonstrated
that the enzymatic potency and specificity of human angiogenin are partly modulated by hydrogen bonding between two distant amino acid
residues (Thr44 and Thr80) that inability to
form the hydrogen bond (made possible by a T80A mutation) increases the
ribonucleolytic activity of angiogenin 11-15-fold (26). This raises
the possibility that substrate-specific ribonucleases other than fungal
ribotoxins may also modulate their specific activity by similar
mechanisms.

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Fig. 7.
Hydrogen-bonding between
-loop 1- and
-loop 6- domains of
restrictocin. Loop 1 residues 11-16 are fully exposed to solvent
and are consequently missing in the atomic model. Hydrogen bonds are
denoted by dashed lines. Amino acid residues not relevant in
forming hydrogen bonds were omitted for clarity.
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Deletions in region Asp56 to Lys88 (loop 3) of
mitogillin do not appear to interfere with the ribonucleolytic activity
or the specificity of mitogillin. The dramatic decrease in RNase
activity in
Arg77-Gln83 may be due to the
deletion of a catalytically important residue(s) or a change in the
structure of the toxin affecting its catalytic activity. The latter
interpretation is more likely since the active site of mitogillin does
not indicate the involvement of any residues in region
Arg77-Gln83 for catalysis (20). However, these
residues being adjacent to Cys75 which forms a disulfide
bond with Cys5 in mitogillin and deletion of
Arg77-Gln83 would presumably induce a change in
the microenvironment of the active site of mitogillin and, as a result,
reduce RNA cleavage activity. Based on available information we cannot
define further the functions of loop 3 of mitogillin. Yang and Moffat
(21) suggest that this region may influence mitogillin/cell surface receptor recognition or translocation of the toxin through the lipid
bilayer of the cell; mutants constructed in this region would be good
candidates to elucidate the functions of this extra loop of the fungal ribotoxins.
In conclusion, we have described the production and properties of 10 deletion mutants of mitogillin, predicated on the hypothesis that
motifs showing little amino acid sequence homology with guanyl/purine ribonucleases are functional domains inserted into T1-like
ribonucleases through natural genetic engineering. Characterization of
the enzymatic properties of the mutant proteins suggests that elements
in loop 4 region of mitogillin are involved in the specific recognition of the toxin to the ribosome and elements in
1-loop 1-
2 region involved in modulating the catalytic activity of the toxin. Our studies
also suggest that loop 3 of mitogillin may be involved in functions
other than targeting mitogillin to the ribosome. It is evident that
these additional elements, which appear to have been engineered
naturally into T1-like ribonucleases, are not required for RNase
activity but have functions specific to cytotoxic activity. The
availability of these mutants will aid in further investigation of the
biochemical and/or physiological roles of ribotoxin function. An
interesting question is whether it will be possible to insert such
targeting motifs into other protein sequences to generate other types
of ribosome-specific toxins.
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ACKNOWLEDGEMENTS |
We thank Dr. M. Better for providing E. coli W3110, plasmid pING3522, purified mitogillin, and rabbit
anti-mitogillin antibody. We also thank Drs. S. B. delCardayre and
V. Webb for helpful discussions.
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FOOTNOTES |
*
This work was supported by the Natural Sciences and
Engineering Research Council.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom reprint requests should be addressed. E-mail:
kao{at}davies.microbiology.ubc.ca.
 |
ABBREVIATIONS |
The abbreviations used are:
rRNA, ribosomal ribonucleic acids;
PCR, polymerase chain reaction;
PAGE, polyacrylamide gel electrophoresis.
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