Document ID: EPA-HQ-OPP-2004-0135-0002
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2004-04-28T04:00Z

1
UNITED
STATES
ENVIRONMENTAL
PROTECTION
AGENCY
WASHINGTON,
D.
C.
20460
JAN
30
2004
OFFICE
OF
PREVENTION,
PESTICIDES
AND
TOXIC
SUBSTANCES
MEMORANDUM
SUBJECT:
Review
of
Product
Characterization
and
Safety
Data
for
Phosphomannose
Isomerase,
an
Inert
Marker
Gene
from
Syngenta
Seeds,
Inc.

TO:
Mike
Mendelsohn
Regulatory
Action
Leader
Microbial
Pesticides
Branch,
Biopesticides
and
Pollution
Prevention
Division
(
7511C)

FROM:
John
L.
Kough,
Ph.
D.,
Biologist
Microbial
Pesticides
Branch,
Biopesticides
and
Pollution
Prevention
Division
(
7511C)

ACTION
REQUESTED:
To
review
the
data
submitted
for
a
determination
of
dietary
safety
for
phosphomannose
isomerase
(
PMI)
an
inert
ingredient
(
or
marker
gene)
in
plant­
incorporated
protectant.
s.

CONCLUSION:
The
data
presented
shows
a
lack
of
acute
oral
toxicity
at
a
high
dose
of
purified
PMI
(
3080mg/
kg
BW).
The
PMI
protein
was
also
shown
to
be
rapidly
degraded
by
simulated
gastric
fluid
or
simulated
intestinal
fluid
and
to
lose
significant
enzymatic
activity
when
incubated
at
65

C.
PMI
is
not
derived
from
a
source
known
to
have
induced
allergy
and
has
no
significant
amino
acid
sequence
homology
to
known
toxins
or
allergens.
These
data
suggest
that
the
is
a
reasonable
certainity
of
no
harm
resulting
from
the
cumulative
exposure
to
PMI.

DATA
REVIEW
RECORD
Active
Ingredient:
Cry1Ab
protein
and
PMI
protein
marker
gene
in
Event
3243M
Product
Name:
Event
3243M­
derived
Cry1Ab
Corn
Company
Name:
Syngenta
Seeds,
Inc.,
Research
Triangle
Park,
NC
2
Company
Number:
67979
Petition
No:
3E6748
Chemical
Number:
706505
Decision
Number:
330482
DP
Number:
292501
MRID
No:
459344­
02;
459344­
05
thru
09
BACKGROUND:
The
phosphomannose
isomerase
(
PMI)
is
a
new
marker
gene
employing
unusual
carbohydrate
metabolism
to
allow
for
selection
of
transformants
in
cell
culture.
Use
of
this
marker
addresses
some
of
the
complaints
received
from
the
public
about
the
possible
adverse
effects
of
using
antibiotic
resistance
genes
as
selection
markers.

DISCUSSION:
The
data
relating
the
PMI
protein
expressed
in
corn
to
the
bacterially
expressed
protein
had
one
curious
difference.
The
bacterial
protein
was
expressed
with
an
additional
17
amino
acids
(
a
14
AA
T7
tag
plus
3
AA
from
a
polylinker
region)
compared
to
that
stated
as
being
in
the
plant.
While
the
N­
termianl
tag
would
not
be
expected
to
alter
the
toxicity
of
PMI,
it
is
unclear
why
there
was
no
detectable
difference
between
these
two
forms
in
SDS­
PAGE.

RECOMMENDATION:
The
company
should
identify
the
92
known
and
hypothetical
proteins
that
are
stated
in
MRID
459344­
05
as
sharing
significant
homology
with
PMI.
This
should
include
the
known
source
of
these
proteins
as
well
as
any
activities
or
functions
that
are
associated
with
them.
It
would
also
be
helpful
for
the
company
to
explain
the
sensitivity
of
the
SDS­
PAGE/
western
blot
assay
to
detect
the
17
AA
difference
between
the
bacterial
and
plant
expressed
proteins.

SUMMARY
OF
DATA
SUBMITTED:

459344­
02­
This
is
a
summary
of
the
data
provided
elsewhere
on
phosphomannose
isomerase
(
PMI)
enzyme
and
the
genetic
material
required
for
production
of
this
protein
in
transgenic
corn
plants
derived
from
transformation
event
3243M.
PMI
is
a
ubiquitous,
essential
enzyme
from
a
nonallergenic
source
and
has
conceivably
always
been
in
the
human
diet
in
low
quantities.
The
results
of
the
equivalence
studies
indicate
similar
molecular
weight
by
SDS­
PAGE
between
the
corn
silk
expressed
PMI
and
that
produced
in
an
E.
coli
expression
vector.
This
is
somewhat
unusual
in
that
the
E.
coli
form
of
PMI
contained
an
additional
17
amino
acids
which
should
be
distinguishable
by
western
blot
analysis.
The
enzymatic
activity
is
also
similar
based
on
the
detectable
level
of
PMI
by
ELISA.
These
data
suggest
that
the
two
sources
produce
PMI
protein
that
is
similar
enough
that
the
bacterial
PMI
source
can
be
used
for
further
biochemical
and
toxicological
tests
to
establish
dietary
safety.
CLASSIFICATION:
Acceptable.

459344­
05­
The
PMI
amino
acid
sequence
showed
no
significant
homology
to
known
protein
toxins.
CLASSIFICATION:
Supplemental.
Can
be
upgraded
to
acceptable
with
identification
of
the
92
known
and
hypothetical
proteins
identified
as
having
significant
amino
acid
sequence
homology
to
PMI
along
with
the
source
of
those
proteins.
If
the
PMI
homologues
have
other
activities
or
3
functions
these
should
also
be
listed
when
identifying
the
92
proteins.

459344­
06­
The
PMI
protein
showed
no
significant
amino
acid
homology
with
known
or
putative
allergenic
proteins
using
either
an
8
amino
acid
sequence
stepwise
comparison
or
and
an
80
amino
acid
fragment
comparison.
CLASSIFICATION:
Acceptable.

459344­
07­
The
oral
LD
50
of
PMI­
0198
protein
for
males,
females,
and
combined
was
greater
than
3080
mg/
kg.
CLASSIFICATION:
ACCEPTABLE
­­
TOXICITY
CATEGORY
III
459344­
08­
Full
length
PMI­
0198
protein
was
degraded
to
undetectable
levels
in
Coomassie
blue
stained
SDS­
PAGE
gels
after
incubation
in
simulated
gastric
and
intestinal
fluids.
CLASSIFICATION:
Acceptable.

459344­
09­
The
phosphomannose
isomerase
(
PMI)
enzyme
derived
from
E.
coli
was
prepared
at
0.44
mg/
mL
in
standard
buffer
(
50
mM
Tris­
HCl,
pH
7.0)
and
incubated
at
ambient
temperature
(
25

C),
37,
55,
65
and
95

C
for
30
minutes.
PMI
enzymatic
activity
was
monitored
by
measuring
NADPH
production
using
a
spectrophotometer.
Results
indicate
that
incubation
at
ambient
temperature
(
25

C)
and
at
37
and
55

C
for
30
minutes
had
little
effect
on
PMI.
However,
incubation
at
65
and
95

C
for
30
minutes
inactivated
PMI.
CLASSIFICATION:
Acceptable.
4
DATA
EVALUATION
RECORD
________________________________________________________________________________
Reviewer:
Anthony
Q.
Armstrong,
M.
S.,
Oak
Ridge
National
Labs
EPA
Reviewer:
John
L.
Kough,
Ph.
D.,
Biologist
________________________________________________________________________________
STUDY
TYPE:
Product
Identity
[
Selectable
Marker
Phosphomannose
Isomerase
(
PMI)
Characterization
and
Safety]
MRID
NO:
459344­
02
DP
BARCODE:
D292501/
3E6748­
TEST
MATERIAL:
Cry1Ab
and
PMI
(
Corn
Event
3243M)
PROJECT
NO:
NA
SPONSOR:
Syngenta
Seeds,
Inc.,
3054
Cornwallis
Road,
RTP,
North
Carolina
27709­
2257
TESTING
FACILITY:
Syngenta
Seeds,
Inc.,
3054
Cornwallis
Road,
RTP,
North
Carolina
27709­
2257
TITLE
OF
REPORT:
Characterization
and
Safety
of
Phosphomannose
Isomerase
(
PMI),
a
Selectable
Marker
Expressed
in
Event
3243M­
Derived
Maize
(
Corn)
Plants
AUTHOR:
Demetra
Vlachos
and
Robert
Joseph
STUDY
COMPLETED:
April
28,
2003
GOOD
LABORATORY
PRACTICE:
Non­
GLP
compliant
CONCLUSION:
This
is
a
summary
of
the
data
provided
elsewhere
on
phosphomannose
isomerase
(
PMI)
enzyme
and
the
genetic
material
required
for
production
of
this
protein
in
transgenic
corn
plants
derived
from
transformation
event
3243M.
PMI
is
a
ubiquitous,
essential
enzyme
from
a
non­
allergenic
source
and
has
conceivably
always
been
in
the
human
diet
in
low
quantities.
The
results
of
the
equivalence
studies
indicate
similar
molecular
weight
by
SDS­
PAGE
between
the
corn
silk
expressed
PMI
and
that
produced
in
an
E.
coli
expression
vector.
This
is
somewhat
unusual
in
that
the
E.
coli
form
of
PMI
contained
an
additional
17
amino
acids
which
should
be
distinguishable
by
western
blot
analysis.
The
enzymatic
activity
is
also
similar
based
on
the
detectable
level
of
PMI
by
ELISA.
These
data
suggest
that
the
two
sources
produce
PMI
protein
that
is
similar
enough
that
the
bacterial
PMI
source
can
be
used
for
further
biochemical
and
toxicological
tests
to
establish
dietary
safety.
CLASSIFICATION:
Acceptable.

This
submission
by
Syngenta
summarizes
data
and
information
relevant
to
the
characterization
and
safety
of
the
phosphomannose
isomerase
(
PMI)
enzyme
and
the
genetic
material
required
for
production
of
this
protein
in
transgenic
corn
plants
derived
from
transformation
event
3243M.
PMI
is
considered
an
inert
ingredient
with
respect
to
pesticidal
activity
and
is
present
in
plants
as
a
selectable
marker.
The
company
stated
that
it
is
not
feasible
to
extract
sufficient
PMI
protein
directly
from
Event
3243­
M
plants
to
conduct
safety
studies.
Therefore,
PMI
was
produced
in
a
5
recombinant
E.
coli
over­
expression
system
for
testing
purposes
and
the
bacterially
produced
substance
was
compared
to
the
plant
version.

The
results
of
the
studies
showing
the
equivalence
of
the
bacterial
and
plant
produced
protein
are
found
in
appendix
A
in
this
submission
and
discussed
below.

Comparison
of
PMI
protein
produced
in
event
3243
corn
and
recombinant
E.
coli
(
appendix
A)
Proteins
purified
from
corn
silks
and
E.
coli
expression
systems
were
compared
by
apparent
size,
immunoreactivity
and
enzymatic
activity.
The
corn
silks
were
collected
from
event
3243M
derived
10­
week
old
corn
(
genotype
H8431
x(
NP2499
x
NP2500)­
3243M)
and
shipped
to
Syngenta
Seeds.
The
silks
were
powdered
with
a
mortar
and
pestle
in
liquid
nitrogen,
lyophilized
and
labeled
3243MPMI
silk.
Similar
corn
silk
powders
were
prepared
from
two
samples
of
isogenic
but
transgenic
negative
corn.
The
bacterial
protein
was
labeled
PMI­
0198
and
consisted
of
a
purified
preparation
isolated
from
E.
coli
strain
BLRDE3
transformed
with
the
pET­
3a
vector
containing
the
manA
(
pmi)
gene.
The
PMI
protein
expressed
is
similar
to
the
corn
protein
except
for
an
additional
N­
terminal
13
amino
acids
in
the
T7­
Tag
 
and
3
amino
acids
from
the
poly
linker
sequence.

The
bacterial
PMI
was
purified
by
differential
ammonium
sulfate
precipitation
followed
by
hydrophobic
interaction
chromatography
(
phenyl
sepharose
or
toyopearl
ether
packed
columns)
then
ion
exchange
chromatography.
The
appropriate
collected
fractions
were
pooled
prior
to
dialysis
against
a
50mM
NH
4
HCO
3
pH
8.0
buffer
then
subjected
to
lyophilization.
The
protein
content
of
the
lyophilized
product
was
found
to
be
approximately
93%
PMI
(

45,000
molecular
weight,
enzymatically
active)
and
was
given
the
designation
PMI­
0198.
The
lyophilized
prep
was
stored
at
­
20

C.

One
tenth
gram
of
the
dry
silk
tissue
preparations
was
suspended
in
3
ml.
of
extraction
buffer
(
500mM
CAPS,
1M
NaCl,
20
mM
EDTA,
1mM
AEBS,
2mM
DTT,
1
µ
M
leupeptin,
pH
10.0)
and
further
extracted
with
a
polytron
homogenizer.
This
extract
was
centrifuged
at
10,000
x
g
for
15
minutes.
The
supernatant
was
used
for
further
PMI
comparison
testing
by
ELISA,
western
blot
and
enzyme
activity.
Protein
concentration
of
these
silk
extracts
were
determined
by
the
BCA
method
after
iodoacetamide
treatement.
The
concentration
of
PMI
enzyme
in
the
preps
was
determined
by
ELISA
using
protein
A
purified
rabbit
polyclonal
IgG
and
an
immunoaffinity­
purified
goat
antibody
preparation
specific
for
PMI.

The
PMI
containing
samples
from
corn
silk
were
separated
by
SDS­
PAGE
followed
by
electroblotting.
The
sample
amounts
from
the
silk
extracts
loaded
into
the
wells
were
matched
for
apparent
PMI
concentration
(
5ng)
with
those
of
the
bacterial
preps
employing
the
results
of
the
ELISA
test.
A
similar
amount
of
non­
transgenic
corn
silk
extract
(
194
µ
g)
was
loaded
as
a
control
in
an
adjacent
well.
After
blotting,
the
membrane
was
probed
with
immunoaffinity­
purified
goat
antibody
generated
with
PMI
from
an
E.
coli
over­
expressing
the
manA
gene.
The
blot
was
visualized
by
reaction
with
a
rabbit
anti­
goat
HRP
conjugate
and
developed
with
a
chemiluminescent
substrate.

PMI
enzymatic
activity
was
measured
by
an
SOP
not
included
that
measured
the
production
of
NADPH
from
the
conversion
of
mannose­
6­
phosphate
to
6­
gluconolactone
and
NADPH
in
the
6
presence
of
phosphoglucose
isomerase
and
glucose
6­
phospahte
dehydrogenase.
This
NADPH
product
was
measured
by
absorbance
change
at
340nm
(
molar
extinction
coefficient
6.2).
A
unit
of
PMI
activity
is
defined
as
1
µ
mole
of
NADPH
reduced
per
minute.

The
results
of
the
western
blot
indicate
that
the
PMI
enzyme
purified
from
both
corn
silk
tissue
and
E.
coli
are
of
similar
molecular
weight
and
immunoreactivity
(
see
attached
blot).
The
enzymatic
activity
results
indicate
similar
activity
levels
per
unit
weight
of
PMI
enzyme
concentration
(
109.78
+
3.55units/
mg
PMI
in
silk
extract
and
93.21
+
18.23
units
/
mg
PMI
in
E.
coli
extract).

Source
and
Function
of
PMI
Protein
in
Event
3243M
Plants
The
PMI
protein
produced
in
Event
3243M­
derived
plants
is
encoded
by
the
native
pmi
gene
(
also
referred
to
elsewhere
as
the
"
manA"
gene)
from
E.
coli
(
strain
K­
12).
The
pmi
gene
(
GenBank
Accession
NO.
M15380;
NCBI,
2003)
encodes
a
protein
(
Genbank
Accession
No.
AAA24109.1)
of
391
amino
acids
and
ca.
45,000
molecular
weight.
PMI
catalyzes
the
reversible
interconverison
of
mannose­
6­
phosphate
and
fructose­
6­
phosphate.
The
PMI
reaction
is
specific
for
mannose­
6­
phosphate
and
fructose­
6­
phosphate
with
a
Keq
near
1.0.

Selection
of
PMI­
Expressing
Plant
Cells
by
Mannose
Plant
cells
that
produce
PMI
are
able
to
convert
mannose
to
a
readily
metabolized
compound,
fructose­
6­
phosphate
thus
improving
the
energy
status
of
cells
and
avoiding
the
accumulation
of
derivatized
mannose.
PMI
has
been
shown
to
be
an
effective
selectable
marker
via
multiple
transformation
methods
in
several
plants
including
maize,
wheat,
barley,
sugar
beets,
tomato,
rice
and
cassava.
The
use
of
PMI
as
a
selectable
marker
and
mannose
as
the
sole
carbon
source
and
selective
agent
offers
an
efficient
alternative
selection
system
for
a
number
of
plant
species
to
the
more
traditional
markers
that
confer
resistance
to
antibiotics
or
herbicides.
However,
use
of
this
selection
marker
is
limited
to
those
species
that
do
not
already
express
a
native
PMI
enzyme.

Lack
of
Unintended
Effects
of
PMI
Expression
in
Plants
Expression
of
the
pmi
gene
in
transformed
plants
does
not
appear
to
adversely
affect
plant
morphology,
growth
or
agronomic
characteristics
in
data
referred
to
for
corn
and
sugar
beets.
Studies
indicate
that
the
presence
of
PMI
activity
in
transformed
plants
does
not
affect
the
concentration
of
simple
carbohydrates.

PMI
Enzymes
in
Other
Organisms
PMI
enzymes
of
varying
degrees
of
amino
acid
homology
occur
widely
among
prokaryotes
and
eukaryotes.
Although
PMI
has
not
been
detected
in
some
plants,
PMI
enzymes
have
been
found
in
such
diverse
plant
species
as
tobacco,
pine,
walnut,
lily
bulbs,
Brassica
and
Arabidopsis
as
well
as
soybeans
and
other
legumes.
Putative
genes
for
PMI
have
been
found
in
rice
and
guar.
PMI
enzymes
have
also
been
purified
and
characterized
from
many
other
organisms
including
bacteria,
yeast,
rats,
pigs
and
humans.
There
are
three
classes
of
PMI
enzyme
with
the
subject
of
this
review
being
type
I.
This
type
of
PMI
is
also
found
in
Salmonella
typhimurium,
Saccharomyces
cerevisiae,
Aspergillus
nidulans,
Candida
albicans
and
humans.
The
amino
acid
sequence
homology
assessment
results
given
in
table
2
(
Attached)
are
unclear
since
the
company
states
the
different
PMI
types
are
determined
by
homology
yet
these
types
do
not
seem
to
relate
to
the
groups
shown
or
the
sequence
overlap.
7
Mammalian
Toxicology
Assessment
of
PMI
Protein
1.
An
extensive
bioinformatics
search
was
performed
to
determine
whether
the
amino
acid
sequence
of
the
PMI
protein
shows
homology
with
proteins
known
to
be
toxins.
PMI
showed
no
significant
homology
with
any
proteins
identified
as
or
known
to
be
toxins.
(
MRID
459344­
05)

2.
Because
it
was
not
feasible
to
extract
sufficient
PMI
protein
from
Event
3243M­
derived
corn
plants
for
toxicology
studies,
PMI
protein
was
produced
in
recombinant
E.
coli
by
over­
expressing
the
same
pmi
(
E.
coli
manA)
gene
that
was
introduced
into
Event
3243M
corn.
Equivalence
studies
between
the
corn
derived
PMI
and
E.
coli
PMI
(
appendix
A)
were
conducted
to
establish
the
similarity
of
the
protein
from
the
two
sources
and
are
reviewed
above.

3.
The
susceptibility
of
PMI
to
proteolytic
degradation
was
evaluated
in
simulated
mammalian
gastric
fluid
(
SGF)
containing
pepsin
and
also
in
simulated
mammalian
intestinal
fluid
(
SIF)
containing
pancreatin.
The
E.
coli
derived
PMI
was
rapidly
degraded
in
SGF
such
that
no
intact
PMI
was
detected
upon
immediate
analysis
of
the
reaction
mixture
by
both
protein
presence
and
enzymatic
activity.
Likewise,
the
E.
coli
derived
PMI
was
also
rapidly
degraded
in
SIF
with
no
intact
PMI
detected
after
2
min.
of
incubation
at
37

C.
These
data
indicate
that
PMI
will
be
readily
digested
as
conventional
dietary
protein
under
typical
mammalian
gastric
conditions.
Furthermore,
in
the
unlikely
event
that
PMI
protein
survives
the
gastric
environment,
it
will
rapidly
be
degraded
in
the
intestinal
environment.
(
MRID
459344­
08)

4.
An
acute
mouse
oral
toxicity
study
was
conducted
using
the
E.
coli
derived
PMI.
PMI
was
administered
to
7
male
and
6
female
young
mice
via
gavage
at
a
dose
of
5050
mg/
kg
body
weight
equivalent
to
ca.
3080
mg
pure
PMI/
kg
body
weight.
No
test
substance­
related
mortalities
or
clinical
signs
of
toxicity
occurred
during
the
study.
Gross
necropsy
of
the
remaining
mice
at
study
termination
revealed
no
observable
abnormalities.
The
NOEL
was
ca.
3080
mg
PMI
protein/
kg
body
weight.
The
estimated
LD
50
was
greater
than
3080
mg
PMI
protein/
kg
body
weight,
the
single
dose
tested.
(
MRID
459344­
07)

Potential
for
Exposure
to
PMI
via
Food
or
Feed
1.
Current
Exposure
to
PMI
Although
Syngenta
Seeds
is
unaware
of
documented
dietary
exposure
to
PMI,
it
is
conceivable
that
small
amounts
of
PMI
proteins
from
various
sources
have
always
been
present
in
the
food
and
feed
supply
due
to
ubiquitous
occurrence
of
PMI
enzymes
in
nature,
including
in
food
plants
and
animals.
Human
PMI,
which
has
significant
structural
and
functional
similarity
to
the
PMI
in
Event
3243Mderived
corn,
exists
in
several
body
tissues
and
is
an
essential
enzyme.
Furthermore,
PMI
is
present
in
naturally
occurring
E.
coli
strains
and
other
gram­
negative
bacteria
that
colonize
the
human
intestine
and
it
is
expected
that
these
PMI
proteins
are
highly
homologous
or
identical
to
PMI
present
in
Event
3243M­
derived
corn.

2.
Potential
for
Dietary
Exposure
to
PMI
via
Event
3243M­
Derived
Corn
PMI
is
present
in
the
grain
of
Event
3243M­
derived
plants
at
a
concentration
of
ca.
2
ppm
on
a
dry
weight
basis.
Although
trace
quantities
of
PMI
were
detected
in
15­
day
old
corn
silage
prepared
from
whole,
chopped
Event
3243M­
derived
corn
plants,
PMI
was
not
detectable
in
29­
day
old
silage.
Cooking
and
most
forms
of
food
processing
would
be
expected
to
degrade
and/
or
inactivate
8
PMI
in
corn
products
and
by­
products.

Evidence
that
PMI
is
Unlikely
to
Become
a
Food
Allergen
Although
the
probability
that
any
specific
novel
protein
will
become
a
food
allergen
is
small,
the
potential
allergenicity
of
the
PMI
protein
was
evaluated
using
a
weight
of
evidence
approach.
First,
food
products
derived
from
Event
3243M
corn
are
expected
to
contain
little
or
no
PMI
protein.
As
previously
noted,
the
level
of
PMI
in
corn
grain
is
not
expected
to
exceed
2
ppm.
Second,
PMI
protein
is
not
derived
from
a
know
source
of
allergens.
The
pmi
gene
was
cloned
from
E.
coli
which
has
no
history
of
allergenicity.
Third,
PMI
does
not
have
amino
acid
sequence
homology
to
known
allergens.
Extensive
bioinformatics
searches
were
conducted
to
determine
whether
the
amino
acid
sequence
of
the
PMI
protein
shows
homology
with
proteins
known
or
suspected
to
be
allergens.
Comparisons
among
protein
databases
containing
allergens
or
putative
allergens
was
compared
by
contiguous
PMI
peptides
of
80
amino
acids
as
well
as
comparisons
using
shorter
sequences
of
8
amino
acids.
Results
from
the
searches
indicate
no
similarity
of
the
PMI
protein
to
any
known
or
putative
allergens
for
which
amino
acid
sequences
were
available.
(
MRID
459344­
06)

Fourth,
PMI
protein
is
not
highly
stable
like
many
food
allergens.
Incubation
of
PMI
at
65

C
for
30
minutes
essentially
inactivated
the
enzyme.
Additionally,
the
PMI
protein
is
unlikely
to
be
stabilized
by
disulfide
bonds
since
there
are
only
two
cysteine
residues
in
the
protein.
Stabilized
bonds
appear
to
contribute
to
the
allergenicity
of
some
food
proteins.
Fifth,
PMI
protein
is
readily
degraded
in
simulated
mammalian
gastric
and
intestinal
fluids
(
see
Mammalian
Toxicity
section).
Sixth,
PMI
protein
is
not
likely
to
be
post­
translationally
glycosylated
because
its
expression
in
corn
is
not
targeted
to
a
cellular
glycosylated
pathway.
PMI
expressed
in
E.
coli
is
not
glycosylated
and
mass
spectrometric
analysis
of
human
PMI
indicates
no
post­
translational
modifications.

In
summary,
PMI
is
a
ubiquitous,
essential
enzyme
from
a
non­
allergenic
source
and
has
conceivably
always
been
in
the
human
diet
in
low
quantities.
The
non­
allergenic
characteristics
of
the
PMI
protein,
combined
with
the
minimal
potential
for
additional
dietary
exposure
via
Event
3243M
corn
indicate
that
the
potential
for
PMI
to
become
a
food
allergen
is
extremely
low.

Reviewer's
Conclusion
The
results
of
the
equivalence
study
are
the
only
new
and
essential
information
provided
in
this
submission.
The
other
parts
are
a
reiteration
of
information
provided
elsewhere
or
derivative
information
used
by
the
company
to
estimate
dietary
exposure
of
PMI
expressed
in
corn.
The
results
of
the
equivalence
studies
indicate
similar
molecular
weight
by
SDS­
PAGE
between
the
corn
silk
expressed
PMI
and
that
produced
in
an
E.
coli
expression
vector.
This
is
somewhat
unusual
in
that
the
E.
coli
form
of
PMI
contained
an
additional
17
amino
acids
which
should
be
distinguishable
by
western
blot
analysis.
The
enzymatic
activity
is
also
similar
based
on
the
detectable
level
of
PMI
by
ELISA.
These
data
suggest
that
the
two
sources
produce
PMI
protein
that
is
similar
enough
that
the
bacterial
PMI
source
can
be
used
for
further
biochemical
and
toxicological
tests
to
establish
dietary
safety.
9
DATA
EVALUATION
RECORD
________________________________________________________________________________
Primary
reviewer:
H.
Tim
Borges,
Ph.
D.,
MT
(
ASCP),
DABT,
Oak
Ridge
National
Lab
EPA
Reviewer:
John
L.
Kough,
Ph.
D.,
Biologist
________________________________________________________________________________
STUDY
TYPE:
Amino
acid
sequence
comparison
to
known
protein
toxins
MRID
NO:
459344­
05
DP
BARCODE:
D292501/
3E6748
TEST
MATERIAL:
Event
3243M
Corn
EUP
PROJECT
REPORT
NO:
SSB­
016­
03
TESTING
FACILITY:
Syngenta
Seeds,
Inc.,
Product
Registration
Group,
P.
O.
Box
12257,
3054
Cornwallis
Road,
Research
Triangle
Park,
NC
27709­
2257
TITLE
OF
REPORT:
Phosphomannose
isomerase
protein:
assessment
of
amino
acid
homology
with
known
toxins
AUTHOR:
J.
Zawodny
STUDY
COMPLETED:
April
23,
2002
CONCLUSION:
The
PMI
amino
acid
sequence
showed
no
significant
homology
to
known
protein
toxins.
CLASSIFICATION:
Supplemental.
Can
be
upgraded
to
acceptable
with
identification
of
the
92
known
and
hypothetical
proteins
identified
as
having
significant
amino
acid
sequence
homology
to
PMI
along
with
the
source
of
those
proteins.
The
other
activities
of
PMI
homologues
should
also
be
listed.
GOOD
LABORATORY
PRACTICE:
Non­
GLP
study
________________________________________________________________________________

A.
STUDY
PURPOSE:
The
study
was
done
to
determine
whether
E.
coli­
derived
phosphomannose
isomerase
protein
(
PMI)
has
any
significant
amino
acid
homology
with
known
toxic
protein
sequences.

B.
MATERIALS
AND
METHODS:
The
391
amino
acid
sequence
of
PMI
was
compared
using
the
v.
2.2.6
BLASTP
program
(
Altschul
et
al.,
1997)
to
known
amino
acid
protein
sequences
contained
in
National
Center
for
Biotechnology
Information
(
NCBI)
GenBank
Database
(
2003
posting).
This
program
develops
an
"
Expect"
(
E)
ratio
where
comparisons
between
highly
homologous
proteins
yield
ratios
approaching
zero
indicating
the
probability
of
similarities
are
not
occurring
by
chance.
To
determine
if
any
of
the
"
hits"
identified
with
E
ratios
<
10
were
had
random
similarities
or
if
they
were
homologous,
the
amino
acid
sequence
of
PMI
was
shuffled
into
five
additional
versions
each
containing
the
same
391
amino
acids.
These
shuffled
sequences
represented
the
background
control
incidence
of
random
hits
for
the
amino
acid
composition.

C.
RESULTS:
The
shuffled
PMI
sequences
returned
E
ratios
ranging
from
0.42
to
8.0.
To
be
considered
potentially
homologous,
the
E
ratio
for
the
intact
PMI
sequence
would
need
to
be
<
0.42.
The
BLASTP
program
identified
92
protein
sequences
in
the
GenBank
database
as
potentially
homologus
with
PMI.
Of
these,
77
proteins
were
PMI
homologs
with
E
ratios
ranging
from
0.0
to
0.42;
one
protein
was
a
Neisseria
menigitidis
RNA
polymerase
 
factor
not
10
known
to
be
toxic,
and
14
hypothetical
protein
sequences.
Therefore,
the
PMI
amino
acid
sequence
showed
no
significant
homology
to
known
protein
toxins.
11
DATA
EVALUATION
RECORD
________________________________________________________________________________
Primary
reviewer:
H.
Tim
Borges,
Ph.
D.,
MT
(
ASCP),
DABT,
Oak
Ridge
National
Lab
EPA
Reviewer:
John
L.
Kough,
Ph.
D.,
Biologist
________________________________________________________________________________
STUDY
TYPE:
Amino
acid
sequence
comparison
to
known
allergens
MRID
NO:
459344­
06
DP
BARCODE:
D292501/
3E6748
TEST
MATERIAL:
Event
3243M
Corn
EUP
PROJECT
REPORT
NO:
SSB­
014­
03
TESTING
FACILITY:
Syngenta
Seeds,
Inc.,
Product
Registration
Group,
P.
O.
Box
12257,
3054
Cornwallis
Road,
Research
Triangle
Park,
NC
27709­
2257
TITLE
OF
REPORT:
Phosphomannose
isomerase
protein:
assessment
of
amino
acid
homology
with
known
allergens
AUTHOR:
J.
Zawodny
STUDY
COMPLETED:
April
23,
2002
CONCLUSION:
The
PMI
protein
showed
no
significant
amino
acid
homology
with
known
or
putative
allergenic
proteins
using
either
an
8
amino
acid
sequence
stepwise
comparison
or
and
an
80
amino
acid
fragment
comparison.
CLASSIFICATION:
Acceptable
GOOD
LABORATORY
PRACTICE:
Non­
GLP
study
________________________________________________________________________________

D.
STUDY
PURPOSE:
The
study
was
done
to
determine
whether
E.
coli­
derived
phosphomannose
isomerase
protein
(
PMI)
has
any
significant
amino
acid
homology
with
known
allergenic
sequences.

B.
MATERIALS
AND
METHODS:
The
391
amino
acid
sequence
of
PMI
was
compared
to
the
Syngenta
Biotechnology,
Inc.
(
SBI)
Allergen
Database.
The
SBI
Allergen
Database
contains
the
amino
acid
sequences
of
known
or
putative
allergens
and
was
initially
compiled
from
the
following
sources:
the
publically
available
GenPept,
PIR
protein
database
(
2001
version);
the
SWISS­
PROT
database
(
2001
version);
the
List
of
Allergens
database
(
International
Union
of
Immunological
Societies,
2001
version);
and
the
Food
Allergy
Research
and
Resource
Program
(
2001
version).
In
addition,
the
database
has
been
updated
with
putative
allergens
identified
in
the
scientific
literature.
The
SBI
Allergen
Database
contains
1114
entries
and
was
last
updated
in
February
2003.

Two
types
of
comparisons
of
the
PMI
protein
sequence
were
done.
For
the
first
comparison,
the
similarity
of
sequential
PMI
protein
80
amino
acid
peptide
blocks
were
compared
to
the
allergens
using
the
FASTA
search
algorithm.
Each
successive
80
amino
acid
block
was
offset
from
the
previous
block
by
one
amino
acid.
Any
sequence
with
>
35%
homology
to
an
allergen
sequence
was
considered
positive.

For
the
second
comparison,
a
computer
program
developed
by
Syngenta
compared
8
or
more
12
contiguous
amino
acids
of
the
PMI
protein
to
every
possible
8
contiguous
amino
acids
of
allergens
in
the
SBI
Allergen
Database.
The
purpose
of
this
study
was
to
screen
for
short,
local
regions
of
amino
acid
identity
that
could
indicate
IgE­
binding
epitopes.

C.
RESULTS:
No
significant
similarities
between
any
of
the
sequential
PMI
80­
amino
acid
peptides
with
peptides
in
the
SBI
Allergen
Database
were
found
using
the
FASTA
algorithm.
In
addition,
no
identical
alignments
of
8
or
more
contiguous
amino
acids
between
PMI
protein
and
known
or
putative
allergens
in
the
SBI
Allergen
Database
were
found.
13
DATA
EVALUATION
RECORD
________________________________________________________________________________
Primary
Reviewer:
Susan
Chang,
M.
S.,
Oak
Ridge
National
Lab
Secondary
Reviewer:
John
L.
Kough,
Ph.
D.,
Biologist
________________________________________________________________________________
STUDY
TYPE:
Acute
Oral
Toxicity
­
Mice
(
OPPTS
870.1100)
MRID
NO:
459344­
07
DP
BARCODE
NO:
D292501
CASE
NO:
Not
reported
SUBMISSION
NO:
Not
reported
TEST
MATERIAL:
PMI­
0198
Protein
containing
~
60%
phosphomannose
isomerase
PROJECT
NO:
4708­
98
SPONSOR:
Novartis
Seeds,
Inc.
­
Field
Crops
­
NAFTA
TESTING
FACILITY:
Stillmeadow,
Inc.,
Sugar
Land,
TX
TITLE
OF
REPORT:
Acute
Oral
Toxicity
Study
in
Mice
AUTHOR:
Janice
O.
Kuhn,
Ph.
D.,
D.
A.
B.
T.
STUDY
COMPLETED:
August
11,
1999
GOOD
LABORATORY
PRACTICE:
GLP
Compliant
CONCLUSION:
The
oral
LD
50
of
PMI­
0198
protein
for
males,
females,
and
combined
was
greater
than
3080
mg/
kg.
CLASSIFICATION:
ACCEPTABLE
­­
TOXICITY
CATEGORY
III
________________________________________________________________________________

I.
STUDY
DESIGN:

1.
Test
material:
PMI­
0198
Protein
containing
~
60%
phosphomannose
isomerase
2.
Test
animals:
Thirteen
male
and
eleven
female
HSD:
ICR
mice
were
received
from
Harlan
Sprague
Dawley,
Inc.,
Indianapolis,
IN,
were
assigned,
and
weighed
22.2­
28.2
g
(
males)
and
18.6­
24.7
g
(
females)
on
the
day
of
dosing.
The
young
adult
animals,
approximately
4
weeks
old,
were
housed
individually
in
suspended,
wire
top,
polycarbonate
cages.
The
animals
were
fed
PMI
Feeds
Inc.
Formulab
N.
5008,
ad
libitum,
except
for
approximately
16
hours
before
dosing.
Municipal
water
was
available
ad
libitum.
The
environmental
conditions
of
the
animal
room
were
as
follows:
temperature,
22

±
3

C;
humidity,
30­
80%;
air
changes,
10­
12
per
hour;
and
photoperiod,
12
hour
light/
dark
cycle.

3.
Methods:
Mice
were
ear­
tagged:
males
(
Test
group
­
Nos.
11­
M
to
16­
M
and
28­
M;
Control
group
­
Nos.
1­
M
to
5­
M
and
27­
M)
and
females
(
Test
group
­
Nos.
21­
F
to
26­
F;
Control
group
­
Nos.
6­
F
to
10­
F).
The
mice
were
quarantined
for
5
days
and
fasted
16
hours
prior
to
dosing.
The
test
material
(
5050
mg/
kg
body
weight)
was
dosed
as
a
20%
w/
v
suspension
in
a
0.5%
w/
v
aqueous
CMC
by
gavage
at
a
volume
of
25.25
mL/
kg
to
seven
test
males
and
five
test
females
(
Table
1).
The
control
group
(
six
males
and
five
females)
was
dosed
with
25.25
mL/
kg
of
0.5%
CMC.
Body
weights
were
recorded
prior
to
dosing,
on
day
14
or
at
death.
The
test
animals
were
observed
for
clinical
signs
of
toxicity
at
least
three
times
post­
dosing
and
at
least
daily
for
14
days.
All
decedent
or
euthanized
animals
were
necropsied.
14
II.
RESULTS:

1.
Mortality:
Mortality
is
given
in
Table
1.
Two
test
males
(
Nos.
11­
M
and
16­
M)
died
on
the
day
of
dosing
and
one
control
male
(
No.
3­
M)
was
dead
on
day
1.
One
replacement
animal
was
available
for
each
group.
All
other
rats
survived
the
study.

TABLE
1.
Doses,
mortality/
animals
treated
Dose
(
mg/
kg)
Males
Females
Combined
0
1/
6
0/
5
1/
11
5050
2/
7
0/
5
2/
12
Data
taken
from
Table
1,
p.
10­
11,
MRID
45934407.

2.
Body
weights:
All
surviving
animals
gained
weight
during
the
study.

3.
Clinical
observations:
No
clinical
signs
of
toxicity
were
noted
from
any
surviving
animal.

4.
Gross
necropsy:
The
decedents
had
perforated
esophaguses
due
to
gavage
error.
No
gross
abnormalities
were
noted
from
any
survivor.

III.
DISCUSSION:
The
mouse
oral
LD
50
for
males,
females,
and
combined
was
greater
than
5050
mg/
kg
of
dosing
solution
or
3080
mg/
kg
of
PMI
protein.
This
places
PMI­
0198
Protein
in
TOXICITY
CATEGORY
III.
The
packet
classification
is
ACCEPTABLE.

Appendix
A.
Characterization
of
the
Test
Substance
PMI­
0198:
Similar
information
is
to
be
found
in
MRID
459344­
02.

The
test
substance
protein
(
sample
PMI­
0198)
consists
of
a
purified
preparation
of
phosphomannose
isomerase
(
PMI)
isolated
from
E.
coli
strain
BLRDE3
transformed
with
the
pET­
3a
vector
containing
the
manA
(
pmi)
gene.
The
PMI
protein
expressed
is
similar
to
that
found
in
corn
except
for
an
additional
16
amino
acids
at
the
N­
terminus
(
13
amino
acids
in
the
T7­
Tag
 
and
3
amino
acids
from
the
poly
linker
sequence).

I.
MATERIALS
AND
METHODS
Purification:
The
bacterial
PMI
was
purified
by
differential
ammonium
sulfate
precipitation
followed
by
hydrophobic
interaction
chromatography
(
Phenyl
Sepharose
®
or
Toyopearl
Ether
®
650M
packed
columns)
then
ion
exchange
chromatography
on
a
Sepharose
Q
®
column.
The
appropriate
collected
fractions
were
pooled
prior
to
dialysis
against
a
50mM
NH
4
HCO
3
pH
8.0
buffer
then
subjected
to
lyophilization.
The
protein
content
of
the
lyophilized
product
was
found
to
be
approximately
93%
PMI
(

45,000
molecular
weight,
enzymatically
active)
and
was
given
the
15
designation
PMI­
0198.
The
lyophilized
prep
was
stored
at
­
20

C.
An
aliquot
of
PMI­
0198
was
dissolved
in
50mM
Tris­
HCl,
pH
7.0
to
a
final
concentration
of
10
mg/
ml.

Protein
quantification:
The
PMI­
0198
prep
was
subjected
to
BCA
 
procedure
for
determining
total
protein
against
an
ovalbumin
standard.

PMI
quantification:
The
actual
amount
of
PMI
enzyme
present
was
determined
by
ELISA
(
SOP
2.43,
not
submitted)
using
immunoaffinity
purified
polyclonal
rabbit
antibodies
and
protein
G
purified
goat
antibodies
specific
for
PMI.

Molecular
weight
and
densitometric
determination:
SDS­
PAGE
(
14%
polyacrylamide)
with
Novex
molecular
weight
markers
followed
by
Coomassie
blue
staining
was
used
to
determine
the
molecular
weight
and
purity
of
the
PMI­
0198
prep.
There
was
a
disagreement
between
the
protein
determinations
by
BCA
and
ELISA
assays
for
the
percent
amount
of
PMI
present.
The
company
claims
this
indicates
that
ovalbumin
is
an
inappropriate
standard
to
use
in
BCA
total
protein
assays
for
the
PMI
prep.
Therefore,
an
additional
test
of
purity
of
the
PMI­
0198
was
undertaken
as
a
densitometric
scan
of
the
Coomassie
blue
SDS­
PAGE
gel
(
SOP
2.6,
not
submitted).

Immunoreactivity:
Aliquots
of
the
PMI­
0198
ranging
from
5
to
25
ng
of
PMI
per
lane
prep
were
electroblotted
after
SDS­
PAGE
(
SOPs
2.4
&
2.3,
not
submitted).
The
membranes
were
probed
with
goat
polyclonal
antiserum
specific
for
PMI.
Rabbit
anti­
goat
IgG
horseradish
peroxidase
(
HRP)
conjugate
was
used
to
detect
the
bound
goat
anitserum.
The
bound
antibodies
were
visualized
by
HRP
reaction
on
the
chromogenic
substrate
diaminobenzidine.

Enzymatic
Activity:
The
verify
the
integrity
of
the
PMI
enzyme,
activity
assays
were
performed
using
production
of
NADPH
from
the
reaction
of
PMI
on
mannose
coupled
to
phosphoglucose
isomerase
(
PGI)
and
glucose
6­
phosphate
dehydrogenase
(
G6PDH)
as
seen
below:

PMI
PGI
G6PDH
Mannose
6­
P

fructose
6­
P

glucose
6­
P
+
NADP

6
gluconolactone
+
NADPH
NADPH
production
is
monitored
spectrophotometrically
at
340nm
(
SOP
2.44,
not
submitted).
The
micromolar
extinction
coefficient
of
NADPH
is
6.2
with
a
unit
of
enzyme
activity
being
defined
as
1
µ
mole
of
NADP
being
reduced
per
minute.

Lipopolysaccharide
measurement:
Lipopolysaccharide
(
LPS)
or
gram
negative
bacterial
endotoxin
was
determined
since
it
is
a
common
contaminant
in
preparations
purified
from
E.
coli
expression
systems.
The
E­
TOXATE
 
kit
was
employed
to
quantify
the
amount
of
LPS
present
in
PMI­
0198
following
the
manufacturer's
16
instruction
(
SOP
2.35,
not
submitted).

N­
terminal
amino
acid
sequencing:
A
sample
of
PMI­
0198
was
analyzed
for
the
N­
terminal
amino
acid
sequence
to
confirm
the
expressed
sequence
was
the
same
as
that
encoded
in
the
manA
gene.
The
PMI­
0198
sample
was
subjected
to
SDS­
PAGE,
electroblotted
athe
stained
with
amido
black
stain.
The
appropriate
band
was
cut
out
and
subjected
to
Edman
degradation,
phenylhydantoin
coupling
followed
by
fractionation
of
the
amino
acid
on
HPLC
(
SOP
2.34
not
submitted)

Solubility
determination:
The
solubility
of
PMI­
0198
was
measured
by
SOP
2.36
(
not
submitted).

II.
RESULTS
AND
DISCUSSION
The
apparent
molecular
weight
of
the
major
band,
assumed
to
be
PMI,
appearing
on
SDS­
PAGE
and
western
blot,
in
both
the
initial
and
repeat
run,
was
approximately
45,000.
The
protein
quantification
of
PMI
by
ELISA
correction
of
BCA
value
yielded
an
initial
finding
of
61%
of
the
PMI­
0198
sample
as
PMI
protein.
Re­
analysis
after
the
acute
oral
study
gave
a
value
of
68%
PMI.
Analysis
of
the
PMI
percentage
by
densitometery
from
a
Coomassie
blue
stained
SDS­
PAGE
gel
yielded
a
PMI
value
of
95%
initially
and
93%
in
the
repeat
analysis
after
the
oral
study.
The
enzyme
activity
test
gave
an
initial
value
of
7.34+
0.17
units/
mg
PMI
protein
and
6.26+
0.81
units/
mg
PMI
protein
after
the
study.
The
LPS
value
for
the
PMI­
0198
sample
was
found
to
be
0.3ng/
mg.
The
company
states
that
the
known
LD
50
values
for
LPS
in
rats
by
the
intraperitoneal
or
intravenous
route
of
exposure
are
107
mg/
kg
or
4.33
mg/
kg,
respectively.
The
N­
terminal
amino
acid
analysis
gave
the
following
sequence:

ASMTGGQQMGRGPIMQKL
This
sequence
would
match
the
expected
AA
sequence
starting
with
the
second
amino
acid
in
the
T7
tag.
It
is
important
to
note
that
this
only
includes
the
first
4
amino
acids
of
the
PMI
protein
as
claimed
to
be
expressed
in
plant
tissue:
MQKL.
The
solubility
test
gave
an
estimate
of
solubility
of
the
PMI­
0198
sample
at
above
150
mg/
ml.
All
these
tests
indicate
that
the
PMI­
0198
sample
was
stable
for
the
duration
of
the
acute
oral
study.
17
DATA
EVALUATION
RECORD
________________________________________________________________________________
Primary
Reviewer:
H.
Tim
Borges,
Ph.
D.,
MT(
ASCP),
DABT,
Oak
Ridge
National
Lab
EPA
Reviewer:
John
L.
Kough,
Ph.
D.,
Biologist
________________________________________________________________________________
STUDY
TYPE:
In
vitro
simulated
digestion
MRID
NO:
459344­
08
DP
BARCODE:
D292501/
3E6748
TEST
MATERIAL:
Event
3243M
Corn
EUP
REPORT
NUMBER:
NSB­
002­
99
TESTING
FACILITY:
Novartis
Seeds,
Inc.,
Product
Registration
Group,
3054
Cornwallis
Road,
Research
Triangle
Park,
NC
27709­
2257
TITLE
OF
REPORT:
In
vitro
digestibility
of
PMI
protein
under
simulated
mammalian
gastric
and
intestinal
conditions
AUTHOR:
Privalle,
L.
STUDY
COMPLETED:
June
24,
1999
CONCLUSION:
Full
length
PMI­
0198
protein
was
degraded
to
undetectable
levels
in
Coomassie
blue
stained
SDS­
PAGE
gels
after
incubation
in
simulated
gastric
and
intestinal
fluids.
CLASSIFICATION:
Acceptable
GOOD
LABORATORY
PRACTICES:
Full
compliance
with
40
CFR
160,
signed
June
24,
1999
________________________________________________________________________________

D.
STUDY
PURPOSE:
The
study
was
done
to
determine
whether
E.
coli­
derived
phosphomannose
isomerase
protein
(
PMI)
underwent
proteolytic
degradation
in
simulated
mammalian
gastric
and
intestinal
fluid
environments.
The
equivalence
studies
for
E.
coli
expressed
and
plant
expressed
PMI
are
found
in
MRID
459344­
02.

B.
MATERIALS:

PMI
Source:
The
test
material
was
prepared
as
sample
PMI­
0198
by
extraction
and
differential
ammonium
sulfate
concentration
followed
by
hydrophobic
interaction
chromatography
and
ion
exchange
chromatography
from
the
inducible
over­
expression
pET­
3a
®
vector
in
E.
coli
strain
BLRDE3.
Dialysis
into
50
mM
ammonium
carbonate
was
done
following
lyophilization.
The
sample
was
~
60%
PMI
protein
by
weight
and
accounted
for
~
95%
of
the
protein
in
the
sample.

SGF:
Simulated
gastric
fluid
(
SGF,
pH
1.0­
1.2)
containing
2
mg/
mL
sodium
chloride,
14

L
6
N
hydrochloric
acid/
mL,
and
3.2
mg/
mL
(
3200­
3400
units/
mg)
pepsin
was
prepared
fresh
for
each
assay.
Simulated
gastric
fluid
was
also
prepared
as
above
without
the
addition
of
pepsin
for
use
as
a
negative
digestion
control.
To
monitor
digestion,
SGF
was
prepared
with
10%,
1%,
0.1%,
and
0.01%
the
amount
of
pepsin
as
contained
in
the
standard
SGF.

SIF:
Simulated
intestinal
fluid
(
SIF,
pH
7.5)
containing
6.8
mg
potassium
phosphate,
10
mg/
mL
pancreatin,
and
tap
water
was
prepared
daily
and
checked
for
proteolytic
activity
using
azoalbumin
as
the
substrate.
SIF
was
also
prepared
with
10%,
1%,
and
0.1%
the
pancreatin
to
18
monitor
digestion.

SDS­
PAGE:
A
10

L
aliquot
from
each
reaction
mixture
was
loaded
on
to
14%
polyacrylamide
gels
and
electrophoresed.
The
gels
were
stained
with
Coomassie
blue
for
protein
detection.

C.
METHOD:

Digestion
Studies:
The
digestion
reactions
were
initiated
by
the
addition
of
20

L
of
10
mg/
mL
PMI­
0198
to
80

L
of
each
of
the
following
prepared
solutions:
full
strength
concentration,
10%,
1%,
or
0.1%
enzyme
concentrations
in
the
SGF
or
SIF
mixture.
An
aliquot
was
immediately
removed
and
the
reaction
quenched
with
an
equal
volume
Laemmli
buffer
followed
by
heating
at
75

C
for
10
minutes.
The
remainder
of
the
reaction
mixture
was
incubated
for
two
minutes
before
quenching.
To
follow
the
time­
course
of
gastric
digestion,
80

L
of
10
mg/
mL
PMI­
0198
was
added
to
320

L
of
0.01%
SGF.
Aliquots,
10

L,
were
removed
immediately
after
addition,
and
after
2,
5,
10,
20,
30,
and
60
minutes
of
incubation
at
37

C.

Enzymatic
Inactivation
Studies:
These
studies
were
done
to
monitor
loss
of
PMI
activity
in
SGF.
The
studies
were
done
by
adding
20

L
of
10
mg/
mL
PMI­
0198
to
80

L
SGF
containing
0.01%
pepsin,
SGF
without
pepsin,
or
50
mM
Tris­
HCl
pH
7.0
buffer.
Ten

L
aliquots
of
each
reaction
mixture
were
removed
0,
2,
5,
10,
20,
30,
and
60
minutes
after
substrate
addition
and
immediately
placed
into
PMI
assay
mixture
and
stored
on
ice
until
time
of
assay.
(
The
control
"
buffer
alone"
reaction
aliquots
were
removed
at
0
and
60
minutes
only.)
The
enzymatic
reactions
were
initiated
by
addition
of
mannose
6­
phosphate
and
the
change
in
absorbance
monitored
at
340
nm.

D.
RESULTS:

SGF:
PMI­
0198
was
rapidly
degraded
at
the
standard
pepsin
concentration
and
at
pepsin
concentrations
of
10%
and
1%.
At
a
pepsin
concentration
of
0.1%,
full
length
PMI­
0198
was
degraded
to
smaller
fragments
within
two
minutes.
(
figure
1,
attached)
With
a
pepsin
concentration
of
0.01%
full
length
PMI­
0198
was
degraded
within
10
minutes.
(
figure
3,
attached)
SIF:
Full
length
PMI­
0198
was
degraded
to
smaller
fragments
within
two
minutes
at
the
standard
pancreatin
concentration.
At
lower
pancreatin
concentrations
degradation
was
not
completed
within
the
two
minute
incubation
period.
(
figure
2,
attached)
Enzymatic
Inactivation:
PMI­
0198
enzymatic
activity
was
relatively
stable
at
37

C
in
SGF
without
pepsin
and
in
buffer.
However,
no
enzymatic
activity
was
detected
after
10
minutes
of
incubation
in
the
presence
of
0.01%
pepsin.
(
figure
4,
attached)

Reviewer's
comments
The
in
vitro
SGF
and
SIF
degradation
of
the
PMI
is
apparent
here
by
the
absence
of
significant
Coomassie
blue
stained
bands
after
introduction
of
the
substrate.
Using
lower
amounts
of
the
digestive
enzymes
in
the
buffer
solution,
a
time
course
of
the
degradation
can
be
visualized
showing
an
accumulation
of
smaller
molecular
weight
bands
over
time
as
the
inital
PMI
substrate
band
becomes
fainter
until
all
the
bands
disappear
(
figure
3
for
SGF
attached).
The
PMI
protein
19
does
not
seem
to
be
as
readily
degraded
by
SIF
(
figure
2
attached).

An
approximately
40,000
MW
band
was
faint
but
apparent
in
the
lanes
with
the
full
strength
pepsin
in
SGF.
The
company
stated
the
band
was
pepsin.
Pepsin
has
a
range
of
apparent
molecular
weights
depending
on
the
source
it
was
isolated
from.

The
full
strength
and
10%
intestinal
fluid
lanes
had
a
heavily
staining
band
at
approximately
50,000
molecular
weight
as
well
as
fainter
bands
at
approximately
30,000
and
25,000
molecular
weight.
These
bands
could
also
represent
the
presence
of
numerous
digestive
enzymes
present
in
pancreatin.
Possibilities
include
pancreatic
lipase
(
49,000
MW),
trypsin
(
24,000
MW)
and
carboxypeptidase
B
(
34,700
MW).
20
DATA
EVALUATION
RECORD
________________________________________________________________________________
Primary
Reviewer:
Anthony
Q.
Armstrong,
M.
S.,
Oak
Ridge
National
Labs
EPA
Reviewer:
John
L.
Kough,
Ph.
D.,
Biologist
________________________________________________________________________________
STUDY
TYPE:
Product
Identity
[
Temperature
Stability
of
PMI]
MRID
NO:
459344­
09
DP
BARCODE:
D292501/
3E6748­
TEST
MATERIAL:
Cry1Ab
and
PMI
(
Corn
Event
3243M)
PROJECT
NO:
SSB­
013­
03
SPONSOR:
Syngenta
Seeds,
Inc.
3054
Cornwallis
Road,
RTP,
North
Carolina
27709­
2257
TESTING
FACILITY:
Syngenta
Seeds,
Inc.
3054
Cornwallis
Road,
RTP,
North
Carolina
27709­
2257
TITLE
OF
REPORT:
Effects
of
Temperature
on
Stability
of
Phosphomannose
Isomerase
(
PMI)
AUTHOR:
Kim
Hill
STUDY
COMPLETED:
April
25,
2003
GOOD
LABORATORY
PRACTICE:
Not
40
CFR
160
GLP
compliant
CONCLUSION:
The
phosphomannose
isomerase
(
PMI)
enzyme
derived
from
E.
coli
was
prepared
at
0.44
mg/
mL
in
standard
buffer
(
50
mM
Tris­
HCl,
pH
7.0)
and
incubated
at
ambient
temperature
(
25

C),
37,
55,
65
and
95

C
for
30
minutes.
PMI
enzymatic
activity
was
monitored
by
measuring
NADPH
production
using
a
spectrophotometer.
Results
indicate
that
incubation
at
ambient
temperature
(
25

C)
and
at
37
and
55

C
for
30
minutes
had
little
effect
on
PMI.
However,
incubation
at
65
and
95

C
for
30
minutes
inactivated
PMI.
CLASSIFICATION:
Acceptable.

I.
TEST
MATERIAL:
Purified
PMI
was
produced
from
over­
expression
of
the
pET­
3A
®
vector
(
containing
the
manA
gene)
in
E.
coli
strain
BLR
(
DE3).
This
test
substance
was
designated
PMI­
0198
by
Syngenta.

II.
METHODS:
For
temperature
stability
analysis,
0.44
mg/
mL
solution
of
PM­
0198
was
prepared
in
standard
buffer
(
50
mM
Tris­
HCl,
pH
7.0)
and
incubated
at
ambient
temperature
(
25

C),
37,
55,
65
and
95

C
for
30
minutes.
PMI
enzymatic
activity
was
measured
by
NADPH
production3
using
a
UV­
1601
Shimadzu
spectrophotometer
set
at
UV
absorbance
of
340
nm.
Samples
were
assayed
in
triplicate
and
results
normalized
to
25

C.

III.
RESULTS:
Table
1
contains
results
from
the
temperature
inactivation
studies.
Results
indicate
that
incubation
at
ambient
temperature
(
25

C)
and
at
37
and
55

C
for
30
minutes
had
no
effect
on
PMI.
However,
incubation
at
65
and
95

C
for
30
minutes
inactivated
PMI.
21
IV.
REVIEWER'S
CONCLUSION:
The
PMI
enzyme
activity
is
demonstrated
by
this
study
to
be
significantly
reduced
with
30
minute
incubation
at
temperatures
above
55

.

TABLE
1.
Effect
of
Temperature
on
PMI
Enzymatic
Activity
Temperature
(

C)
Specific
Activity
(
U/
mg
PMI)
Std.
Dev.
(
U/
mg
PMI)
%
Activity
(
normalized
to
25

C)

25
11.74
2.59
100.00
37
11.10
3.01
94.55
55
10.24
0.92
87.22
65
0.27
0.09
2.30
95
0.10
0.07
0.85