Production and purification of formyl peptide receptor : explorations of protein-protein interactions
by Maria Renata Kohler
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Microbiology
Montana State University
© Copyright by Maria Renata Kohler (1997)
Abstract:
Neutrophils play an essential role in fighting bacterial infections. Many neutrophil functions are the
result of an interplay between cell surface receptors and their ligands. Formyl peptide receptor (FPR),
through the action of its ligand, fMLF, has been shown to mediate neutrophil migratory, as well as
cytotoxic functions. This receptor is believed to be organized into seven-transmembrane regions and is
coupled to guanosine triphosphate-binding protein (G protein), which is a key component in
FPR-mediated signal transduction. Previously, numerous attempts have been made to purify the
receptor. In this work, we describe the expression and partial purification of FPR expressed in insect
cells infected with recombinant baculovirus. The receptor expressed in these cells had a significantly
decreased ligand binding affinity with a dissociation constant of 70 nM, as compared to the receptor on
neutrophils with dissociation constant of 3 nM, suggesting that the processing of the receptor may be
different. We, therefore, shifted our effort to purify FPR from Chinese hamster ovary (CHO) cells,
which were found to bind ligand with a similar affinity as human neutrophils.
The purified receptor, as well as intact CHO cells expressing FPR (CHO-FPR), was used in the
selection of phage peptide library in order to identify sequences that bind to the receptor. Although no
consensus sequence was identified, the use of CHO-FPR cells in affinity purification of phage peptide
library allowed the selection of phage that had binding characteristics different from the phage selected
using wild-type CHO cells. This was demonstrated by flow cytometry, which proved to be a rapid and
efficient method for screening the selected phage.
Finally, to further explore the protein-protein binding sites on FPR, and more specifically the
interaction between the receptor and G protein, two cytoplasmic tail deletion mutants of FPR were
constructed and analyzed for their signal transduction capabilities. Partial calcium release and
suppressed chemotaxis by the deletion mutants suggest that the deleted regions are not absolutely
necessary in eliciting the FPR-mediated response. Our results support the notion that there are several
binding sites between FPR and G protein. PRODUCTION AND PURIFICATION OF FORMYL PEPTIDE RECEPTOR:
EXPLORATIONS OF PROTEIN-PROTEIN INTERACTIONS
by
■M aria R e n a ta Kohler
A th e s is su b m itte d in p a rtia l fulfillm ent
of th e re q u ire m e n ts for th e degree
of
M aster of Science
in
Microbiology
MONTANA STATE UNIVERSITY-BOZEMAN
B ozem an, M o n tan a
J u ly 1997
APPROVAL
of a th e sis su b m itte d by
M aria R e n ata Kohler
T his th e s is h a s been re a d by eac h m em b er of th e th e s is com m ittee
a n d h a s b een found to be satisfacto ry reg ard in g c o n te n t, E nglish u sag e,
form at, c itatio n s, bibliographic style, a n d consisten cy , a n d is ready for
su b m issio n to th e College of G ra d u a te S tu d ies.
Dr. Heini M iettinen
(Signature)
(Date)
A pproved for th e D e p artm en t of M icrobiology
Dr. Keith Cooksey
A pproved for th e College of G ra d u a te S tu d ies
Dr. R obert Brow n
(Signature)
(Date)
Ill
STATEMENT OF PERMISSION TO USE
In p re se n tin g th is th e s is in p a rtia l fulfillm ent of th e re q u ire m e n ts
for a m a s te r’s degree a t M o n ta n a S ta te U niversity-B ozem an, I agree th a t
th e L ibrary sh a ll m ak e it available to borrow ers u n d e r ru le s of th e
Library.
If I have indicated, m y in te n tio n to copyright th is th e sis by
in clu d in g a copyright notice page, copying is allow able only for scholarly
p u rp o se s, c o n siste n t w ith “fair u s e ” a s p rescrib ed in th e U .S . C opyright
Law.
R e q u ests for p erm issio n for ex ten d ed q u o ta tio n from or
re p ro d u c tio n of th is th e s is in w hole or in p a rts m ay be g ra n te d only by
th e co p y right holder.
S ig n a tu re
I dedicate th is w o rk to m y p a ren ts,
A ntoni a n d B arbara Balon, w ho gave
m e determ ination in p u rsu in g m y goals.
Dedykuj^ tq. pracQ moim rodzicom,
Antoniemu i Barbarze Balonz
ktorzy obdarowali mnie wytrzymatosciq.
dobrniQda do wyznaczonych celow.
(tra n sla te d from E n g lish by M aria Kohler)
ACKNOWLEDGEMENTS
I would like to express my appreciation to my mentor, Dr. Heini
M iettinen, for her guidance, support and patience. And m ost of all, for
dem onstrating to me, through her own example, w hat real scientists are made
of. Thank you! I also th an k Dr. Al Jesaitis for his expert advice and
opportunity to work in his laboratory.
I am grateful to all laboratory members: Dr. Jo h n Mills for his help with
FPR purification and flash courses in biochemistry; Jeannie Gripentrog for
introducing me to various laboratory tasks; Kathy Billings-Sandoval for being a
friend; Dan Siemsen (the man) for-showing me better ways to do things; Tom
Foubert for patient explanations of concepts of physics and computers; Heather
Edens for being a great resource of information; Dr. Jim Burritt, for answering
m any questions; Dave Barnidge for his chemistry-oriented input; Michael
Vlases for introducing me to net search; Connie McDonald for creating a good
working atm osphere; and finally to our undergraduate students, Karen Hix,
Tori Holloway and Rubi Khaleel, for m aking me feel like I know something.
Next, I th an k the graduate students: Wendy Cochran for introducing me
to m any aspects of graduate school; Robin Williams for inspiration (if she can
do it, I can); D eanna Nash, Qinfang Qian, Tresa Goins for motivation in my
thesis writing; Scott Kobayashi for hum or and also serious advice; Mike Ferris
for his unique ways; Barb Frederick for visions beyond grad school; Ace Baty
for his singing talent; Karen Xu, A nastassia Litvintseva, Eric Kern, Steve Nold,
Stacey Anderson, David Davidson, David Walker, Fintan Van Ommen Kloeke,
Elinor Pulcini, Thane Papke, Dmitri Kazmin, Brian Ellis and Malcolm Warnecke
for their friendship.
Many thanks to Patti Scarrah, Elizabeth Ferris, Fatmi T aha and Joanne
Schons for adm inistrative support; Pat Kraft for her im portant work in the
sterilization room; and Tim Carlson for putting up with my late night rehearsals
for next day presentations.
I extend my gratitude to Dr. Cliff Bond and Bridgid Petersen for letting
me u se their equipm ent and their limited space; Dr. Seth Pincus for advice on
the phage selection method; Dr. Gill Geesey for help with my very first journal
club presentation; and Dr. Vance Thurston for donosy.
Special th an k s to LTC Jo h n Kretzschmar, who h as awakened within me
the dream of graduate school and who supported me along the way.
Lastly, and m ost importantly, I th an k my family: my husband Lee and
m y children Patricia, Matthew and Anna, w ithout whom none of this would be
possible; Jo h n and Marjorie Kohler, as well as Michelle, Rob and Cathy, for
being there.
vi
TABLE OF CONTENTS
C h a p te r
1. INTRODUCTION
Role of n e u tro p h ils in im m u n e re sp o n se
Form yl p ep tid e rec ep to r .
.
.
.
P u rp o se of th is stu d y
.
.
.
.
Page
.
.
.
I
.
.
.
I
3
9
.
.
.
.
2. ATTEMPTS TO PRODUCE AND PURIFY FORMYL PEPTIDE
RECEPTOR USING BACULOVIRUS EXPRESSION SYSTEM
11
In tro d u c tio n
.
.
.
.
•
•
•
M aterials a n d M ethods .
.
.
.
.
•
Cell lin es a n d an tib o d ies
.
.
.
.
P ro d u ctio n of re c o m b in a n t b acu lo v iru s
.
.
R eco m b in an t plaq u e p u rificatio n
.
.
.
Im m u n o flu o rescen ce sta in in g of FPR e x p re sse d in
Sf9 in se c t cells
.
.
.
.
.
S odium dodecyl sulfate-polyaciylam ide gel
electro p h o resis a n d W estern blot a n a ly sis .
P re p a ra tio n of Sf9 m e m b ra n e s a n d ex tra ctio n of FPR
FA C Scan an aly sis of form yl p eptide b in d in g to FPR
ex p re ssed in Sf9 cells
.
.
.
.
R e su lts
.
.
.
.
.
.
.
.
Im m u n o flu o rescen ce of FPR .
.
.
.
Tim e c o u rse for m axim al ex p ressio n of FPR in
Sf9 cells
.
.
.
.
.
.
E x tractio n of FPR from th e m e m b ra n e s of Sf9 cells
FA CScan re s u lts .
.
.
.
.
.
D isc u ssio n .
.
.
.
■
•
•
•
3. PURIFICATION OF FORMYL PEPTIDE RECEPTOR
EXPRESSED IN CHINESE HAMSTER OVARY CELLS
.
In tro d u c tio n
.
.
.
•
•
•
•
M aterials a n d M ethods .
.
.
.
.
.
Cell lin es a n d an tib o d ies
.
.
.
.
P re p a ra tio n of CHO-FPR m e m b ra n e s a n d ex tra ctio n
of F P R ............................................................ 29
11
14
14
14
15
16
16
17
18
19
19
19
23
23
24
27
27
28
28
V ll
, TABLE OF CONTENTS - C o n tin u ed
C h a p te r
Page
Octyl glucoside ex tractio n of FPR a n d p u rificatio n
by h igh p re s s u re liquid ch ro m a to g ra p h y (HPLC)
W estern blo t a n d silver s ta in a n a ly se s of HPLC .
fractio n s
.
.
.
.
•
•
R e su lts
HPLC p u rificatio n of ^MBpaFYK-F luor p h o to cro sslinked FPR from tra n sfe c te d CHO cells
.
W estern blo t a n d silver s ta in a n a ly se s
.
.
D isc u ssio n .
.
.
•
•
•
•
4. FORMYL PEPTIDE RECEPTOR SCREENING
WITH PHAGE-DISPLAY PEPTIDE LIBRARY
.
.
.
31
32
32
37
37
40
In tro d u c tio n
M aterials a n d M ethods
P hage p ep tid e library screen in g u sin g FPR co u p led
to C N B r-activated Sepharo.se 4B b e a d s
.
A m plification of selected p h ag e
.
.
.
Phage lib rary screen in g u sin g CHO-FPR cells
.
Flow cytom etry of selected p h ag e b o u n d to
CHO cells .
.
.
•
•
S eq u en cin g of selected p h ag e clones .
.
.
R e su lts
.
.
•
•
•
•
■
Affinity p u rificatio n of p hage u sin g FP R -coupled
C N B r-activated S ep h a ro se 4B .
.
.
P hage selection on CHO cells expressing FPR
.
D isc u ssio n .
.
.
■
•
•
•
5. ANALYSIS OF CYTOPLASMIC TAIL DELETION MUTANTS
OF FORMYL PEPTIDE RECEPTOR IN CHO CELLS .
.
In tro d u c tio n
M aterials a n d M ethods .
.
•
•
O ligonucleotide-directed m u ta g e n e sis of FPR
Ligation a n d tra n sfe ctio n
.
.
.
.
FA C Scan an aly sis of FPR ex p ressio n of e a c h
m u ta n t clone
.
.
•
•
30
40
43
43
44
45
46
47
48
48
51
53
57
•
.
57
58
58
60
•
50
viii
TABLE OF CONTENTS - C o n tin u ed
C h a p te r
Page
A nalysis of m u ta n t FPR ex p ressio n by im m u n o ­
fluorescence
.
.
.
.
*
In tra c e llu la r calcium release a s s a y .
.
.
C hem otaxis a s s a y .
. . .
.
.
R e su lts
.
.
.
.
.
.
.
.
Isolation of h ig h e st e x p ressin g CHO clones
.
R elease of in tra c e llu la r calciu m by th e cells
e x p ressin g m u ta n t FPRs
.
.
.
^M LF-m ediated chem otaxis of CHO cells ex p re ssin g
m u ta n t FPRs
.
.
.
.
.
D isc u ssio n .
.
.
. . .
.
6. CONCLUSIONS
.
.
.
.
.
LITERATURE C I T E D ............................................................
.
61
61
62
63
63
63
64
67
70
71
IX
LIST OF TABLES
Page
Table
I.
P hage tite rs afte r e ac h ro u n d of selection
49
2.
R an d o m region am ino acid se q u en c es of selected p h ag e
50
LIST OF FIGURES
F igure
Page
1.
S ch em atic figure of th e pBlueBaciTzs vector
2.
Im m u n o flu o rescen ce a n aly sis of FPR ex p ressed in Sf9 cells
20
3.
-SDS-PAGE a n d W estern blot of Sf9 cells infected w ith
re c o m b in a n t baculO virus (His-FPR) .
.
21
4.
.
.
13
.
SDS-PAGE a n d W estern blot of m em b ran e p re p a ra tio n of
Sf9 cells infected w ith re c o m b in a n t b acu lo v iru s,
afte r ex tra ctio n u n d e r v a rio u s cond itio n s .
22
5.
HPLC of p h o to cro sslin k e d FPR ex tra cted from CHO cells
33
6.
Second ste p in HPLC purificatio n of FPR
7.
HPLC of m e m b ra n e e x tra c ts of CHO-WT cells
8.
SDS-PAGE of FPR ex tra cted from CHO cells a n d p urified
by H P L C ........................................................................ 36
9.
Flow cytom etry a n aly sis of b in d in g of selected p h a g e to
to CHO-FPR a n d CHO-WT cells
.
.
.
52
S ch em atic re p re se n ta tio n of FPR b a se d on th e
B aldw in m odel
.
.
.
.
.
59
10.
11.
12.
.
.
.
34
.
.
35
.
R elease of in tra c e llu la r calciu m by CHO ex p ressin g
w ild-type FPR, u n tra n s fe c te d CHO cells,
a n d th e CHO cells e x p ressin g th e cytoplasm ic
d eletion m u ta n ts , CT9 a n d CT29
.
.
.
65
C h em o tax is a s s a y of CHO-FPR a n d th e cytoplasm ic
deletion m u ta n ts , CT9 a n d CT29
.
.
.
66
ABSTRACT
N eu tro p h ils play a n e sse n tia l role in fighting b a c te ria l infections.
M any n e u tro p h il fu n ctio n s are th e re s u lt of a n in te rp la y betw een cell
su rface re c ep to rs a n d th e ir ligands.
Form yl p ep tid e recep to r (FPR),
th ro u g h th e a ctio n of its ligand, yMLF, h a s b een show n to m ediate
n e u tro p h il m igratory, a s well a s cytotoxic fu n ctio n s. T h is recep to r is
believed to be organized into se v e n -tra n sm e m b ra n e regions a n d is
coupled to g u a n o sin e trip h o sp h a te -b in d in g p ro tein (G protein), w hich is a
key c o m p o n e n t in F P R -m ediated signal tra n s d u c tio n .
Previously,
n u m e ro u s a tte m p ts have b een m ad e to purify th e recep to r. In th is work,
we d escrib e th e e x p ressio n a n d p a rtia l purificatio n of FPR ex p ressed in
in se c t cells infected w ith re c o m b in a n t bacu lo v iru s.
The recep to r
ex p re ssed in th e se cells h a d a significantly d e crea se d ligand bind in g
affinity w ith a d isso ciatio n c o n s ta n t of 70 nM, a s co m p ared to th e
rec ep to r o n n e u tro p h ils w ith d isso ciatio n c o n s ta n t of 3 nM , suggesting
th a t th e p ro ce ssin g of th e rec ep to r m ay be different. We, therefore,
sh ifted o u r effort to purify FPR from C hinese h a m s te r ovary (CHO) cells,
w hich w ere fo u n d to b in d ligand w ith a sim ilar affinity a s h u m a n
n e u tro p h ils.
T he p urified receptor, a s well a s in ta c t CHO cells ex p ressin g FPR
(CHO-FPR), w as u s e d in th e selection of p hage peptide lib ra ry in o rd er to
identify se q u e n c e s th a t b in d to th e receptor. A lthough no c o n se n s u s
seq u en ce w a s identified, th e u s e of CHO-FPR cells in affinity p u rification
of p h ag e p ep tid e lib rary allow ed th e selection of p h ag e th a t h a d binding
c h a ra c te ristic s different from th e p h ag e selected u sin g w ild-type CHO
cells. T h is w as d e m o n stra te d by flow cytom etry, w h ich proved to be a
ra p id a n d efficient m eth o d for screen in g th e selected phage.
Finally, to fu rth e r explore th e p ro tein -p ro te in b in d in g sites on FPR,
a n d m ore specifically th e in te rac tio n betw een th e re c ep to r a n d G protein,
two cy toplasm ic tail deletion m u ta n ts of FPR w ere c o n stru c te d a n d
an aly zed for th e ir signal tra n s d u c tio n capabilities.
P artial calcium
release a n d s u p p re s s e d chem otaxis by th e deletion m u ta n ts su g g est th a t
th e d eleted regions a re n o t a b so lu tely n e c e ssa ry in eliciting th e FPRm ed iated resp o n se . O u r re s u lts s u p p o rt th e notion t h a t th e re are several
b in d in g sites b etw een FPR a n d G protein.
I
CHAPTER I
INTRODUCTION
R ole o f N eu trop h ils in Im m u ne R esp o n se
The im m u n e system , w ith its in n a te a n d a cq u ired c h a ra c te ristic s,
p lay s a very im p o rta n t role in p ro tectin g th e h o s t from invading
m icro o rg an ism s.
In ' c o n tra s t
to
a cq u ired
im m u n ity
re su ltin g
in
m o u n tin g a re sp o n se to a specific p ath o g en , th e in n a te im m u n ity
involves nonspecific factors, w hich p rev en t th e p a th o g e n from en terin g
a n d s u b s e q u e n tly rep licatin g in th e h o st. The "noncellular" co m p o n en ts
of th e in n a te im m u n ity in clu d e th e p h y sical b a rrie rs form ed by sk in a n d
m u c o u s m e m b ra n e s,
te m p e ra tu re ,
and
and
v a rio u s
th e
physiological b a rrie rs,
b actericid al
su b s ta n c e s.
such
The
a s pH,
"cellular"
co m p o n e n t of nonspecific im m u n e re sp o n se include m a n y cells capable
of en d o cy to sis a n d few er specialized cells capable of phagocytosis, s u c h
a s m onocytes, m ac ro p h ag e s a n d n e u tro p h ils. U tilizing th e ir exceptional
m ig rato ry
m e c h a n ism s,
th e
n e u tro p h ils,
w hich
based
on
th e ir
m orphological fe a tu re s a re also referred to a s p o ly m o rp h o n u clear cells
(PMNs), a re u s u a lly th e first leukocytes to e n c o u n te r th e invading
o rganism .
2
In th e n o rm al non-infec.ted sta te , th e n e u tro p h ils m arg in ate from
th e c e n te r of th e co lu m n of blood to w ard s th e p e rip h e ry to sc a n for a
ch em o tactic signal, in d icatin g th e p re sen c e of in flam m atio n in th e body.
C h e m o a ttra c ta n ts
d am ag ed
tis s u e
are
im m u n o reg u lato ry
cells,
end o th elial
cells
s u b s ta n c e s ,
and
th e
derived
from
m icro o rg an ism s
th em selv es, w hich c a n in d u ce im m u n e cells to m ig rate along th e ir
g rad ie n ts. If d u rin g m arg in atio n n e u tro p h ils d e te ct th e c h e m o a ttra c ta n t,
th ey
s ta r t rolling by tem porarily b in d in g to
e n d o th e liu m
th ro u g h
a d h e sio n m olecules, s u c h a s selectins. The binding of c h e m o a ttra c ta n t
to its rec e p to rs on th e su rface of n e u tro p h ils, re s u lts in in c re a se d affinity
of n e u tro p h il in te g rin s for th e en d o th elial receptors. T h is lead s to a rre s t
of n e u tro p h ils a n d c o n se q u e n t tig h t a d h eren c e to e n d o th e liu m followed
by tra n s m ig ra tio n betw een th e en d o th elial cells.
squeeze
th ro u g h
followed
by
n e u tro p h ils
in te rce llu lar ju n c tio n s
cytoplasm ic
c o n tin u e
to
stream in g .
m igrate
by
O ut
th ro u g h
e x ten d in g
of th e
th e
following th e g ra d ie n t of c h e m o a ttra c ta n ts.
T he n e u tro p h ils
p se u d o p o d s
circu latio n ,
e x tra ce llu lar
th e
m atrix
O nce th e n e u tro p h ils
physically e n c o u n te r th e b acteria, th e y begin th e ph ag o cy to sis p ro cess
followed b y th e form ation of phagolysosom e a n d d e g ra n u la tio n , d u rin g
w hich th e g ra n u le s fu se w ith th e phagocytic vesicle a n d release th e ir
c o n te n ts.
T he
p rim a ry
(azurophilic)
g ra n u le s
c o n ta in
m ainly
3
m yeloperoxidase,
e la sta se ,
lysozym e
and
d efen sin s,
th e
seco n d ary
(specific) g ra n u le s also c o n ta in lysozym e, in ad d itio n to collagenase a n d
lactoferrin.
T he d e g ra n u la tio n is acco m p an ied by e n h a n c e d oxidative
m etab o lism , w hich re s u lts from NADPH oxidase re d u c tio n of oxygen to
su p ero x id e an io n . T his rad ic al u ltim ately is tra n sfo rm e d into a variety of
toxic oxygen species, s u c h a s hydrogen peroxide, hydroxyl rad ic als a n d
sin g let oxygen.
T hese oxygen -d ep en d en t toxic species, along w ith th e
o x y g e n -in d ep en d en t b actericid al c o n te n t of g ran u le s, re n d e r n e u tro p h ils
very effective in m icrobial killing.
F orm yl P ep tid e R ecep tor
T he different activities of n e u tro p h ils re s u lt from th e in terp lay
betw een cell su rface rec ep to rs a n d th e signaling m olecules, called
ligands.
T he recep to rs, a ctin g a s tra n s d u c e rs , relay th e signal a c ro ss
p la s m a m e m b ra n e a n d m ed iate in tra c e llu la r re sp o n se s lead in g to specific
cell
activity,
such
as
chem otaxis.
The
c h em o tactic
abilities
of
n e u tro p h ils have b een observed a s early a s th e tu rn of tw e n tieth c en tu ry ,
how ever th e evidence for c h e m o a ttra c ta n t recep to rs on n e u tro p h ils w as
n o t provided
s u p e r n a ta n ts
u n til
from
th e
1970s.
c u ltu re s
S chiffm ann
of
Escherichia
et a l
coli
observed
had
th a t
p o te n t
c h e m o a ttra c ta n t effect on ra b b it in tra p e rito n e a l n e u tro p h ils (54).
T his
4
o b serv atio n led to th e p ro d u ctio n of sh o rt form ylated p e p tid e s a n d th e
a n aly sis of th e ir fu n ctio n s a s c h e m o a ttra c ta n ts (58).
T he m o st p o te n t
c h e m o a ttra c ta n t form ylated peptide sy n th esized w as form yl-m ethionineleu cin e -p h en y la la n in e
(/MLF).
Interestingly, th e
sa m e peptide w as
iso lated from E. coli c u ltu re filtrates by M arasco et ah (34). T hrough th e
b in d in g of rad io lab eled JMLF, it h a s b een show n th a t n e u tro p h ils have
specific rec e p to rs for th e form ylated p e p tid es (65).
In su b s e q u e n t
stu d ie s, it h a s b een d e term in ed th a t, in ad d itio n to chem otaxis, th e
form yl p ep tid e re c ep to r (FPR) is resp o n sib le for m a n y o th e r n e u tro p h il
fu n ctio n s a sso c ia te d w ith h o s t p ro tectio n from invading b a cteria. T hese
fu n ctio n s in clu d e lysosom al d e g ra n u latio n , superoxide p ro d u ctio n , a n d
p h ag o cy to sis (59).
A lth ough o th e r c h e m o a ttra c ta n t rec ep to rs have b een identified
(C5aR, I1-8R, LTB4R), th e FPR h a s b een th e m o st stu d ied .
d e m o n s tra te d
th a t
affinity-labeled
receptor,
p a rtially
Niedel et al.
purified
from
h u m a n n e u tro p h ils, electro p h o resed a s a b ro ad b a n d w ith a n a p p a re n t
m o lecu lar w eight betw een 55,000 D a a n d 7 0 ,0 0 0 D a on so d iu m dodecyl
su lfate-p o lyacrylam ide gel, sugg esting h ete ro g en o u s glycosylation of th e
rec ep to r (41). Niedel a n d cow orkers su b se q u e n tly fu rth e r ch aracterized
th e c ellu lar a n d m o lecu lar c h a ra c te ristic s of FPR in h u m a n n e u tro p h ils.
They show ed th a t th e recep to r c a n be specifically labeled w ith b o th
5
flu o resc en t a n d
ad d itio n ,
th e y
rad io io d in ated
w ere
rad io io d in ate FPR.
th e
first
derivatives of form yl p eptides.
to
su ccessfu lly
covalently
In
affinity
T h e 'deglycosylation tre a tm e n ts of th e recep to r w ith
en d o -b eta-N -acety lg lu co sam in id ase F re su lte d in 33 k D a polypeptide
b ack b o n e, w hich re ta in e d its ligand bin d in g activity, a s rep o rted by
M alech et a t
(33).
T hey co n clu d ed th a t th e re c e p to r polypeptide
b ack b o n e c o n ta in e d a t le a st two N -Iinked oligosaccharide c h a in s th a t
w ere n o t re q u ire d for ligand binding.
T he stu d ie s of th e effect of n u c le o tid e s on th e b in d in g of form ylated
p e p tid es to FPR on n e u tro p h il m e m b ra n e s led to th e id entification of two
affinity s ta te s exhibited by th e rec ep to r (31). The a d d itio n of GTP a n d its
n onhydrolyzable derivative, g u anylylim idodiphosphate, to th e m em b ran e
p re p a ra tio n s, re s u lte d in th e conversion of high-affinity sta te to lowaffinity s ta te of th e receptor.
T h ro u g h th e ir detailed b in d in g stu d ie s of
oligopeptide c h e m o a ttra c ta n t rec ep to r on g u in ea pig m ac ro p h ag e s a n d
m acro p h ag e
m e m b ra n e
p re p a ra tio n s,
S n y d erm an
and
cow orkers
su g g ested t h a t GTPase or a g u a n in e nucleotide reg u la to ry u n it m ay be
involved in tra n s d u c tio n m e c h a n ism s of th e recep to r (30,31). L ater, th is
n u cleo tid e reg u la to ry u n it w as referred to a s G T P-binding pro tein (G
protein).
6
The early know ledge of th e role of G p ro tein in signal tra n s d u c tio n
cam e from stu d ie s of h o rm o n e a n d n e u ro tra n s m itte r re c e p to rs a n d th e ir
reg u latio n of adenylyl cyclase (for review see (4)). GTP w a s show n to play
a n e sse n tia l role in rec ep to r b in d in g a s well a s in m ed iatin g effector
sy stem s.
S u b s e q u e n t stu d ie s led to th e identification of G p ro tein
tra n s d u c in g sy ste m s in d e p e n d e n t of cyclic AMP g en eratio n .
T hese
sy ste m s involve, am o n g o th ers, p h o to rec ep to rs (64) a n d c h e m o a ttra c ta n t
rec ep to rs, in clu d in g FPR (59). The G p ro tein classification w as initially
b a se d o n th e ir ability to e ith er stim u la te (Gs) or in h ib it (Gi) adenylyl
cyclase. Interestingly, it h a s b een d e term in ed th a t Gs c a n be activated by
c h o lera toxin (39), on th e o th e r h a n d , Gi c a n be in h ib ite d by p e rtu s s is
toxin (27).
The FP R -m ediated signal tra n s d u c tio n , chem otaxis a n d
d e g ra n u la tio n w as show n to be p e rtu s s is toxin sensitive, suggesting th a t
th e G p ro tein t h a t in te ra c ts w ith FPR is of Gi su b ty p e (3,5,32,42).
F u rth e r
c h a ra c te riz a tio n
of FPR,
as
a
G
p ro te in -d e p e n d e n t
recep to r, cam e from th e identification of its DNA seq u en c e (8).
The
iso latio n a n d seq u en cin g of cDNA clones of FPR e x p re ssed in COS-7 cells
revealed a tra n s la tio n p ro d u c t of 350 am ino acids.
T he sequence w as
d ete rm in e d to have sim ilarities w ith o th e r m em b ran e p ro te in s co n tain in g
c h a ra c te ristic h ydrophobic polypeptide stre tc h e s p ro p o sed to form seven
a lp h a helical
tra n s m e m b ra n e regions w ith a n e x tra ce llu lar am ino
7
te rm in u s
and
in tra c e llu la r
carboxyl
te rm in u s.
The
seven
tra n s m e m b ra n e m otif w as first observed in b a c terio rh o d o p sin (16).
Later, th is s tru c tu re w as show n to be c h a ra c te ristic of th e larg e st fam ily
of cell su rface re c ep to rs called G p ro tein -co u p led re c e p to rs (GPCR) (for
review
see
(53)).
n e u ro tra n s m itte rs ,
ch em o k in es.
The
light,
GPCR
fam ily
o d o ran ts,
and
in clu d e
m ore
recep to rs
rec en tly
for
describ ed
A lthough v a rio u s GPCRs resp o n d to different signaling
a g en ts, a m odel for th e com m on c h a in of events of G p ro tein -m ed iated
signal tra n s d u c tio n h a s b een d escrib ed (I). The ligand-occupied recep to r
in te ra c ts w ith a specific, h e tero trim eric G pro tein , followed by th e
exch an g e of G p ro te in -b o u n d GDP for GTP. The G T P-bound a -s u b u n it of
G p ro tein d isso c iates from (By d im er a n d activ ates a ta rg e t p ro tein leading
to th e p ro d u c tio n of seco n d m essen g ers.
T he c h a in of ev en ts following th e bind in g of th e c h e m o a ttra c ta n ts
to th e ir rec ep to rs, a lth o u g h n o t com pletely u n d e rs to o d , h a s b een
ex am in ed by v a rio u s gro u p s. M cPhail et a l d escrib ed th e events leading
to th e fu n ctio n al re sp o n se s of phagocytic cells (36). Briefly, th e bind in g
of th e lig and to th e rec ep to r in itia te s th e activation of one or m ore
p h o sp h o lip a se s,
m e sse n g e rs
w hich
such
as
in
tu r n
m ed iate
1,2-diacylglycerol,
th e
p ro d u c tio n
of second
1,2-diradylglycerol,
inositol
trip h o s p h a te , p h o sp h a tid ic acid, ara ch id o n ic acid, a n d calcium ions.
8
T h ese in tra c e llu la r m esse n g e rs m ed iate th e activation of cellu lar pro tein
k in a s e s able to p h o sp h o ry la te ta rg e t p ro tein s. T h ro u g h ev en ts y et to be
defined, th e p h o sp h o iy la te d p ro tein s reg u late specific cellu la r re sp o n se s,
su c h a s ch em o tax is, d e g ra n u la tio n or NADPH oxidase assem bly.
T his
sim plified d e scrip tio n of signal tra n s d u c tio n is becom ing m ore a n d m ore
com plex w ith
co m p o n en ts.
th e
findings
of new
signaling
p a th w a y s
and
th e ir
Stoyanov et a t d e m o n stra te d th a t th e rec ep to r-a sso c iated
G p ro tein m ay a c t directly on p h o sp h o in o sitid e-3 k in a se , w hich lead s to
th e g en eratio n of p h o sp h o in o sitid e second m e sse n g e rs (61).
More
recently, B row ning et al. rep o rted th a t _/MLF stim u la te s th e activation of
n u c le a r
facto r-k ap p aB ,
w hich
is
c e n tra l
to
th e
reg u latio n
of
p ro in flam m ato ry g en e-expression (9).
U n d e rsta n d in g
FP R -m ediated
signal tra n s d u c tio n
events
and
re su ltin g n e u tro p h il fu n ctio n s is c ru c ial for developing new strateg ies to
p rev en t a n d to tre a t v a rio u s clinical d iso rd e rs c h arac terize d by ab n o rm a l
F P R -related n e u tro p h il fu n ctio n s.
N eutrophils w ith a b n o rm a l FPR-
m ed iated c h em o tactic activity have b e en identified in a p a tie n t w ith
ju v en ile p erio d o n titis (JP) (44), a d ise ase of b a cterial p a th o g e n esis, w hich
lead s to alveolar bone reso rp tio n a n d p re m a tu re to o th loss.
PMNs from
th e J P p a tie n t failed to m igrate in re sp o n se to I O 9-IO-7 M JMLF, b u t
ex h ib ited n o rm al ch em o tax is u p o n ex p o su re to C 5a.
The a u th o rs
9
c o n clu d ed th a t J P n e u tro p h ils have defective FPR, how ever th ere are
p ossibly o th e r re a s o n s for failed chem otaxis. One of th o se re a so n s could
be a m issin g co m p o n e n t of th e F P R -d ep en d en t signal tra n s d u c tio n . The
role of c h e m o a ttra c ta n t receptor, h a s also b een im p licated in rh e u m a to id
a rth ritis. In th is a u to im m u n e d isorder, th e n e u tro p h ils becom e activated
and
m ig rate to w a rd s chronically inflam ed
synovial cavities, w hich
p ossibly c o n ta in form ylated p e p tid es rele ased from in ju re d h o s t cells.
The localization of n e u tro p h ils to th e affected jo in ts lead s to th e
d e stru c tio n of cartilage a n d s u b s e q u e n t d e stru c tio n of th e jo in t (for
review see (45)).
P urpose o f th is S tu d y
In o rd er to stu d y FPR, we se a rc h e d for a n e x p re ssio n system th a t
w ould allow th e p ro d u c tio n a n d pu rificatio n of fu n ctio n al receptor. After
u n s u c c e s s fu l a tte m p ts to purify FPR u sin g a b a cu lo v iru s expression
sy stem (C h ap ter 2), we decided to u s e a m am m alia n e x p ressio n system .
In th is w ork, we describ e a m eth o d for th e isolation a n d purification of
FPR e x p re ssed in C hinese h a m s te r ovary cells (CHO) (C h ap ter 3),
The
purified re c ep to r c a n be utilized in v a rio u s a ssa y s to s tu d y th e receptor.
T he u n d e sire d effects of n e u tro p h il activation, re su ltin g in tis su e
d am ag e observed in rh e u m a to id a rth ritis, led to a se a rc h of w ays, by
10
w hich th is a ctiv atio n c a n be prevented.
b in d in g
of ligand
to
FPR,
O ne a p p ro a c h is to block th e
by designing
m im icking th e FP R -iriteracting m olecule.
a
pharm acological a g en t
In o rd er to design su c h a n
ag en t, it is u se fu l to d eterm in e p eptide seq u en ces, o th e r th a n JMLF, th a t
b in d to FPR.
H ere, we em ployed p h ag e peptide lib rary screen in g u sin g
two different m e th o d s of selection of F P R -interacting p e p tid e s (C hapter
4).
The first m eth o d involved th e u s e Of FPR -coupled CNBr-activated
S ep h a ro se 4B b e ad s. T he second m eth o d involved th e u s e of in ta c t CHO
tra n s fe c ta n ts th a t e x p re ss FPR.
O ur
seco n d
a p p ro a c h
to
o b ta in
inform ation
reg ard in g
FPR-
m ed iate d signaling involved site-d irected m u ta g e n e sis of th e receptor. In
o rd er to ex am ine th e role of FPR carboxyl tail in signal tra n s d u c tio n a n d
ch em o tax is, two cytoplasm ic deletion m u ta n ts w ere g en erated a n d
fu n ctio n ally analyzed (C hapter 5).
11
CHAPTER 2
ATTEMPTS TO PRODUCE FORMYL PEPTIDE RECEPTOR
USING BACULOVIRUS EXPRESSION SYSTEM
In tro d u ctio n
T he b a c u lo v im s ex p ressio n sy stem h a s b een w idely u s e d for large
scale p ro d u c tio n of v a rio u s p ro te in s in clu d in g m e m b ra n e receptors.
N aray an
and
cow orkers
(40) w ere
able to e x p re ss
fu n ctio n al r a t
lu tro p in / ch o rio g o n ad o tro p in rec ep to r (LH/CG-R) in Sf9 in se c t cells u sin g
th e b a cu lo v im s' e x p ressio n system .
p ro tein -co u p led rec ep to r family.
LG /C G -R is a m em b er of th e G
T he functio n ality of th e recep to r w as
d ete rm in e d by th e d etectio n of in c re a se d in tra c e llu la r c o n c e n tra tio n of
cAMP in re sp o n se to h igh affinity b in d in g of h u m a n choriogonadotropin.
B aculoyirus, also know n a s A utographa califom ica m u ltiple n u c le a r
p o ly h ed ro sis v iru s (AcMNPV), p ro d u ce s two viral form s d u rin g infection:
e x tra ce llu lar v iru s p a rticle s (ECV) a n d occlusions m ad e m ainly from th e
p o ly h ed rin pro tein . T hese o cclu sions, also called p o ly h ed fa, p ro te c t a n d
tra n s m it th e v iru s.
A viral gene encoding th e p o ly h ed rin pro tein is
n o n e sse n tia l for infection or replication. T his gene c a n be rep laced w ith
a gene of in te re s t a n d will re s u lt in a n occlusion negative v im s. T here
12
a re several ex p re ssio n vectors th a t a re designed for effective p ro tein
e x p ressio n a n d p u rificatio n u sin g th e b acu lo v iru s e x p re ssio n system .
In th is stu d y , we u s e d a n ex p ressio n vector pB lueB acffzs coding for
six h istid in e re s id u e s u p s tre a m from th e in itia to r m eth io n in e of FPR
(Fig. I). H istidine a n d try p to p h a n re s id u e s are know n for th e ir m etal ions
c h elatin g p ro p erties w hich c a n be utilized in p ro tein isolation.
The
h istid in e tagged p ro te in s c a n be s e p a ra te d from th e r e s t of th e solubilized
m e m b ra n e p ro te in s on a m etal affinity colum n.
T he im m obilized
m ateria l c a n th e n be e lu ted from th e co lu m n by e ith e r low ering th e pH or
by tre a tm e n t w ith im idazole.
The p B lu eB acfiis vector also c o n ta in s a n
e n te ro k in a se cleavage site d o w n stream from th e polyhistidine sequence.
T his cleavage site c a n be u s e d d u rin g purificatio n to rem ove th e h istid in e
tag.
In o rd er to u s e th e purified p ro tein for fu rth e r ex p erim en ts, it is
n e c e ssa ry to d ete rm in e if th e e x p ressed pro tein is pro p erly folded.
In
case of cell su rfa ce recep to rs, a p ro p er tra n s p o rt of th e m olecule to th e
p la sm a le m m a a n d a n o rm al ligand b in d in g affinity a re good in d icato rs of
p ro p er
p ro tein
folding.
The
fluorescence
a ctiv ated
cell
sc a n n e r
(FACScan) c a n be utilized to c alcu late th e d isso ciatio n c o n sta n ts of
flu o resc ein a te d form ylated p ep tid es to FPR e x p ressed o n th e su rface of
Sf9 cells.
13
BamHI FPR Hind III
pBlueBacHis
10.3 kb
Fig. I . S ch em atic figure of th e pB lueB acH is vector. T he FPR cDNA w as
in se rte d into th e BamHI-HindIII cloning site of re a d in g fram e B.
A d ap tatio n from a figure sup p lied by Invitrogen (San Diego, CA).
14
M aterials and M ethod s
C ell L in es an d A n tib o d ies
Sf9 cells a re derived from th e p u p a l ovarian tis s u e of th e fall arm y
w orm , Spodoptera frugiperda.
T hese cells double a b o u t every 24 h o u rs
a n d c a n e ith e r be grow n in m onolayers or in s u s p e n s io n in com plete
TNM-FH in se c t m ed iu m (Sigma, St. Louis, MO) su p p le m e n te d w ith h e a t
in ac tiv a te d fetal bovine seru m . They grow b e s t a t te m p e ra tu re s betw een
27 a n d 30°C.
A nti-FPR se ru m w as p ro d u ce d by im m u n izin g a ra b b it
w ith a p ep tid e encoding th e carboxyl-term inal 14 am in o acid s of FPR
co u p led to Keyhole lim p et hem ocyanin. The anti-F P R a n tise ru m w as
fu rth e r p u rified u s in g th e carb o x y -term in al peptide c o u p led to cyanogen
b ro m id e-activ ated S e p h a ro se beads.
P ro d u ctio n o f R eco m b in a n t
B acu loviru s
Polym erase c h a in reactio n (PCR) w as perform ed to am plify th e FPR
cDNA (kindly provided by Dr. R ichard Ye, S cripps R e se arch In stitu te , La
Jo lla, CA; 47).
The p rim e rs w ere c o n stru c te d w ith o verhanging BamHI
a n d HindIII re stric tio n sites. The FPR cDNA seq u en ce w a s th e n in se rte d
in to th e tra n s fe r vector, pB lueB acH isB (Invitrogen, S a n Diego, CA)
lin earized a t Bam HI a n d H indlll sites. T his vector allow ed th e FPR in s e rt
15
to be ligated in fram e.
The pBlueBacH zsB w ith th e in s e rt w as th e n
tra n sfe c te d into Sf9 cells along w ith com m ercially a cq u ired lin e ar
AcMNPV DNA u sin g th e cationic liposom e tec h n iq u e (18).
T hrough
hom ologous reco m b in atio n , th e FPR seq u en ce w as in se rte d into th e viral
DNA.
R eco m b in a n t Plaque P u rification
To o b ta in p laq u e s, th e Sf9 cells w ere p lated to cover a b o u t 50% of
th e p late
su rface • a n d th e re c o m b in a n t b acu lo v iru s w as ad d ed a t
m u ltip licity of infection (MOI) 5.
After a sufficient a m o u n t of tim e
allow ed for infection (approxim ately I h), th e cells w ere overlayed w ith
agarose.
In 5-7 d ay s th e p laq u e s s ta rte d to a p p ea r.
T he ad d itio n of
chrom ogenic dye 5-brom o-4-chloryl-3-indolyl-p-D -galactopyranoside (Xgal) to th e p la te s p ro d u ce d a blu e color a ro u n d th e p la q u e s in dicating
su c ce ssfu l infection
by th e
re c o m b in a n t v iru s w h ich
allowed th e
p ro d u c tio n of p-galactosidase by th e cells. The selected p laq u e lifts w ere
u s e d ,to infect fresh cells a n d th e e x p ressio n of FPR w a s d eterm in ed by
im m u n o flu o rescen ce.
R eco m b in an t b acu lo v iru s t h a t re s u lte d in th e
h ig h e st ex p re ssio n level of FPR w as p ro p ag a ted for fu rth e r experim ents.
16
Im m u n o flu o r esce n c e S ta in in g o f
FPR E x p ressed in S£9 In se c t C ells
The infected- cells w ere p lated on coverslips tre a te d w ith poly-Lly sin e. T hey w ere allow ed to a tta c h for approxim ately 30 m in a n d fixed
for 2 h in 2.5% p arafo rm ald eh y d e in p h o sp h a te buffered saline so lu tio n
(PBS).
Cells w ere perm eabilized w ith 0.01% sa p o n in a n d non-specific
b in d in g w a s blocked by 0.2% gelatin.
in c u b a te d
first w ith
anti-F P R
P rep ared in th is w ay cells w ere
an tib o d y a n d
th e n
w ith fluorescein
iso th io c y an a te (FITC) c o n ju g ated goat a n ti-ra b b it antibody.
C ell-bound
flu o rescen ce w as observed w ith a fluorescence m icroscope.
S od iu m D o d ecy l S u lfateP o ly a cry la m id e Gel
E le c tro p h o r esis (SDS-PAGE)
and W estern B lot A n alysis
T he Sf9 cells w ere infected w ith th e re c o m b in a n t b acu lo v iru s a n d I
m l a liq u o ts w ere rem oved a t different tim e p oints. The cells w ere pelleted
a n d re s u s p e n d e d in 20 mM PBS. After 4 cycles of freeze-thaw tre a tm e n t,
th e in so lu b le p ro te in s w ere pelleted by m icro cen trifu g atio n (14,000 rpm ,
10 m in). B oth, th e pellets a n d th e s u p e rn a ta n ts w ere re s u sp e n d e d in
Laem m li buffer a n d loaded onto a 10% polyacrylam ide gel.
After th e
com pletion of th e electro p h o resis, th e p ro tein s w ere tra n s fe rre d onto a
n itro cellu lo se filter. T he filter w as blocked w ith B lotto Tw een (5% n o n -fat
17
m ilk , 0.2% Tw een 20 in PBS pH 7.4), in c u b a te d in d ilu tin g buffer (3%
n o rm al g o at se ru m , 1% BSA, 0.2% Tw een 20 in PBS pH 7.4) w ith an tiFPR polyclonal an tib o d y (1:200) o vernight a t 4°C, w a sh e d w ith w a sh
buffer (250 mM NaCl, 10 mM HEPES pH 7.4, 0.2% T w een 20 in dH 20),
in c u b a te d w ith a n alk alin e p h o sp h a ta s e co n ju g ated se c o n d a ry antib o d y
(1:2000), I h a t room te m p e ra tu re , w a sh e d a n d developed u sin g th e 5B rom o-4-chloro-3-indolyl p h o s p h a te / N itroblue tetraz o liu m (BCIP/NBT)
k it acco rd in g to m a n u fa c tu re r’s rec o m m e n d atio n s (K irkegaard & Perry
L ab o rato ries, G a ith e rsb u rg , MD).
P reparation o f S f9 M em branes
and E x tra ctio n o f FPR.
Sf9 cells w ere infected for 48 h o u rs a n d rin se d in H a n k s ’ B alanced
S a lts so lu tio n (Sigma, St. Louis, MO).
T hey w ere th e n re s u sp e n d e d in
Relax (+) (10 mM HEPES pH 7.4, 100 mM KC1, 10 mM NaCl, 3.5 Mm
MgCl2 a n d I mM ATP) a n d a p re s s u re of 400 p si w a s applied for 15
m in u te s to lyse th e cells.
The cavitate w as cen trifu g ed a t slow speed
(900 x g, 15 min) to rem ove cell n u clei a n d som e organelles.
The
rem a in in g s u p e r n a n ta n t w as centrifuged a t (228,000 x g, 30 m in), to
pellet cell m em b ra n e s.
A sim ilar m eth o d for p re p a ra tio n of n e u tro p h ils
a n d fib ro b la st m e m b ra n e s w as d escrib ed previously by S ch reib er a n d
cow orkers (56). T he m e m b ra n e s w ere re s u sp e n d e d in p h o sp h a te buffer
18
(20 mM, pH. 7.8) a n d I mM phenylm ethylsulfonyl fluoride (PMSF) w ith
a d d itio n of e ith e r 60 mM octylglucoside (OG) or 1% T rito n X -100 (TX100),
in
th e
p re sen c e
or
in
th e
a b sen c e
of
IM
NaCL
After
u ltra c e n trifu g a tio n (900 x g, 5 m in u tes), th e o b tain ed pellet (insoluble
proteins) a n d s u p e rn a ta n t (containing e x tracted FPR) w ere su b jected to
SDS-PAGE a n d W estern blo t analysis.
FACScan A n a ly sis o f Form yl
P ep tid e B in d in g to FPR
E x p ressed in S£9 C ells
T he cells w ere infected (MOI 5) for 48 h o u rs.
After w ashing, th e
cells w ere re s u s p e n d e d in PBS co n ta in in g C a2+, Mg2+, a n d 5% FBS
followed by in c u b a tio n (40 m in on ice w ith o ccasio n al mixing) w ith
different d ilu tio n s of /NleLFNleYK-fluorescein derivative (fluoresceinlabeled an alo g of yMLF) a n d FA CScan analysis. To m e a s u re unspecific
binding, p ara lle l tu b e s w ere in c u b a te d w ith a 1000-fold excess of ^MLF.
Specific b in d in g w as calcu late d a s to ta l bind in g s u b tra c te d by binding in
th e p re se n c e of _/MLF. The disso ciatio n c o n s ta n t (Kd) w a s d eterm in ed by
le a st s q u a re s a n aly sis of th e m e a n fluorescence u s in g th e G rap h P ad
P rism softw are.
19
R e su lts
Im m u n o flu o r esce n c e o f FPR
Fixed a n d perm eabilized Sf9 cells w ere tre a te d w ith anti-FP R
an tib o d y followed by FITC co n ju g ated seco n d ary antibody. T here w as a
visible
difference
betw een
e x p re ssin g FPR (Fig.2).
th e
u n in fec te d
Sf9
cells
and
th e
cells
The labeled FPR seem ed to be evenly d istrib u te d
th ro u g h o u t th e cells, w hich m ade it difficult to d eterm in e if th e ex p ressed
rec ep to r w as properly in se rte d into th e m e m b ra n e s of Sf9 cell.
T im e C ourse for M axim al
E x p ressio n o f FPR in S f9 C ells
Infected
Sf9
cells
w ere
lysed
and
th e
re s u ltin g ' solubilized
(su p e rn a ta n t) a n d n o t solubilized (pellet) p ro tein s w ere su b je cted to gel
electro p h o resis.
W estern blo t w as perform ed in o rd e r to determ in e a
tim e p o in t a t w hich th e Sf9 cells p ro d u ce th e m o st FPR.
In ad d itio n to
th e b a n d m ig ratin g a ro u n d th e expected 69 kD a, th e re w a s also a b a n d
a p p e a rin g j u s t above th e 28 kD a m o lecu lar w eight s ta n d a rd (Fig.3).
band
w ith
a
sim ilar
relative
m obility
w as
re p o rte d
earlier
A
by
Q u e h en b e rg er a n d h is cow orkers (49) a s a n ungly co sy lated form of FPR.
T his b a n d w as n o t p re s e n t in th e negative control (uninfected cells) (not
B
Fig. 2. Im m u n o flu o rescen ce a n aly sis of FPR e x p ressed in Sf9 cells. A.
U ninfected Sf9 cells (control).
B.
Cells infected w ith rec o m b in a n t
b acu lo v iru s. U ninfected a n d infected cells were tre a te d identically; th e
cells w ere fixed a n d perm eabilized, in c u b a te d w ith a n a n tib o d y a g a in st
FPR a n d a se co n d ary fluo rescein -co n ju g ated antibody. The difference in
th e b a c k g ro u n d s of th e two p h o to g ra p h s is du e to th e e x p o su re tim e (20
s for A a n d 2 s for B) being c o m p e n sa te d for by th e c am era , i.e., if th e
b a c k g ro u n d in A w ould be a s d a rk a s in B , th e sta in in g of th e u n in fected
cells w ould be b arely visible.
21
kDa
s
I
2
3
4
5
105
6
7
8 9
s 10 1 1 1 2 13 14 15 16 17 18 s
v
69
43
28
*
/S I
Fig. 3.
SDS-PAGE a n d W estern blot of Sf9 cells infected w ith
re c o m b in a n t b acu lo v iru s (His-FPR). The cells w ere infected for 18 h
(lanes I a n d 10), 24 h (lanes 2 a n d 11), 30 h (lanes 3 a n d 12), 42 h (lanes
4 a n d 13), 45 h (lanes 5 a n d 14), 49 h (lanes 6 a n d 15), 53 h (lanes 7 a n d
16), 67 h (lanes 8 a n d 17) a n d 73 h (lanes 9 a n d 18) a n d lysed by freezeth aw tre a tm e n t. The ly sa te s w ere re su sp e n d e d in 20 mM PBS a n d
sed im en ted . L anes 1-9 re p re se n t pellets after ex tractio n a n d lan es 10-18
re p re se n t e x tra ctio n s u p e rn a ta n ts . The top arrow p o in ts tow ards th e
p u tativ e glycosylated form of FPR. The bottom arrow p o in ts tow ards th e
p u tativ e u nglycosylated form of FPR. B ased on th e blot, th e two form s of
FPR are b e s t e x p re ssed a t 67 h o u rs p o st infection (lane 8). s = m o lecular
w eight sta n d a rd .
22
kDa
s
I
2
s 3 4 5 6 7 s 8
9
10 11
12
s
105
69
43
28
I S .j U
.
Fig. 4. SDS-PAGE a n d W estern blot of m em b ran e p re p a ra tio n (mp) of
Sf9 cells infected w ith re c o m b in a n t bacu lo v iru s, after ex tractio n u n d e r
v ario u s co nditions: lane I - m p low speed pellet, lan e 2 - m p high speed
s u p e rn a ta n t, la n e s 3 a n d 8 - buffer su sp e n sio n of cell m e m b ra n e s (high
speed pellet), la n e s 4 a n d 9 - ex tractio n w ith 1% TX -100, la n e s 5 a n d 10
- ex tra ctio n w ith 1% TX-100 a n d IM NaCl, lan e s 6 a n d 11 - ex traction
w ith 30 mM OG a n d la n e s 7 a n d 12 - ex tractio n w ith 30 mM OG a n d I
M NaCL
L anes 3-7 re p re se n t pellets after e x tractio n , lan es 8-12
re p re se n t ex tra ctio n s u p e rn a ta n ts . The top arrow in d ic a te s th e p u tativ e
glycosylated form of FPR. The bo tto m arrow in d ic a te s th e putative
u n g ly co sy lated form of FPR. B ased on th e blot, th e FPR a p p ea re d m ostly
in th e final pellet (lanes 3-7) in d icatin g u n su c c e ssfu l ex tractio n from th e
m e m b ra n e s of Sf9 cells, s = m o lecu lar w eight s ta n d a rd .
23
show n).
T he m axim al p ro tein ex p ressio n w as e sta b lis h e d to o ccu r
betw een 4 5 -6 7 h o u rs p.i.
E x tra ctio n o f FPR from th e
M em branes o f S£9 C ells
It is n e c e ssa ry to be able to e x tra c t th e FPR from th e m e m b ra n e s of
th e Sf9 cells before it c a n be purified a n d u s e d in fu rth e r experim ents.
The following ex p erim en t w as c arried o u t to exam ine th e solubility
p ro p erties of FPR ex p re ssed in in se c t cells. The Sf9 cells w ere infected
for 48 h a n d th e m e m b ra n e s w ere p re p a re d by Na cavitation.
V arious
FPR e x tra ctio n co n d itio n s w ere c arried o u t in o rd er to determ in e th e
m o st effective one.
W estern blot of th e e x tra c t show ed a b a n d slightly
below th e 69 kD a m a rk e r (Fig. 4), w hich c o rre sp o n d s to th e glycosylated
form of th e receptor.
However, it a p p e a re d m ostly in th e final pellet,
w hich in d ic ate d th a t th e recep to r w as n o t su ccessfu lly e x tra cted from th e
m e m b ra n e s of th e in se c t cells.
FACScan R e su lts
T he FA CScan re s u lts in d icated th a t th e b in d in g affinity of FPR
ex p re ssed in Sf9 cells w ith Kd of 70 nM w as significantly lower th a n th e
b in d in g affinity of FPR ex p ressed in n e u tro p h ils (Kd=2.7-40 nM) (48).
Several fac to rs could have c o n trib u te d to th e low b in d in g affinity.
The
24
difference in p o sttra n s la tio n a l m odification in in se c t cells a s co m p ared to
n e u tro p h ils, a s well a s im p ro p er folding of FPR in th o se cells, could have
led to a lte re d glycosylatio n a n d lower b in d in g affinity. Finally, Fay et al.
(17) have show n th a t th e u n c o u p lin g o f th e rec ep to r from G pro tein
re s u lts in two o rd e rs of m ag n itu d e d e crea se in ligind b in d in g affinity a s
co m p ared to th e b in d in g affinity of th e recep to r a sso c ia te d w ith G
p ro tein . It h a s b e en also rep o rted (50) th a t th e Gi p ro te in s a re a b s e n t in
th e Sf9 cells. T his could possibly lead to a lower ligand b in d in g affinity of
th e FPR ex p re ssed in Sf9 cells, w hich we a n d o th e rs (50) have observed,
D isc u ssio n
T he b a cu lo v iru s ex p ressio n sy stem h a s b een effectively u se d for
th e
p ro d u c tio n
of large a m o u n ts
of m am m alia n
p ro tein s.
M any
re s e a rc h e rs re p o rt su c ce ssfu l ex p ressio n a n d s u b s e q u e n t ex tractio n of
v a rio u s
m e m b ra n e
p ro tein s
such
as
th e
rat
olfactory
seven-
tra n s m e m b ra n e -d o m a in recep to r (21) a n d th e h u m a n ep id erm al grow th
factor re c ep to r (23). A lthough, we have observed th e e x p ressio n of FPR
in Sf9 cells, we w ere n o t able to effectively e x tra ct th e re c e p to r from th o se
cells.
It h a s b e en previously show n by Klotz a n d J e s a itis (29) th a t th e
energy d epletion of h u m a n n e u tro p h ils d ecrea se d th e a sso c iatio n of FPR
w ith th e s tru c tu re s u n d erly in g th e p la sm a m e m b ran e possibly m ak in g
25
th e FPR ex tra ctio n easier.
In th e ir w ork th e d epletion of ATP w as
achieved by th e p re tre a tm e n t of n e u tro p h ils w ith NaF p rio r to m em b ran e
p re p a ra tio n .
P e rh a p s p re tre a tin g th e Sf9 cells w ith N aF w ould allow
m ore effective e x tra ctio n of FPR. A nother m ethod, sh o w n by Iizuka a n d
Fukuda
to
be
effective in
th e
ex tractio n
of th e
bovine nicotinic
acetylcholine rec e p to r a -s u b u n it from b a cu lo v iru s infected Sf9 cells, w as
th e e x tra ctio n w ith Z w ittergent (25). T his m eth o d co u ld be investigated
w ith FPR extraction.
T he FA CScan a n a ly sis revealed th a t th e FPR e x p re ssed in Sf9 cells
did n o t b in d ligand efficiently.
It h a s b een show n th a t large com plex-
type o lig o saacch arid es, w hich are p ro d u ce d by v e rte b ra te cells, a re
a b s e n t in in se c ts cells (24).
T his could have lea d to th e altered
glycosylation of th e rec ep to r a n d c o n se q u e n t low er ligand bin d in g
affinity.
A p ossible m isfolding of th e FPR ex p ressed in Sf9 cells could
have also c o n trib u te d to th e altere d disso ciatio n c o n s ta n t of th e receptor.
F u rth e rm o re , th e a b se n c e of Gi p ro te in s in th e Sf9 cells, a s rep o rted by
Q u eh en b e rg er et a t (50), c a n lead to a single low b in d in g affinity of th e
rec ep to r a s opposed to G p ro te in -d e p e n d e n t low a n d h igh b in d in g
affinities of ^MLF observed in n e u tro p h ils (30).
26
D ue to p ro b lem s in purificatio n of His-FPR from Sf9 cells a s well a s
a ltere d lig an d b in d in g we decided to investigate th e possibility of FPR
p ro d u c tio n in a m am m a lia n ex p ressio n system .
27
CHAPTER 3
PURIFICATION OF FORMYL PEPTIDE RECEPTOR
EXPRESSED IN CHINESE HAMSTER OVARY CELLS
In tro d u ctio n
E arly d evelopm ents in te c h n iq u e s of m am m alia n tis su e c u ltu re
a n d s u b s e q u e n t cell c u ltu re d a te b a c k to th e b eg in n in g of th e 2 0 th
c en tu ry .
S an fo rd et a t (52) investig ated th e p ossibility of obtaining a
p u re , C3H m o u se L cell c u ltu re sta rtin g w ith j u s t a single cell. They w ere
able to isolate single cells from a m ixed p o p u latio n of cells, th a t
o rig in ated from connective tis su e ex p lan t, a n d s u p p o rt th e ir grow th a n d
proliferation.
Since th e n , m am m alia n cells have b e en u s e d in m an y
re s e a rc h a p p lica tio n s s u c h a s vaccine p ro d u ctio n , v iral propagation,
h y b rid o m a
g en eratio n ,
as
well
as
th e
p ro d u c tio n
of
desirable
tra n s fo rm a n ts .
It h a s b e en previously rep o rte d th a t FPR c a n be functionally
ex p re ssed in tra n sfe c te d m am m alia n cell lines (47).
In o u r fu rth e r
ex p erim en ts, we have u s e d C hinese h a m s te r ovary (CHO) cells, w hich
have b e en u s e d extensively for th e ex p ressio n of re c o m b in a n t p roteins.
T he CHO cell line e x p ressin g FPR w as g en erated in o u r lab o ra to ry by Dr.
I
28
M iettin en .
T he ligand b in d in g of FPR e x p ressed by CHO cells w as
d e te rm in e d by flow cytom etry.
d isso ciatio n
c o n s ta n t
The CHO-FPR tra n s fe c ta n ts exhibited a
(Kd) of approxim ately
4
nM
for binding
of
/NleLFNleYK-fluorescein (M iettinen, u n p u b lish ed ), w h ich is co m parable
to th e h ig h affinity Kd of n e u tro p h il FPR of 1-3 nM , a s rep o rted by
P ro ssn itz et al. (48).
M aterials and M ethod s
C ell L in es an d A n tib o d ies
T he h u m a n FPR cDNA seq u en ce w as ligated in to pBGSA p lasm id
(62) a n d tra n sfo rm e d into E. coli Top 10. A p lasm id c o n ta in in g th e in s e rt
in th e p ro p e r o rien tatio n , a s verified by re stric tio n m apping, w as
tra n sfe c te d into CHO-wild type (WT) cells. G eneticin (G418) re sistan c e,
,
con ferred by th e p lasm id , w as em ployed in th e selection of cell clones.
CHO tra n s fe c ta n ts e x p ressin g FPR w ere grow n in m onolayers in a m odified E agle’s m ed iu m (Sigma, St. Louis, MO) su p p le m e n te d w ith 5%
FB S, 50 U /m l penicillin a n d 50 p g /m l strep to m y cin .
m a in ta in e d w ith 0.5 m g /m l G eneticin (G418).
They w ere
To in d u c e in cre ased
e x p ressio n of FPR, 6 mM so d iu m b u ty ra te w as ad d ed to th e m edium 1624 h o u rs before e a c h ex p erim en t (22).
Anti-FITC a n tis e ru m w as
29
p ro d u ce d by in jecting ra b b its w ith fluorescein linked to keyhole lim pet
h em o cy an in . T he a n tis e ru m w as fu rth e r purified on anti-FIT C colum n.
P reparation o f CHO-FPR
M em branes an d E x tra ctio n o f FPR
N ear co n flu e n t cells grow n on 15 cm tis su e c u ltu re p lates were
placed o n ice a n d rin se d 3 tim es w ith cold PBS before th e ad d itio n of 100
nM
^M B enzoylphenylalanineFY K -A uorescein
p ep tid e
(/MBpaFYK-
fluorescein, a g e n ero u s gift from Dr. Mills). T his photoaffinity analog of
^MLF w as u s e d to label FPR a n d to p e rm it th e d etectio n a n d th e
p u rificatio n of th e receptor. The cells w ere in c u b a te d for 10 m in on ice
a n d su b je c te d to UV light for 15 m in to allow for photo cro sslin k in g ,
d u rin g w h ich a covalent b o n d w as form ed betw een th e rec ep to r a n d th e
peptide. After th e crosslinking, th e cells w ere in c u b a te d for 10 m in w ith
a d d itio n of 10 pM JMLF, to com pete off th e non-specifically b o u n d ligand
a n d lig an d t h a t w as n o t cro sslin k ed , I p g /m l le u p e p tin e a n d 200 pM
DTT.
Next, th e cells w ere rem oved from th e p la te s by scrap in g a n d
sed im e n te d by c en trifu g atio n (100,000 x g, 30 m in, 4°C).
The pellets
w ere re s u s p e n d e d in I m l of h igh sa lt buffer (0.8 M N aC l, 50 mM Na 2 COs
pH 10-11, 200 pM DTT, I p g /m l leupeptine), so n ic ated a n d su b jected to
a n o th e r h ig h sp e ed sp in (100,000 x g, 30 m in, W ) . T his p ro ced u re w as
re p e a te d a n d th e new pellets w ere re s u sp e n d e d in PBS c o n tain in g 200
30
p,M DTT a n d I j-ig/ml leu p ep tin e, so n icated a n d cen trifu g ed (100,000 x g,
30 m in, 4°C). T he re su ltin g pellet w as sto re d a t - 2 O0C for la te r u se.
O cty l G lu cosid e E x tra ctio n o f
FPR a n d P u rifica tio n b y H igh
P erform ance Liquid
C hrom atography (HPLC)
'
T he m e m b ra n e pellets w ere re s u sp e n d e d in 5 mM H EPES, pH 7.4,
3% OG, 2 0 0 pM DTT a n d I p g /m l leu p ep tin e, so n ic ated a n d sed im en ted
(100,000 x g, 30 m in, 4°C). The s u p e rn a ta n t c o n ta in in g solubilized
m e m b ra n e e x tra c t w as su b jected to HPLC by ru n n in g th ro u g h a n io n
exch an g e c o lu m n VYDA C-300U H P575P followed by gel filtration colum n
TSK G 3000SW . The buffers u s e d were: Buffer A; 5 mM H EPES, pH 7,4,
1% OG, 0 .02 % NaNz a n d Buffer B; 2 M a m m o n iu m ace ta te .
The r u n
w as c arrie d o u t a t a 97:3 ratio of Buffer A a n d B uffer B. Total p ro tein
w as m o n ito red by observing em ission in te n sity a t 340 n m w ith excitation
a t 280 nm . The fluorescence w as m o n ito red by ex citatio n a t 490 n m a n d
em issio n a t 520 nm . T he sam p les w ere collected in 30 seco n d fractio n s
(beginning w ith
th e
sam p les
show ing in cre ased
fluorescence)
and
analyzed on a H uorim eter to d eterm in e th e ratio of try p to p h a n (by
ex citatio n a t 280 n m a n d em ission a t 340 nm) to fluorescein (by
ex citatio n a t 4 9 0 n m a n d em ission a t 520 nm) in d ic atin g th e p u rity of
e x tra cted FPR.
31
W estern B lot an d S ilver S ta in
A n a ly ses o f HPLC F ra ctio n s
The
HPLC
fractio n s
w ere
c o n c e n tra te d
u s in g
centricom
(A m icon, Beverly, MA) a n d 4x Laem m li sam ple buffer w a s added.
30
Two
id en tical se ts of sam p le s w ere r u n on a 10% S D S-polyaciylam ide gel.
O ne se t w a s tra n s fe rre d to nitrocellulose a n d W estern an aly sis w as
perfo rm ed a s d escrib ed before (C hapter 2) except for th e p rim ary
a n tise ra , w hich in th is ex p erim en t w as an ti-flu o re sc ein a n tise ru m
(1:200). T he seco n d se t of sa m p le s w as u s e d for th e silver s ta in analysis.
The gel w a s fixed for I h in 50% MeOH a n d 12 % acetic acid, w a sh ed 3
tim es (20 min) w ith 50% E tO H , so ak ed in th io su lfate so lu tio n (0.2 g/1) for
I m in, w a sh e d 3 tim es (30 s) w ith dH 20 , soaked in silver solution (2 g/1
silver n itra te , I m l/1 form aldehyde) a n d w a sh ed 2 tim es (20 s) w ith dH 20 .
Next, th e gel w as developed u n til b a n d s becam e visible (2-3 min) a n d th e
rea ctio n w a s sto p p e d w ith C oom assie d e sta in (25% isopropanol, 10%
acetic acid ).
32
R e su lts
HPLC P u rifica tio n o f
JM BpaFY K -fluorescein
P h o to c r o sslin k e d FPR from
T r a n sfected CHO C ells
In o rd er to purify FPR5 th e CHO-FPR cells w ere in c u b a te d w ith a
flu o rescein -co n ju g ated photoaffinity an alo g u e of JMLF. T he crosslinking
of th e lig and to th e rec ep to r w as in d u c e d w ith UV light a n d th e
p h o to cro sslin k e d FPR w as ex tra cted from th e iso lated m e m b ra n e s w ith
OG.
In o rd er to isolate th e FPR from cell ex tract, a n a n io n exchange
followed by a gel filtration, in a n HPLC system , w ere utilized.
M ost
solubilized m e m b ra n e p ro tein s w ere re ta in e d on th e a n io n exchange
co lu m n , allow ing th e positively ch arg ed p ro tein s, in clu d in g FPR, to flow
th ro u g h .
In th e ta n d e m gel filtration co lu m n th e e lu te d p ro tein s w ere
.
th e n se p a ra te d by size.
A ccording to th e differential d istrib u tio n s of
flu o rescein a n d try p to p h a n (Fig. 5A), th e fractio n s c o n ta in in g FPR w ere
e lu ted from th e co lu m n j u s t before th e m ajo r p ro tein p e a k (Fig. SB). The
seco n d HPLC ru n , c arried o u t w ith fluorescein p e a k frac tio n s of th e first
ta n d e m a n io n exchange, gel filtration ru n , revealed only one p e ak of to ta l
p ro tein w h ich c o rre sp o n d e d to th e fluorescence p e a k (Fig. 6). To o b tain
co n tro l (non-FPR) fractio n s, u s e d for la te r ex p erim en ts, th e WT cell
m e m b ra n e s w ere also
su b jected to two HPLC r u n s
a n d fractio n s
33
A
52.7
Fig. 5. HPLC of p h o to cro sslin k ed FPR ex tracted from CHO cells. OG
m e m b ran e e x tra c t w as purified on a n io n exchange co lu m n followed by
gel filtratio n colum n. A. F luorescein fluorescence in th e elution profile
identifies fluorescein-labeled m olecules, p resu m ab ly m ostly FPR.
B.
T ry p to p h a n fluorescence in th e e lu tio n profile identifies to tal protein.
The n u m b e rs in d icate th e tim es (min) of e lu tio n s of th e m ark e d peak s.
34
A
19.0
A
B
19.0
Fig. 6. Second step in HPLC purificatio n of FPR. T he fractio n s e lu ted
from th e first HPLC ru n co rresp o n d in g to th e fluorescein fluorescence
p eak w ere su b je cted to a second HPLC ru n . A. F lu o rescein fluorescence.
B. T ry p to p h a n fluorescence. B ased on th e differential d istrib u tio n of
flu o rescein a n d try p to p h a n , to tal p ro tein fractio n s c o n ta in m ostly
flu o rescein -labeled FPR. The n u m b e rs in dicate elu tio n tim es (min) of th e
m ark e d p eak s.
35
A
43.0
Fig. 7. HPLC of m e m b ran e e x tra c ts of CHO-WT cells. A. F luorescein
fluorescence. B. T ry p to p h an fluorescence. The differential d istrib u tio n
of flu o rescein a n d try p to p h a n in d icate th a t th e fluorescein fluorescence
is a sso c ia te d w ith th e to tal pro tein fraction. The n u m b e rs indicate th e
tim es (min) of e lu tio n s of th e m ark ed p eak s.
36
A
kDa
s
1
b
2
3
4
5
6
7
8
9
b
s
4
5
6
7
8
9
s
215
105
69
B
k Da
s
b
1 2 3
Fig. 8. SDS-PAGE of FPR e x tracted from CHO cells a n d purified by
HPLC, a s d escrib e d in th e text. Lane n u m b e rs c o rre sp o n d to th e o rd er of
th e collected fractions.
A. W estern blot u sin g anti-FITC antib o d y
followed by alk alin e p h o sp h a ta se -c o n ju g a te d se co n d ary antibody. B.
Silver sta in . A bbreviations: s = m o lecu lar w eight s ta n d a rd , b = b la n k
lane.
37
co rre sp o n d in g to th e flu o resc en t fractio n s isolated from FPR ex p ressin g
cells w ere collected (Fig. 7).
W estern B lot and
S ilv er S ta in A n a ly ses
To fu rth e r analyze w h e th e r th e flu o rescen t-lab eled HPLC fractio n s
c o n ta in e d FPR, th e selected fractio n s w ere su b jected to SDS-PAGE a n d
th e se p e ra te d p ro te in s w ere analyzed by W estern blo t a n d silver stain .
The anti-FIT C a n tis e ru m w as utilized to d e te ct th e p h o to cro sslin k e d FPR
w hich a p p e a re d a s a b ro ad b a n d ~65 k D a (Fig. 8A, la n e s 4-7) w ith th e
h ig h e st
in te n sity
c o rresp o n d in g
to
th e
fractio n s
rep re se n tin g
th e
flu o rescen ce p e a k (Fig. 6A). The silver s ta in also revealed a b a n d a t ~65
k D a in
th e
earlier fractio n s
(Fig.
SB, la n e s 4-7) w ith
no
m ajor
c o n ta m in a tin g p ro tein s. The la te r fractio n s co n ta in e d a low er m olecular
w eight p ro tein c o n ta m in a tio n (Fig. SB, la n e s 8 a n d 9).
Silver sta in
re s u lts su p p o rte d by th e im m u n o b lo t strongly in d ic ate d th a t we h a d
b een able to o b ta in p u re FPR from th e CHO cells.
D isc u ssio n
CHO cells a re frequently u s e d for ex p ressio n of h u m a n G p roteinco u p led recep to rs.
S a m a m a et al. (SI) u s e d CHO cells for ex p ressio n of
Pa-adrenergic rec ep to r a n d w ere able to o b tain cell lin es sta b ly expressing
38
w ild-type a n d m u ta n t recep to rs.
More recently, Fiore et al. (19)
ex p re ssed fu n ctio n al FPR, a s well a s FP R -related lipoxin A4 recep to r in
CHO cells.
In a d d itio n to e x p ressio n in CHO cells, fu n c tio n a l FPR h a s
b een e x p re ssed in several different cell lines, su c h a s m o n k ey COS 7 cells
(43), m o u se L cell fib ro b lasts (49), a n d h u m a n k id n ey 293 cells (15).
The stu d y of signal tra n s d u c tio n does n o t req u ire iso lated recep to rs a n d ,
in m o st c a se s, in ta c t ex p ressin g cells or th e ir m e m b ra n e p re p a ra tio n s are
su fficien t to perform th e ex p erim en ts.
Several m e th o d s of
p a rtia l
p u rificatio n of FPR from p re p a re d m e m b ra n e s h av e b een reported.
J e s a itis et al. (27) o b ta in e d p artially purified, photoaffinity labeled FPR by
se d im e n ta tio n of OG solubilized m e m b ra n e s of n e u tro p h ils on a su cro se
d e n sity g rad ien t.
S ch reib er et a t (56) im m u n o p rec ip ita te d th e OG
solubilized FPR, from th e FP R -expressing, tra n sfe c te d m o u se fibroblast
(TX2 cells) m e m b ra n e s, w ith anti-F P R antibody.
Im m u n o p recip itatio n
w as also em ployed by Q u e h en b erg er et al. (50) to iso late FPR from OG
solubilized
m e m b ra n e s
of
Sf9
cells
infected
w ith
re c o m b in a n t
b acu lo v iru s.
In th is stu d y , we u s e d CHO cells ex p ressin g FPR to p ro d u ce
recep to r. T he p h o to cro sslin k e d FPR w as su ccessfu lly e x tra cted from th e
m e m b ra n e s of CHO cells w ith OG a n d purified by HPLC. O u r W estern
b lot a n d silver s ta in a n a ly se s in d ic ate d th a t th e HPLC p e a k fluorescence
39
fractio n s .co n tain ed purified FPR.
The relative m o le cu la r w eight of FPR
e x p re ssed in CHO cells w as com p arab le to th e re p o rte d size of FPR
p artially e x tra c te d from th e m e m b ra n e s of n e u tro p h ils (41).
T his
su g g e sts th a t th e glycosylation p ro c e sse s in th e two cells lines are
sim ilar.
To o u r know ledge, th is is th e first re p o rt of FPR purification.
T he p u rified re c ep to r will be u s e d in fu rth e r experim ents.
40
CHAPTER 4
FORMYL PEPTIDE RECEPTOR SCREENING WITH
PHAGE-DISPLAY PEPTIDE LIBRARY
In tro d u ctio n
T he fu n ctio n s of FPR, a s well a s m an y o th e r recep to rs, are
m ed iated by p ro tein -p ro te in in te ra c tio n s.
T hese in te ra c tio n s involve
specific am ino acid se q u en c es d isplayed on th e in te ra c tin g molecules-.
The id en tification of th e p ro te in -p ro te in binding site s h a s been m ade
p o ssib le by th e u s e of ra n d o m p ep tid e libraries, in p a rtic u la r phaged isp lay p ep tid e lib raries.
Up to billions of u n iq u e , sh o rt peptide
se q u e n c e s c a n be displayed on th e su rface of fila m e n to u s phage.
T h ro u g h v a rio u s affinity p urification m eth o d s, ph ag e d isplaying p ep tid es
of in te re s t c a n be iso lated from th e lib rary a n d p ro p a g a te d in b acterial
cells.
T he sin g le -stra n d e d DNA (ssDNA) of F-specific bacterio p h ag es,
s u c h a s M 13, encode two m ajor c o m p o n e n ts of th e v iral coat: pro tein III
a n d p ro tein VIII (37).
To c o n s tru c t th e library, th e oligonucleotides
encoding th e ra n d o m am ino acid se q u en c es are ligated to th e DNA
encoding one or th e o th e r of th e co at p ro tein s. The lib ra ry u s e d in th is
stu d y e n co d es th e ra n d o m am ino acid se q u en c es on th re e to five copies
41
of p ro tein III, w hich a re located a t one en d of th e virion (for review see
(10)). T his p ro tein is n e c e ssa ry for th e infection, d u rin g w h ich it a tta c h e s
to th e F p ilu s of th e b a c te riu m allow ing th e in se rtio n of th e viral genom e.
T h ro u g h th e cell’s m o lecu lar m achinery, th e c o m p lem en tary DNA s tra n d
is p ro d u c e d form ing a viral d o u b le -stra n d e d DNA (dsDNA), referred to a s
replicative form (RF).
ssDNA sy n th e sis.
T he RF is req u ire d for mRNA tra n s c rip tio n a n d
T he virions asse m b le a t th e p erip lasm ic space a n d
e x tru d e from th e cell w ith o u t killing th e b a c te riu m (37).
The single-
s tra n d e d genom e of virions facilitates th e DNA seq u en c in g of th e affinityselected, am plified phage. The am ino acid se q u en c es th a t are o b tain ed
from th e DNA se q u en c es m ay form a c o n se n s u s se q u en c e of a m olecule
th a t in te ra c ts w ith th e purified ta rg e t protein.
T here are several
n u cleo tid e, am ino acid, a s well a s p ro tein seq u en ce d a ta b a s e s , w hich c a n
be h elp fu l in th e se a rc h a n d th e identification of a m olecule co n tain in g
th e c o n s e n s u s seq u en ce.
P h ag e-d isp lay p ep tid e lib raries have been su c ce ssfu lly u se d in th e
id en tification of in te ra c tin g sites betw een p ro tein s a n d a n tib o d ies (12,57),
a s well a s th e identification of p e p tid es th a t b in d to p ro te in s a n d play a
role in m ed iatin g th e ir fu n ctio n s (14). Je llis et a l (26) c o n stru c te d a 20am ino acid lib rary (PDL-20) u s e d to identify th e recognition seq u en ce of a
m o n o clo n al an tib o d y (mAb) raised a g a in st a HIV-I isotype MN envelope
42
p ro tein .
T he recognition seq u en ce c o n sisted of 4 am in o acid re sid u e s
lo cated a t different p o sitio n s w ith in th e ra n d o m se q u en c e of selected
clones from PDL-20.
More recently, B u rritt et a l (11) c o n stru c te d a
n o n a p e p tid e library.
T his library, in c o n ju n c tio n w ith a hexap ep tid e
lib ra iy (57), w as em ployed to m ap epitopes of m A bs specific for th e
cytochrom e
b
co m p o n en ts,
p22Phox a n d
gp9lP hox.
The
identified
c o n s e n s u s p ep tid e se q u en c es w ere n e arly id entical to th e co rresp o n d in g
regions of p rim a ry s tru c tu re seq u en ce of cytochrom e b.
The sam e
n o n a p e p tid e a n d h ex ap ep tid e lib raries w ere em ployed by DeLeo et al. (13)
for id en tification of five p o ten tial sites of in te rac tio n on cytochrom e b
s u b u n its w ith
a
cytosolic c o m p o n en t of NADPH oxidase, p47Phox.
S y n th etic p e p tid e s th a t m im icked two identified se q u e n c e s found on
gp9 IPhox cytochrom e b s u b u n it, w ere able to in h ib it NADPH oxidase
activity w h en a d d ed p rio r to assem b ly of th e oxidase, d e m o n stra tin g th e
biological significance of th e identified sequences.
In th is stu d y , th e p h ag e-d isp lay peptide lib ra rie s w ere u s e d to
select p h ag e th a t b in d FPR . The affinity purificatio n of th e phage w as
perfo rm ed u s in g two different m eth o d s.
The first m eth o d involved
co lu m n sc ree n in g of th e lib rary u sin g purified FPR co u p led to CNBractiv ated S e p h a ro se 4B b ead s. The goal w ith th is m eth o d w as to identify
G p ro tein se q u e n c e s involved in in te ra c tio n s w ith FPR a n d p e rh a p s to
43
discover novel FPR -interacting, p ro tein s.
The seco n d m eth o d of lib rary
screen in g involved in ta c t CHO-FPR cells a tta c h e d to tis s u e c u ltu re plate.
A sim ilar m e th o d w as em ployed by Dr. P in c u s for th e selection of phage
th a t b in d to H IV -infected H9 cells (personal com m unication). The goal of
our
p h ag e
lib rary
screen in g
w ith
in ta c t
cells
w as
to
identify
n o n fo rm y lated am ino acid se q u en c es th a t b in d FPR.
M aterials an d M ethod s
Phage P ep tid e Library
S c r ee n in g U sin g FPR C oupled
to CNBr- A c tiv a te d
S ep h a ro se 4B B eads
HPLC fractio n s c o n tain in g a to ta l of ~5(j,g of FPR w ere ad d ed to 100
m g of b e a d s w a sh e d w ith I mM HC1. The b e ad s w ere th e n in c u b a te d for
24 h a t 4°C u n d e r ro ta tio n a n d 1% OG a n d I M T ris a t pH 8.0 w ere
a d d e d to block th e rem a in in g reactive g ro u p s on th e b e a d s.
The FPR-
co u p led b e a d s w ere sto re d in glycerol a n d w ash ed w ith p h ag e buffer (50
nM T ris-C l pH 7.5, 150 mM NaCl, 0.5% Tween 20, I m g /m l BSA) p rior to
p h ag e selections.
For e a c h selection, approxim ately one th ird of th e
b e a d s w a s p laced in a 5 m l p lastic colum n b a rre l (Evergreen) a n d
in c u b a te d 24 h a t 4°C w ith 75 pi of n o n a p ep tid e p h ag e lib rary (I x IO12
phage) in p h ag e buffer. After in cu b a tio n , th e b e ad s w ere w a sh e d w ith 50
44
m l of p h ag e buffer a n d phage w ere e lu ted w ith e lu tin g buffer (0.1 M
glycine, pH 2.2) im m ediately followed by ad d itio n of 2 M T rizm a b a se (to
final m o larity of 150 mM) to n e u tra liz e th e pH en v iro n m en t of th e phage.
The e lu a te w as collected for titra tio n of th e phage a n d fu rth e r selection.
A m p lifica tio n o f S e le c te d Phage
T he e lu te d p h ag e w ere am plified by in c u b a tin g w ith a fresh c u ltu re
of K91 E. coli for 15 m in before m ixing w ith 35 m l of soft ag ar a n d
sp re ad in g on a sterile 9 x 2 4 -in ch g lass p late w ith LB.
T he p late w as
in c u b a te d 24 h in 37°C a n d p hage w ere lifted from th e b a cte ria l law n by
gently ro ta tin g w ith TBS (50 mM Tris-Cl, pH 7.5, 150 mM NaCl) for 3 h a t
RT.
T he collected p h ag e w ere p rec ip ita te d by in c u b a tio n w ith 0.15
volum es of 16.7 % P E G /3 .3 M NaCl on ice for 24 h o u rs. The p recip itated
p h ag e w ere sed im e n te d by c en trifu g atio n (25 m in, 4°C, 10,000 rpm ,
SS34 rotor), re s u sp e n d e d in TBS a n d m icrocentrifuged for 3 m in a t
12,000 rp m .
The s u p e rn a ta n t w as in c u b a te d for I h on ice w ith 0.15
v o lu m es of 16.7 % P E G /3 .3 M NaCl a n d m icrocentrifuged for 2 m in. The
pellet w as re s u s p e n d e d in 300 pi of TBS. The am plified p hage w as u s e d
for s u b s e q u e n t ro u n d s of selections a n d phage am plifications.
p h ag e w ere o b ta in e d by two m eth o d s.
C ontrol
F irst m e th o d involved phage
selection on u n c o u p le d (blank) C N B r-activated S e p h a ro se 4B b e ad s,
45
seco n d m e th o d involved selection on b e a d s reacted w ith HPLC fractio n s
of CHO-WT e x tra c ts co rresp o n d in g to fluorescence frac tio n s of CHO-FPR
e x tra cts.
T he selections a n d am plifications of co n tro l phage w ere
perfo rm ed in th e sam e m a n n e r a s w here phage w ere selected w ith FPR.
The p h ag e tite r w as a s s e s s e d after e a c h ro u n d of selection. Briefly, 100
pi of p h ag e serial d ilu tio n s w ere m ixed w ith 250 pi of fresh K91 c u ltu re, 4
m l of soft a g a r a n d p la te d on LB p la te s a n d in c u b a te d in 37°C for 24 h.
The re s u ltin g p la q u e s w ere c o u n te d a n d th e a m o u n t of p h a g e /m l w as
d eterm in ed .
Phage Library S cree n in g
U sin g CHO-FPR C ells
M ixed library, c o n sistin g of 50 pi of lin e ar 9 -m er (6.6 x IO11 phage)
a n d 50 pi of c irc u la r 10-m er (I x
IO12 phage) lib ra rie s, d ilu ted in
TB S/B SA , w as p re a b so rb e d for I h a t 37°C on a c o n flu e n t 15 cm plate of
CHO-WT cells.
The p re a b so rp tio n w as rep e ate d tw ice, e a c h tim e on a
fresh p late of CHO-WT cells.
The p h ag e b o u n d to cells after th e th ird
p re a b so rp tio n w ere u s e d a s control phage.
The u n b o u n d phage w ere
selected o n N a-b u ty ra te in d u ce d CHO-FPR cells by in c u b a tin g I h a t
37°C. After th e in c u b a tio n th e cells w ere w ash ed 5 tim e s w ith TBS a n d
lysed w ith 1% NP40 for 10 m in. The collected lysate w a s m ixed w ith soft
a g a r a n d fre sh c u ltu re of K91 E. coli a n d sp re ad on LB (9 x 24-inch)
46
plate.
T he p late w as in c u b a te d o vernight a t 37°C a n d am plified phage
w ere collected a n d p rec ip ita te d a s d escrib ed above. T he am plified phage
w ere u s e d for th e n e x t ro u n d of selection.
Total of fo u r ro u n d s of
selectio n s a n d am plifications w ere perform ed.
Flow C y to m etry o f S e le c te d
Phage B ou nd to CHO C ells
CHO-FPR a n d CHO-WT cells w ere rem oved from th e tis su e c u ltu re
p la te s w ith
I mM EDTA in PBS a n d re s u sp e n d e d in cold PBS++
(Ca2+/M g 2+) su p p le m e n te d w ith 5% FBS a n d aliq u o ted into ep p en d o rf
tu b e s.
V arious c o n c e n tra tio n s of selected a n d co n tro l phage w ere
in c u b a te d w ith th e cells for I h a t 4°C u n d e r ro tatio n .
rec ep to r ra tio s u s e d w ere 10:1, 1:1 a n d 1:10.
The phage to
The average n u m b e r of
re c ep to rs in CHO-FPR cells h a d b een previously d e te rm in e d from bind in g
of
form ylated,
flu o rescein -co n ju g ated
peptide
to
th e
cells
u sin g
flu o resc en t b e a d s a s a s ta n d a rd (Flow C ytom etry S ta n d a rd s C orporation,
S a n J u a n , PR). A pproxim ately I x IO6 recep to rs w ere ex p re ssed on Nab u ty ra te in d u c e d CHO-FPR cell.
After th e in c u b a tio n th e cells w ere
m icro cen trifuged (2000 rpm , 3 m in), w a sh e d one tim e w ith I m l PBS++,
m icro cen trifuged ag ain a n d ra b b it anti-M 13 se ru m w a s ad d ed a t p ro p er
d ilu tio n
(1:10,000).
ex am in in g
th e
The
b a c k g ro u n d
d ilu tio n
levels
w as
previously
p ro d u ced
by
d e term in ed
different
by
se ru m
47
c o n c e n tra tio n s.
After in c u b a tio n a n d w a sh , FIT C -conjugated goat a n ti­
ra b b it a n tib o d y w as a d d e d (1:200).
Following th e la s t in cu b a tio n , th e
cells w ere w a sh e d a n d tra n s fe rre d into FA CScan tu b e s .
C ell-associated
m e a n flu o rescen ce w as reco rd ed w ith flow cytom eter a n d analyzed u sin g
th e G ra p h P a d P rism c o m p u te r program .
S e q u en cin g o f S e le c te d
Phage C lo n es
Single
iso lated
p laq u e s
w ere
picked w ith
sterile
p ip et tips,
in o cu late d into 2 m l of 2 x YT (tryptone, y e a st e x tract, NaCl) co n tain in g
75 p g /m l of k a n a m y c in a n d in c u b a te d in 37°C overn ig h t w ith vigorous
sh ak in g .
After th e
in cu b a tio n , th e
b a c te ria w ere
sed im en ted
by
m icro cen trifu g atio n a t 12,000 rp m for 5 m in a n d 138 pi of 40% PEG8 0 0 0 alo n g w ith 138 pi of SM NaOAC pH 7.0 w as a d d e d to 1.25 m l of
s u p e r n a ta n t a n d p laced on ice for 24 h.
m icro cen trifu g ed
at
12,000
rp m
for
The p re c ip ita te d phage w ere
15 m in
and
th e
pellet w as
re s u s p e n d e d in 30 pi of TE. The p urified phage w ere th e n im m ersed in
boiling w a te r b a th for 5 m in to release phage DNA.
T he sequencing
re a c tio n s u sin g iso lated phage DNA w ere perform ed following th e
d irectio n s of th e S e q u e n ase version 2.0 k it (U. S. B iochem ical Corp.)
u sin g th e seq u en c in g p rim e r 5 '-g ttttg tc g tctttc ca g ac g -3 ’, w hich allowed
th e sy n th e sis of th e ra n d o m oligonucleotide region of th e phage.
The
48
se q u e n c e s w ere re a d from th e gel im age o b tain ed u s in g a M olecular
D ynam ics m odel 4 0 0 E P h o sp h q rIm a g er.
R e su lts
A ffin ity P u rifica tio n o f Phage
U sin g FPR -C oupled CNBrA c tiv a te d S ep h arose 4B
To select FP R -binding ph ag e from n o n a p ep tid e p h a g e library, th e
HPLC p u rified re c ep to r w as im m obilized on C N B r-activated S ep h aro se 4B
b e a d s a n d th e lib rary w as in c u b a te d w ith th e b ead s. P hage th a t b o u n d
to FPR w ere e lu ted from th e colum n w ith low pH, am plified, u s e d in two
m ore ro u n d s of selection on previously u n u s e d FPR -coupled b e ad s, a n d
seq u en ced .
W ith e a c h ro u n d of selection a sim ilar in c re a se in tite rs of
th e p h ag e selected on FPR -coupled b e a d s, a n d th e p h a g e selected on
b la n k b e a d s, w as observed (Table I).
M oreover, th e re w as n o t a
sig n ifican t difference betw een th e FPR -selected a n d co n tro l phage tite rs
o b tain ed after th e final selections of two ex p erim en ts u tilizing two types
of co n tro l b ead s; th e b la n k b e ad s a n d th e b e ad s th a t w ere reacted w ith
HPLC frac tio n s of CHO-WT e x tra c ts co rresp o n d in g to fluorescence
fractio n s of CHO-FPR cell ex tracts. T hese o b serv atio n s su g g est th a t th e
selection of th e p hage w as n o t FPR specific. The o b ta in e d ran d o m region
se q u en c es of th e FPR -selected p h ag e revealed a c o n s e n s u s seq u en ce
Table I. Phage titers (phage/m l) after each round of selections.____________________
S e le c tio n #1
S e le c tio n #2
W ash
E lu ate
W ash
WT3
FPR1
B lan k 2
FPR 1
E lu ate
W ash
E luate
W ash
E lu ate
3.5 x IO12
ND
2 x IO12
4 x IO8
I x IO1O
5.4 x IO8
6 x IO11
6.5 x IO1O
I x IO1O
R ound I
3 x IO7
I x IO6
9 x IO8
2 x IO6
3.6 x IO12
Rou nd 2
1.3 x IO1O
7.2 x 109
1.4 x IO1O
5.7 x IO8
I x IO1O
Rou nd 3
2 x IO1O
1.5 x IO1O
1.4 x IO1O
I x IO1O
I x IO11
iS election c arried o u t u sin g FPR -coupled C N B r-activated S ep h aro se 4B beads.
2Selection c arried o u t u sin g b la n k C N B r-activated S epharose 4B beads.
3Selection carried o u t u sin g HPLC fractions of CHO-WT m em b ran e ex tra cts coupled to C N B r-activated
S ep h a ro se 4B b ead s. The fractions co rresp o n d ed to HPLC elu tio n profile of FPR.
50
Table 2. R andom region am ino acid se q u en c es of selected phage. Amino
acid s a re in single le tte r code.___________________________________________
Control phage
FP R -selected phage
Selection
W HL
W H M G N G K P
W
L RKP V
WH
L R G WG
W H L R G G G
P A
T P
S WF G
MNAP
A WA
AYRH
STL
S
R
GF KG
QGS A
S F K N L
T Y A W H
L R T Q
Q V T N
M M R M S
\M M
H G H H P H E A W
WH L
WHLK
KPV
W H K G R P A
W H Q K F
L Y
H H L R I V Q
T
X
L
I
L
S
Y
V
M
A
D
F
L
L
X
A
R
G
L
S
I
T
N
P
A
L
R
R
P
P
L
V
A
F
R
T
V
I
E
G
T
S
V
L
M
H
T
A
S
V
K
Q
Y
N
E
R
P
V
N
E
G
L
V
P
K
M
H
V
F
K
H
H
T
R
R
TNPD
THLK
G
K N
R G
G N
L G R P E L
G
S
K
G
F
Q
L
I
T
T
N
V
S
H
K
P P
G
T
V
P
R
L
V
Q
K
V
Q
V
Q
V
W
W
I
S
L
R
K
MSVV
E
G
P
H
P
Y
A
F
X
Y
X
R
R
K
X
S
M
L
K
S
X
Q
S
Q
V N K
A P V
N F T L
Q N L V
G
Y
T
V
G M V S
A G T A
G
Q E Y L
A
F
I
G
T
R
D
S
L F D
X P S T G V
b e ad s a n d b la n k b e a d s for control selection.
bSelection c arried o u t u sin g FPR -coupled C N B r-activated S ep h aro se 4B
b e ad s a n d a s a control, b e a d s coupled w ith HPLC frac tio n s of CHO-WT
c o rre sp o n d in g to th e elu tio n profile of FPR.
cSelection c arried o u t u sin g in ta c t CHO-FPR cells a n d in ta c t CHO-WT
cells a s a control.
51
WHLR (Table 2).
A lthough th is seq u en ce a p p e a re d in two se ts of
ex p erim e n ts (notice th e sam e WHLRKPV seq u en ce selected in two
different .experim ents), it also a p p e a re d in th e co n tro l p hage b u t w ith
lower freq u en cy w h en th e b la n k b e a d s w ere u s e d in th e control p hage
selection.
T his could su g g est th a t th e WHLR e x p re ssin g phage w ere
specific for a c o n ta m in a n t, w hich m ay have elu ted from th e gel filtration
co lu m n along w ith FPR .
The p resen c e of th e WHLR seq u en ce in th e
co n tro l p h ag e e lu ted from th e b la n k b e a d s c a n be explain ed by th e fact
th a t, a lth o u g h fresh b la n k b e a d s w ere u s e d for e a c h new ro u n d of
selection, th e p h ag e u s e d w ere th e p h ag e e lu ted from th e FPR -coupled
b e a d s in th e p revious selection.
Phage S e le c tio n o n CHO C ells
E x p ressin g FPR
In o rd e r to identify phage th a t b in d to in ta c t cells, m ixed lin e ar 9m er a n d c irc u la r 10-m er lib raries w ere selected on tis s u e c u ltu re p la te s
c o n ta in in g n e a r co n flu e n t m onolayers of CHO-FPR a n d CHO-WT cells.
The cells w ith th e b o u n d phage w ere lysed a n d th e ly sa te s w ere u s e d in
p h ag e am plification.
A to ta l of four selections a n d fo u r am plifications
w ere p erform ed a n d th e selected p h ag e w ere analyzed for cell b in d in g
affinity o n FA CScan before sequencing.
FA CScan re s u lts revealed
in c re a se d b in d in g of FPR -selected p h ag e to CHO cells e x p ressin g FPR a t
52
3 0 0 -,
] CHO-WT cells
] CHO-FPR cells
8
200
-
nn
phage:receptor 1:100
1:10
1:1
CHO-FPR
selected phage
C
<u
O
CO
2B
O
^
Rn
10:1
nn
n i~i FI n
Hn
nr-,
1:100
1:10
10:1
BK
1:1
nn
CHO-WT
selected phage
100J
] CHO-WT cells
] CHO-FPR cells
200
-
phage: receptor
CHO-FPR
CHO-WT
selected phage
selected phage
Fig. 9. Flow cytom etry an aly sis of b in d in g of selected p h ag e to
a n d CHO-WT cells. In ta c t cells w ere u se d in th e selection
p eptide library. A. Only th e CHO-FPR cells w ere in d u c e d
b u ty ra te to stim u la te in cre ased FPR p ro d u ctio n . B. B oth, th e
a n d th e CHO-WT cells w ere indu ced.
CHO-FPR
of phage
w ith NaCHO-FPR
53
1:1 p h ag e to re c ep to r ratio, a s co m p ared to CHO-WT cells (Fig. 9A). The
sam e tre n d b u t to a le sse r degree w as observed w ith CHO -W T-selected
phage.
However, w h en th e FA CScan w as rep e ate d u s in g CHO-WT cells
in d u c e d w ith N a -b u ty rate, th e b in d in g of phage selected w ith CHO-FPR
w as h ig h er to CHO-WT cells th a n to cells ex p ressin g FPR (Fig. 9B). T his
su g g e sts th a t th e p h ag e m ay have b een selected on su rfa c e m olecules
ex p re ssed by b u ty ra te -in d u c e d cells.
The se q u e n c e s o b tain ed from
selected p h ag e did n o t reveal a n y stro n g c o n se n s u s se q u en c e b u t several
com m on m otifs w ere observed. The TVQ seq u en ce w a s selected on CHOFPR cells a s well a s on th e FP R -coupled beads.
T he LF a n d th e RV
m otifs a p p e a re d in th re e o u t of 15 seq u en ces.
D isc u ssio n
T he u s e of p h ag e-d isp lay peptide lib raries h a s b e en su ccessfu l in
id en tification of p ep tid e bind in g sites of v ario u s p ro tein s. H ere, we have
u s e d n o n a p e p tid e a n d decap ep tid e lib ra rie s to select p h a g e th a t b in d
FPR.
T he re s u lts of phage selections u sin g th e F P R -co u p le d ' CNBr-
activ ated S e p h a ro se b e a d s revealed a stro n g c o n se n s u s seq u en ce, WHLR,
b u t th e significance of th is finding is u n c le a r b e c a u se th is seq u en ce w as
also ob served in co n tro l phage.
More ex p erim en ts n e e d to be done to
d eterm in e th e so u rce of selection of th e frequently a p p e a rin g WHLR
54
seq u en ce in b o th th e FPR -selected ph ag e a n d th e co n tro l phage.
m odification
of th is
su c ce ssfu l re s u lts .
p hage
selection
m eth o d
could
lead
to
The
m ore
For exam ple, if th e phage ex p re ssin g WHLR in th e ir
ra n d o m region seem ed to be selected on th e S ep h aro se b e a d s, m aybe th e
u s e of a d ifferent m a trix w ould allow for m ore FPR-specific selection a n d
e lim in atio n of S ep h a ro se-b in d in g phage.
If, on th e o th e r h a n d , th e
WHLR selection re s u lte d from th e c o n ta m in a n t w hich e lu ted in th e sam e
fractio n a s th e HPLC purified FPR, p e rh a p s a p re a b so rp tio n of p hage on
CHO-WT coupled b e a d s w ould elim inate th e c o n ta m in a n t-b in d in g phage.
In a d d itio n to th e coupling to C N B r-activated S e p h a ro se b ead s, , th e
p urified re c ep to r c a n be utilized in o th e r m eth o d s of p hage selection,
s u c h a s b io p an n in g .
B esides th e WHLR seq u en ce, th e selection of LF m otif is in te restin g
a s it rese m b le s /MLF. F u rth e rm o re , a tru n c a te d p h ag e peptide, ILFS, is
sim ilar
to
CHO-ILF-OH,
th a t
along
w ith
several
o th e r
p ep tid es
resem b lin g fWLLF w ere analyzed for th e ir ability to in d u c e lysosom al
enzym e rele ase a n d chem o tactic activities (20).
T he fiL F peptide w as
show n to be biologically m ore active th a n m o st of th e o th e r peptides. A
m odification of p h ag e selection m eth o d involving th e u s e
of h igh
c o n c e n tra tio n of JMLF (mM range) in elu tin g th e p h a g e could re s u lt in
55
th e iden tification of a d d itio n al p e p tid es th a t b in d to th e ligand binding
site.
T he selection of p hage u sin g in ta c t cells is a novel a n d prom ising
a p p ro a c h .
In o u r ex p erim en ts th e u s e of different cells in th e selection
p ro d u c e d p h ag e w ith different b in d in g profiles to th o se cells.
However,
th e FA C Scan a n a ly sis of p hage b o u n d to N a-b u ty rate in d u c e d cells gave
u n e x p e c te d re s u lts , su g g estin g th a t th e selected p h a g e m ay n o t have
b een specific for FPR.
screen in g
w as
also
T he u s e of in ta c t cells in p h ag e peptide libraryem ployed
by
L.
M azzucchelli,
MD
(personal
com m u n ication) to identify h igh affinity b in d in g p e p tid e s to PM Ns. The
selected p h ag e w ere
in c re a se d
b in d in g
specific for n e u tro p h ils,
to
PMNs
in
co m p ariso n
as
d e m o n stra te d
to
p e rip h e ral
by
blood
lym phocytes, m onocytic cells, a s well a s h u m a n epithelial, en d othelial
a n d fib ro b la st cell lines.
In a n effort to exam ine th e specificity of th e
p h ag e iso lated u s in g in ta c t CHO-FPR cells, we analyzed th e binding by
flow cytom etry. T his allow ed u s to fu rth e r select th e p h a g e p o p u latio n s
o r in d iv id u al clones th a t a p p ea re d to have th e h ig h e s t b in d in g affinities
to w ard
FPR in
in ta c t CHO cells.
T his a p p ro a c h
elim inated
seq u en c in g of p h ag e th a t m ay have b o u n d unspecificalfy to th e
th e
CHO
cells. In su m m ary , a lth o u g h we w ere u n su c c e ss fu l in th e identification
of F P R -binding p e p tid es or p ro tein s, d u rin g th e c o u rse of th is w ork, we
56
w ere able to e sta b lis h co n d itio n s a n d m e th o d s for fu tu re m ore extensive
lib rary screening.
57
CHAPTER 5
ANALYSIS OF CYTOPLASMIC TAIL DELETION MUTANTS
OF FORMYL PEPTIDE RECEPTOR IN CHO CELLS
In tro d u ctio n
T he a d v a n c e m e n t in th e re c o m b in a n t DNA te c h n iq u e s h a s led to
th e d ev elo pm ent of new , pow erful tools for stu d y in g fu n c tio n s of gene
p ro d u c ts.
O ne of th o se tools, com m only u s e d in th e an aly sis of th e
s tru c tu re -fu n c tio n
m u ta g e n e sis.
re la tio n sh ip s
of
v ario u s
p ro te in s,
is
in
vitro
It is referred to a s “reverse genetics”, a s it allows u s to
ch an g e th e gene DNA seq u en ce a n d th e n analyze th e gen e’s fu n ctio n
(63).
Site-specific m u ta g e n e sis h a s b e en u s e d extensively to stu d y th e
s tru c tu re -fu n c tio n rela tio n sh ip of FPR in o u r la b o ra to ry (38) a n d o th e r
lab o ra to ries (46,48).
In th is stu d y , we exam ined th e role of th e
cy toplasm ic tail in cell re sp o n se s m ed iated by FPR. T he cytoplasm ic tail
w as of in te re s t b e c a u se it h a s b een previously show n in two in d e p e n d e n t
stu d ie s th a t th e m iddle region of th e tail m ay in te ra c t w ith G p ro tein
(7,55). O ligonucleotide-directed m u ta g e n e sis w as c a rrie d o u t to p ro d u ce
two cy to p lasm ic tail deletions of FPR, CT9 a n d CT29.
T he stop codons
w ere in se rte d a t L316 (CT9) a n d T336 (CT29) p o sitio n s , of th e FPR cDNA
58
seq u en ce (Fig. 10) re su ltin g in th e tru n c a te d carb o x y l-term in al tails.
T his c h a p te r d e scrib e s th e a n aly sis of th e deletion m u ta n ts to provide
in sig h t
into
th e
im p o rtan ce
of th e
deleted
se q u e n c e s
in
signal
tra n s d u c tio n a n d c h em o tax is by CHO cells th a t ex p re ss FPR.
M aterials and M ethod s
O lig o n u cleo tid e-D irected
M u ta g en esis o f FPR
T he FPR cDNA seq u en ce w as in se rte d into B lu e sc rip t SK+ a n d
tra n sfe c te d in to a s tra in of E. coli (Cd 236), w hich la c k s two enzym es,
dU TPase
p ro cessin g .
and
u ra c il
N -glycosylase,
n e c e ssa ry
for
p ro p er
u rac il
T he infection of th e b a c te ria w ith M 13K07 h elp er p hage
re s u lte d in th e p ro d u c tio n of u rac il-c o n ta in in g single s tra n d e d (ss) DNA
(U-DNA). T he h e lp e r p hage allow ed th e U-DNA to be properly packaged
in to p h a g e p article (35).
The U-DNA w as iso alted a n d m u tag en ic
oligonucleotides c o n ta in in g stop codons (+10 n u cle o tid e s com plem entary
in b o th directions) w ere a n n e a le d a n d c o m p lem en tary s tra n d w as
sy n th esized w ith T4 DNA polym erase. The d o u b le -stra n d e d DNA m ix tu re
w as tra n sfe c te d in to a n E. coli s tra in w ith n o rm al N-glycosylase activity,
w h ich allow ed th e U -containing s tra n d , w hich does n o t co n ta in th e
m u ta tio n , to be destroyed. The m u ta tio n s w ere verified by sequencing.
59
Extracellular side
N-terminus
A)WTYG
180(F
T ) VIi
Cytoplasmic side
CT29
;50
C-terminus
340
]^XAXTADXSJ(T)S%
Fig. 10. S ch em atic re p re se n ta tio n of FPR b a sed on th e B aldw in m odel
(2).
S h a d e d a re a re p re se n ts th e deleted region.
T he stop codons
re su ltin g in CT9 a n d CT29 deletions w ere in se rte d a t L316 a n d T336,
respectively.
60
L igation an d T ra n sfectio n
The cDNA w a s ligated into th e BcoRI site of pBGSA.
pBGSA is a
vector d esig n ed for stab le ex p ressio n of exogenous genes in m am m alian
cells (62). It confers th e G eneticin (G418) re sista n c e to tra n sfe c te d cells.
The ligation m ix tu re w as tra n sfe c te d into E. coli a n d individual clones
w ere an aly zed to identify ex p ressio n vectors c o n tain in g th e in s e rt in rig h t
o rien tatio n .
T he vector w as tra n sfe c te d into wild type CHO cells by
lipofection (18). After a w eek of c u ltu re , twelve G 4 1 8 -re sista n t colonies of
e ac h m u ta n t w ere picked a n d p lated into se p a ra te w ells for an aly sis of
e x p ressio n levels.
FACScan A n a ly sis o f FPR
E x p ressio n o f E ach M utant C lone
The cells w ere rin se d in PBS a n d in c u b a te d in PBS c o n tain in g 1.1
mM EDTA a n d ~4 pM try p sin . Cells w ere allowed to ro u n d u p a n d w ere
rem oved from th e p la te s in cold PBS ++ w ith 5% FBS a n d I m l aliq u o ts
w ere p laced in FA CScan tu b e s on ice. 10 nM of jNleLFNleYK-fluorescein
w as a d d e d to eac h tu b e a n d in c u b a te d for I h. The m e a n fluorescence of
th e ex am in ed clones w a s com pared to th e m ea n flu o rescen ce of CHO-WT
a n d CHO-FPR cells.
The average n u m b e r of m u ta n t re c ep to rs p e r cell
w as c a lc u la te d from two (CT9 a n d CT29) b e st ex p re ssin g cell lines by
61
c o m p arin g
th e m
w ith
th e
m ea n
fluorescence
of CHO-FPR.
The
b a c k g ro u n d fluorescence w as d ete rm in e d u sin g CHO-WT cells.
A n a ly sis o f M utant FPR E xp ression
by Im m u n o flu o r esce n c e
T he cells w ere grow n on g lass coverslips u n til a b o u t 70-80%
co n flu en t, rin se d w ith cold PBS a n d in c u b a te d 15 m in on ice w ith 200
nM yM BpaFY K-fluorescein peptide. Next, th e cells w ere su b je cted to UV
lig h t for 10 m in to allow for p h o to cro sslin k in g of th e peptide to th e
recep to r. G ently rin se d coverslips w ere m o u n te d on slid es a n d observed
w ith flu o rescen ce m icroscope.
In tra cellu lar C alcium
R elea se A ssay
T he cells w ere w a sh e d in PBS, in c u b a te d w ith I mM EDTA,, gently
sc rap e d off th e p la te s a n d re s u sp e n d e d in PBS++ w ith 5% FBS a n d 2 pM
F u ra -2AM.
The cells w ere in c u b a te d in 37°C for 40 m in, sed im en ted
(B eckm an 1000 rp m for 5 m in), a n d re s u sp e n d e d in PBS++ w ith 5% F B S .
C alcium rele a se d by th e cells w as detected by fluorom eter.
The
m e a su re m e n ts w ere o b tain ed u n d e r c o n s ta n t stirrin g w hile adding I pM
yMLF a t 50 s, I pM ATP a t 100 s, 0.2 % TX-100 a t 150 s, a n d 30 mM
EGTA a t 2 00 s. The fluorom eter d a ta w a s analyzed w ith FeliX Softw are
(Photon Technology In tern a tio n a l, So. B runsw ick, Nd).
62
C h em o ta x is a ssa y
T he cells w ere rem oved from th e
p lates w ith
trypsin-ED TA ,
re s u sp e n d e d in m ed iu m w ith 6 mM N a-b u ty rate a n d in c u b a te d I h a t
37°C. Next, th e cells w ere re s u sp e n d e d in m ed iu m w ith o u t se ru m w ith 6
mM N a -b u ty ra te a n d p la te d on 8 pm pore T ransw ell in s e rts a t d en sities
of
SxlO 5
cells
per
in se rt.
The
following
c o n c e n tra tio n s
of
c h e m o a ttra c ta n ts w ere a d d e d into lower wells: I nM _/MLF, 10 nM _/MLF,.
100 nM jfMLF a n d 20 p g /m l fibronectin (FN). The in s e rts w ith th e cells
w ere p laced in th e w ells a n d in c u b a te d 4 h a t 37°C.
After th e
in c u b a tio n , th e cells th a t h a d n o t m ig rated th ro u g h th e in se rts w ere
rem oved w ith co tto n sw abs. The in s e rts w ere placed in w ells co n tain in g
2.5% PFA a n d fixed overnight.
Next, th e in s e rts w ere placed in
H em atoxylin Gill's s ta in for 30 m in a n d w a sh e d w ith PBS. S tain ed filters
w ere rem oved from th e in se rts , p laced on slides a n d observed u n d e r
m icroscope. T he degree of ch em o tax is w as d eterm in ed from th e average
su rface a re a of m ig rated cells o b tain ed from six different 40x fields
ex am in ed by a n im age an aly sis sy stem (Im aging R e se arch M4 T rue Color
Im age A nalysis S ystem , Im aging R esearch , St. C a th e rin e s, O ntario).
63
R e su lts
Iso la tio n o f H ig h est E xp ressin g
CHO C lo n es
E a c h m u ta n t clone w as exam in ed by flow cytom etry for its ability
to b in d th e ^NleLFNleYK-Iluorescein ligand. The clones t h a t exhibited th e
h ig h e st m e a n fluorescence w ere iso lated a n d th e average n u m b e rs of CT9
a n d CT29 m u ta n t re c e p to rs w ere e stim a te d to be 1.08 x IO4 a n d 9.5 x
IO3 p e r cell, respectively, t h e n u m b e rs of recep to rs p e r cell were b a se d
on th e c alc u la te d n u m b e r of rec ep to rs on CHO cells e x p re ssin g WT FPR,
a ssu m in g th a t th e ligand b in d in g affinity of th e m u ta n ts e q u al th a t of WT
FPR.
T he selected clones w ere su b c u ltu re d a n d u s e d in fu rth e r
ex p erim en ts.
R elea se o f In tracellu lar C alcium
b y th e C ells E xp ressin g
M utant FPRs
To exam ine th e coupling of m u ta n t FPR to G p rotein, jfMLF
stim u la te d cells w ere observed for th e ir ability to rele ase calcium from
in tra c e llu la r sto res. T he cells w ere in c u b a te d w ith Fura-2A M , w hich c a n
p a s s th ro u g h cell m em b ran e s. U pon b in d in g to calciu m , F u ra -2 exibits
sp e c tra l re sp o n se , w hich c an be u tilized in d etectio n of c h an g es in
calciu m c o n c e n tra tio n in th e cytoplasm . The a ssa y re s u lts revealed th a t
64
u p o n stim u la tio n w ith JMLF b o th m u ta n t cell lines w ere able to release
c alciu m (Fig. 11), su g g estin g th a t th e re w as coupling of G p ro tein to th e
m u ta te d rec ep to rs.
However, th e h ig h c o n c e n tra tio n of /M LF (I pM)
req u ire d for calciu m m obilization by CT9 a n d CT29 su g g e sts th a t th e re
w as only p a rtia l coupling of G p ro tein to m u ta n t re c ep to rs or th e
re c ep to rs exhibited low b in d in g affinity for th e ligand. B oth m u ta n t cell
lines, how ever, w ere able to release calciu m u p o n stim u la tio n w ith ATP,
w h ich w a s u s e d a s a control to d eterm in e cell signal tra n s d u c tio n
cap ab ilities.
/M L F -M ediated C h em o ta x is o f
CHO C ells E x p ressin g M utant FPRs
T he CHO cells e x p ressin g wild type FPR a re able to m igrate
to w a rd s JMLF, w ith th e m axim al m igration a t 0.1-1 nM , a s d eterm in ed
by Dr. M iettinen (personal com m unication).
To ex am in e w h e th er th e
cells e x p re ssin g m u ta n t FPRs w ere also able to
m ig rate to w ard s
c h e m o a ttra c ta n ts , th e chem otaxis a s s a y w as carried o u t. The cells w ere
in c u b a te d in
seru m -free m ed iu m
and
allowed to m ig rate th ro u g h
sem ip erm eab le filters to w ard s fibronectin, u s e d a s a positive control, a n d
d ifferent c o n c e n tra tio n s of JMLF.
A ccording to th e a s s a y re s u lts , CHO
cells e x p re ssin g CT9 m ig rated to w ard s /M LF (Fig. 12) w ith th e m axim al
m ig ratio n a t 10 nM , b u t th e re w as no FP R -m ediated c h em o tax is of CHO
65
CHO-WT
Ratio A340
CHO-FPR
CHO-FPR CT29
340/380
CHO-FPR CT9
Fig. 11. R elease of in tra c e llu la r calciu m
FPR (A), u n tra n s fe c te d CHO cells (B), a n d
cy toplasm ic deletion m u ta n ts , CT9 (C) a n d
a final c o n c e n tra tio n of I pM, ATP to I pM,
30 mM.
by CHO e x p re ssin g w ild-type
th e CHO cells ex p ressin g th e
CT29 (D). fM LF w as ad d ed to
TX-IOO to 0.2% , a n d EGTA to
66
] medium
50000-,
I
11 nM flVILF
10 nM WlLF
■ i 100 nM flVILF
40000-
f
Q
FN (20 jutg/ml)
30000.
CO
0)
0)
H 20000.
10000
-
5
CHO-FPR
CT9
I1I i i i
CT29
Fig. 12. C h em o tax is a ssa y of CHO-FPR a n d th e cytoplasm ic deletion
m u ta n ts , CT9 a n d CT29. The cells w ere allowed to m igrate to w ard s
different c o n c e n tra tio n s of JMLF or 20 p g /m l fibronectin (FN), a s m arked.
B ars re p re s e n t s ta n d a rd deviation calcu late d from six different ran d o m
fields of a sem ip erm eab le filter.
67
cells e x p re ssin g CT29. Since CT9 h a d a larger deletion th a n CT29, th e se
re s u lts w ere so m ew h at u n e x p ec te d a n d
could be d u e to th e low
e x p ressio n of m u ta n t F P R s. The low ex p ressio n co u ld also explain th e
in ab ility to effectively c arry o u t th e GTPyS a ssa y on th e m u ta n ts , th e
in ab ility to d ete rm in e KdS of m u ta n t FPRs, a s well a s th e difficulty
visualizing photoaffinity labeled cells.
The re s u lts from th e chem otaxis
a ssa y su g g e st th a t th e deleted regions of FPR carboxyl te rm in u s m ay
play a role b u t a re n o t a b so lu tely n e c e ssa ry in m ed iatin g m igration of
cells e x p re ssin g th e m u ta n t FPRs to w a rd s JMLF.
D isc u ssio n
In th is c h a p te r, we d escrib ed th e fu n ctio n al a n aly sis of two
cytoplasm ic tail deletion m u ta n ts of FPR. The stop c o d o n s w ere in se rte d
in p o sitio n s L316 a n d T336 to p ro d u ce re c ep to rs w ith 9- a n d 29-am ino acid
long cy toplasm ic tails, respectively.
B o m m a k an ti et al. th a t a sy n th e tic
It h a s b een d e m o n stra te d by
peptide c o rre sp o n d in g to th e
322RALTEDSTQTSDTAT336 seq u en ce of FPR com peted w ith n e u tro p h il
rec ep to r for b in d in g to bovine G p ro tein , suggesting t h a t th e carboxylte rm in a l tail of FPR w as involved in coupling to G p ro te in (7). Since th e
cellu lar re s p o n se s to fi/ILF observed w ith th e m u ta n ts w ere n o t of th e
sam e m a g n itu d e a s w ith th e WT FPR, it m ay su g g e st several possibilities:
68
I.
T he cytoplasm ic tail m ay be im p o rta n t for G p ro te in coupling b u t
o th e r reg io ns of th e re c ep to r m ay also p a rticip a te in th is in te rac tio n in a
fash io n sim ilar to ligand bin d in g to m u ltiple different resid u e s.
T his
p o ssib ility
and
is
su p p o rte d
B o m m a k an ti et al. (6).
by re s u lts
by
S ch reib er
et al.
(55)
T h ro u g h com petitive ELISA, a s well a s FPR-G
p ro tein re c o n stitu tio n a ssa y s, S ch reib er et al. provided evidence th a t, in
a d d itio n to th e carboxyl-term inal tail, th e FPR second in tra c e llu la r loop
also c o n ta in e d a c o n ta c t site w ith G p ro tein (55).
More recently,
B o m m a k an ti et al. exam ined th irte e n sy n th e tic FPR p e p tid e s for th e ir
ability to d is ru p t FPR-G p ro tein com plexes a n d co n clu d ed th a t all th re e
cytoplasm ic loops a n d p a rts of cytoplasm ic tail a re involved in th e
p h y sical in te ra c tio n betw een th e re c ep to r a n d G p ro tein (6).
2.
It is
p ossible th a t th e poor re sp o n se of th e m u ta n ts w a s d u e to a low
e x p ressio n level of th e m u ta n t recep to rs.
B ased on th e flow cytom etry
re s u lts of v a rio u s m u ta n t clones, we w ere able to e stim a te th e n u m b e r of
re c ep to rs p e r cell a t a b o u t I x IO4, a 100-fold d e crea se in ex p ressio n a s
co m p ared to th e WT recep to rs.
3.
Finally, th e m u ta tio n s m ay have
re s u lte d in a s tru c tu ra l ch an g e in th e rec ep to r a lterin g th e ligand bind in g
affinity.
T h ro u g h
a n aly sis
of FPR
c h im eras,
Perez
et al.
have
d e m o n s tra te d th a t th e carboxyl-term inal tail w as c ap ab le of m o d u latin g
rec ep to r affinity for th e ligand (43).
P e rh ap s th e cytoplasm ic tail
69
d eletio n s re s u lte d in rec ep to rs w ith very low ligand b in d in g affinity,
w hich co u ld explain th e difficulty in o b tain in g th e d isso c iatio n c o n sta n ts
of th e m u ta n t recep to rs.
A dditional site-d irected m u ta g e n e sis stu d ie s
m ig h t allow th e d e te rm in a tio n of th e re sid u e s th a t play critical role in th e
FPR-G p ro tein in te rac tio n . F u rth e rm o re , p hage p ep tid e lib rary screening
u sin g p e p tid e s th a t m im ic th e FPR se q u en c es co u ld lead to th e
id en tification of specific re sid u e s involved in th is in te rac tio n .
70
CHAPTER 6
CONCLUSIONS
1.
The ex p re ssio n of formyl p ep tid e recep to r (FPR) by Sf9 in se c t
cells re s u lts in a rec ep to r w ith d e c rea se d ligand b in d in g affinity (Kd=70
nM), a s c o m p ared to FPR in n e u tro p h ils (Kd=3 nM).
2.
P h o to cro sslin k ed FPR ex p re ssed in C hinese h a m s te r ovary
(CHO) cells c a n be effectively e x tra cted from cell m e m b ra n e s w ith
octylglucoside a n d p urified by h igh p erform ance liquid chro m ato g rap h y .
3.
P h ag e-d isp lay p eptide lib rary selection u s in g eith er FPR-
co u p led cy anogen b ro m id e-activ ated S ep h aro se 4B b e a d s or in ta c t CHO
cells e x p re ssin g FPR p ro d u ce d inconclusive re su lts.
M odifications of
p h ag e lib rary selection m eth o d s m ay allow th e identification of p ep tid es
th a t b in d to th e receptor.
4.
F u n c tio n a l a n aly se s of two FPR cytoplasm ic tail deletion
m u ta n ts , CT9 a n d CT29, revealed p a rtia l calcium release a n d su p p re sse d
ch em o tax is.
T his su g g e sts th a t th e cytoplasm ic region of FPR is n o t
ab so lu te ly n e c e ssa ry for FP R -m ediated signal tra n s d u c tio n .
71
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