IDENTIFICATION OF POTENTIAL EXOSITE IN CATHEPSIN V NECESSARY FOR ELASTIN DEGRADATION
by
LI HSUEN CHEN
B. Sc. McMaster University (2005)
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE
in
THE FACULTY OF GRADUATE STUDIES
(Biochemistry)
THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) September 2008
© Li Hsuen Chen, 2008 Abstract
Besides vertebrates Typical the Among Despite cathepsin
whereas and of needed from proteins against those cathepsin Glyl the L
this barrier 112-119)
were
cathepsin
Zn-dependent
residues
very
peptide
18
amino mutant
which
constructed
for
the elastolytic
a
from elastin-Rhodamine
collagen,
cathepsin
involving
contributes
shared L
similar
V
elastin
cysteine and
with
V
from
acids
region
cathepsin
displaying
cathepsin
may
and
similar amino
residues
matrix
subsite
L
112
elastin proteases
act
degradation.
89
cathepsin
and
mutual
is
lacks
cathep
approximately
as to
to
proteins located
V,
evaluated
acid
the an metalloproteinase
to
119
119
specificities,
TVVAPGK
is
it.
and
sins,
exosite
conjugates
swapping
are
collagen
sequence
the
L
highest
A
in
of
insertion
were below
support
It
series
the
cathepsins cathepsin
cathepsin
most
for
was
for
serine-dependent
60%
constructed
their
activity
of
relatively
subsite identity
(amino
the
common
found of
only
whereas the
of
chimera
of
V. productive
elastolytic
residues
Gly
V
12, importance
S,
activity
cathepsin
Based
that
preserves
binding acids among
K
of
and
between
in
connective
the
resistant
and
over mutants
retaining
several
an
VDIPK
113-119)
on
retention.
region
activities.
pancreatic V
binding
all
attempt
80%
of
V
the
are the
Prol
the
containing known
has
to
lysosomal
3-D
between highly
the
mutant’s
tissue
FTVVAPGK
(amino
16
amino
of of
non-specific
to
a S2
Several
The
structure peptide
and and
cross-linked
cathepsin
potent
define
mammalian
and
potent
structural
results
acid
Lysi
leukocyte
cathepsins
various
acids cysteine
elastolytic
forms
additional
a
elastase
sequence
region
of
17
specific
elastases
V,
obtained
(amino
degradation.
cathepsin 113-117)
in
proportions
elastin.
a deletion
protein
proteases.
elastases,
elastases.
cathepsin
spanning
wall-like
V
activity
activity
mutant
region
region
and
acids
with
with
V, in
of
of
L 11 Abstract
Table List
List List
Acknowledgements CHAPTER
Reference
CHAPTER
Reference
Reference CHAPTER
of of
of
of
Tables
Figures Abbreviations
1.1 1.2
1.3 2.2 2.1 2.3
2.4
Contents
Human
Human
Elastin
1.2.1
1.2.2
1.2.3 Expeimental 1.3.2 Introduction 1.3.3 1.3.lElastin Result 2.3.2 2.3.3 2.3.1 Discussion
I
II
III
Overview
Manuscript
Conclusion
Human
Tissue Crystal
The Human
1.2.2.2 1.2.2.1
Dissection Initial A 2.3.3.1 Cathepsin 2.3.3.2
Degradation
Cathepsin
Cathepsin
Parallel
Composition
Approach
Distribution
The and
Overall
Structure Cathepsin
Bridge Procedure The
Cathepsin
V
Approach
and
in Introduction
Substrate
Involvement
V Family
Region
TABLE
by
Exchange
Structure
Recommendation
to
of
Human
of
V
V
and
the
Human
Human eDNA
in
to
Binding from
Atherosclerosis OF Identification
Functions
Probe
and
of
Between
Cathepsin
Amino
CONTENTS
and
Cathepsin
Glycine
Cathepsin
Catalytic
the Pocket
Chromosomal
for
Bridge
of
Acid
Cathepsin
V
118 Human
Future
of
V
Machinery
V
Elastin-Binding 54
in
Region
and
to
Elastolytic
Work
Cathepsin
V
Cathepsin
121
Gene
and
in
L
Localization
Cathepsin
Activity
V
L
Site
in
V
viii
iii
vi
ix
23 10 12 23 14 25 27 18 ii 28 29 18 30
19 55
57
64 V
111
3
3 1 5 8 1 1 9 9 1 APPENDIX .65
iv List
Table Table
of
2.1 2.2
Tables
Enzyme PCR
Primers
kinetics
of
mutant
of
both
cathepsins
wildtype
and
mutant
cathepsins
37 38 V List of Figures
Figure 1.1 Multiple amino acid sequence alignment between human cathepsin V. human cathepsin L and mouse cathepsin L 10
Figure 1.2 Ribbon model of human cathepsin V 11
Figure 1.3 Superposition of human cathepsin V (Purple) with human cathepsin K (yellow), and S (red), and the mature part of the human procathepsin L (blue) (9) 12
Figure 1.4 View of the cathepsin V binding site with the structure of bound APC-3316 representsed as a stick model 13
Figure 2.1 A schematic diagram showing the various proportions of cathepsins V and L in mutants 39
Figure 2.2 kcat/Kmcomparison of selected mutant and wildtype cathepsins against Z-FR-MCA 40
Figure 2.3 The elastolytic activity of mutant and wildtype cathepsins using elastin-rhodamine conjugates at 120 minutes 41
Figure 2.4 The HPLC profiles of wildtype cathepsin V and Ml 42
Figure 2.5 The elastolytic activity of mutants 2, 4, 5 and wildtype cathepsins using elastin-rhodamine conjugates at 120 minutes 43
Figure 2.6 The HPLC profiles of wildtype cathepsins V, L and M5 44
Figure 2.7 The HPLC profiles of wildtype cathepsins V, L and M4 45
Figure 2.8 Multiple amino acids sequence alignment of human cathepsins K,S,VandL 46
Figure 2.9 The superposition of human cathepsins V and L 47
Figure 2.10 The elastolytic activity of mutants 8, 9 and wildtype cathepsins using elastin-rhodamine conjugates at 120 minutes 48
Figure 2.11 The HPLC profiles of wildtype cathepsins V, L and M8 49
Figure 2.12 The HPLC profiles of wildtype cathepsins V, L and M9 49
vi Figure Figure
Figure Figure
Figure
Figure
2.13
2.14
2.15 2.16
A. 2.17
1
The
elastin-rhodamine
cathepsin of The
The The The A
cathepsin schematic
Michaelis-Menten
elastolytic
HPLC HPLC
distribution
V
profiles
profiles
V
representation
activity
of
conjugates
potential
of
of
wildtype wildtype
Graph
of
mutants
of
elastin
at
for
the
cathepsin
cathepsin
120
Mutant
proposed
6,
binding
minutes
7
and
6
V, V,
wildtype
elastolytic
domains
L L
and
and
M6
M7
cathepsins
in
mechanism
human
using
vii
50
51
52 53
54 65 List of Abbreviations a.a. Amino Acids
Cat V cathepsin V
Cat L cathepsin L
EDTA ethylenediaminetetraacetic acid
HPLC High Performance Liquid Chromatography
microlitre mL millilitre
micromolar mM millimolar
PCR Polymerase Chain Reaction
Z-FR-MCA Carbobenzoxy-(Z)-Phe-Arg-Methylcoumarylamide
viii Acknowledgements
been to project. rose
extend without and comments time cloning to basis;
cloning were guidance Lavalle Kruglyak and
attempted, received learning Furthermore, which friendship especially PCR. energy
first
ensure
Raymond
suggestions
and of
given,
Throughout
nowhere
Dr.
Finally,
carried
my
to thank
doubts
and
His the
techniques,
for
and
obstacles
from
on
go
Marcio
smooth for
Summer and
gratitude and
regardless
and
help
his supports; strong
kinetics
on.
perseverance
my
I
me
Pan being
and
to
advices,
a
I
heartfelt I
friendship,
would
from
handful would
throughout
be
want these
through supervisor,
Alves
proceeding
for
depressions;
support,
were
Liu
his
to
seen,
a
study
Dr.
of
members
their
my
good
heartfelt
to like
Morgan
two
for
like
how for
encouragements. encountered
Andriy
of
a
and
thank
and
committee
help easy-going-ness
in
personable
her
major
to
his
to
years,
amazing
listener
my
Dr.
silly
research
of
use the
Dr. supports;
constant
of
mention
instruction
on
Martin
encouragements
Samokhin
my
Dieter
my
projects;
the
obstacle
it this
mouthwatering
yeast
Susan
countless
may
and
project;
people are
members:
Brömme families
manner
opportunity
both
for
attentiveness,
Brömme,
expression
the
Saadat
Wilson
be;
very
a
Moreover,
in
her on
for
and
and
wonderful
of
helps,
outside
my
advices, Alexander
Xiii
yeast
and his
much great for lab:
which
Hashamiyan the
Dr.
Dr.
and
for
research;
steak encouragement
for
to
Du
thorough
their
Dr.
and
not-so-often
suggestions, expression;
Gary
her
help
Francois
of
express
supports
appreciated.
this
for
such encouragements
aided
for
Dong
audience
subsequent
from
BrOmme
help
Geidman
undivided
in
without
Brayer,
project
and
his
a
ligation
guidance
for the
and
my
Wei
the
wonderful
when
helps
Jean,
Jie
Jadwiga
her
French
project gratitude.
whenever
warm
and
Keg;
lab:
and
for
it,
for Zhang Li
I
will
procedures;
supports would
role
the
and
I
and
for
for
his
his
comments
whenever comments
Meng will
Paul.
and
not
smile
positive
jokes;
her and in
Kaleta transformation as
for helpful
his
comments
enthusiasm
I
not
also
be a great a
helps
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insights
insights
A.
Li
intriguing
on
facilitator insightful
and
joke
have
realized
Natasha
Vincent
for
Lythgo
like for
doubts a
during
clones
critics
that
ways.
daily
have
love,
like
was
her
her
the
on
on
to
in
in
ix
I CHAPTER
1.1 cysteine keeping cathepsins are H,
degradation Other bone
pool a 1.2 degenerate putative identified 1.2.1 Full-length calculated
search
X,
Human
expressed
Human
resorption,
Human
was
cathepsins F,
The
The
functions
for
17 protease
L
which
whose
screened
molecular
primers.
human
and Cathepsin
cDNA
amino
or Cathepsin
cDNA
novel
either
I Cathepsin
turnover —
0
and
displayed
such
functions
to Overview
papain-hike
acid originally are cathepsins of
were
ubiquitously
Out
antigen for
specific
human
mass
found Family
as
V
signal
of
of
regions
V
later
K,
motifs
proteins,
the
cDNA
of
encompass
presentation
cathepsin
V, roles
in
are
found
peptide, cathepsin
and
37329
twenty
constructed
many
and
or
surrounding
that
a
and
in
family
Introduction
tissue-
activation
S
in
localized
Da are
eight
organs
a
V
Chromosomal
are
a
sequences.
papaya
96
and
characteristics
(also
(2).
whole
of
or
involved
encoding
amino
hits,
processing.
cysteine
cell-specifically.
and
The
the
of
known
parts
one
spectrum
seeds.
proenzymes,
may
conserved
acid
open
A
of
in
unique
human
a
as
proteases
Gene
propeptide
the
fuffihl more
of 334
reading In
cathepsin
ranging papain-hike
body
humans, cathepsin-like
Localization
amino
kidney
active specific
generic
and
Cathepsins
that
frame
(1).
and
hormone
L2) from
acid
Quick
resemble
site
Human
there
functions functions
cysteine
a
was
translated
221
generic
protein residues
such
fragment
Clone
identified
maturation.
are
amino
cathepsins
papain,
proteases.
as
such such
house
eleven
with
cDNA
into
B,
using
acid
was
C,
as as
in
a
a
a
1 mature peptide that contained the characteristic active site residues, C138, J-{277and
N301. This open reading frame was deposited in GeneBank as cathepsin V (3).
The amino acid sequence of cathepsin V is highly homologous to human cathepsin L. They share 77.5% identity for the preproenzymes and 79.5% identity for the mature proteins. Sequence identities are also shown to a lesser extend between human cathepsin V and other cathepsin members ranging from 58.6% with cathepsin K to 30.8% with cathepsin B. Futhermore, a comparison of the human preprocathepsins V and L with murine preprocathepsin L demonstrated that human cathepsin V, but not human cathepsin
L, is more closely related to murine cathepsin L (Fig 1.1)(2).
The chromosomal localization of the cathepsin V gene was mapped using FISH analysis and found to be on chromosome 9q22.2, a site that is adjacent to cathepsin L (3).
Both genes consist of eight exons that code for analogous regions of the cathepsins V and
L structures. Moreover, the fact that cathepsin V shares almost 80% identity to cathepsin
L suggests their recent evolution by gene duplication from an ancestral cathepsin-(V-L) like gene. This close homology between two cathepsin members can also be seen in other cathepsin pairs such as cathepsins W and F, and cathepsins S and K; hence hinting on the mechanisms through which diversity within a family can be achieved (5, 6).
1.2.2 Crystal Structure of Human Cathepsin V: Overall Structure, Catalytic
Machinery and Substrate Binding Pockets
1.2.2.1 Overall Structure and Catalytic Machinery
Activation of cysteine cathepsins is a crucial step in controlling the enzymes’
2 proteolytic
pepsin cathepsins conditions
sequencing that with Ser-Val,
helices,
homologous sheets
splits superposition utilizes (12), peptide
to deacylation functioning depending The
159
help
are
exerts
Leu
side
K
the
resembles
treatment
Cathepsin
(13),
roughly
Cys
maintain demonstrates
suggesting bonds
114
chain
two
in
is
revealed
activities. on several
steps
as
25,
to the
indeed
and
of
of
Gin
lobes
the
the
of
the
(Fig equal
His
lysosome
or
cathepsin
the
S
with
the
V,
Gin
functions
base
(14)
papain 19,
autoactivation
preproenzyme.
that
that
a
159
R-domain
leads
deprotonated
Recent
like
combination 1.3).
structural
in
the
a
19
reveals
in
the
the
residue
and
size
stabilizes (7,
assistance
family
V other
the
to
This
first
activation
in
Asn with
(Fig
8).
studies
the
deacylation
their
catalysis.
of
homology
five
that
members
Experimentally,
catalytic
of
active at
papain 175
papain
1.2).
The of
state
the
structural
acidic
cysteine
of
amino
is
have
inter-
(papain
leads
morphology
The
stabilization
carbonyl
strictly
of
site.
These
whereas
(11),
reaction
pH of to
triad suggested
acids
His
and
domain
to
proteases.
The
the
similarity.
the
numbering)
to
a
and
conserved
include
intramolecular
159,
221-residue
oxygen
of
remove
initializes the
papain
the
papain
structure
(15). the
the
of that
of
protonating active
other
that
cathepsin
intermediate
more
mature
forming
As
consists
The
of
the
L-domain
throughout family
to
the
it
enzymes
domain, the
of
a
closely
propeptide
mature
carry
oxyanion
can
protein
process cathepsin series
activation
intermediate
an
V
of
the
be of
consists
out
ion
primarily
in
(10).
containing
can related
protein
seen cysteine
leaving
the
of
the
are
the
pair under
hole
(9).
be
V
papain
The acylation
in of Leu-Pro-Lys
oxyanion.
hydrolysis
of
obtained with
therefore
cathepsin
is that N-terminal
substrate Figure
lysosomal
amine
twisted
acidic
two
proteases,
cleft
primarily
three
Cys family.
begins
lobes
that
3,
pH
and
and
His
by
a
25 B-
of
is
in
L
a
3 both
Asn
Other protonated
solvent.
177
1.2.2.2 differentiates cathepsin arrangement
irreversible inhibitor’s benzenesulfonyl- pockets,
this The Cys whose floor and
lies
the 175
the
63
site
S2
constructed
studies
The While methyl
The ,
carbonyl
acylation
takes Additionally,
directly
respectively,
are is
V
form
rather
hPhe,
Substrate
Si
formed
of
determined show
them
the inhibitor
group
all
pocket, the
on
of
1
above
oxygen
shallow,
from
-phenethylallyl)carbamoylJ-2-phenylethyljamide) and
indirect
Phe,
His
the
that
cysteine
amino
is
by
“closes”
whereas
deacylation
the
Binding
evidence
159 other Ala
Trp
and formed
Lys His
of
using
APC-33
role
which acids
substrate
(18,
133,
Gly
177
N-methylpiperazine
159
155,
hand
proteases
the
the
in
Pockets
by
19).
that x-ray
may
66.
Gly
suggests
and
suggests
the
Asn
16 steps
sulfone wall
is
Asn
Finally,
specificity. form
160
well catalysis also
Asn
crystallographic
156,
of (4-Methylpiperazine-
along
64,
employ
and
play a
phenyl the
this
Trp
defined.
175
Leu
small
Gly
lying
the
with
substrate
by
site.
a
and
177
This
157,
23,
role
side
group residues
contribution the positioning
on
the
It
A
seems
may
Gly
the
in
Phe67,
is chain
is
unique
backbone
same
binding
methods
binds
catalysis.
situated
determined
far
65
play
to
reside
of
1
side
and
the
-carboxylic
the
catalytic
shield
feature
in
to
Met
a
pockets.
the
of
main the
of in
the
in His
role
in
The
demonstrates
68,
Cys
the
the
Si’
the these
substrate
by
the
carbonyl
of
159
indole
in
chain
while
R-domain
apparatus,
region 25
the
the
presence
The
Si,
stabilizing correctly
residues
acid (16).
S2
identity
of
ring
the
structure
S2
specificity.
oxygen is
(Fig
Asp
Finally,
that
walls Ala
and
of
[1
of
with
(17). from
what
1.4).
-[(3-
Trp
158,
and
205 the
the
the
S3
of
of of
of
4
a cathepsin
length cathepsins, cathepsins residues pocket
Furthermore,
serves chain. chain demonstrates of
might leading 59. which papain chain involved
specificity
this
Additionally,
carbonyls
and
Its
of
serve
as
site,
is
of
family.
plays
to
that
V
formed this
sides
a cathepsin
in
the a
S
of
the
and
is
potential
to spectrum
may
and
since a the
Si’
utilization
carbonyl
a
be
are
different
S2
This
also
of
major
by
K.
binding the otherwise
an
catalytic
created
pocket Asn
the
V
because
the
interesting
Due is
conservation target is
of
has
variability
carbon likely
64
shape defined
properties
side role
of
to
by
its of
and
Phe
machinery,
only
in
of
its
chain
the
bottom cathepsin
due
in
mainly
of
Gly
the
the
point
67
depth,
by
will
weakly
side
Asp the
of
to
of
associated to
difference
design
59. of
the
S2
be the defined for
determine
chain
S3 because
substrate Trp
158
the
When
which V
position among
similar
associated inhibitor
fact
and is of
177,
S2
of
rather
by
with
in
that specific comparing
of
Phe
binding
Ala
is
throughout cathepsins
the
the
the
across
Gly specificity
of the
design.
highly
residues
this
67,
136,
to
width
side positioning
Ala
narrower
three
65,
other
Arg inhibitors
pocket.
the
is
chain 205.
to
66
Finally,
conserved.
of
side
is
conserved
the
70,
papain
forming
other
cathepsins;
and
of
this
rather
When
and
papain
As
of chains and
can
of
papain-like
part
(10).
S2
cathepsins,
His
the
a
the
family longer
Gln
accommodate
obvious,
result,
this
compared
of
across Si’
that family
This
main
159,
61,
therefore
The
the
binding
than is
form
the
and
both
suggests chain
whereas
Asn
much
rather
it
(10). S3
cathepsins.
this
S3
therefore
those the
the to
binding
are
the
60
the
pocket,
of
larger
other
of
main
little,
sides
side
also
side Gly the
S2
the
the
of
5 the
addition, 1.2.3.
epithelium
suggests cathepsin
Class-Il might playing described
1.3.1. 1.3. mechanism as explained
found composed groups is
made
transcript
skin,
Elastin
Tissue
Elastin
in be Brömme
Elastin
a
are
complex
a
by
elastin
lung, role V others
as
in a
mainly role
(3,
is
enzymatically is
Degradation
cross-linking
Distribution
valuable
terms
for participating
in
compatible 20).
involved
is
in
and
et
atherosclerosis
are
had
the
human
of
in the
al.
The
of
responsible
large
extracellular
neutral
human
have
claimed
regulation entropic
diagnostic
specific in
cathepsin
with by
numerous
and
the
screened oxidized blood
in
amino
Human
thymus
degradation
elastolytic
Functions
the
the contribution.
along
and
for
of
matrix marker vessels.
classical
V
acids
expression
the
autoimmunity.
several
to
high
soluble
is
Cathepsin (21). with
form
cross-link
uniquely
protein
such
activity
expression
of
for
of
The lysosomal
In
theory
immune
Human
It
tropoelastin
invariant
a—aminoadipic
as colon
pathological has
of
elastic
that
V
glycine,
expressed
of
formation
of
also human
In
tissue-related
imparts tumors
level Cathepsin
cathepsins activated
rubber
fact,
chain
properties
been
molecules.
alanine,
of
cathepsin
as
in
circumstances,
elasticity
(Ii) where
elastic
demonstrated
(3),
acid cathepsin
Tolosa
thymus
macrophages
L,
V
that
valine
and
organs
of &—semialdehyde
K
some
recoil
The
stabilizes
and
V
elastin et
(24,
it
and
V
and
al.
in
had
lysine S and
lysine
to
in
25).
that
testis
(22,
cathepsin
pointed
the
tissues
proline,
the
as
found
have
also
the
Elastin
the
23).
E—amino
residues
cornea! thymus
well
(2).
MHC
basic
been
been (26).
such
that
out,
and
In
as
V
is
6 desmosine, Subsequently, protein
cross-linked
its conditions.
confers cross-linking elastin.
inflammatory
adequate accumulation the density
formation (the contributes of enzymes
1.3.2.
detailed
these
arteries.
innermost
Human
in Atherosclerosis
an Atherosclerosis
lipoproteins
cell
such
mammalian
removal
u—helical
of
and
To
structure
to
nature
sites
plaques.
types
through
of
response
the date,
as
Cathepsin isodesmosine
layer
macrophages.
matrix
formation
in
of
and
(35
conformation it
(HDL).
is
of
tropoelastin,
bodies
had
fats
The either
not
is
is —
extreme
in
artery)
metalloproteinases,
V generally
41).
been
a
and infiltration
known
the
in
Atherosclerosis
cross-links
(31
of disease aldol
Atherosclerosis
These
It
reported atherosclerotic walls
and
cholesterol —
hydrophobicity,
is
(34).
33). and primarily
condensation
characterized
then
subsequent
affecting
extra-cellular
of
of
However
thereby, While
are
that
promoted
macrophages
arteries.
chemically
from
and
is
due
the
its
commonly lesions.
arterial
deposition
the
further
or
cysteine
elasticity
building
to
the it
by
It
by
degrading
Schiff
its
possible
is
the
occurs
macrophages
synthesized low
and believed
Notably
insolubility
research blood
enlargement
proteases
block
has referred
reactions,
of
density
smooth
confirmation
in
been
lipids enzymes
vessels.
extra-cellular
of
is to
large
needed
(27
to
elastin,
under
are
lipoproteins
be muscle known
by
which
lysino-norleucine,
as
of —
part the
secreted
It
destroy
functional
30).
“hardening”
arterial
physiological
to
is
tropoelastin,
for
of
most
cells
leads
due
predict
Due
a
degrading
insoluble
decades,
without chronic
by
the
(SMC)
to
stable
intima
to
to
high
both
two the
the
its
the
of
7 main
degradation,
rupture abovementioned
for degradation most is
epithelium, 1.3.3. enzymes
suggests sequence cathepsin site proteases also substrates and cathepsin
associated
--‘60%
adjacent
V
potent
seems
underlying
The
are
While
Nonetheless,
of
a display
from
L V
highly identity
to Comparison
of
close
elastolytic occurs the
human
demonstrates
to
to
the
and
to
probe
the the
cathepsin
the
point
the
degradation
atherosclerotic
differing linkage blood
L
components similar
expression
total
extracellularly
papain
with
cathepsin from
cathepsin
the
cathepsin
in
activity
vessels
elastin
of specificity
and
as
the
a cathepsin V.
only
expression
family
Human
cathespin
cathepsin
Among
are
exhibit
same
of
L
L
of (22).
a
V
degradation.
are
is
locus
cathepsins
minimal
plaque. cathepsin
reported
by is arterial
a
of Cathepsin
direction.
L.
weakened,
these described
similar
ubiquitously
cathepsin
V,
patterns,
V
(2).
each
Moreover,
L-like has
elastolytic
The
three
walls,
that This
V
V,
substrate
substrate
been
Of
is
Studies cathepsin
S
as
V
the
K
cysteine
ancestor.
is cathepsins, facilitating
restricted
and
this,
the
and
and
the
mapped expressed
a
Si,
their
suggestion
activity
K
preferences
most elastins
binding
S,
Cathepsin
approximately
S2, employing
(42,
V
whereas
proteases
Furthermore,
chromosomal
to
to and
shares
potent
cathepsin
the
43).
(22).
thymus,
chromosome
and
S3
of
formation
These
the L
(44).
almost
Based
subsites possible elastase
a
the
responsible
remaining
library
V
Even
of
two testis,
substrate
enzymes collagens.
demonstrates
localization
on
80%
several
of
known,
and
9
thirds
divergence
their
though
of
and
at
cathepsin
amino
the 9q22.2, one
synthetic
profiling
identical
account for
cysteine
cornea!
of
Upon
where
these
third
later
acid
also
the
the
the
of
L
a
8 mode
a rendering
specific
of
catalysis
its
exosite
elastolytic
and
in
similarity
cathepsin
activity.
in
V
substrate
which
facilitates
preferences,
the
we
binding
hypothesize
of
elastin
the
presence
and
thus
of
9 ______S ______E ____T ______
112 V 33 *3 93 hCTSV - ii V St N DTlç A cN Wr4ti hCTSL P Tt 1’ L 1 II S.EQ I M N r - mCTSL I Lk[14 Vi eTfr?F H s F
02 712 82 82 102 hCTSV - MCC hCTSL ‘ rn CTSL €WE
110 Ix iX 140 1120 horsy 7 hCTSL rnCTSL Iktk
210 240 hCTSV V1 T\ V A P ü K - =L KL1fl ASSfl ‘ 1
202 270 .282 323 IiCTSV “- A N hCTSL C L IP C E UM S 93 D mCTSL 01*5 P SL S N - * t::y LVI1EiGY.’ 1) ,K iflittjI
370 336 I 310 340 hCTSV $iP -. S N — K Nfln$nItJ() SGTSL tn1L C, 1.4 RR*-oS*4&j’ I1 CTSL 1) 1. *‘W’W N
Figure 1.1 A multiple amino acid sequence alignment between human cathepsin V, human cathepsin L, and mouse cathepsin L. Dark shaded background represents identical amino acids whereas similar amino acids are light shaded, and unrelated residues have a white background. Arrowheads mark the putative cleavage sites between the signal sequence and the propregion, A17-V18, and between the pro- and the mature regions of the protease (Dl 13-LI 14). Typical for cathepsins, the second amino acid residue adjacent to the processing site of cathepsin V is aproline. (P115) (1)
10 Figure 1.2 Ribbon model of human cathepsin V (PDB code IFHO).The coil represents a—helixwhereas the arrow stands for 13—strands.The black stick molecule in the middle represents the inhibitor APC-3316. The smudge yellow portion at the bottom represents the bridge area (aa 113 to 119), and the brown region on the left is from an 89—104.
11 Figure 1.3 Superposition of human cathepsin V (Purple) with human cathepsin K (yellow), and S (red), and the mature part of the human procathepsin L (blue) (Figure Adapted from Ref 10).
12 Figure
the stick shown inhibitor
Si,
model.
as 1.4
S2
are
dotted
and
View
structurally
The
S3
of
white
pockets
three
the
lines.
hydrogen
cathepsin
homologous
of
this
It
can
protease,
bonds
V
be
binding
to
the seen
between
respectively.
P1,
that
site
P2,
with
the
main-chain
and
hPhe,
the
The
P3
structure
residues
sulfone
Phe,
atoms
and
phenyl
of
of
of
N-methylpiperazine
a
bound
the
natural
group
inhibitor
APC-33
substrate
binds
and
16
in
and
represented
the
the
moieties
Si’
are
enzyme
bound
region.
of
as
are the
13
in
a References
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17 2.1 CHAPTER turnover function thymus, (1). the Il-associated resistance numerous been as mammalian contribution Zn-dependent Necessary human along A atherosclerotic directly Chen, of studies version Introduction skin, elastin generation Among LH. solved with The Elastin cathepsin have related testis, may of to for Geidmen, lung, cross-linked this through to cathepsins highly human Elastin elastases as the invariant indicated to range
II- chapter of proteolytic cornea is matrix to plaques an and tissue’s the eleven antigen-presentable V A. the Degradation. specialized ct—helical
Manuscript from cathepsin a had and extracellular will large and formation K, series such metalloproteinase chain that Brömme molecules where be been human generic and elasticity epidermis degradation submitted blood elastolytic as Ii of S, conformation functions reported family into serine-dependent D. they cathepsin adsorption of cathepsins, housekeeping matrix The vessels of and and CLIP for (2 cLB—CLIP contribute Identification is soluble cathepsins publication. — the as such tensile largely group 4). protein 12, suggests V one (5). subsequent (6). and Its was cathepsin as and tropoelastin of in involvement functions of Similar strength bone The to owing desorption that also pancreatic of the K human several papain-like cathepsin the Potential and result imparts most expressed resorption rupture V degradation to to S thymus such is had lysosomal may potent whose Exosite is collagen, its to evident. in and V elastic an of as cysteine converting been elastin is structure. by carry and plaque leukocyte (3). protein insoluble the elastase in structure macrophages recoil Cathepsin antigen elastin found Among protease out cysteine elastin in proteases (4). degradation the the a It to (4). elastin elastases, expressed non-catalytic had that While presentation V tissues consists degradation MHC/class confers the regulating Moreover, proteases, found recently may known whose whose recent such and the its be in of 18 in manner
concerning elastin’s
conjugates counterpart,
cathepsins highly against
specificity relates of acid
2.2 lying PurUication. polymerase pPIC9 recombinant
constructs
both
Experimental
sequence
directly
While
(Invitrogen, to similar
Recombinant
these
as
cathepsins.
insolubility
the
were
a
V
characterization
such the
(Fig. human
mean
Wildtype
under human cathepsin mutant
substrates.
and
differences
substrate
mechanism
ligated
as
Procedures
2.1)
L
to CA)
In
the
in
cathepsin
cathepsin
elastin-
share
cathepsins
this commence
aqueous
Mutant
cathepsin
S2
V’s
(Fermentas,
into rendering
preferences
are
An
subsite
study,
studies
potent
78%
employed
the
Congo
responsible
amino
V
solution.
L,
X7zoI
Cat
V
we
were
demonstrated
the
identity
contributes
direct
however
elastolytic
recombinant
using
hepsins:
ON)
(8).
have
proteolysis acid Red,
and
by
constructed
fusion
This
for
peptide and
NotI determined
in
sequence
these
and
oniy
to
the their
activity,
Cloning,
information
primers of
potent
sites
the
cDNA
elastin-rhodamine,
different
the of
elatases
exhibits
substrate
majority amino
by
elastin
of
alignment
proenzyme
a
elastolytic
and
was
the
outlined
region
PCR
Expression,
could
elastin-degrading
acids
within
minimal
suggests
P.pastoris
previously
(7),
of
libraries
amplification
its
whose
has
no
sequence
in sequence,
activity
be
elastolytic
which
further
Table
shown that
elastolytic
its
elucidated
has
existence
expression
prepared
Activation
a highly
distinct against
also
with
short
1.
information
and that
activity. capabilities
using
The
the revealed
(8).
directly activity
domain due
similar
subsite human
elastin
amino
vector
yeast
PCR
Pfu
The
and
to
19 secretory and
level selected selected mutants
30°C, nitrogen extract,
at
nitrogen extract, O.D.600
subjected 4th supernatant membrane pepsin and adjusted mixture
30CC.
day
subsequently
the
of
followed
The
The
to reached
of
2% were
and activity 2%
for
base,
base,
was a-factor
clear to The
to
induction
a (Millipore,
resulted
were
concentrated
pH (w/v)
(w/v) expression tested
final
first induction
incubated 4x
cells
4x
supematant
by
6.0
4.0
against
electroporated
withdrawn
10-5%
10-5%
pheromone
inoculated
peptone,
peptone,
for concentration resuspending
His+
were
before
using
(96hrs
MA)
productivity.
through
with
the
(w/v)
(w/v)
at Mut+
yeast
again
they
glacial
to
was post
37°C,
100mM
synthetic
100mM
to
in
signal
0.5%
about
biotin,
biotin,
clones
test
into
supernatant
were
5
resuspended
fermentation.
first
the
concentrated
of
thL
acetic
and
for
P.pastoris
(vlv)
30
sequence.
Among
potassium
potassium cell
0.6
resuspended
0.5%
1%
induction). that
substrate, of
mE. enzyme
monitored
MD
acids mg/mL pellet (v/v)
methanol
carried
(vlv)
containing
which,
into
(Dextrose
GS1 The
glycerol))
Briefly,
50-fold
(Fisher
activity.
phosphate
into
phosphate
Z-FR-MCA,
methanol)).
Cells
the
(Sigma-Aldrich,
into
500
vector 15
for
the
every
50
recombinant
using
1.5
mL
were the
activity the Scientific,
using
mL clone
2%
The
followed
was
L
(pH
(pH
recombinant
24hrs
clones of
a
v/v)
of
At of
cells separated
(Alexis,
standard
that
an
then
BMGY
BMGY
BMMY 6.0),
6.0),
this
using
and
Amicon while
mutant were
NH)
by
produced
bearing
MS).
linearized
point,
grown
1.34%
1.34%
a
Switzerland) protocol
Z-FR-MCA
by
and
((1%
harvested l2brs
and
((1%
proenzyme
aliquots
The
enzymes
centrifugation
the
Ultrafiltraion
recombinant
grown
for
(w/v)
mixed (w/v) the
w/v)
incubation
w/v)
with
cells
activation
(9).
24hrs
highest
of
on
yeast were
yeast
yeast
yeast
Sad
were
until
as was with
was
the
the
20
at
a fluorogenic
to 2.5mM
centrifugation column
reached containing
linear
mutant acetate, sulphate
against further
0.5mM aliquots
cuvettes Initial Carbobenzoxy-(Z)-Phe-Arg-Methylcoumaiylamide 1.5mL
2.5mM
380
5.5
and
gradient
EDTA
Recombinant and Z-FR-MCA. rates concentrated
and
enzymes
pH
(GE
EDTA baseline.
for
EDTA and
at
450
2M
25°C
storage diafiltered
substrate
5.5,
animonium
0.5mM
Healthcare,
of
at
and
nm,
starting
ammonium
and
and
containing
in
4,000g,
methylcoumarylamide
were
Recombinant
2.5mM
at respectively. a
The
0.5mM
10-fold EDTA
2.5mM Perkin-Elmer
Cat
-80°C. in
with
with
the
eluted
hepsin
100mM sulfate
the
U.K.)
dithiothreitol.
sulfate,
major
100 0.5mM
and
100mM
using
dithiotbreitol.
cleared
dithiothreitol
protease
mutant
mM
0.5mM
and
The V,
was
sodium
280
Amicon
0.5mM fluorimeter
EDTA
Cat
sodium
sodium
washed supernatant
assay
added
nm
proteins hepsin
activity
dithiothreitol
The
substrate
acetate
EDTA
The
Ultra
and at
absorbing
buffer
acetate, acetate
activation
to
with
pH5.5.
at
L,
0.5mM
containing
fmal
were
excitation
concentrator
a
was
and
buffer,
hydrolysis
and
contained
Substrate
final 100
buffer,
pH
solution
and
The
loaded peaks, eluted
0.5mM
was
Mutant
dithiotbreitol.
mM
5.0,
concentration
ending
pH
fractions
and
active
stopped
pH
containing
from
as
on sodium
100mM were (Millipore,
was
dithiothreitol 5.5,
and
emission
5.5,
Cat
monitored
an
with
site
the
hepsins divided
supplemented
by
were
monitored
supplemented
n-butyl
Enzyme
The
acetate,
100 sodiuam
bringing
concentration
colunin
2M
wavelengths of
combined
MA)
recombinant
mM
ammonium into Assay
until
2M. by
Sepharose
Kinetics.
pH
to
using
in activity sodium the
acetate,
lOOuL
A280
After
about
with
with
with
5.0,
1cm
pH
and
21
of
of
a the purified
measured. cathepsins
concentrations: Z-FR-MCA. assay
The remnant with thus
Km intercept
The appropriate using buffer Appendix to
(EMD, Conjugates
appropriate
incubate
of
diluted titration
suggesting
diluted
buffer,
nonlinear
recombinant
to
Elastin
cathepsins
Germany)
initial
which
construct
for
The
(5
cathepsins
and
dilutions
and
with
cathepsins The was
iL)
detail
range
range
100
Degradation
represents activity
the
HPLC
incubated
regression
selected
commenced were
either
(final
was
a
wildtype concentration on
j.iM,
Michaelis-Menten
of
were
of
were
processing
Degradation determined
diluted
inhibitor
would
and
concentration
E-64
10
elastin-congo
range
the
for
made
mixed
program
iM
assay
Assay
and
concentration
an
was
with
appropriately
then
then
and
hour concentrations.
on
mutant
a
of
with
by
buffer pre-determined Michaelis-Menten
the
Profile.
Using
cathepsin
consisted
cathepsins
be
1
Prism
at E-64
1
FM),
representation
different
red
abovementioned Omg/mL)
plotted
25°C
cathepsins
in
Elastin-Congo
titration.
(Sigma-Aldrich,
of
to
20
and
the
The
before
of
active
E-64
obtain
to
jiL
concentrations on
Briefly,
10
same initial
and
cathepsins
achieve
by
of to
a
were
needed
A
testing
site.
graph).
of
assay
spreadsheet
mixing
the 12
diluted
pretest
fashion
rates
cathepsins
concentrations portions
points
determined
The
Red
kinetics
appropriate
for
to
buffer
MS)
were
against
cathepsins
kinetics
20
was
achieve
of
remnant
and
as
of
Z-FR-MCA
of
iL
or
to E-64 diluted
in
in
done
which
constants
purified Elastin-Rhodamine
Z-FR-MCA
by
MaxQ elastin-rhodamine determine
the of
constant
initial
100%
of activity
concentrations.
first
E-64
the
and
was
pre-test.
appropriately
E-64
5000
following: aliquot
to
inhibition
40
and activities.
(Refer
analyzed
(various
kcat
against
isolate
the
mixed
p.L
were
assay
floor
The
and
of
of
x 22
to shaker at activity precipitate
speed appropriate
(Molecular measurements neck 570
elastin MaxQ500
The and for column
resulted acid 2.3 2.3.1
efforts
time
analysis.
Results
the
supernatant
nm
elastin
and
Initial
at on
(EPC,
points
In was focused
(Phenomenex, mixture
spectra
37°C
and a
ending
order
floor
undigested
Devices,
plates also
was
bench-top
Approach
The
MS)
0’,
at
were
590
shaker were
to
on
was was
200
recorded
carried
degraded
30’,
with
identif’
(final
for
dividing
carried
CA)
nm
rpm
injected
centrifuged analyzed
60’,
CA) at
particles
accuSpin
to 90%
O.D.490
out
concentration
37°C or
using
(Geneq
at the
the
90’
fragments
and
out fluorescence
as
the
the
acetonitrile
into
Identification
structural with
at
and
follow.
eluted
in
measurement of
Gemini cathepsin same
at
200 mc,
Microcentrifuge
System
triplicates.
the
120’
elastin-conjugates. top
rpm were
QC).
time
with
Diluted
of
32
speed
region
and
measurement
plate
supplemented
Karat for
1 Gold
V
loaded
Omg/mL) Samples
points.
a
of mixed
linear
sequence
1
The
to
using
8hrs.
cathepsins
responsible
Elastin-Binding
Detector
reader
program
precipitate
onto
machine
HPLC
The
gradient
with
of
The
and
Vmax
reaction
with
Supernatants
a into samples
(Molecular
with
reaction
HPLC E-64 (Beckman
reversed
assay
were
degradation
for
Kinetic
excitation
undigested
four starting
(Fisher
0.1%
elastin
to
mixture
mixed
buffer,
were (Beckman
Site
was roughly
stop
phase
Coulter,
trifluoric Microplate with
Device,
Scientific,
in
were
degradation,
centrifuged
stopped
and
the
with profile
and
were
elastin
Cathepsin
C-
0.1%
equal
reaction.
absorption
Coulter,
loaded
18
incubated
bovine
CA).
withdrawn
CA).
with of
acid.
analytical
particles.
trifluoric
NH)
regions.
Reader
bovine
at
initial
V
E-64,
neck unto
The
CA)
The top
All
to
of
23
in corresponding Since
M2 cathepsin boundary
half and until
confirmed
fluorogenic
in site L,
V. carried demonstrate potent elastolytic cathespin and same
the
is
In
and
the
as
of
responsible L,
a.a.
range
level it other
depicted
elastin The
cathepsin
rest We
M3,
out
cathepsin
is
V
of
V.
with
171
hypothesized
activity
from
with
to known
words, substrate,
of
catalytic
the were
using It
elastolytic
regions
degrading
observe
the
can
automated while
by
divided
wildtype cathepsin
for
(a.a.
constructed
parent
the
be the V
that
elastin-rhodamine
of
binding
activity
Z-Phe-Arg-MCA. of
the
seen specificity
clearly
121
for
chimeric
both
regions
that activity. cathepsin activity. cathepsin
cathepsins
sequence
cathepsin
loss
rest
V.
to
that
wildtype of
an
220). with
M2
of
exhibits
of
being
Ml,
elastin
mentioned
exosite
mutant
constant,
M2
the
elastolytic contains
Evaluation
standard
L
L Finally,
analysis
V
in
L.
based
and
chimera
and
demonstrates cathepsin
thus and
comparison
as
present When
roughly
that
All
M3,
L.
a roughly kcat/Km
above.
mutant on rendering cloning
M3
to
three
activity.
contains
substrate.
comparing
of
sequence
on contain
proteins
in
contains
5-times
V.
the
obtained
the
cathepsin
Ml constructs
half to
protocols
cathepsins
only
All
Three
M2
the the
other
mutants’
has
the
of
Figure
was
alignment
the
three
elastolytic
potential
and
more the
cathepsin
correct a.a.
a
(Fig.
hand,
mutant
V,
minor
possessed
fragment
using tested
two
M3,
1
but
based
mutant
2.3
activity
elastolytic 2.2).
to
perform
sequences.
wildtype
demonstrates
absent
exosite were
54 primers
vectors,
L
elastolytic
using activity
shows
The
on
(a.a.
from
from
functional
constructs
substituted
against
the
from
activities
in
sequence
the
1
spanning
cathepsins
activity cathepsin namely
the
of cathepsin
to
roughly
activity
cathepsin cathepsin
synthetic
121)
activity,
the
residual
elastin
active
were
were
most
Ml,
into
and
will
was
the
the
L,
24
of L
V rhodamine.
activity experimental
mutant’s
cleavage be
elastin elastin cathepsin reversed
profiles fragments, mechanisms
other. wildtype responsible
2.3.2
two amino
M5
compared
more
was
Dissection
followed in Moreover, The
acid
Further
when
between
elastolytic
sites
the
phase
cathepsin
V
created
primers
and
Furthermore,
residues
same
for
error
while same
using
and
in compared
the
the
dissection
by
c-i
elastin.
in cathepsin
observation
Mi of while
fashion
intensity
were
based elastolytic
HPLC
fluorogenic
degrading
ability,
Region
L. 8
responsible
cathepsin
column.
obtained
These
In to
designed:
the
while
analysis
on of
as
V
it
other
wildtype
from
represents
the does
wildtype, amount
and
activity
both
elastin
data was
V.
elastin it
The
for
by
54-121
words,
Amino
can
Mi
not
was
M4
also
the indicate
structure
running
cathepsin
peaks
as
of in
be
suggests provide
the conjugates.
carried elastin
divides
formation
cathepsin
obtained the
the
amino
fluorescent
Acid
seen
quantity
correspond
mechanism
that
peaks
degraded
degradation
any analysis
that
out.
54
they
the
V,
acid
the
of
to
from V.
information Hence,
the
region
Figure
of Mi
in
both
the
region 121
rhodamine
amino
fragments.
both
activity
displays
and
putative fragments
HPLC
of
to
in
employ
2.4 further
to
against
elastin
Cathepsin
profiles
was
different
acid
sequence ensure
shows
regarding
actually
profiles
carried an
exosite.
A certainly
similar,
into
unlabeled
sequence
degradation
of
comparison
increased
the
almost
the
sizes
two
elastin
V
alignment.
lies
mutant
out
of HPLC
the
For
if
points
segments,
M2,
not
overlap
of
to
close
54 bovine
locations
this
elastolytic
through
zoom could
profile
degraded
identical,
of degrades —
M3
purpose,
to
to
HPLC
121
Upon
neck
each
the the
into
not
and
and
of
of
25
is
a examining
This is
M4 119 conjugate. (Fig. elastolytic compared
specific majority The M4
the
by from suggests other, resulting 2.6).
demonstrates
therefore
M5
even
region
of
parental
result
region
2.2).
This
a.a.
few
The
cathepsin
to Although
bridge
displays
of
a
HPLC
to
89
the
activity
M4,
suggests
in
The
peaks (a.a.
further
potentially
partial defmes expressed
the
M2,
cathepsin
to
crystal
such
full
area,
while
elastolyic
89 wildtype’s
113.
analysis,
V
are
has
the
an
when
confirms
to
or
the
the
elastolytic
lies
is
incomplete exceptionally
either
After containing the
bridge structure
113)
mutant
incomplete
interesting.
only
bottom
involvement
V’s
directly
compared
cathepsin
activity
it
for
missing elastolytic
subjecting
able
can
elastolytic
that
region
cathepsins
the
of
of
activity
be
portion
to
under
a one
cathepsin
remaining digestion
mechanism
of
to
recover
L high
seen
compromised
or
is
of
both sequence
wildtype
of
activity
certainly
bovine
shown
the
its
activity,
value the that from
was
M4
mutants
bridge S2
60%
V
entrances
elastolytic
while
pattern.
and
cathepsin in expected
neck (11),
of might sub
and
cathepsin
till
significant
of
lower
its
specificity M5
region
binding site
112
both
the
the
was
it
elastin
inability
be
both
can
that
On and
elastin
and intensity
participation
activity.
L
profiles
to
assessed
employed
V.
(a.a.
until
be
lead
mechanism
exhibit
forms
thus the
fragments
in
employ
On
constant
seen
to
degrading
112
retaining
amino
to
the
contains
other
in
are
recover
a with
the
that
to functional
the
other
bridge-like due
highly
of
a
118)
against
active
acid
degraded
profile amino
might
hand,
elastin-rhodamine
complete
residues
to a
activity
the
hand,
the
vast
in
89,
similar
loss
site
cathepsin
retaining
Z-FR-MCA
active
full acid
be
of
M4
retains
amount
M5,
structure.
overnight
of
cleft
(Fig
from
M5
in
binding activity
to
112
region
which
when
sites;
effect
(Fig.
each
and 2.4).
full
the
the
V
to
26
of mechanism profile to
assay pertaining thus
2.3.3
cathepsin between further binding that
This deletion directly rather, outward characteristic the the and
M5,
entrance
involvement
generating
possibly
there
trend
A
and
of
there
As
it
Parallel
characterize
domain.
followed
connecting
whereas
cathepsin
appears M4
V’s full
HPLC
it is
is
resembling
were
into
has
creating
however a
suggested
small
elastolytic
elastolytic
a
proline
Approach
In
slightly
of
been
the that profiles,
such
still
by
L
another,
size,
glycine
to
this
active
a
either the
and
few
orientation not
a shown
that lysine
residue that
activity, different the
glycine
lysine
region.
activity,
observed
it
peaks cathepsin
to
site
an
even 118 sequence
used
lysine
can
Probe
that residue.
antenna
cleft followed
side
in
residue
that
other
be though
degradation
of by
In
retaining
the
that
cathepsin
in
the
inferred
the
for chain
V
one the are
alignment
cathepsin residues
Due
next
for
follows
to
lysine
elastin.
Bridge
which
fewer
missing
wildtype by
instance,
elastin
further is
to
round the
either
pointing
V that
pattern.
proline’s
side
discrepancies
must
the
lies
in
Region To
of
bridge
L
(Fig.
binding
while
assess
orienting
cathepsins
as glycine
cathepsin of
chain
test
ahead
a
the
have
the
negatively
inward
mutations
2.7).
region this
unique
both
bridge
is
proline its
aided
while
of
seems
not
hypothesis,
Combining
the
were
role
a
V
regions
(Fig.
S,
recovers
lysine
observed
in
structure
opening
region
subsequent
(Fig.
K,
were
charged
in
to
as
the seen
2.9).
and
the
orient
a
residue
are
binding
2.5).
mainly
when
potential
glycine
both
V was
bridge
the
It
up
in
and
significant
amino
demonstrated
thus
its
cathepsin
the
The lysine
majority
the
exchanged
compared
(Fig.
side
glycine
of
region
made
width suggests
118
elastin HPLC
elastin
elastin
acid
chain
away
2.8).
was
in
of 27
to
L;
of
is
is
a deleted from cathepsin V while an extra glycine residue was inserted in between proline
116 and lysine 117 in cathepsin L.
2.3.3.1 Bridge Exchange Between Cathepsins V And L
The results obtained from M4 and M5 suggests that the bridge is directly involved in retaining approximately 60% of the wildtype elastolytic activity in cathepsin V. It was thus designed to mutually swap the residues VDIPK (amino acids 113 — 117) in cathepsin
L with residues TVVAPGK (amino acids 113 — 119) from cathepsin V to assess its effect in either creating or diminishing the elastolytic activity, respectively. The mutants were cloned and expressed using standard protocol. The kinetics analysis on both mutants against synthetic substrate Z-FR-MCA was carried out, and the resulted kt/Km values suggest the proper formation of active site and retention of subsites preferences found in their corresponding wildtype cathepsins. Mutant cathepsin V bearing the residues VDIPK from cathepsin L (M8) was assayed against elastin-rhodamine. The swapping of the bridge area seemed to contribute to a 60% loss of elastolytic activity (Fig. 2.10), and to alter the degrading pattern as is evident from the HPLC profile (Fig. 2.11). Several characteristic elastin fragments seen in the profile of cathepsin V are not found in that of
M8 which suggests incomplete degradation. On the other hand, however, mutant cathepsin L carrying residues TVVAPGK of cathepsin V (M9) did not seem to reflect the mirror-image effect one would expect. The introduced bridge region in M9 was unable to generate any significant increase in elastolytic activity as the amount of fluorescence generated by rhodamine upon cleavage was comparable to the result obtained from degradation by wildtype cathepsin L (Fig. 2.10). Upon examining the M9’s HPLC profile
28 on the degraded elastin fragments however, it can be seen that while most of the characteristic peaks seen in cathepsin L’s degradation profile were not observed, a characteristic peak resembling one from cathepsin V’s profile was found (Fig. 2.12). So even though no observable elastolytic activity was concluded through this mutation, pattern of elastin degradation was certainly altered which supports the region’s involvement in elastin degradation.
2.3.3.2 Glycine 118’s Involvement In Elastolytic Activity
In parallel to the bridge exchange, the effect of glycine 118 was evaluated, and two mutants were created. M6 contains a single glycine 118 deletion from the wildtype cathepsin V, and M7, serving as the counterpart in this assessment, carries a glycine insertion in between proline 116 and lysine 117 in cathepsin L. Both mutants’ activities against Z-FR-MCA were assessed and kinetics study was carried out; both results suggested the retention of parental wildtype cathepsins’ specificity constant (Fig. 2.2).
Further elastin degradation assay showed the same trend found in M8 and M9. Glycine deleted M6 demonstrated a 25% decrease in elastolytic activity (Fig. 2.13), and the HPLC profile generated indicates a slight change in the size of fragments obtained (Fig. 2.14).
These results pointed toward the involvement of glycine 118 in elastin degradation; however, the expected effect of glycine insertion was not manifested in the case of M7.
The elastin degradation assay by M7 using elastin-rhodarnine demonstrated that inserting
a glycine is not sufficient to restore the activity (Fig. 2.15). This is compatible with results obtained from M9; since swapping of the bridge region did not restore any
elastolytic activity, glycine insertion should not as well. Furthermore, much like the
29 bovine
effect inserted
2.4
of has reported
presentable invariant V
known While mammalian comparison other preferences raise specificity elastolytic for
has
cathepsin
Discussion
the
been
the
brought
also hand,
there
The
neck high
Nonetheless,
certainly
about
that chain
question
described
been
activity
amino profiling
to
of are
elastolytic xl3
L,
elastin
only
elastase
by
cathepsin
other
both
its
—CLIP
Ii
and
a
solved
altered
the
into
growing
acid
what demonstrated
elastolytic
observed
has
human
also
enzymes
as
one
bridge
studies
CLIP
complexes
activity
sequence
to-date, (11),
been
a
structural
the
V
potential
pointed
major
number
cathepsins
is
and
region
local
and
to
found
on
was
activity.
involved
of
the therefore
of
its its
protease
a
various
conformation in cathepsin toward
found
difference
concern of
of
minor cathepsin
active
in
human structural
elastolytic reports
cathepsin
V
testis,
Cathepsin
to in
and
the
suggesting human
sites
in
elastolytic
that
be
thymus V.
the
on
cornea,
L
between
V
involvement
corneal
the
difference
are
and
of
lies
V.
cathepsin’s bears
conversion
activity
cathepsins
highest
that
the
highly
V
(3).
sub-pockets
in
and
its
approximately
activity.
epithelium
is cathepsins area
this
degradation
The
role
but thymus
described
(4).
similar
of
against study
(Fig.
function
crystal
of
in
reported not
that Human
the
Furthermore,
MHC/class
were
2.16).
is
to (9,
V
(8).
(2).
region
generation
profile
how structure
elastin-conjugate
the
and
as
and
80%
12,
thoroughly
These
cathepsin
Moreover,
that
the
difference
to
2).
L
structure,
identity as
of
is
contribute
the
TI-associated
Cathepsin
most
observations
of
the
M7
responsible
a
of
cathepsin
L,
substrate
substrate
antigen-
studied.
glycine
against
to
it
potent
on
less
in
was
that
the
the
the
30
V
in
is enzyme’s protease activity in general. This question can be answered with the kinetic behaviors of the mutants. First, both cathepsins V and L employ an identical catalytic triad to carry out the proteolysis. While cathepsins V and L have different turnover rates, the enzyme kinetics of all mutants are compatible with their parental cathepsins. So, a mutant enzyme behaves more like either one of the parental cathepsins when the proportion of that parental cathepsin becomes greater. However, the elastolytic activity observed is independent of such trend in enzyme kinetics. When comparing the kcat/Km value of MS with wildtype cathepsin L against synthetic Z-Phe-Arg-MCA, M5 and cathepsin L demonstrate comparable specificity constants (Fig 2.2). However, their elastolytic activities against elastin-rhodamine display a 2-folds difference (Fig 2.5). This suggests that while the mutant may behave like wildtype cathepsin L, the added region from cathepsin V has “created” the elastolytic activity. This independence of elastolytic activity to the enzyme’s protease activity can also be seen when comparing M5 and M3 where they clearly behave similarly in the Z-Phe-Arg-MCA hydrolysis, but differ greatly in their elastolytic activity (Fig 2.2, 2.3 and 2.5). This observation supports our original hypothesis that an elastin binding domain must exist to render cathepsin V its elastin degrading activity. Furthermore, it has been reported that human cathepsin L demonstrates potent activity against soluble ETNA-elastin but displays minimal adsorption to insoluble elastin (7). This fmding is compatible with our hypothesis in that human cathepsin L must lack the elastin binding domain thereby is unable to adsorb to and degrade polymerized elastin efficiently. Moreover, human cathepsin L’s lack of an elastin binding domain and its potent elastolytic activity against soluble elastin also helps
31 distinguishing the insoluble elastin degrading ability of cathepsin V as a structure related phenomenon but less as a general protease activity issue.
The investigation on elastolytic activity has not been an easy task mainly due to elastin’s insolubility in aqueous phase, and its random pattern of linking and cross- linking of tropoelastins. As a result, one cannot predict the overall structure of the polymerized elastin; and for this reason, elastolytic activity of enzymes are assayed either using fluorogenic or chromogenic elastin conjugates or by labeling degraded elastin fragments with fluorogenic chemicals such as fluorescamine. In this study, we set out to pinpoint the potential elastin binding domain in human cathepsin V by cloning chimeric mutants and mutants involving point-mutation and region swapping. Our sequential analysis has pinpointed the potential elastin binding domain to amino acids 89 to 119 in cathepsin V with the TVVAPGK (amino acids 113 — 119) region contributing most to the elastolytic activity found in cathepsin V (Fig. 2.16). The TVVAPGK domain which resembles a bridge-like structure is located beneath the S2 subsite pocket. Being located right at the entrance leading to the active site cleft, it is tempting to speculate that the bridge directs the bound elastin directly into the cleft where the active site residues could carry out the cleavage reaction. We assessed the importance of this domain by mutually swapping this domain between human cathepsins V and L, designated M8 and M9. It was clearly seen that without the bridge domain, M8 lost 50’—60%of its elastolytic activity whereas switching the bridge domain to cathepsin L did not rescue any noticeable elastolytic activity (Fig. 2.10). The inability to generate elastolytic activity in cathepsin L led us to consider the involvement of other domain; however, the decrease in elastolytic activity found in M8 indicated that the TVVAGPK domain played a role in elastin
32 degradation. Furthermore, this domain is also interesting because the extending arms of lysine residue 119 pointing away from the active site cleft (Fig. 2.9) may service two possible purposes. First, the lysine side chain may act as an antenna that inserts into and hold the elastin in place; secondly, its orientation may also clear up the “passage way” leading to the active site. The opposite is observed in human cathepsin L where the side chain of 7lysine” is oriented inwardly facing the active site cleft. This configuration may have contributed to the inability of cathepsin L to bind to elastin molecule. From the overlay of the bridge domains and sequence alignment of human cathepsins V and L, it was found that a glycine residue immediately following a proline residue is present in cathepsin V but not L. Due to the unique structure of proline and the size of glycine, such arrangement may have resulted in the orientation of the succeeding lysine. So, a glycine deletion and insertion was carried out in cathepsins V and L, respectively, and designated as M6 and M7 mutants to evaluate the impact of such arrangement. It was found that the deleted glycine correlates with a 25% decrease in cathepsin V’s elastolytic activity (Fig.
2.13) suggesting its potential role in elastin degradation. On the other hand, while inserting a glycine residue into cathepsin L did not generate an increase in elastolytic activity, the HPLC profile of elastin degradation by M7 revealed a elastin degradation pattern that differs from that of wildtype cathepsin L and resembles that of wildtype cathepsin V (Fig. 2.15). Taken together, even though no information concerning the mechanism employed by these mutants during elastin degradation could be inferred, the participation of the TVVAPGK domain and of the glycine residue in the reaction was evident. Other residues that may play a role in elastin degradation are found within amino acid 92 to 104 (Fig. 2.8 and 2.16). Residues that differ between cathepsin V and L within
33 that
elastins
cathespin serve explore nonproductive degradation
form enzyme was adsorptionldesorption
been potent is demonstrate
catalytic compatible that However, cathepsin elastolytic adsorptionldesorption
an
region
noted
since
shown
a
as
interaction
It
catalytically
elastolytic
are
the
may
a
step
L
had
that
elastin potential
if
activity
L consisted
involvement
show
with
through
would
to
high
either
that
(4). manner,
such should
demonstrate
been
such
more
elastolytic
is between
is
activity.
after not
remain
adsorption
Therefore
productive
the binding
mainly
the
mainly
reported
option
proceeds
commence
be
favor
and
neutral
following
of
swapping
case,
contributed
this
adsorbed
cathepsin
high the
The
consisted
of
activity
as
patch
such
how
step
region
that neutral
adsorbed only complex.
one in
amino
degrading
result
rapid
steps: spontaneously
the
interaction.
whereas
was
do
minimal cathepsins
on
if
may
L in
of
potential
amino
acids
to such
we
reported
the
not
cycle.
elastin first
and
enzyme
neutral
Upon
its
consider
activity
explain
surface
observed
the
speculation
in
the
elastin
the
acids,
lack
One
It
degradation.
cleavage
cathepsin elastin-binding
charged K,
amino enzyme
from
then
is
elastin and
against would of
of
the
therefore a
S degradation
in that
neutral
elastin
rapidly interacts
a
our
and
inability cathepsin
acids,
is
and
residues
adsorbs molecule,
strong
however V
true.
the
soluble
laboratory
L
than
surface
or
release
of
thus
as
may domain? with
inability
Because
dissociates
was
elastin-binding great
to
to
L
well
in
in
elastin,
possibly
expect
probably
rescue
elastin
then
other
of
the
of
observed
proceed
cathepsin
interest
however
thus
the
the
It
cathepsin
same
the
to
elastin
if
cathepsin
can
molecule
region
cathepsin
from
products,
possessing producing there
because
detect
subsequent
in for
to
region
be
L.
was
it
domain.
sites
further
elastin
human would
indeed argued
L Since
(7).
L
in
had
any
the
L’s the
not
of
to 34
to
It
a
a
a possess
surface hydrophobic including
is elastin allow
other elastin-binding L, weaken binding elastolytic tropoelastins
“pods” domains make to elastin adsorptionldesorption cathepsin facilitated cathepsin
negative
demonstrated elastin,
hand,
strong
perfect is
binding of
only with
elastin
domains
relatively
on
the
tropoelastins
V
by
as
V
cathepsin in
activity
different
adsorption interaction
a
adsorbs substrate
neutral
the
are
well
extended
mechanistic
few
“dives”
domain,
binding.
by
compromised
insertion
in
vastly
large
cathepsin localized
as
cathepsin
can
electrostatic
V,
on
facets
model
binding
elastin
and
of
hence
with regions
after comparing
into
the
Upon
cross-linked
be
the
into
is
sense
to
surface
negatively
proposed
elastin.
V
swapping
so
interaction
explained
the enzyme the region
involve V
and
more
elastolytic
may
of
much
are
potential.
observed
for
the
to
gaps
subsequent
of
However,
involve
is
considerations,
cathepsin
not
cathepsin
to
to
larger in
in surface
of
mostly
elastin
charged
the
as found another
between each a
the
adjacent
activity
lack
three-dimensional
Human
in surface
well.
the
bridge-region
neutral
fastening
in
including
the
L,
size other,
on
patches
of
V
following
study
a
the
electrostatic
to increased
because the
Since
non-catalytically to
in
tropoelastins.
cathepsin
of
the
negative
thereby
each
may comparison
possess
(7), neutral elastin
on
of
the
fact
elastin,
E-amino
(TVVAPGK)
an steps. other
its
possess
the
elastolytic active
potentially
interaction that
in
overall surface
V
surface
patches
multiple potential
introduced
spite
has but
First,
to
two
So,
being
site
side
multiple
cathepsin
productive
still
been
putative potential
of
while
potential
in
activity.
weakly
cleft
combining
chain
of
elastin-binding facilitating
the
with with
the
polymers
contribute
cathepsin
region reported
introduced its
(9).
extending
cathepsin
region elastin
of
V,
model
may
charged
surface
manner
On
elastin
lysi
it
Since
may
will
the
not
the
the
to
35
19
of to
as
of L of neutral amino
presentable for such perhaps (3). regular the
approaches
inhibitor
first
Therefore,
as
acids
The
active
functions
through
step.
atherosclerosis
to
cd3—CLIP
identification
design.
91
site
As
develop
the
to
interruption
cleft the
might
design
104,
Cathepsin
mechanistic
complexes
where
elastolytic
be
followed
(4);
of
of
beneficial.
elastin-binding
a
the
however,
of
specific
V
in
catalytic
elastin
activity-specific
was
information
by
immune
found the
Identifying
inhibitor
its
binding
step
spontaneous
involvement
system
domains
to
takes
of
degrade
that
while
elastin
inhibitor
the
suggests
place
will
in
elastin
docking
elastin
still
cathepsin
degradation
in
diminish
(Fig.
will
its
allowing
the
binding
in
2.17).
eventually
important
of
pathological generation
V
its
tropoelastin
might slowly
domains elastolytic
it
to
role
be
carry
be
unfolds,
of
realized.
conditions
in
exploited
is
antigen- into
humans
activity
simply
out
the
the
its
36 Table 2.1
PCR Primers for mutant cathepsins A compilation of all primers used in constructing mutant cathepsins. PCR reactions were carried out using a standard protocol. The melting temperature was 95°C, annealing temperatures vary according to the individual primers and the extension temperature was 72°C. An initial 5 minutes heat-start and final 8 minute extension step was also applied. Mutant Annealing Cathepsins Primer Sequences Mutation Ternperatu re (°C) Mutanti CatL:aalto54 Forward 5’ -CAC TGA GCG AGC AGA ATC TGG- 3’ Cat V: aa 47 to 57.9 Reverse 5’ -CCA GAT TCT GCT CAC TCA GTG- 3’ 221 57.9 Mutant2 CatL:aaltol27 Forward 5’- GGA GAA GGC CCT GAT GAA AGC AG -3’ Cat V: aa 119 to 60.0 Reverse 5’ —CTGCCT TCA TCA GGG CCT TCT CC- 3’ 220 60.0 Mutant 3 CatL: aalto 171 Forward 5’ -GAT CAT GGT GT1’CTG GTG OTT GG- 3’ CatV: aa 163 to 58.1 Reverse 5’ -CCA ACC ACC AGC ACA CCA TGA TC- 3’ 220 58.1 Mutant4 CatL:aalto9O Forward 5’ -GGC CTG GAC TCT GAG GAA TCC TAT CC- 3’ Cat V: aa 82 to 60.1 Reverse 5’ -GGA TAG GAT TCC TCA GAG TCC AGG CC- 3’ 221 60.1 Mutant5 CatL:aaltoll2 Forward 5’ -TCT GTT GCT AAT GAC ACC GGC TTT- 3’ Cat V: aa 105 to 59.0 Reverse 5’ -AAA 0CC GOT GTC ATT AGC AAC AGA- 3’ 221 59.0 Mutant 6 Forward 5’ - ACA GTG GTC GCA CCT AAG GAG -3’ Cat V 18gly’ 58.9 Reverse 5’ - CTC Cr1’ AGG TGC GAC CAC TGT -3’ Knock-out 58.9 Mutant 7 Cat L glycine Forward 5’- GTG GAC ATC CCT GGA AAG CA -3’ Insertion between 57.4 Reverse 5’—TO CTT TCC AGO GAT GTC CAC —3’ 6Pro” and 7Lys” 57.4 Flanking Primers Forward 5’-GAC TGG TEC CAA HG ACA AGC-3’ N/A 54.3 Reverse 5’-GCA AAT GGC AH CTG ACA TCC-3’ 54.8
37 Table The The regression condition CathepsinV Enzyme Ml M2 M8 M6 M4 M5 M3 M7 M9 CathepsinL k0, enzyme 2.2 and outline algorithm. Km kinetics value under of of The “experimental both each substrate kcag(sec) wildtype enzyme 25.18 26.82 24.92 24.76 18.19 11.24 16.18 17.72 11.57 23.5 7.72 procedure”. used was and Enzyme mutant calculated was ±0.83 ±0.24 ±0.48 ±0.86 ±1.43 ±0.35 ±0.03 ±0.42 ±0.86 ±2.37 ±0.1 the Kinetics cathepsins. synthetic using Km(.tM) GraphPad 11.45 11.20 7.04 3.00 3.12 2.54 8.29 2.00 1.15 1.89 1.33 fluorogenic Prism ±0.08 ±0.83 +0.29 ±0.99 ±1.56 ±1.54 ±0.07 ±0.20 ±0.22 ±0.12 ±0.33 substrate program kcag/Km through Z-FR-MCA 2.20±0.07x 2.58 2.17±0.31x 3.24±0.lOx 5.91 7.54±0.02x 3.86 6.36 1.00±0.04x 8.72± 1.31 (M ± ±0.48x ± ± + sec’) 0.03 0.17 0.45 0.01 1.78x non-linear x x x x using 106 106 10 lO 106 106 106 106 106 i07
38 Residual Elastase Activity L69 Cf5 162H1 187N1 Cat L —27%
Mutant I 5r”______—120% I — — — — • 89_ — Mutant 4 —100%
113_ Mutant 5 1x —60%
Mutant 6 —75%
Mutant 7 .1. —20%
Mutant 8 .f —50%
C25 P69 H163 N188 100% Cat V
Figure 2.1 A schematic diagram showing the various proportions of cathepsins V and L in mutants. Both cathepsins L (blue) and V (red) have their catalytic units marked and named in red. The only difference to their S2 subsite is also marked and identified in black. (Leucine in cathepsin L and proline in cathepsin V). The box indicates regions that were shown to be responsible for any elastolytic activity. A cross-mark (x) in Mutant 6 indicates 8glycine” deletion and a plus sign (+) in Mutant 7 represents a glycine addition.
39 mixed kinetics representation Figure Appropriate 0 0 with 2.2
constants.
O.OOE+OO
5.OOE+06
2.OOE+07 2.50E+07
1.50E+07 1.OOE+07 The dilution different of kcatlKm cathepsins All was - - - values concentrations comparison made are
which CatV on derived cathepsins was of of from analyzed Z-FR-MCA
selected
M6 kcatlKm to measurements achieve using
mutant Ml
and Corn decent nonlinear and assay
of
M4 parison triplicates. initial wildtype buffer regression activity. to cathepsins construct program
The M3 diluted against a
Prism CatL Michaelis-Menton cathepsins to Z-FR-MCA. obtain were
the 40
taken
LandM3. dithiothreitol Figure
stirring
120 120
. minutes, 0
>
minutes.
in
2.3
of
triplicates.
140
120
100
40
20
60
80
The
0-
200
then
at lOmgImL ------
-
elastolytic
p115.5.
rpm
E64
Thep
in
was
Aliquots
of
activity
>0.31
activity
elastin-rhodamine
added CaV
between
of
buffer
and
of
sample
mutant
Elastin-Rhodamine
the
Cat
containing
mutants
were
V Ml
and
and
was
withdrawn
wildtype
M1;p
tested
incubated
100mM
>0.05
for
cathepsins
from
absorbance
sodiuam
at
between
37
the
°C
using
reaction
M3
Cat
with
acetate,
at
L
590
elastin-rhodamine
I
and
iM
mix
2.5mM
nm.
M2,
of
at
All
andp enzyme t
=
EDTA
measurements
L
0,
>0.08
30,
with
conjugates
and
60,
between
constant
90,
2.5mM
were
and
41
Cat at Figure 2.4 The HPLC profiles of wildtype cathepsin V and Ml. Cathepsins (final concentration 1 jiM) were mixed with bovine neck elastin (EPC, MS) (final concentration of IOmg/mL) and assay buffer, and incubated in MaxQ500 floor shaker at 37 °C at 200 rpm for l8hrs. The reaction was stopped with E-64, and the mixture was centrifuged at top speed to precipitate undigested elastin particles. The supernatant was injected into System Gold Detector (Beckman Coulter, CA) for HPLC profile. The degraded fragments were loaded onto a reversed phase C-18 analytical column (Phenomenex, CA) and eluted with a linear gradient starting with 0.1% trifluoric Acid and ending with 90% acetonitrile supplemented with 0.1% trifluoric acid. The chromatograms were analyzed with 32 Karat program. It can be seen that both profiles almost superimpose each other suggesting Ml retains all the residues needed to exercise the full elastin binding mechanism.
42 Elastin-Rhodamine
120 Cat V M4
100 -
80 - 0 60 -
40 - U, a, 20 - 0-
Figure 2.5 The elastolytic activity of mutants 2, 4, 5 and wildtype cathepsins using elastin-rhodamine conjugates at 120 minutes. lOmg/mL of elastin-rhodamine was incubated at 37 °C with 1 tM of enzyme under constant shaking of 200 rpm in activity buffer containing 100mM sodiuam acetate, 2.5mM EDTA and 2.5mM dithiothreitol at pH5.5. Aliquots of sample were withdrawn from the reaction mix at t = 0, 30, 60, 90, and 120 minutes, added with E64 and tested for absorbance at 590 nm. Enzyme activities against Z FR-MCA were also recorded at the above time points to monitor their residual activities. All measurements were taken in triplicates with standard deviation shown as error bar. While M4 retains full elastolytic activity, M5 only possesses 60% of the original elastolytic activity. However, when compared to M2, M5 which has only 8 amino acids more from cathepsin V demonstrates, a dramatic increase in elastolytic activity. P >0.96 betweenCatV and M4;p <0.02 betweenCatV and M5;andp> 0.05betweenCatL and M2.
43 M5 CatV CatL .60660IITth 0,0a?
350 90
00 302
30 250
02
* 56
156
40
ISO
30
50
lb
2 4 6 8 10 12 14 16 10 20 191MM,
Figure 2.6 The HPLC profiles of wildtype cathepsins V, L, and M5. Several peaks that can be seen from the profile of human cathepsin V are missing from the profile of M5. Also, the highest peaks that overlap each other in both profiles seem to be different in intensity. This suggests that while the bridge region is important in generating 60% of elastolytic activity, it does not provide full binding mechanism resembling one employed by wildtype human cathepsin V.
44
other might of
elastolytic Figure
M4,
peaks
be
2.7
M4
yet
involved
The
activity.
are
M4
lower
HPLC
still
in
Also,
demonstrates
for
achieving
profiles
M4.
it
CatV
can
of
a
be
wildtype
complete
to
seen
retain
that
cathepsins
binding
full
the
elastolytic
CatL
highest
mechanism,
V,
L
peaks
and
activity.
M4.
are
they
Few
almost
It
may
suggests
peaks
not
equal
are
be
that
in
missing
as
intensity
crucial
while
from
other
in
even
generating
the
residues
though
profile 45
the
between glycine region were
dots Figure
alignment
V
V K V
L K V
L V
L
S
S K
S S K L K V
L
S L K
S
CLUSTAL
Sequence
region
and
extracted
and
2.8
residue
cathepsins
those
algorithm.
highlighted
the
Multiple
W
Alignment
mature
of
(1.83)
through
in
less
L
the
amino
and
Identical
enzyme.
NKYWLVKNSWGEEWGMGGYVKMAKDRRNHCGIASAASYPTV NKHWIIKNSWGENWGNKGYILMARNKNNACGIANLASFPKM
KEYWLVKNSWGHNFGEEGYIRNARNKGNHCGIASFPSYPEI KALMKAVATVGPISVAIDAGHESFLE’YKEGIYFEPDCSSEDMDHGVLVVGYGFESTESDN
homology KALMKAVATVGPISVAt4DAGHSSFQFYKSGIYE’EPDCSSKNLDHGVLVVGYGFEGANSNN MIELHNQEYREGKHSFTMAMNAFGDMTSEEFRQVMNGFQNRKPRKG---KVFQEPLFYE
KALKRAVARVGPVSVAIDASLTSFQFYSKGVYYDESCNSDNLNHAVLAVGYGIQ-----KG GNEGCNGGLMDYAFQYVQDNGGLDSEESYPYEATEESCKYNPKYSVANDTGFVDI MIELHNGEYSQGKHGFTMANNAFGDMTNEEFRQMMGCFRNQKFRKG---KVFREPLFLD
GNQGCNGGFMARAFQYVKENGGLDSEESYPYVAVDEICKYRPENSVANDTGFTV7 MNPTLILAAFCLGIAS--ATLTFDHSLEAQWTKWKAMF{NRLYG-MNEEGWRRAVWEKNMK
SKYWLVKNSWGPEWGSNGYVKIAKDKNNHCGIATAASYPNV DVLKEAVANKGPVSVGVDARHPSFFLYRSGVYYEPSCT-QNVNHGVLVVGYGDL----NG GNKGCNGGFMTTAFQYIIDNKGIDSDASYPYKAMDLKCQYDSKYRAATCSKYTEI MNLSLVLAAFCLGIAS--AVPKFDQNLDTKWYQWKATHRRLYG-ANEEGWRRAVWEKNMK MKR---LVCVLLVCSSAVAQLHKDPTLDHF{WHLWKKTYGKQYKSKNEEAVRRLIWEKNLK
MWG---LKVLLLPVVS--FALYPEEILDTHWELWKKTHRKQYNNKVDEISRRLIWEKNLK
YISIHNLEASLGVHTYELAI4NF{LGDMTSEEVVQKMTGLKVPLSHSRSNDTLYI
-NDGCGGGYMTNAFQYVQKNRGIDSEDAYPYVGQEESCMYNPTGKAAKCRGYREI
PKSVDWRKKGYVTPVKNQKQCGSCWAFSATGALEGQMFRKTGKLVSLSEQNLVDCSRPQ—
PDSVDWREKGCVTEVKYQGSCGACWAF’SAVGALEAQLKLKTGKLVSLSAQNLVDCSTEKY
PRSVDWREKGYVTPVKNQGQCGSCWAFSATGALEGQMFRKTGRLISLSEQNLVDCSGPQ- * PDSVDYRKKGYVTPVKNQGQCGSCWAF’SSVGALEGQLKKKTGKLLNLSPQNLVDCVSE-- FVMLHNLEHSMGJ?4HSYDLGI?4NHLGDMTSEEVMSLMSSLRVPSQWQR--NITYKSNPNRI * .::*::*****
•
in
blue multiple *****
.* CBI
***:*:**
V
blue of
:**
acids that
.***
region.
Cathepsins database.
*
are
The resides
*
may
with sequence
*
the letters
**:**
sequence contribute ** *
*
The one ****:
::*
Pro-X-Gly-X
are The *
**
residues highlighted
*
dot.
marked
K, alignment
;** sequences **: *
:.** 5,
.*
to
All alignment
**:*****:*****:
V the
*:**:
with
amino
and shown **
:****.**. segments.
:
elastin
in
of were
:*
L
an red,
*: human
* acids
:***
in
asterisk, degrading *:*::
****.
are :*
aligned
bold
Notice
sequences the .
. cathepsins **
* and
:
first related
•. using
*:*
ability that
*
red residue ***:*:** *
.:
contain
all
the
K,
residues colour *
.
of
333 329
334 331
but
S,
Clustal
cathepsin
of
V
cathepsin the
:*
the
are
and
are pre-pro-region,
******
mature
the multiple
**
marked
L.
V.
ones
PEWEG
All
L
enzyme
possess
sequences
sequence
with
differing
pro-
232 233
292 293 235
two 290 232
288
173 115 55
175 173 114 115 57 57
173 114 57
and
46 the Figure 2.9 The superposition of human cathepsins V and L. These two structures were obtained from the RCSB Protein Data Bank with the designated number IFHO (human cathepsin V) and 1ICF (human cathepsin L). The overlay was done using PyMol program. The bridge region is represented as a stick model for both cathepsin V (yellow) and cathepsin L (red). It can be seen that the region mostly superimposes each other as it moves from left to right until proline (in white box). The lysine side chain immediately follows the proline residue in cathespin L orients inward (red arrow) while the lysine side chain in cathepsin V following the glycine (gray) residue is imposing outwardly (yellow arrow).
47 ______
Elastin-Rhodamine
140 120 cv
M8 60
40 i CatL— 20
0
Figure 2.10 The elastolytic activity of mutants 8, 9 and wildtype cathepsins using elastin-rhodamine conjugates. lOmgImL of elastin-rhodamine was incubated at 37 °C with 1 M of enzyme under constant shaking of 200 rpm in activity buffer containing 100mM sodium acetate, 2.5mM EDTA and 2.5mM dithiothreitol at pH5.5. Aliquots of sample were withdrawn from the reaction mix at t = 0, 30, 60, 90, and 120 minutes, added with E64 and tested for absorbance at 590 nm. All measurements were taken in triplicates with standard deviation shown as error bar. M8 retains approximately 60% of activity where M9 demonstrates comparable activity to cathepsin L. P <0.04 betweenCat V and M9, and p < 0.04 betweenCat L andM9.
48 ______
M8 CatV CatL 6644s6
10 12 04 16 14 20 Ufla.
Figure 2.11 The HPLC profiles of wildtype cathepsins V, L and M8. The profile of M8 displays high similarity to wildtype cathepsin V with only a few peaks that are lower in intensity relative to other peaks. The highest peak is also significantly lower for M8.
4 or 2, a. I CatV CatL
350
70
60
200
60
40
100
30
0 2 4 6 6 10 62 14 II 10 20
Figure 2.12 The HPLC profiles of wildtype cathepsins V, L and M9. M9 which contains the bridge region of cathepsin V demonstrates an increase in intensity of the highest peak even though no other degraded fragments are observed.
49 Elastin-Rhodamine
120 V 100
80
C.) 60
(U 0 40 U, L 20 0
Figure 2.13 The elastolytic activity of mutants 6, 7 and wildtype cathepsins using elastin-rhodamine conjugates at 120 minutes. lOmgImL of elastin-rhodamine was incubated at 37 °C with 1 M of enzyme under constant shaking of 200 rpm in activity buffer containing 100mM sodium acetate, 2.5mM EDTA and 2.5mM dithiothreitol at pH5.5. Aliquots of sample were withdrawn from the reaction mix at t = 0, 30, 60, 90, and 120 minutes, added with E64 and tested for absorbance at 590 nm. All measurements were taken in triplicates with standard deviation shown as error bar. It can seen that M6 retains approximately 75% of the wildtype elastolytic activity, whereas insertion of gicyine in cathepsin L (M7) did not rescue elastin degrading activity. P < 0.03betweenCatV andM6,andp > 093 between M7andCatL.
50 MG CatV CatL Apr 17th 00420
70 250
270
50
ISO
110
10
10
2 4 6 6 10 12 14 16 20 MirwA.s
Figure 2.14 The HPLC profiles of wildtype cathepsins V, L and M6. Carrying a glycine deletion, the HPLC profile of M6 demonstrates a degradation pattern that is highly similar to the wildtype but with few missing peaks and a less intense highest peak. This confirms the deletion of glycine compromises the elastolytic activity possibly because the mutant was unable to bind to certain residues of elastin.
51
proline
elastin-rhodamine
chain Figure suggests 250 200 150 300 50
00
thus
2.15
116
M7
the
allowing
and
The importance
2
lysine
HPLC
assay, partial
4
117
profiles
of
it
binding
is
in
glycine
CatV
evident cathepsin
6
of
and
wildtype
in
that
degradation the
I’ L,
6
the
possibly
the
j
cathepsins
binding mutant 0 M0to, 60
\
of
CatL
changing
elastin.
although
pattern V,
62 L
_-
the
is
and
did
more orientation
64
not
M7.
like
generate
After cathepsin
16
of
inserting
the
any
succeeding detectible
18
V
than
a
glycine cathepsin 20 30 60 20 50 50 40 SO
35 90
results
lysine
between
from
L.
side
52 It Figure 2.16 The distribution of potential elastin binding domains in human cathepsin V. Shown here is a PyMol rendered surface representation of human cathepsin V. The domains potentially correlate with the elastolytic activity are colored in steel blue, royal blue and yellow whereas the rest was left as red. The steel blue domain spans amino acid 113 to 119, TVVAPGK, in cathepsin V is located directly at the entrance to the active site cleft. The royal blue patch represents 118glycine which may involve in elastin binding by orienting the subsequent 19lysine’ The yellow domain spanning amino acid 89 to 104 is where the neutral patch is located. Notice how the two domains are not adjacent to each other which is mechanistically reasonable as elastin in .its natural form is a polymerized tropoelastin with complicated cross-linking, therefore by placing the elastin binding domains on different facets of the enzyme it allows the enzyme to interact the macromolecule in a three-dimension fashion.
53 the bridge-region; Figure Catalysis extending initiated elastin 2.17 through takes mo’ecule
pod ( A schematic red: of place binding elastin occurs neutral as a of representation pod molecule immediately patch) the of side elastin of (gray). chain cathepsin after falls of of Adsorption the the into Iysine 119 Binding proposed catalysis V the contact active (dark happens elastolytic leaving site with blue) cleft. when other a of cleaved mechanism The cathepsin the extending final elastin-binding product. dissociation V of pod (light cathepsin of blue) /CatalYsis elastin domains of V. enzyme to Binding one molecule. (green: of from the 54 is References
1. Lecaille, F., Kaleta, J. and Bromme, D. (2002) Chem Rev, 12, 4459 — 4488
2. Adachi, W., Kawamoto, S., Ohno, I., Nishida, K., Kinoshita, S., Matsubara,
K.,and Okubo, K (1998) Invest. Ophthalmol. Vis.Sd. 39, 1789—1796
3. Tolosa, E., Li, W., Yasuda, Y., Wienhold, W., Denzin, L. K., Lautwein,
A.,Driessen, C., Scbnorrer, P., Weber, E., Stevanovic, S., Kurek, R., Meims,
A.,and Bromme, D. (2003) .1 Clin. Investig. 112, 517—526
4. Yasuda, Y., Li, Z., Greenbaum, D., Bogyo, M., Weber, E., and Bromme, D. (2004)
J Biol. Chem., 35, 36761 — 36770
5. Debellem L., and Tamburo., A. M. (1999) Int. JBiochem. Cell boil, 31, 261 —
272
6. Tamburro, A. M., Pepe, A, and Bochicchio, B. (2006) Biochemistry,45, 9518 —
9530
7. Novinec, M., Grass, R. N., Stark, W. J., Turk, V., Baici, A. and Lenarcic, B.
(2007) 1 Biol. Chem., 11, 7893 — 7902
8. Choem Y., Leonetti, F., Greenbaum, D. C., Lecaille, F., Bogyo, M., Bromme, D.,
Eliman, J. A., and Craik, C. S. (2006) J. Biol. Chem., 18, 12824—12832
9. Brömme, D., Li, Z., Barnes, M., and Mehler, E. (1999) Biochemsitry. 38, 2377 —
2385
10. htt:p:!/tools.invitrogen.corn/content/sfs/rnanuals/pichrnan.pdf
11. Somoza, J.R., Zhan, H., Bowman, K.K., Yu, L., Mortara, K.D., Palmer, J.T.,
Clark, J. M., McGrath, M. E. (2000) Biochemistry, 39, 12543 — 12551
55 12. Santamaria, I., Velasco, G., M., C., Fueyo, A., Campo, E., and Lopez-Otin,
C.(1998) Cancer Res. 58, 1624—1630
56 perform CHAPTER the identified;
only
epidermis, thoroughly 80% crystal
function other described
between specificity observations, V
the elastolytic enzyme’s
However,
sites
and
later
kinetic
carried
identity
to
hand,
L
The
structure
Nonetheless,
carry
must
an
the additions
and
as
protease
some
the
activity and
profiling
behaviors
characterized.
human
out
array
substrate the
to
only
it
exist III out
structure,
kinetics
thymus
prompts
in
that members
of
most
proteolysis —
the
to
of
activity
cathepsin
that observed demonstrated
cathepsins
Conclusion
of
one
study
the
of
potent
preferences
functions. past
of
cathepsin causes
(2,
the
us
less
potential
cathepsin
of
While
all
ten
in
on
to
3,
mutants.
the
mammalian
to
V
general?
even
mutants is
hypothesize
years
the 4)
are various
has
the
family
known
While
there
L,
suggesting
of
minimal potent
concern
and
a
though
family been
structural
(2).
and
both
family
Both
This
is
human
are
are
Recommendations
The
for
about
elastolytic its
studied elastase
compatible
human
that
cathepsins their as that
rather
a
question
elastolytic
expression
of
some,
amino
growing
a
its
difference
cathepsins a
potential
lies papain-like
its
substrate
structural
(5),
identification
cathepsins new.
to-date
elastolytic
the in acid activity
can
with and
V
this
number
(1).
has
activity.
functions
be
and
sequence
but
role
(6).
its
preference
revealed
their
study difference
Human
answered
been
of
cysteine
V
active
L
not
Human
in
cathepsin
and
of employ activity.
and
for
parental
is
immune
to found
Moreover,
reports
of
and
cathepsin
L how sites
significant
characterization
Further
the
cathepsin
proteases
taking may
cathepsin
(7). between
identical
distributions
in
difference
to
and
V. Cathepsin
cathepsins.
Based
on
response.
differ
testis,
contribute
advantage
sub-pockets
V
a
cathepsin’s
Work cathepsins
V
similarity
found
L,
is
substrate
on
catalytic
slightly.
cornea,
bears
one
on
in
these
V
was
The
The
are
the
the
the
of
to
57
of
is
a •
mutant proportion behavior,
reported
elastin unable with cathepsin
of pattern investigation soluble
carried degraded structure
cathepsin the domain-swapping. TVVAPGK domain
entrance
119)
cathepsin
active
our
seems
but
In behaves
of
to
elastin
out
to
that
hypothesis
of
leading
the elastin
L’s
this
cross-linking
displays
V
site
adsorb of
amino
the
either
to
domain
V
of
elastolytic by human
lack
either
study,
altogether
cleft
correlate
polymerized as
elastolytic
more
fragments
cloning
to
acids
a
of
using to
minimal
Our
and
the
structure
that
parental
we (bridge-like)
an cathepsin
and
like
89 of
active
sequential
the
elastin set activity
with
human fluorogenic
also
chimeric
tropoelastins
with degrade to
activities:
one
out adsorption
orientation
elastin
cathepsin.
related
119 majority helps
site
to binding
L
fluorogenic
parental
cathepsin
is
pinpoint
is
in
demonstrates
cleft, analysis
independent
and
mutants
distinguishing
polymerized
an
cathepsin
phenomenon.
or
1)
to
of
the
of
domain interesting
is
elastin
While
chromogenic
and
insoluble
cathepsin
random. elastolytic
the
L
the
assay
chemicals has
and
therefore must
lysine
potential
V
there
is
and
of
pinpointed
potent
of
mutants
where
elastin
insoluble
the
lack elastin
one
such
One
However,
or
elastolytic
its
side
activity
is
such
insoluble
elastin
may
the
potent
because
activity
elastin the
hence
a
trend. TVVAPGK
efficiently.
chain.
(8).
trend
involving
other
as
the
elastin in
direct
found
two
This fluorescamine.
conjugates
elastolytic
activity cannot
aqueous
binding
Furthermore,
potential
of
elastin
against
in
It
along
the
obstacles
its
is
finding
binding
the
in
point-mutation
(amino
located
Therefore,
predict
close
bound
are
domain
cathepsin
degrading
mutants’
phase,
with
soluble
activity
elastin
or
almost
is
domain
proximity
lying
by
it acids
compatible
right elastin
the
increasing
and
has
in
labeling
ETNA
binding
kinetic against
human
V. overall
always
human
ability
in
at
113
2)
been
thus
The
into
and
the
the
its
58
to — the
mutual
clear not loss
cathepsin
chain
orientation as specific the access
have
the residue cathepsins
succeeding potential correlates L
into
to
an
active
E-amino
rescue
of
size
cathepsin that
contributed
evaluate
of
“antenna” swapping of elastolytic
One
Following
is
orientation
the lysine”9 L
role
of
elastin
present
site
V
with may
any
led
lysine.
other
group
glycine, loss
and
in
L cleft
the us
clear
noticeable
elastin between
a to
for
did
point
of activity.
L,
that
to
in
the
as
So
of
impact
25% the
may
for
the
consider
cathepsin
degradation.
it
not
up
it lysine’17
a
such
was
inserts sequence inability
of
degradation.
glycine
domain
degradation.
points
the service
generate
decrease
human
interest
However,
of
found elastolytic
arrangement
“passage
the
such
V
into
is
away
deletion of
two
from
cathepsins
involvement
but
alignment
orienting
The
in
that
an
cathepsin
in
arrangement.
and
this
possible
On
implementing
not
increase
The
way”
cathepsin
from
opposite
cathepsin
a
activity
glycine
the and
bridge-like
hold
in
may
importance
inwardly
leading L. V
the
other
L
in
insertion
purposes:
in of
and
the Due to
have
is
the
V
active This
residue
another
detectible
V’s adsorb
It
observed
hand,
L, constitutes
elastin
to
this
to
bridge-like
domain
was
facing
designated
resulted
absence
the
the elastolytic was
of
site
same first,
while
immediately to
domain.
found
unique
this
active
carried
in
elastin
elastolytic
cleft. in
the
is
the
domain
an
place;
human
inserting
in domain
the
of
active
domain
M8
that
site
approximately
£-amino
structure
activity
the We molecule.
elastolytic
out
orientation
and
thus
the
and
following
in
activity, cathepsin
in
orientation believed
site
was
a
cathepsin
M9.
cathepsin
of
deleted allowing
glycine
group
secondly,
suggesting of
cleft
assessed
both
And
proline
activity
of
the
5O6O% a that
and
L
may
the
glycine
residue proline
human
It
of
where
HPLC L
easier
V
was
may
side
this
this
did
and
and
by
act
the
in
its 59 the profile wildtype
the
degradation that suggest found
elastins
exploring serve of
nonproductive degradation form
the was enzyme had L catalytic indeed
to
cathespin
mechanism
adsorptionldesorption participation
region
been
pointed
demonstrate
a as
within
of
It
catalytically
is
the are
a
may
cathepsin
step
the potential shown had
an elastin
consisted
involvement
show
was
through
L
that
either interaction
amino
involvement
been
would manner,
should
employed
evident.
to
such
high degradation
more of
V
binding
remain
demonstrate
productive
hypothesized
the
acid
mainly
but
the
not
adsorption
elastolytic
commence
and
neutral
following
between
of
not
92
favor
Furthermore,
by
TVVAPGK
proceeds
of
adsorbed
patch
other
the
of to
these
this
L.
by
neutral
such adsorbed complex.
104.
amino
Taken
a
region whereas
activity
step
residues
this
cathepsin
high
that steps:
mutants
spontaneously
in
interaction. The on
was
mutant rapid
amino
together,
acids
domain
cathepsins
degrading as
the
in
first
enzyme
Upon difference
if
the
was
in
elastin
not
during
surface
such
L
cycle.
in
the
elastin
mainly acids,
suggested and
observed
mentioned
cleavage
and cathepsin
even
It
speculation
enzyme
then
degradation.
activity
and
elastin
K,
is
elastin
One
of
a
between
degradation,
of therefore
charged
neutral
though
elastin
S,
interacts
rapidly
would
in the
a and
adsorbs
and
degradation
against molecule,
V
earlier,
cathepsin
degrading
is
glycine than
surface
or cathepsins
release no
residues
L
of
however
true.
thus
with
dissociates
may information
to
great
and
soluble
our
in
elastin
Because
other
displaying then
of
of
L
cathepsin
residue
proceed
pattern
in could these
results
interest
probably
the
the
expect
V
the
elastin,
the
elastin
and molecule
region
from
products,
residues
same
be
concerning
cathepsin
subsequent
seemed
similar
in
to in
cathepsin
L
L. inferred,
a
because it
sites if
further elastin
within
region
elastin
would
potent
Since
(8).
there
in
are
the
to
to
60
to
It L
a elastolytic with
L
be how the potential.
consisted negatively
in site relatively
binding strong hand,
weaken demonstrated
Upon elastin not in
other,
(6).
contributed
cathepsin extended
cleft
potential
impossible.
do
such
Therefore
cathepsin
more
adsorption
may
Taken we
degradation,
elastin
domain,
is
of
option
large
activity.
Human
charged
entirely explain
possess
neutral
considerations,
V
region
elastin-binding
to
together
compromised
binding.
are
comparing
Since
V,
one
as
its
hence
of
the The
not patches
cathepsin
neutral.
only
amino after
lack multiple
the
of
one
may
elastin,
inability
adjacent
result the
the
the enzyme
minimal of
swapping
may
consider
acids,
on
to
However,
a
surface observed
however,
results
extending
strong
reported V
elastolytic
being domain? cathepsin
its
question
to
to
to
has it
surface
elastin
rescue
each
the
possibly
that
of
and
including
elastin-binding
polymers
the
lack
been
the
the
surface
from
other
“pods” the It
the L,
the
cathepsin while degradation
electrostatic
activity
fact
bridge-region
of can
the
inability
reported
possesses
possibility
our
potential
but
increased
of
of that
the
majority negative
be
of
laboratory
elastin tropoelastins
still
tropoelastins
because
active
two
L’s
argued
domain.
to
was
to
contribute
potential
a
participation
elastolytic
potential
detect
of
elastolytic
in surface
is
surface
(TVVAPGK)
possess
site
observed
weakly
spite
multiple
that
however
the
However,
vastly any
cleft
of
with
of
and
potential since to
introduced
elastin
activity
cathepsin
only
positive
the
adsorptionldesorption
activity.
elastolytic
for
(2).
elastin-binding
of
is was
neutral
cross-linked
introduced
elastin if
so
with
human
other
a
binding
Since
that
not
after
may
much
few
and
L
On
cathepsin
electrostatic
region
compatible
is
residues
activity is
is
cathepsin
the swapping
not
elastin
localized
the
the
negative
larger
domains
elastin mainly
to
active
allow
case,
other
sites.
each
may
are
L,
61
is in
in to size
tropoelastins. dimensional
following (8), non-catalytically
fastening on
spontaneous step carried through acids
binding adsorptive purposes
activity degradation through (9).
functions
possess
the
in
an
takes
Therefore,
91
comparison
neutral
The out
overall
by in
patches
interruption
to
of
steps. in
might
pathological multiple
place.
step
alanine
in
identification 6-amino of 104
docking
interaction
inhibitor
So,
order
patches
MEC-Il
through
First, putative
a
should
be
combining
of productive
As
to
specific
substitution.
to
elastin-binding
highly
neutral
cathepsin
side
of
a
of
weakly
in
confirm
design.
condition
result
complex
its
tropoelastin
be
with
elastin
the
model
chain
of
side
examined beneficial. inhibitor
the
domains.
region
maimer
elastin-binding
charged
of
elastin
V, or
Cathepsin
chain,
of
adsorptionldesorption
binding
in of
the
such to
it
lysl
immune
domains of will
alternate elastin
into
that proposed
facilitated
to as
cathepsin
as
it
amino
Secondly,
Through
19
make
will
while evaluate
atherosclerosis the
cathepsin
to
will
V
binding
system
elastin,
neutral
domains on
the be
was acids
perfect
diminish model,
still
the V
interesting
different
by
model.
as
the
found
adsorbs
91
suggests
V
allowing identification
the
Lys”9 by as active
mechanistic
involvement
model
to
in
“dives”
several
well
(6); First,
cathepsin
insertion
its
to
104,
cathepsin
facets
on
to site
is
its
carry
however, as elastolytic
proposed
it
the assess the
followed
suggested
further
important
elastin
cleft
into
to
to
region
surface
sense
of
out
into
V of
carry
V
involve
where its
the
the
elastin
may
its
by
serves interaction
elastin
works
by
activity and
for effect
between
involvement
elastin gaps
role
Novinec
out
to
of
a
the cathepsin
involve
in
elastin
subsequent
subsequent
initiate
interaction
significant
its
should
in
degrading on
between
a catalytic
perhaps
humans
binding
regular
elastin three-
amino
found
et
in
the
the
al.
V
be
62
in
a understood. domain
in
Altogether, cathepsin
V,
these
studies
findings
on
shall
the
mechanisms
shed
light
on
the
of
design elastin
of
degradation
specific
inhibitor.
can
be
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B. 64 — APPENDIX thesis substrate Figure program, Procedure, used obtained graph a enzyme above Michaelis-Menten A. was by 35. 30 25 15 10 for was The at 5 0 I in for various The data 0 the Mutant determined the I followed enzyme
I constructed, kcat Michaelis-Menten were reaction spectrophotometer. concentrations. will I 6 10 kinetics first graph by where again as would the input appropriate following.
Michaelis-Menten is need utilization Vmax of based The Graph be 20 I to both I to enzyme and input construct be on for Therefore, the The Km
[Z-FR-MCAJ further of information the Mutant in concentration wildtype the 30 data will order absorbance I a Prism converted 6. Michaelis-Menten were be appropriate to The calculated. and program calculate
Graph such collected was 40 data
I I mutant measured
(pM) 1tM. to were as the (GraphPad
for the the unit After collected as cathepsins correct 50 kcat outlined over graph concentration
M6 conversion the from 1 unit using Michaelis-Menten minute Inc.). as described 60 in I Vm. I as the Z-FR-MCA Experimental the In one which would The the of y-axis shown in active Prism 70 value I was this as 65 be of a commenced
substrate
degraded
to
convert
per
second,
the
absorbance
i.e.,
iMJsecond.
measured
over
1
minute,
to
concentration
of
66