for chemotactic activity. Increasing the length of the synthetic tripeptide from 5 to 10 subunits decreased its chemotactic activity, while the single tripeptide subunit.
Journal
of Leukocyte
Biology
39:255-266
(1986)
Chemotactic Activity of Collagen-Like Polypeptides for Human Peripheral Blood Neutrophils Debra L. Laskin, Richard A. Berg
Terutoshi
Kimura,
Shumpei
Sakakibara,
David
J. Riley,
Department of Pharmacology and Toxicology, Rutgers University (D.L.L.), Departments of Biochemistry (D.L.L.,R.A.B.), and Medicine (D.J.R.), UMDNJRutgers Medical School, Piscataway, New Jersey, and The Peptide Institute, Protein Research Foundation, Osaka, Japan (TK.,S.S.) Damage to interstitial connective tissue is associated with the rapid accumulation of monocytes and neutrophils at the site of injury. To study the role of collagen fragments in neutrophil migration, we analyzed the chemotactic properties of peptide fragments of bovine collagen digested with bacterial collagenase or cyanogen bromide and small molecular weight synthetic polypeptides containing proline (Pro), hydroxyproline (Hyp), and glycine (Gly), the major amino acids that comprise collagen. Using the Boyden chamber and under agarose techniques, we found that collagen fragments were as potent in inducing chemotaxis in neutrophils as the bacterial-derived peptide f-met-leuphe. The synthetic polytripeptides (Pro-Pro-Gly)5 and (Pro-Hyp-Gly)5 were found to be equipotent in inducing chemotaxis, producing a maximal induction of chemotaxis at 5-10 nM. This suggests that Hyp, the unique imino acid found in collagen, is not required for chemotactic activity. Increasing the length of the synthetic tripeptide from 5 to 10 subunits decreased its chemotactic activity, while the single tripeptide subunit (Pro-Hyp-Gly)1 was the least active peptide, inducing a maximal response at 100 nM. To study the structural requirements for chemotaxis, Pro-Hyp-Gly tripeptides were synthesized with modifications at the N and C terminals ends. Addition of a methyl group to the carboxyl of Gly to form an ester enhanced the chemotactic activity of the peptide by 50%, while substitutions on the amino terminus with an acetyl group decreased the chemotactic activity by 50%. Substitution on the amino terminus with a Boc group decreased the chemotactic activity by 100%. These results indicate that there are specific structural requirements for chemotaxis induced by peptides having a collagen-like sequence of amino acids.
Key words:
chemotaxis,
Received
June
26,
Reprint
requests:
©
1986
Alan
1985; Debra
R
Liss,
collagen,
accepted L.
Laskin,
Inc.
September Rutgers
neutrophils
Il,
1985.
University,
P.O.
Box
789,
Piscataway,
NJ 08854.
and
256
Laskin
et al
INTRODUCTION Collagen is composed
is one primarily
hydroxyproline
of the most abundant of the amino acids
(Hyp)
in repeating
triplet
connective tissue proteins in the body. It glycine (Gly), alanine, proline (Pro), and units
of Gly-X-Y,
in which
Pro and Y is frequently of all tissues and organs
Pro or Hyp. Collagen is essential for and is also involved in tissue remodeling
injury lular
Since collagen is the capacity to undergo
or inflammation. matrix, it has
the
integrity following
major structural protein in the extracelchanges in response to injury. These
changes include dismantling of the extracellular connective tissue, often in the form of a scar. Damage to interstitial collagens by chemicals, associated with the rapid migration injury [4]. Phagocyte accumulation
X is frequently
the structural and repair
of monocytes is mediated
matrix
and
oxidants,
resynthesis
of
or physical
and neutrophils by chemotactic
new
insults
is
to the site of tissue factors released by
damaged tissues and cells. A number of factors have been described that are chemotactic for phagocytes including complement fragments [3,20,27,30,], products of the kinin and coagulation pathways [9,10], arachidonic acid metabolites [7-9,13,31], and synthetic peptides related to bacterial-derived products [24]. Postlethwaite and his coworkers fragments
have reported that of digested collagen
human types are chemotactic
I,
[171 but not neutrophils [17]. Stecher [28] found ase was a potent chemoattractant for neutrophils, were inactive. In fact, these products appeared collagenase. In contrast native soluble collagen activity
In support
skin
concentration like fragments.
collagen fragments for human peripheral
dependent These
may
and results
be mediated,
connective
related suggest
in part,
AND
Preparation Insoluble
here
with cyanogen bromide neutrophils. Chemotactic
are potent activity is
structure of the synthetic accumulation in tissue
of chemotactic
peptides
collagenfollowing
derived
from
bovine
Peptides skin
collagen
M Tris
buffer
(pH
were
calculated from was determined fetal
calf
pooled
and
weighed by gel skins
with
obtained
lyophilized.
pepsin
5mM
collected acetate,
samples filtration
from
a dispersion
from Seton Company bacterial collagenase
7.5) containing
The dialyzable peptides were eluted with 0. 1 M ammonium
from
we report
METHODS
of Collagenous
untanned cowhides, was purchased was treated with one-tenth (w/w)
volume
release
observations,
consisting of triplet units of Pro, digested by bacterial collagenase or
tissue.
MATERIALS
0.01
latter
to the size and that neutrophil
by the
[5] observed that both possessed chemotactic
and Houck products
of these
produced blood
collagens and peptide [18] and monocytes
while purified bacterial collagendegradation products of collagen to inhibit the chemotactic activity of
molecular weight synthetic polypeptides Gly, as well as bovine dermal collagen
type I calf chemoattractants
injury
in vivo.
and III fibroblasts
that
to these findings, Chang and collagen degradation
for neutrophils
that small Hyp, and
II, for
CaC12
of
(Newark, and dialyzed and
2.5
mM
washed,
N-ethylmaleimide.
and chromatographed on a Biogel pH 5.0. The fractions composing The
concentration
using an average [1]. Soluble calf followed
of peptides molecular skin type
by purification
split,
NJ). The collagen at 22#{176}Cagainst
in this
P2 column the void digest
was
weight of 1,500, which I collagen was extracted
using
a filtration
procedure
Chemotactic
[25]
Cyanogen
.
bromide
Activity peptides
of Collagen-Like
were
prepared
Polypeptides from
type
the method of Sage and Bornstein [21]. The concentration was calculated from weighed samples using an average which was based on peptide analysis [2]. Preparation
of Synthetic
The Gly)5,
synthesis
Collagen
and
(Pro-Pro-Gly)10,
described reacting
characterization
of the and
[22,23]. The tripeptide it with hydrogen fluoride
Boc-Pro-Hyp-Gly the synthetic
performance
chromatography
Cell
peripheral
sedimentation
gradient washing
collagen-like
polypeptides
(Pro-Hyp-Gly)10
Pro-Hyp-Gly and methanol.
was
by treatment tripeptides was and
blood
paper
neutrophils
in dextran
(6%)
have
(Pro-Pro-
been
previously
converted to the methyl ester The tripeptide Pro-Hyp(benzoyl)-
by
with trifluoroacetic acid 1141. The examined by reverse phase high
chromatography
were
followed
as previously described [12]. three times in phosphate-buffered
Measurement modified
[I].
of Chemotaxis
Chemotaxis Boyden
prepared by
from
on
Neutrophils were saline (PBS).
in Microwell
Chemotactic
of neutrophils through chamber technique [3].
normal
centrifugation
millipore For our
used
healthy
the upper neutrophils containing 7.2) was removed
chamber was (approximately
immediately
Chemotaxis
was
after
Chambers
filters studies,
into place. Following 1 x l0 cells) in Hanks’
0.5% bovine serum added to each well. and stained in Camco
in ten oil immersion Measurement
set
donors
a Ficoll-Hypaque
was measured using the we used a 48-well micro-
chemotaxis chamber (Neuro Probe, Inc., Cabin John, MD). Twenty-five of the chemoattractants or control reagents were placed in each well chamber. A 5-pm pore size polycarbonate filter was then placed over
NJ).
to
Preparation Human
by
according
by Berg et al [1] and deblocked with hydrogen (Boc)-Pro-Hyp-Gly was prepared from Boc-Prohydrogenation. The unblocked peptide Pro-Hyp-Gly
was produced from purity of each of liquid
I collagen
of peptides in this digest molecular weight of 10,000,
Peptides
(Pro-Hyp-Gly),
Gly was acetylated as described fluoride [14]. Butyloxycarbonyl Hyp(benzoyl)-Gly by catalytic
257
microliters of the bottom the wells and
thermal equilibration, balanced salt solution
50 of (HBSS)
albumin (BSA) and 2.4 mg/ml HEPES buffer (pH After incubation for 30 mm at 37#{176}C,the filter was Quik Stain (American Scientific Products, Edison,
measured
as the number
of cells
that
migrated
through
the filter
fields.
of Chemotaxis
Under
Agarose
Chemotaxis of neutrophils under agarose was measured using a modification of the technique described by Nelson et al[15] and Orr and Ward [16]. A 1 % agarose (CalBiochem, San Diego, CA) solution in HBSS containing HEPES buffer (2.4 mg/ ml, pH 7.2) was mixed with an equal volume of 1 % BSA and pipetted (I ml/plate) into
35-mm
tissue
dish was removed mately 5 mm from
culture
dishes.
by aspiration the center
After
solidifying,
and a horizontal of the dish. Thirty
l0 cells) was then added to the well. culture dish not covered with agarose. were fixed with 4% formaldehyde.
the agarose well was microliters
Chemotactic factor After incubation The agarose was
covering
half
of each
cut in the agarose approxiof a cell suspension (5 x was added to the half of the for 2 hr at 37#{176}C,the plates then removed and the cells
258
Laskin
stained migrating
et al
with Camco Quik Stain. Chemotaxis from the edge of the well toward
was measured the chemoattractant.
as the
number
of cells
RESULTS Induction
of Chemotaxis
In our the
initial
chemotactic
by Digested
experiments, activity
Collagen
using
of
the
collagenase
collagen with the well-characterized We found that both collagenase
Boyden and
chamber
neutrophil and cyanogen
Induction
of Chemotaxis
We next peptides pentameric, both tion
examined
the
of these peptides range of 0.1-1,000
maximum observed
induced
two
at all of the long fragments,
Induction
Containing
activity
Proline
of two
more
cells
to migrate
for neutrophils were dose
than
the
by Hydroxyproline-Containing
Gly)1,
as a chemoattractant was observed with
was
chemotactic
for
for neutrophils. The 10 nM. Interestingly,
neutrophils,
but
only
were similar the shorter longer
peptide,
not just
a
to those synthetic (Pro-Pro-
due to cyclization before their use. Peptides
the amino acids (Pro-Hyp-Gly)5, potent of the
Pro, and Hyp-
stimulation at 5 nM (Fig. 5 to 10 subunits decreased
maximal stimulation the single subunit,
in the
poly-
Pro, and Gly in We found that
in the concentrarelated, reaching
Synthetic
containing synthetic peptides tested, producing a maximal 3). Increasing the length of the Pro-Hyp-Gly fragment from
Glycine
collagen-like Pro, forms.
We also examined a series of synthetic peptides containing Hyp, and Gly in repeating triplet sequences of(Pro-Hyp-Gly)1, (Pro-Hyp-Gly)10. (Pro-Hyp-Gly)s was found to be the most
its activity Hyp-Gly)10
and
synthetic
triplet amino acid sequences decameric, (Pro-Pro-Gly)10,
concentrations tested. These effects were since all peptides were boiled for 5 mm
of Chemotaxis
bovine
[24,26]. chemoat-
of 0. 1-1,000 nM (Fig. 1). In fact, to that observed with f-met-leuof chemotaxis by the collagenous with 10 nM. Tryptic peptides of
potent chemoattractants nM (Fig. 2). The effects
times
of
f-met-leu-phe were potent
induction of chemotaxis at 10 nM. These results with digested bovine collagen (Fig. 1). (Pro-Pro-Gly),
peptide, Gly)10, of the
Peptides
chemotactic
repeating and were
we compared digests
no chemotactic activity for neutrophils, demonstratpeptides in inducing cell migration (Fig. 1).
by Synthetic
containing the (Pro-Pro-Gly)5,
bromide
chemoattractant bromide digests
tractants for neutrophils in the concentration range the activity of the collagenase digest was comparable phe in the same concentration range. Induction peptides was dose related, reaching a maximum bovine serum albumin displayed ing the specificity of collagenous
technique,
cyanogen
Concentration
with (Pro(Pro-Hyprange
of
100
nM. Induction
of Directed
To demonstrate movement, and
we
Hirsch
[33].
Cell that
performed This
filter in the of (Pro-Hyp-Gly)5,
Migration the
by Synthetic
response
a modified involved
varying
across gradient
the
directed the top
cell movement also increased well of the chamber to decrease
Peptides
to the synthetic checkerboard the
chemotactic chamber. (Pro-Pro-Gly)5,
peptides assay
concentration We found or digested
involved
as described gradient that as collagen
directed of the
the
(Table 1). However, as peptides the chemotactic gradient, the
cell
by Zigmond peptides
concentration was increased, were number
added to of cells
Chemotactic
Activity
of Collagen-Like
Polypeptides
259
8
I:F S8A
-
o1
-f I
lao
10
ONENTRAT1ON Fig.
I.
Dose-related
collagen. increasing gen
of chemotaxis
x
l0)
were
removed,
incubation stained,
cells!
10 oil
30
mm
counted.
(H-
1.
at 37#{176}C,the
Each
eollagenasein the
point
cyanogen
wells
of
(X-X).
8%). When which was
the
average
chamber
bromide-digested
adhered,
of
buffer
migrated
18 oil
bovine
chemotactie
(#{149}-#{149}). or
containing the
bromide-digested
the
eyanogen
f-met-leu-phe
filter
represents
(standard error ISEI wells, random migration.
and
upper
collagen
albumin
for
and
experiments ehemotactie
by
incubated
of collagenase-digested
trypsin-digested
After
separate in the
(1
concentrations
(O-U),
wells.
3.2
induction
Neutrophils
(nil)
with colla-
in the
lower
neutrophils
immersion
fields
buffer alone (0.5% BSA in HBSS) subtracted from each data point,
from
was three
was placed was 69. 1 ±
fields.
8 x 0 .
0 0 0 =
0
a
Cl)
>(
I
oi
I 100
I
lb
(nM)
CONCENTRATiON Fig. x
2. l0)
Dose-related were
(Pro-Pno-Gly)5 30
mm
counted. (SE migration.
induction
incubated
in the
(X-X),
at 37#{176}C,the Each 8%).
point When
which
wells
filter
containing
represents
the alone
subtracted
synthetic
the
(0.5%
polypeptides.
chamber
or buffer
with
in the
lower
adhered,
migrated
neutrophils
of
immersion
fields
BSA each
collagen-like
ehemotaetie
(#{149}-S).
average
from
by
of the
(Pro-Pro-Gly)10
buffer was
of ehemotaxis upper
A
io#{243}o
data
18 oil
in HBSS) point,
was was
72.9
placed ± 5.9
in the cells,
After removed,
three
separate
chemotactie 10 oil
(I
concentrations
wells. was
from
Neutrophils
increasing
fields.
of
incubation
for
stained,
and
experiments wells,
random
Laskin
260
et al
0 0
x
0 1 .
0
= 0
0
(I)
100
10
(nM)
CONCENTRATiON Fig.
3.
Dose-related
Neutrophils
(I
concentrations buffer
in the
induction
x
l0)
of chemotaxis
were
incubated
of(Pro-Hyp-Gly)1 lower
wells.
(X-X). After
was
removed,
stained,
fields
from
three
separate
experiments
in the
chemotactic
5.1
cells!lO
migrating
oil
random
Hyp-containing
upper
wells
synthetic
of the
(Pro-Hyp-Gly)
incubation
neutrophils
wells.
by
in the
for 30 mm
and
counted.
Each
(SE
8%).
migration.
(S-#{149}).
at 37#{176}C.the point
When
which
was
filter
containing the
alone
subtracted
polypeptides.
chamber
with
Pro-Hyp-Gly)10
represents butTer
collagen-like
chemotactie
from
or
the adhered,
average
(0.5%
increasing
(0-U). of
BSA each
18 oil
in HBSS)
data
point,
migrated immersion was was
placed 70.6
±
fields.
through
a concentration Stimulation was
the
filters
gradient of dose related,
decreased.
It is of interest
chemoattractant, cell reaching approximately
to note
that
movement 1.5 times
in the absence
of
was also stimulated. control values with
5
nM (Pro-Hyp-Gly)5 and 10 nM (Pro-Pro-Gly)5. This suggests that these peptides induce chemokinesis as well as chemotaxis. Similar results were obtained when (ProPro-Gly),0 and (Pro-Hyp-Gly),0 were used in the checkerboard assays (not shown). Cells morphology
migrating in response to a chemoattractant characteristic of locomotory cells [32].
display This can
an oriented, polarized readily be observed by
light microscopy using the under-agarose technique. In this assay, cells migrate under a layer of agarose toward a chemoattractant placed in a well cut in the agarose. Using this technique, we found that in a gradient of (Pro-Pro-Gly) or (Pro-Hyp-Gly)5, neutrophils migrated toward the chemotactic well (Fig. 4). The magnitude of the response was similar to that observed for f-met-leu-phe. In addition, cells oriented with
large
heads
in the direction
shown) 1121. Similar and digested collagen confirm the induction chamber
technique
Chemotactic
erties
and
Activity
Using the Boyden of the individual
collagenous in neutrophils Hyp was the
peptides.
of the chemoattractant
results were obtained when were used in the under-agarose of chemotaxis by synthetic checkerboard of Pro,
and
small
knob-like
(not
assay.
Hyp,
and
Gly
chamber technique, we also examined the chemotactic amino acids Gly, Pro, and Hyp that make up collagen We found
tails
(Pro-Pro-Gly)10, (Pro-Hyp-Gly),0, assay (not shown). These results peptides observed using the Boyden
that
(Table 2). These effects most potent of the amino
all three were acids
of these
amino
acids
induced
propand the migration
dose related in the range of 1-1,000 tested. However, it should be noted
nM. that
Chemotactic
Activity
of Collagen-Like
Polypeptides
261
a) a) 0
I 0
AWAY
FROM
CA
TOWARDS
Fig.
4.
Induction
technique.
of neutnophil
Neutrophils
culture
dish
Gly)5.
the
of cells
was per
chemoattractant
were
5 mm
from
unit
(CA) in a gradient
point
TABLE
represents
counted
of
cells
0 “
fixed
with
with
Quik
Stain.
0. 1 mm)
from
the
well
toward
#{149}-U. in a gradient cells
(SE
migrating
Cells of
formalde-
The
number
or away
migrating
from
randomly
f-met-leu-phe;
in a gradient
4%
#{149}-.. of
the
in
the cells
(Pro-Hyp-Gly)5.
8%).
of collagenous
peptides
10”
in top
wells
(M)
l0
IO
732h
78.6
80.4
793
187.4 298.8
130.0 204.4
144.3 175.6
410.4
384.6
360.2
238.1
68.8
71.8
73.4
70.2
l0” l0
101.3 249.6
67.7 204.1
58.3 136.7
61.2 123.3
l0
111.3
84.0
63.2
46.4
0
78.4
80.4
79.6
84.3
183.0 346.7
83.4 243.3
74.2 183.8
86.9 177.6
l0
414.3
aNeutrophils
(I
concentrations and
neutrophils 9%).
were
tissue
(Pro-Pro-
272.1 343.3
0
l0
“Each
10 nM
stained
Concentration
l0M
upper
M f-met-leu-phe.
cell
under-agarose
in a 35-mm
were
0
l0
C
layer
Assay#{176}
inlowerwells(M)
B
10
by the
agarose
cells
migrating
samples
as measured
a 0.5%
2 hr at 37#{176}C.cells the
Li-U,
of three
x
5
microscopically.
peptides
l0 l0
into
of
collagenous
A
After and
(Pno-Pro-Gly)5: mean
peptides cut
containing
alone.
0-C,
1. Checkerboard
Concentration
a well
(approximately
then
the
in a well
by aspiration,
distance
was
by collagenous
placed
or buffer
removed
of a chemoattractant;
migrating Each
l0)
(Pro-Hyp-Gly)5,
agarose
migrated
absence
chemotaxis
x
approximately
10 nM
hyde,
(5
CA
(m)
DISTANCE
l0)
were
placed
wells.
After
removed,
represents
collagen
incubation
stained, the
average
367.0
in the
of collagenase-digested lower was
point
x
for
and of
upper
wells
(A), 30 mm
of
246.6 the
ehemotactie
(Pro-Hyp-Gly) at 37#{176}C.the
(B), filter
199.9
chamber or
with
(Pro-Pro-Gly)5
containing
the
adhered.
increasing (C)
in the
migrated
counted. 12 oil
immersion
fields
from
two
separate
experiments
(SE
262
Laskin TABLE
c Activity
2. Chemotacti
et al
of Am mo Acida Chemotaxis (cells!
Amino
acid
l0
41.3
166.1
20.2 90.5
155.0
Alanine
0.0
0.0
0.0
Methionine
0.3
2.0
0.0
3.4 13. 1
10.0
3.4
6.8
70. 1
point
was
placed
from
each
Concentration
represents
were
placed
mm
8%). 78.4
upper
wells
of
acids
in the lower
filter
stained,
chemotactic the
wells.
adhered,
and counted.
18 oil
immersion
When
buffer
alone
random
± 6.3
the
containing
of
wells,
was
35.0
of amino
37#{176}C, the
average
(SE
point,
at
removed,
in the chemotactic data
in the
concentrations
the
experiments
3. Checkerboar
migration.
cells!
10 oil
fields
(0.5%
from
BSA
which
three
in HBSS)
was
subtracted
fields.
d Assay5
of Concentration
amino
acids
in
lower
wells
(M)
of amino
0
0 lOb 108
100.5
0 10 ‘#{176} l0’ 106 0 l0b0 108 106
61.2 96.3 119.2 203.4 59.4 96.4 142.8 228.9 (I
x
l0)
were
placed
acids
in upper
wells
(M) lO6
100
56.9” 66.4 84.3
106
aNeutrophils
10-6
2.3 25.5
separate
Hyp
10-8
32.0”
bEach
Gly
(M)
Proline Hydroxyproline
Neutrophils (I x l0) chamber with increasing After incubation for 30 migrated neutrophils was
Pro
‘#{176}
fields)
Glycine
Glutamine Phenylalanine
TABLE
10 oil
70.1
78.4 84.1 95.2 112.3 93.3
74.3
88.6 109.1
89.4 100.3
wells
91.7 100.1
118.4 123.6 128.1 207.7
109.2
123.6 226.3 88.4 99.5 135.6 205.2 in the upper
89.3 95.9
109.4
206.1 116.2
144.3
129.0 145.9 207.8
104.6
133.1 213.7
of the chemotactic
chamber
with
increasing
concentrations of Pro, Gly, or Hyp in the upper and lower wells. After incubation for 30 mm at 37#{176}C. the filter containing the adhered, migrated neutrophils was removed, stained, and counted. bEach point represents the average of 12 oil immersion fields from two separate experiments (SE 8%).
maximal
chemotactic
activity
was
observed with the synthetic peptide (Figs. 2, 3). Alanine, methionine,
at
10 nM,
containing glutamine,
which
was
1.5-2-fold
Hyp in the same and phenylalanine
less
concentration were also
chemoattractants. Phenylalanine (1 zM) was found to induce chemotaxis phils, but it displayed only about one-tenth of the activity of synthetic peptides neutrophils. using Gly
(Table The
a checkerboard were
placed
2). None of the other amino activity of the amino acids Gly, assay on either
(Table side
3).
of the
When filter,
acids Pro,
equal movement
tested were and Hyp was concentrations was
than
that
range tested as
in neutrocollagen-like
chemotactic for further analyzed of Hyp,
stimulated
Pro,
to the
or
same
Chemotactic
Activity
of Collagen-Like
Polypeptides
263
8
I CONCENTRATION Fig.
5.
Effects
Neutrophils
of modification
(1
concentrations (O-0), 30
x
l05)
of
(Pro-Hyp-Gly),
on the
were
at
counted. ments
37#{176}C.the
Each
data
(SE
random
point
migration.
extent as although
in the these
containing
the
adhered,
the
average
buffer
(0.5%
BSA
was
presence amino
subtracted
end
of(Pro-Hyp-Gly),
wells
(s-U).
represents
When which
upper
of
the
of in
from
in the
migrated
neutrophils
immersion
HBSS)
each
data
of a concentration acids stimulated
chamber
(X-X).
or buffer 18 oil
on chemotactic
chemotactic
Boc-Pro-Hyp-Gly
(oMe)
filter
8%).
C terminal
in the
(S-S).
Pro-Hyp-Gly-omethyl
mm
N and
incubated
(nil)
alone point,
acetyl
lower fields
was was
73.4
gradient movement,
After
incubation
for
removed,
stained,
and
from
placed
three
in the
± 5.8
increasing
(Ae)-Pro-Hyp-Gly
wells. was
potency.
with
cells!
separate
experi-
chemotactic 10 oil
wells.
fields.
of these amino acids. Thus, it appeared to be random
(chemokinesis). Effects
of Modifications
Chemotactic
on the
To study analyzed modified
the
active
the chemotactic at the N and
peptide,
N and
Ca Terminal
End
structural
requirements
activity C terminal
(Pro-Hyp-Gly)1,
was
of
found
a series ends. As
of the
to be chemotactic
alpha
amino
group
of Pro
acetyl
group
decreased
acetyl
ment
on
of the
group Boc
in (Pro-Hyp-Gly)1
to the group
molecule
with
activity
produced
resulted
in a total
a 50% loss
neutrophils
we
peptides subunit
(Fig.
5). The
on this peptide resulted in a (Fig. 5). Substitution of the
a butyloxycarbonyl
of the
molecule,
(Pro-Hyp-Gly)1 above, the single for
group to the carboxyl end of Gly chemotactic activity of the molecule the chemotactic
Pro-Hyp-Gly
of synthetic mentioned
addition of a methyl 50% increase in the
the
of (Pro-Hyp-Gly)1
Potency
molecule
decrease
(Fig. in activity,
in chemotactic
(Boc)
or
5). Addition while
an of
attach-
activity.
DISCUSSION
human
In this paper neutrophils.
to be as potent
we demonstrate Collagenaseand
chemotactic
agents
chemotactic factor f-met-leu-phe for chemotactic activity of the
that collagenous peptides cyanogen bromide-digested
for human
neutrophils
(Fig. 1). To determine collagenous peptides, we
as was
are chemotactic collagen were
for found
the bacterial-derived
the structural requirements began a systematic analysis
Laskin
264
of the
chemotactic
taming Pro, checkerboard induced both Similar were
properties
molecular
weight
synthetic
polypeptides
con-
Hyp, and Gly, the major amino acids present in collagen [29]. Using and under-agarose assays. we demonstrated that the synthetic peptides directed and nondirected migration of neutrophils (Table I ; Figs. 2, 3).
results have also been reported for f-met-leu-phe [26]. In our initial studies, synthetic peptides containing Pro and Gly, (Pro-Pro-Gly)n, compared to peptides containing Pro, Hyp, and Gly, (Pro-Hyp-Gly). We found
that these peptides et al I 17] reported recognized
were that
equipotent in inducing chemotaxis Hyp is an essential constituent
by the chemotactic
demonstrate
that
chemotactic
responsiveness
Alternatively, neutrophils found
of small
et al
Hyp
receptor
is not
on human
required may
for be
distinct receptors may to the different peptides.
Peptide length that peptides
appeared containing
fibroblasts.
neutrophil
due
(Figs. 2, 3). Postlethwaite of the amino acid sequence
to differences
be
In contrast,
chemotaxis.
involved
in the in
the
our
These cell
results
differences types
chemotactic
to play a significant role in chemotactic five monomeric triplet sequences of
in
examined. response
of
potency. Pro-Pro-Gly
We or
Pro-Hyp-Gly were more active in inducing chemotaxis than the peptides containing ten subunits or the single subunit, (Pro-Hyp-Gly)1 (Figs. 2, 3). This suggests that critical length of the peptide may be required for maximal chemotactic activity. However, receptor
it is also
possible
binding through We also found that
cyanogen
bromide
albumin specific
were
that steric bovine
the
chemotactic
was inactive (Fig. for digested collagen.
peptides
interfere
digested for
with
neutrophils,
I). This indicates The fact that both
chemotactic activity suggests that acid sequences capable of inducing with those reported by Postlethwaite 1181 and are supported Using substituted
longer
hindrance. collagen
with
chemotactic
either
bacterial
while
trypsin-digested
a
factor
collagenase
or bovine
that the chemotactic response was types of digested collagen displayed
many regions of the collagen neutrophil chemotaxis. These et al for human monocytes
chains contain amino results are consistent [17] and fibroblasts
by our studies using the synthetic peptides (Figs. 2, 3). derivatives of (Pro-Hyp-Gly)1, we were able to deduce several
additional characteristics about the neutrophil membrane receptor for collagen peptides. The addition of a methyl group to the carboxyl end of Gly to form an ester enhanced the chemotactic activity of the peptide by 50% (Fig. 5). This suggests that a free carboxyl group possible that the effects degradation. Substitutions
is not essential for chemotactic activity, although it is also were due to an increased stability of the molecule to enzymatic on the amino end of the molecule decreased the chemotactic
potency of (Pro-I-lyp-Gly)1 by 50% with an acetyl group These results indicate that a charged group at the amino required for chemotactic activity. It is also possible that addition is due to steric when the amino terminus chemotactic are currently
activity and in fact became underway in our laboratory
be an antagonist The tides was neutrophils
hindrance by the bulky of f-met-leu-phe was
substituents. Freer blocked with Boc,
chemotaxis
chemotactic
activity
also analyzed. (Table 2).
Pro, Gly, and Hyp were We found that movement
the chemotactic
chamber
with
each
et al [61 found that this peptide lost its
a potent antagonist of f-met-leu-phe. to determine if Boc-Pro-Hyp-Gly
of (Pro-Hyp-Gly)1-induced of the
and 100% with a Boc group. end of the tripeptide may be the loss in activity after Boc
amino
of the
acid
amino
in neutrophils.
residues
contained
all found to stimulate of neutrophils through acids
Studies may also
was
the
same
within
the
pep-
migration the filter in the
presence
of in
Chemotactic or absence
of a chemotactic
neutrophils
was
to
stimulate
Activity
of Collagen-Like
gradient.
not directed.
This
indicated
Furthermore,
movement
was
tenfold
Polypeptides
265
that
the stimulated
the concentrations
of amino
greater
than
with
the
migration acids
synthetic
of
required
collagen-like
peptides containing the amino acids. These results suggest that directed migration neutrophils toward synthetic peptides requires a specific sequence of amino acids. Breakdown of interstitial connective tissue is associated with the accumulation of neutrophils
and
this accumulation from collagen
is due, breakdown
monocytes
at the in part, products
site
of injury
to the
generation [5]. Our data
[4].
It has
been
of
hypothesized
that
of chemotactic stimuli generated are consistent with the idea that the
collagen molecule contains a chemotactic signal for neutrophils and that chemotactic factors are released during the process of collagen degradation [5]. In previous studies we found that intratracheal instillation into rats of the synthetic collagen peptide (ProHyp-Gly)5 as well as collagenase-digested collagen caused a migration of neutrophils
1191.
into the lungs chemoattractants
Our findings in vitro suggest
of phagocytic synthetic response
cells
in the
lung
that these a potential
and structurally mechanism
related underlying
following
acute
[4].
injury
peptides are potent the accumulation
Small
collagen peptides provide a novel tool for the study of neutrophils. Further studies with these peptides are
terize
their
precise
role
in interstitial
was
supported
collagen
molecular
weight
of the inflammatory necessary to charac-
disease.
ACKNOWLEDGMENTS This
research
by the American
Lung
Association
of New
Jersey.
REFERENCES I. Berg,
R.A.,
Prockop.
2.
Gly)
and
ylase.
Evidence
in the
enzyme.
Y., Sakabibara,
Hydroxylation for and
collagen
Sage.
types.
49,957,
1980.
Boyden.
S.
prolyl
hydroxactive
16,1615, H.
Annu.
Rev.
8.
site
of
tures of antibody
chemotactic and
effect
antigen
leukocytes.
of
Med.
mix-
E.J., and Senior,
Chang,
C., and
of
chemotactic
the
Proc. Soc. Exp. 6. Freer, Ri.. and
Houck,
J.C.
of
requirements
Further for
Goetzl,
E.,
Woods,
ulation
of
human
J.,
E., and
Gorman, and
polymorphonuclear migration
leukocyte by
K. The
plasma.
II. The
by activation
proactivator.
J. Clin.
of
Invest.
1973. A., Kay, A., and Austen,
K. A preal-
ance
of
for
H.J.,
neutrophils
struc-
kallikrein.
che-
12.
Laskin, human
1980. R. Stim-
chemotactic by
J. Exp. D.L.,
and
neutrophilic
monoclonal
activity
conversion
of
Med.
135,387,
Rovera,
G.
to
1972. Stimulation
of
chemotaxis
by
granulocyte
antibodies.
human
prekallikrein
J. Immunol.
134,1
146,
1985.
neutrophil
chemotactic
12-L-hydroxy-5.8,
Austen,
of human
J.A.. Schiff-
peptide
and
Med.
III. Appear-
19,2404.
eosinophil
chemo-
B. J. Exp.
activation of prekallikrein.
on the
synthetic
Biochemistry
structural
bumin
Showell,
studies
Novel
1970.
134,22. S..
52,2591, 11. Kaplan.
collagen.
acids
1980.
neutrophil
of ehemoactivity
plasminogen
Demonstration
properties
Biol. Med.
E.L.
moattractants.
random
Pickett. W.
A., Goetzl,
generation
1981.
polyactivity
1981.
10. Kaplan,
Cell injury
2,357,
Day. A.J., Radding.
Becker,
tural 7.
Med.
E., Aswanikumar,
man,
R.M.
human
125, 1789,
of the human
fibrinolytic pathway
and repair. Clin. Chest 5.
E.. and
153,482,
115,453,
The
tactic activity of leukotriene
1962. 4. Campbell.
59, 179,
hydroxy-eicosatetranoic J. Immunol.
9. Goetzl,
on polymorpho-
J. Exp.
Invest.
chemotactic
leukocyte
complex
determinants The
J. Clin.
E., and Pickett, W.
(HETEs).
dis-
Biochem.
Goetzl,
morphonuclear
1977.
Structurally
acid.
1977.
(Pro-Pro-
an asymmetric
Biochemistry
P.,
nuclear
by
eicosatetranoic
S., and
of
(Pro-Pro-Gly)1()
Bornstein, tinct
3.
Kishida, D.J.
and 10,14-
13. Lewis, Austen.
R.,
Goctzl,
E.,
K., and Corey,
Drazen,
J., Soter, N.,
E. Functional
charac-
Laskin
266 terization of synthetic sterochemical 154.1243,
leukotriene
isomers.
I.
B and
Exp.
Biochim.
of
L., and
D.J.
Synthesis
chain
of type I procollagen:
fragments
required
lagen.
In
New
York:
of
Vol.
proalpha
Synthesis
Press.
E.,
pp.
method
Quie, under
491-494.
115, 1650. 16. Orr, W..
20.95.
collagen
J.
A.E..
Chemotactic
blasts
to type I. II.
Seyer.
J.M..
attraction and
DL..
NatI.
Guss. and
Berg.
of collagen
R.A.
Sci.
Intratra-
A..
and
Mayer.
component
cleaved
of complement.
and
from
the
Science
V.J. Ann.
Stryer,
30. Ward, munol. 31. Turner.
162.361,
H., and
Bornstein,
P. Preparation
of procollagens
gen-collagen zymol. 82.96,
intermediates. 1982.
Sakakibara, Y..
Synthesis
S.. Inouye. Koboyashi. of weights.
Y..
donate
and for
and
32.
procolla-
Methods
En33.
K.. Shudo.
(Pro-Hyp-Gly) Evidence
and
K.. Kish-
Acad.
and
upon and
of selected cell degradation
Sci.
prod-
256,178. 2nd
1975.
ed.
San
Fran-
and Co., p. 186, 1981.
Newman,
L.
factor from
human
A
neutrophil C5a.
I. Im-
W.
Bioge-
1969.
S., Tainter, J.. and lipoxygense
Lynn,
molecules system
by the arachi-
of platelets. Nature
1975. S. Ability
leukocytes
to orient in gradients
of polymorphonuclear of chemotac-
tic factors. I. Cell Biol. 75,606, 1977. Zigmond, S., and Hirsch, J. Leukocyte motion
in-
endo-
1969.
chemotaxis tissue
Gew-
neutrophil
CS
serum
Zigmond,
and
evaluation
of defined
mo-
chemotactic 1973.
seMed.
I..
from
pig
Freeman
102,93,
D.J.
stabilization
enzyme Exp.
S. A
Biochemistry,
A.,
Prockop, the
activity
as chemotactic
Philips.
103,413.
The
N.Y.
L.
H..
derived
nesis of chemotactic
fifth
S.H.,
structure
J.
guinea
to connective
257.680,
characterization
lecular
Stecher,
cisco: W.H.
1968. 21. Sage,
The
peptides
Shin.
factor with
chemotactic
M. Chemotactie
fragment
29.
induces
E.,
Mellors,
I colla-
S., Corcoran,
Mergenhagen,
toxin. J. Immunol. 28.
ucts.
R., Friedman,
anaphylactic
Sci.
255,9427,
of lysosomal
R.,
H., and
teraction
Assoc.
Snyderman,
S.
1976.
ehemotactie
Curran,
peptides
Type
Chem.
neutrophils.
Snyderman.
fibro-
a neutrophil influx in rat lungs. Trans. Am. Phys. XCVII, 1984. HR..
27.
and col-
H.N.,
Acad.
R.J., Zigmond,
EL.
inducers
for
Kang.
Acad.
Freer,
Becker, and
types
75,871, 1978. Di., Kerr, iS., instillation
and
of human
Proc.
J. Biol.
E., Assanikumar,
143,1154,
Med.
III collagens
peptides.
S.F., Laskin,
Exp.
urtz,
A.H.
lagen-derived
Hi.,
cretion
Colla-
ehemotaxis
monocytes.
Natl.
Proc.
relations of synthetic factors
A.H.
WahI,
as chemoattrac-
1975.
in solution.
B., and
I.
1978. Kang.
41,1273,
B., and
peptides
72.1059.
26. Showell,
of leuko-
peptide-induced
1976.
Postlethwaite,
and
molecular
Jpn.
1980.
lmmunol.
P. Quantitation
A.E.,
143. 1299.
ido.
Soc.
Y.,
of poly-
of defined
Chem.
E., Corcoran,
Schiffman,
Methods
blood
20. Shin,
gen
polymorphonuI.
1973. Kikuchi,
25. Silver, F.H., and Trelstad, R.L.
spon-
by three different methods.
human
cheal
and simple and
monocytes.
and Ward,
Immunol.
22.
Schiffman,
U.S.A.
R.
1975.
17. Postlethwaite,
U.S.A. Riley,
human
and
taxis in agarose
and
A new
Y.,
K. Synthesis
N-formylmethionine
Simmons,
chemotaxis
of
leukocytes
and
agarose:
migration
clear
P.,
for measuring
taneous
19.
Kakiuchi,
tants for leukocytes. R..
Chemotaxis
18.
Kishida,
Bull.
hydroxyproline.
1968.
1980.
of
and
weights. 24.
Ed.)
S.,
R.,
by
Acta 303,198,
(L-prolyl-L-polyglycl)
I
of the of col-
II (Gross.
Academic
Sakai,
Prockop.
for the synthesis
Peptides.
15. Nelson,
gen-
triple helix
Biophys.
23. Sakakibara,
T.. Tuderman,
N-protease
of collagen
its
Med.
1981.
14. Morikawa,
et al
chemotaxis.
New
and demonstration factor.
J. Exp.
loco-
methods
for
of a cell derived Med.
137,387,
