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(Received for publication, January 26, 1996) From the
The dissection of the activities mediated by type I collagen
requires an approach by which the influence of triple-helical
conformation can be evaluated. The
Collagens are important structural components of the
extracellular matrix (ECM). ( Type I collagen, the most
abundant protein in higher vertebrates, is composed of two identical
Several
distinct sequences derived from type I collagen CB fragments have been
identified as cell adhesion sites. The adhesion of Chinese hamster
ovary cells to type I collagen is inhibited by the 757-791
sequence located within the There may be two
types of active sequences within type I collagen. The activity of some
sequences may require triple-helicity, while others might be
nonfunctional when contained in native, triple-helical conformation but
revealed in the denatured state(33) . Only single-stranded
peptides (SSPs) have been utilized in prior studies on the cellular
activities of type I collagen sequences. However, the triple-helical
conformation of collagen has been shown to be important (if not
crucial) for influencing cell
adhesion(2, 7, 13, 15, 22, 23, 24, 25) ,
cell spreading(26) , cell migration(1) , matrix
metalloproteinase (MMP) binding(27) , and human platelet
adhesion and aggregation(13, 28, 29) .
Conversely, cell adhesion to denatured, but not native, collagen can be
inhibited by linear peptides containing Arg-Gly-Asp
sequences(15, 30, 31, 32, 33) .
Denatured collagen Arg-Gly-Asp sites are bound by the
In
the present study, we have examined potential cellular recognition
sites within type I collagen and studied the significance of
triple-helical conformation. Sequences were derived from
Alternatively, the branched peptide-resin was
synthesized by using HBTU/HOBt as coupling reagents. 1.95 mmol of the
above-mentioned Fmoc-amino acids were coupled to 1 g of
Fmoc-Gly-Sasrin After assembly, branched peptide-resins were washed several times
with DMF followed by dichloromethane. A small quantity of the branched
peptides was deprotected and cleaved with water-TFA (1:19) for 1 h.
After precipitation and washing twice in methyl t-butyl ether,
the branched peptides were lyophilized to a colorless powder. The
branched peptides were analyzed by either ESMS or FABMS, giving an (M
+ H)
The large-scale
synthesis of peptide Preparative RP-HPLC purification
was performed on a Rainin AutoPrep System with a Vydac 218TP152022
C
Edman degradation sequence analysis was performed on an
Applied Biosystems 477A Protein Sequencer/120A Analyzer as
described(42, 43) for ``embedded''
(noncovalent) sequencing. FAB mass spectra were obtained on a VG
7070-HF mass spectrometer and ES mass spectra on a Sciex API III double
quadrupole mass spectrometer. Laser desorption mass spectrometry was
performed on the Kratos Kompact MALDI matrix-assisted laser desorption
time-of-flight mass spectrometer. Circular dichroism (CD) spectroscopy
was performed on a Jasco 710 spectropolarimeter using a 0.01-cm cell.
Thermal transition curves were obtained by recording the molar
ellipticity ([
Inhibition of NHDFs was monitored in the presence of
soluble synthetic peptides or mAbs generated against the
The 10 homotrimeric THPs and a generic THP
(designated GPP*) containing only eight repeats of Gly-Pro-Hyp were
examined for promotion of NHDF adhesion. At a peptide concentration of
0.1 mg/ml, only the THP incorporating
Figure 1:
Promotion of NHDF adhesion by type I
collagen, GPP*, or crude THPs incorporating type I collagen sequences.
Substrate concentrations were 0.1 mg/ml for peptides and 0.02 mg/ml for
type I collagen. Cells were allowed to adhere to peptide- or
protein-coated Immulon 1 plates for 30 min at 37 °C. All assays
were repeated in triplicate. Conditions are given under
``Experimental Procedures.''
The triple-helical
conformation of
Figure 2:
CD spectra of purified
Figure 3:
Thermal transition curves for purified
Figure 4:
Structures of
Figure 5:
Promotion of NHDF adhesion by crude
To study the significance of collagen triple-helical structure on
cellular recognition, we compared the NHDF adhesion-promoting ability
of the
Figure 6:
Promotion of NHDF adhesion by purified
The dissection of the various biological activities mediated
by type I collagen requires an approach by which the influence of
triple-helical conformation can be evaluated. More specifically, the
mechanisms of collagen catabolism may require two types of cellular
recognition sites, those that are dependent upon triple-helical
conformation and those that are revealed upon denaturation of the
triple-helix. Our initial interest is in identifying sites that are
recognized in triple-helical conformation. The
The 10 THPs were
screened for cell adhesion activity without prior purification of the
peptides. Edman degradation sequence analysis indicated that each crude
THP contained a substantial amount of the desired peptide. Thus, the
crude THPs were adequate for screening purposes. Of the 10 THPs,
The large scale synthesis and
purification of the The Adhesion of NHDF to the One curious result is
the ability of the Fibroblast interaction with collagen has tremendous
implications for understanding the regulation of collagen metabolism
and hence processes such as wound healing. Degradation of collagen may
proceed (i) intracellularly following phagocytosis or (ii)
extracellularly by MMPs (3, 4, 61) .
Fibroblasts both phagocytize type I collagen (3, 4) and produce MMP-1(62) . For
fibroblasts, the two mechanisms of collagen catabolism may be inversely
correlated(4) . For example, interleukin 1 We view the studies
presented here as an encouraging start to understanding the variety of
biological activities mediated by type I collagen. As stated by
Tuckwell et al.(21) , ``crucially, the
demonstration of conformation dependence suggests that linear peptides
may be unsuitable (for studying) cell-collagen interactions and implies
that more sophisticated methods may be necessary for future
studies.'' Our triple-helical peptide approach appears to be a
logical one for the identification of conformationally dependent
collagen-mediated functions.
Volume 271,
Number 21,
Issue of May 24, 1996 pp. 12234-12240
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and 
integrin binding sites
within type I collagen are dependent upon triple-helical conformation
and contained within residues 124-822 from 1(I). Seven
1(I)-derived triple-helical peptides (THPs) were synthesized based
on charge clustering (1(I)256-270, 1(I)385-396,
1(I)406-417, 1(I)415-423, 1(I)448-456,
1(I)496-507, and 1(I)526-537). Three additional
THPs were synthesized (1(I)85-96, 1(I)433-441,
and 1(I)772-786) based on previously described or proposed
activities (Kleinman, H. K., McGoodwin, E. B., Martin, G. R., Klebe, R.
J., Fietzek, P. P., and Wooley, D. E.(1978) J. Biol. Chem. 253, 5642-5646; Staatz, W. D., Fok, K. F., Zutter, M. M.,
Adams, S. P., Rodriguez, B. A., and Santoro, S. A.(1991) J. Biol.
Chem. 266, 7363-7367; San Antonio, J. D., Lander, A. D.,
Karnovsky, M. J., and Slayter, H. S.(1994) J. Cell Biol. 125,
1179-1188). Of the ten THPs, 1(I)772-786 THP had the
greatest activity, with half-maximal normal dermal fibroblast adhesion
occurring at a peptide concentration of 1.6 µM.
Triple-helicity was essential for activity of this sequence, as the
non-triple-helical peptide analog (1(I)772-786 SSP)
exhibited considerably lower levels of cell adhesion promotion even at
peptide concentrations as high as 100 µM. Within the
sequence itself, residues 784-786 (Gly-Leu-Hyp) were important
for cellular recognition, as the 1(I)772-783 THP had greatly
reduced cell adhesion activity compared with 1(I)772-786
THP. Preliminary studies indicate that the integrin
subunit mediates fibroblast adhesion to 1(I)772-786 THP. The
identification of fibroblast integrin binding sites within type I
collagen may have important implications for understanding collagen
metabolism.
)They are distinguished by
their triple-helical conformation composed of three chains with a
repeating Gly-X-Y sequence motif. In addition to
providing the structure of connective tissue, collagens can mediate
intracellular communication. Cell-collagen interactions play a role in
a number of processes including cell migration(1, 2) ,
collagen catabolism(3, 4) , and the aggregation of
platelets(5, 6, 7, 8, 9, 10, 11, 12, 13) .
One approach for developing novel therapies for diseases linked to ECM
interactions, such as tumor cell metastasis, atherosclerosis,
inflammation, and thrombosis, or to better understand normal processes
such as wound healing, is to identify cellular recognition sites within
collagen molecules and dissect the structure-activity relationship for
receptor-ligand binding(14) .1 chains and an 2 chain. Cyanogen bromide (CB) fragments of
the 1 chain have been used to locate integrin-mediated cell
binding sites within type I collagen. The 1(I)CB3 fragment
(residues 403-551) and the 1(I)CB8 fragment (residues
124-402) contain binding sites for integrin(2, 15) . 1(I)CB3 also contains a
binding site for the hepatocyte integrin(15) , while 1(I)CB3, 1(I)CB7 (residues
552-822), and 1(I)CB8 contain binding sites for the platelet
integrin(12, 13, 16) .1(I)CB7 fragment(17) . A
peptide incorporating residues 1(I)769-783 supports human
fibroblast adhesion and migration and inhibits human fibroblast and
human T-lymphocyte attachment to type I
collagen(18, 19) . By using short synthetic peptides,
an integrin binding site could be
identified containing the minimal sequence
Asp-Gly-Glu-Ala(20) . This sequence corresponded to residues
435-438 within the 1(I)CB3 fragment. The
1(I)434-438 peptide inhibits adhesion of platelets and
breast carcinoma cells to type I collagen in a concentration-dependent
manner (20) and, not surprisingly, was not effective at
inhibiting -mediated chondrosarcoma
cell adhesion to type II collagen(21) .
(15) or
integrin(30) . To fully
understand the role of collagen in ECM remodeling requires delineation
of native collagen active sites from denatured collagen sites.1(I)CB
fragments that possess integrin binding sites. To utilize relatively
short sequences (
15 residues) and yet ensure triple-helical
conformation under biological assay conditions, we applied a
methodology developed specifically for the assembly of collagen-model,
triple-helical peptides (THPs)(34, 35) . A total of 11
THPs have been synthesized. Cellular recognition was studied by
assaying normal human dermal fibroblast adhesion to these THPs. The
influence of triple-helicity was examined by comparing the activity of
a SSP and THP containing the same collagen-derived sequence. The
potential involvement of integrins in mediating cell adhesion to a
specific THP was evaluated by screening monoclonal antibodies (mAbs)
against integrin subunits as inhibitors of cell adhesion assays.
Materials
All standard peptide synthesis
chemicals and solvents were analytical reagent grade or better and
purchased from Applied Biosystems, Inc. (Foster City, CA) or Fisher
Scientific (Pittsburgh, PA). Fmoc-Gly-Sasrin
resin
(substitution level = 0.7 or 0.65 mmol/g) and Fmoc-Gly-Pro-Hyp
were purchased from Bachem Biosciences Inc. (Philadelphia, PA).
Fmoc-DMPAMP resin (substitution level = 0.46 mmol/g) was from
Novabiochem (La Jolla, CA) and Fmoc-Met, Fmoc-Hyp(tBu), and
Fmoc-Ahx from Advanced ChemTech (Louisville, KY). All other Fmoc-amino
acid derivatives were from Novabiochem or Perseptive Biosystems
(Framingham, MA). All amino acids are of L-configuration. HOBt
was purchased from Novabiochem, HBTU from Richelieu Biotechnologies
(St. Hyacinthe, Quebec), DIEA from Fisher Scientific, and DBU and
DIPCDI from Aldrich. The synthesis and characterization of a generic
THP containing 8 repeats of Gly-Pro-Hyp (designated GPP*) has been
described previously (34) . Monoclonal antibody (mAb) P5D2 was
prepared against the integrin subunit using methods
described previously (36) . A mAb prepared against the
integrin subunit (5E8D9) was obtained from Upstate
Biotechnology (Lake Placid, NY). mAbs prepared against the integrin
subunits (P1E6), (P1B5), and
(P1D6) were purchased from Chemicon International
(Temecula, CA). The anti- mAb was obtained as ascites
fluid (
2.5 mg of IgG/ml of ascites), while the anti- mAb was obtained as both ascites fluid (2.5 mg of IgG/ml of
ascites) and purified IgG; all other mAbs were purified IgGs. Purified
normal mouse IgG was purchased from Organon Teknika Corp. (Durham, NC). Preparation of
(N-Tris(Fmoc-Ahx)-Lys-Lys)-Tyr(tBu)-Gly-Sasrin Resin
0.97 g of
Fmoc-Tyr(tBu) (2.1 mmol), 1.96 g of Fmoc-Lys(Dde) (2.1 mmol),
and 1.96 g of Fmoc-Lys(Dde) (2.1 mmol) were sequentially coupled to 1.0
g of Fmoc-Gly-Sasrin resin (substitution level =
0.7 mmol) in a shaker. Fmoc-protected amino acids were preactivated
with 0.32 g of HOBt (2.1 mmol) and 0.33 ml of DIPCDI (2.1 mmol) in 15
ml of DMF at room temperature for 15 min. The preactivated amino acid
solution was then added together with 0.30 ml of DIEA (2.1 mmol) to the
resin, and coupling proceeded for 1 h at room temperature. Fmoc removal
was by two treatments with 20 ml of piperidine-DMF (1:4) for 3 and 10
min. After coupling of the second Fmoc-Lys(Dde) and following Fmoc
removal, cleavage of Dde protecting groups was achieved with 15 ml of
hydrazine-DMF (1:49) for 2 h. 2.23 g of Fmoc-Ahx (6.3 mmol) was then
coupled for 45 min to the free N
- and N
-amino groups after preactivation with 0.97 g
of HOBt (6.3 mmol) and 0.99 ml of DIPCDI (6.3 mmol) and following
addition of 0.89 ml of DIEA (6.3 mmol). The peptide-resin was washed
three times with DMF. After each coupling and deprotection step, the
completion of the reaction was examined by the ninhydrin test (37) . resin (substitution level = 0.65
mmol/g) with 0.26 g of HOBt (1.95 mmol), 0.67 g of HBTU (1.76 mmol),
and 0.63 ml of DIEA (3.71 mmol) in DMF without preactivation. The
coupling was complete within 1 to 2 h. Fmoc and Dde removal were as
described above. 2.07 g of Fmoc-Ahx (5.85 mmol) was coupled for 2 h to
the liberated N
- and N-amino groups by using 0.82 g of HOBt (6
mmol), 2.00 g of HBTU (5.3 mmol), and 1.90 ml of DIEA (11.05 mmol). 
= 834.4 Da by ESMS and (M +
H) = 834.6 Da by FABMS (theoretical (M +
H) = 834.6 Da).Triple-helical Peptide Synthesis and
Purification
Incorporation of individual amino acids onto the
branched peptide resin was performed initially by Fmoc solid-phase
methodology on a Gilson AMS 422 Automated Multiple Peptide Synthesizer.
The cycles for the Gilson AMS 422 were modified from those described
previously (38) by using double couplings with 6-fold excess of
Fmoc-amino acids. Addition of the 6 final repeats of Gly-Pro-Hyp was
done manually in a shaker vessel. The couplings of the tripeptides to
0.11 g of peptide-resin (substitution = 0.26 mmol/g) were
performed with 57 mg of Fmoc-Gly-Pro-Hyp (0.112 mmol), 17 mg of HOBt
(0.112 mmol), 32 mg of HBTU (0.1 mmol), and 30 µl of DIEA (0.212
mmol) in DMF for 2-3 h. Deprotection of the Fmoc-peptide-resin
was performed with DBU-piperidine-DMF (1:1:48) for 30 min, and the
resin was subsequently washed three times with DMF.1(I)772-786 THP was on an Applied
Biosystems 431A Peptide Synthesizer. Peptide assembly, including final
stepwise incorporation of individual Fmoc-Gly, Fmoc-Pro, and
Fmoc-Hyp(tBu) residues, was performed by Fmoc solid-phase
methodology as described (34) with several modifications.
Coupling utilized 0.45 M HBTU, 0.50 M HOBt, and 0.95 M DIEA in DMF for 1 h, while Fmoc removal was achieved with
0.1 M HOBt in piperidine-1-methyl-2-pyrrolidinone (1:4) for 24
min and 6 min. The following cleavage procedure was applied for the
large-scale synthesis of 1(I)772-786 THP and was modified
for other THPs according to their side-chain protection(39) .
203 mg of the peptide-resin was Fmoc-deprotected with
DBU-piperidine-DMF (1:1:48). Side-chain deprotection and cleavage was
by treatment with water/thioanisole/TFA (1:1:18). The crude
1(I)772-786 THP was precipitated with methyl t-butyl ether, lyophilized, and 70 mg (from a total of 100 mg)
was dissolved in 2.0 ml of water.
column (15-20 µm particle size, 300 Å
pore size, 250 
22 mm) at a flow rate of 5.0 ml/min. The elution
gradient was 0-100% B in 100 min where A was 0.1% TFA in water
and B was 0.1% TFA in acetonitrile. Detection was at 229 nm. A second
purification was performed on the same system with a semipreparative
Vydac 219TP510 diphenyl column (5 µm particle size, 300 Å
pore size, 250 10 mm) at a flow rate of 2 ml/min. The elution
gradient was 0-70% B in 70 min with the described eluents. The
purification of 1(I)772-786 THP was described
recently(40) ; yield was 18.1 mg (15.2% of theoretical).Single-stranded Peptide Synthesis
A peptide
representing the 1(I)772-786 sequence of type I collagen was
synthesized on Fmoc-DMPAMP resin (substitution level = 0.46
mmol/g) using Fmoc methodology on an Applied Biosystems 431A Peptide
Synthesizer(41, 42) . Following synthesis, the
peptide-resin was cleaved with
ethanedithiol/thioanisole/phenol/water/TFA (2.5:5:5:5:82.5) (39) and purified by RP-HPLC(42) . Analysis by FABMS
gave (M + H) = 1420.4 Da (theoretical (M
+ H) = 1420.6 Da).Peptide Analysis
Analytical RP-HPLC was performed
on a Hewlett-Packard 1090 Liquid Chromatograph equipped with a Vydac
219TP54 diphenyl column (5 µm particle size, 300 Å pore size,
250 4.6 mm). The flow rate was 1.0 ml/min. Eluants were 0.1%
TFA in water (A) and 0.1% TFA in acetonitrile (B). The elution gradient
was 0-50% B in 50 min with a flow of 1 ml/min. Detection was at
229 nm.
]) in the range of 10-80 °C at
= 225 nm.Cell Adhesion Assays
Adhesion assays were as
described previously (44) with some alterations. Normal human
dermal fibroblasts (NHDF) (Clonetics, San Diego, CA) were cultured on
fibroblast growth medium from the same company. Cell attachment assays
were performed in 96-well polystyrene Immulon 1 microtiter plates
(Dynatech Laboratories Inc., Chantilly, VA). Peptides were dissolved in
PBS and absorbed onto wells overnight at 37 °C in a humidified
incubator with 5% (v/v) CO. Cells were labeled overnight
with 200 µCi/ml 
S (DuPont NEN). Nonspecific binding
sites were blocked with 2 mg/ml bovine serum albumin (Pentax, Miles
Laboratories, Naperville, IL) in PBS for 2 h at 37 °C. NHDF cells
were detached with trypsin/EDTA solution and, after release of the
cells, the flask was rinsed quickly with HEPES-buffered saline solution
and trypsin neutralizing solution (all from Clonetics). The cells were
resuspended to 5 10
/ml in fibroblast growth medium.
Aliquots of 75 µl of the cell suspension were added to the plate
wells and allowed to adhere for 30 min at 37 °C. For adhesion
assays, wells were washed 3 times with 2 mg/ml bovine serum albumin in
Ca
/Mg PBS. 150 µl of
Microscint-40 (Packard Instrument Co.) were added and the plates were
read on a Beckman LS 6500 scintillation counter (Beckman Instruments).
Adhesion percentages were based on total counts of radioactivity added
to each well., , ,
, and integrin subunits to determine
the cell surface receptor recognizing the 1(I)772-786 THP.
Competition of cell adhesion was performed on surfaces coated with 5
µg/ml type I collagen (half-maximal cell adhesion) or with 10
µM 1(I)772-786 THP using methods described
previously(44, 45) . Cells were preincubated for 20 or
30 min at 37 °C with various concentrations of potential inhibitory
peptides or antibodies. The cells were then, in the continued presence
of the potential inhibitor, added to the wells and allowed to adhere
for 30 min at 37 °C. The cells remained viable in the inhibitory
peptide-cell solution based upon exclusion of trypan blue dye.
Synthesis and Cellular Activities of Crude
Triple-helical Type I Collagen Model Peptides
Four criteria were
used for the identification of potential active sequences from collagen
1(I). First, sequences would be from CB fragments that contained
integrin binding sites (1(I)CB3, 1(I)CB7, and 1(I)CB8)).
Second, sequences containing charge clusters were considered, as
several active sequences from type IV collagen have a clustering of
charged residues (35, 44, 46, 47, 48) .
Third, sequences described previously to have binding activities, such
as 1(I)85-96(49) ,
1(I)433-441(20) , and 1(I)769-783 (17) were utilized. Fourth, sequences were 9-15 residues
in length, as prior studies on type IV collagen had shown this length
to be sufficient to establish cellular
activities(35, 44) . For initial screening purposes,
10 sequences from the 1(I) chain (50, 51, 52, 53) were examined (Table 1). THPs containing these sequences were assembled using a
methodology for the solid-phase synthesis of branched triple-helical
peptides(34, 35) . Three nascent peptide chains were
carboxyl-terminally linked through one N
-amino
and two N-amino groups of Lys and stabilized
by 6 repeats of Gly-Pro-Hyp. Edman degradation sequence analysis
indicated that each of the 10 crude THPs contained at least 50% of the
desired product.
1(I)771-786 showed
substantial (50%) cell adhesion activity (Fig. 1). None of
the other crude THPs showed >10% cell adhesion activity (Fig. 1). The high adhesion of NHDFs to the
1(I)772-786 THP was thus examined further.
Synthesis and Characterization of
The 1(I)772-786
THP1(I)772-786 THP was resynthesized on a larger
scale to allow for extensive structural and biological
characterization. Several modifications of our previously described
procedure (34, 35) were used. The final 6 repeats of
Gly-Pro-Hyp, which had previously been coupled manually using
Fmoc-Gly-Pro-Hyp(34, 35) , were added stepwise (as
Fmoc-Gly, Fmoc-Pro, and Fmoc-Hyp(tBu)) using an automated
peptide synthesizer. The THP was purified by a two-step method
developed recently(40, 54) . Edman degradation
analysis of the purified 1(I)772-786 THP gave the sequence
(Gly-Pro-Hyp)
-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg-Gly-Val-Val-Gly-Leu-Hyp.
Laser desorption mass spectrometry analysis gave (M +
K) = 9868.7 Da (theoretical (M +
K) = 9865 Da). During the purification of
1(I)772-786 THP, we isolated a peptide which, by Edman
degradation analysis, had the sequence
(Gly-Pro-Hyp)-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg-Gly-Val-Val (40) . This peptide was missing the first three amino acids
next to the branch (1(I) residues 784-786) and was thus
designated 1(I)772-783 THP.1(I)772-786 THP and 1(I)772-783
THP was evaluated by CD spectroscopy. At low temperatures,
1(I)772-786 THP exhibited typical features of a
collagen-like conformation (Fig. 2), such as a large negative
molar ellipticity ([
]) at = 200 nm and a
positive [
] at = 225 nm(55) . As
the temperature was increased, [
]
increased whereas [
]
decreased (Fig. 2). The negative [
] at high
temperatures indicates a melted triple-helix. The
1(I)772-783 THP had similar CD spectral characteristics
(data not shown). The thermal stabilities of 1(I)772-786 THP
and 1(I)772-783 THP were studied by measuring
[
] as a function of temperature. When the
temperature was increased from 10 °C to 80 °C,
1(I)772-786 THP showed a structural transition (triple-helix
⇔ coil) with a midpoint (T
) of 43 °C (Fig. 3). The 1(I)772-783 THP also showed a
transition over this temperature range, with T = 36 °C (Fig. 3). Both THPs had sufficiently
stable triple-helical structures to allow for cellular assaying.
1(I)772-786 THP in acetic acid/water (1:99) at 10 °C and
80 °C. Spectra were recorded at 1(I)772-786 THP =
0.13 mM.
1(I)772-786 THP (solid line) and
1(I)772-783 THP (dashed line) in acetic acid/water
(1:99) at 1(I)772-786 THP = 0.13 mM and
1(I)772-783 THP = 0.11 mM. Molar
ellipticities ([
]) were recorded at = 225
nm while the temperature was increased from 10 °C to 80
°C.
Adhesion of Normal Human Dermal Fibroblasts to
Biological studies
proceeded with the following peptides: 1(I)772-786 THP and Analogs1(I)772-786 THP,
1(I)772-783 THP, 1(I)772-786 SSP, and GPP* (see Fig. 4for structures). Cellular recognition of the
1(I)772-786 sequence was studied initially by comparing NHDF
adhesion to the crude and purified 1(I)772-786 THP over a
concentration range of 0.2-10 µM (Fig. 5).
Both the crude and purified 1(I)772-786 THP showed
concentration-dependent promotion of NHDF adhesion. At a peptide
concentration of 2 µM, the crude THP promoted cell
adhesion to a level of 26% while the purified THP promoted cell
adhesion to a level of 52% (Fig. 5). Purification of the
1(I)772-786 THP thus enhanced the promotion of NHDF
adhesion. Interestingly, the purified peptide containing the
carboxyl-terminal deletion of 3 amino acids (1(I)772-783
THP) had low levels (<10%) of adhesion-promoting activity over the
concentration range of 0.2-10 µM (Fig. 5).
1(I)772-786 THP,
1(I)772-783 THP, 1(I)772-786 SSP, and GPP*. The
THP is composed of a carboxyl-terminal branch generated from 2 Lys
residues, one 1(I)772-786 sequence per chain and six
Gly-Pro-Hyp repeats per chain. GPP* is composed of the
carboxyl-terminal branch and eight Gly-Pro-Hyp repeats per
chain.
1(I)772-786 THP (closed circles), purified
1(I)772-786 THP (closed squares), and purified
1(I)772-783 THP (closed triangles). Cells were
allowed to adhere to peptide-coated Immulon 1 plates for 30 min at 37
°C. All assays were repeated in at least triplicate. Conditions are
given under ``Experimental
Procedures.''
1(I)772-786 THP to the 1(I)772-786 SSP (Fig. 6). NHDFs showed a profound preference for binding and
adhesion to the THP compared with the SSP. Half-maximal fibroblast
cellular adhesion occurred at a THP concentration of 1.6
µM, while less than 10% cell adhesion was seen for the SSP
up to a concentration of 10 µM. At a peptide concentration
of 100 µM, cell adhesion to the SSP was only 40% of
the level promoted by the THP (data not shown). There was no NHDF
adhesion to the generic THP containing 8 repeats of Gly-Pro-Hyp (GPP*) (Fig. 6).
1(I)772-786 THP (closed squares),
1(I)772-786 SSP (closed diamonds), and GPP* (closed circles). Cells were allowed to adhere to
peptide-coated Immulon 1 plates for 30 min at 37 °C. All assays
were repeated in at least triplicate. Conditions are given under
``Experimental Procedures.''
Inhibition of Cell Adhesion to
The inhibition of NHDF cell adhesion to
1(I)772-786 THP
by Anti-integrin mAbs1(I)772-786 THP was compared for different anti-integrin
mAbs (Table 2). Initial experiments compared inhibition of
adhesion by normal mouse IgG and the anti- and
anti- mAbs at varying THP coating concentrations.
Inhibition of adhesion was seen for the anti- mAb at
[THP] = 2.0, 5.1, and 10 µM. The
anti- mAb results were somewhat inconclusive. Studies
at [THP] = 10 µM suggested that the
anti- and anti- may inhibit adhesion
to the 1(I)772-786 THP and that the anti- integrin subunit mAb was not an inhibitor.
Inhibition of Cell Adhesion to Type I Collagen
To
determine the ability of 1(I)772-786 SSP and
1(I)772-786 THP to inhibit cell adhesion to type I collagen,
NHDFs were incubated with increasing peptide concentrations in the
range of 0.5-10 µM. Neither the
1(I)772-786 SSP nor the 1(I)772-786 THP inhibited
NHDF adhesion to type I collagen in a concentration-dependent fashion
(data not shown). In contrast to the SSP and THP, type I collagen
inhibited cell adhesion in a concentration-dependent fashion (data not
shown).
and integrin binding sites are dependent upon triple-helical
conformation (56) and contained within 1(I)CB3,
1(I)CB7, and 1(I)CB8 (encompassing residues 124-822
from
1(I))(2, 12, 13, 15, 16) .
From the 124-822-residue region, we selected sequences that
contained ``clusters'' of charged residues. Charged residues
are often found in clusters in type I collagen(57) , and
cellular activities have been ascribed previously to collagen-derived
synthetic peptides that have clustered charged
residues(35, 44, 46, 47, 48) .
Seven THPs were synthesized based on charge clustering
(1(I)256-270, 1(I)385-396,
1(I)406-417, 1(I)415-423, 1(I)448-456,
1(I)496-507, and 1(I)526-537). Three additional
THPs were synthesized (1(I)85-96, 1(I)433-441,
and 1(I)772-786) based on previously described or proposed
activities(17, 20, 49) .1(I)772-786 THP had the greatest cell adhesion-promoting
activity. The other 9 THPs exhibited low levels of activity (<10%),
similar to the generic triple-helical peptide GPP*. Although the other
THPs may represent active sequences, only the 1(I)772-786
THP was pursued in this study.1(I)772-786 THP proceeded as described
for other THPs(34) , with two important modifications. First,
the (Gly-Pro-Hyp) region of the THP was assembled stepwise
using individual Fmoc-amino acids, not Fmoc-Gly-Pro-Hyp tripeptide
blocks. Stepwise assembly had not been possible previously using
Fmoc-Hyp, but was successful with the Fmoc-Hyp(tBu) derivative
used here. We believe that tBu side-chain protection of Hyp
minimizes interstrand hydrogen bonding. Interstrand hydrogen bonding
can be detrimental for efficient peptide assembly (for a recent review,
see (58) ). Second, a recently developed two-step RP-HPLC
method was used for the purification of 1(I)772-786 THP (40) which also allowed for the isolation of the deletion
peptide 1(I)772-783 THP.1(I)772-786 THP
was highly active, with half-maximal cell adhesion occurring at a
peptide concentration of 1.6 µM. Triple helicity was
essential for activity of this sequence, as the non-triple-helical
peptide analog (1(I)772-786 SSP) exhibited considerably
lower levels (40%) of cell adhesion even at peptide concentrations
as high as 100 µM. The triple-helical dependence for cell
binding to the 1(I)772-786 sequence is even more pronounced
than for the 1(IV)1263-1277 sequence described
previously(35) . Within the 1(I)772-786 sequence
itself, residues 784-786 (Gly-Leu-Hyp) were important for
cellular recognition, as the 1(I)772-783 THP had greatly
reduced cell adhesion activity compared with 1(I)772-786
THP.1(I)772-786 THP is inhibited
by an anti- integrin subunit mAb. It thus appears that
an integrin mediates NHDF binding to 1(I)772-786 THP. Our
preliminary results were inconclusive, suggesting the
, ,
or integrin may be involved. It has
been shown that fibroblasts use the integrin for collagen, but not laminin, binding(59) .
Binding of the integrin to the
1(I)CB7 fragment (residues 552-822) is conformationally
dependent(13) , consistent with the conformationally dependent
binding of NHDF to 1(I)772-786 THP. Alternatively,
fibronectin has been proposed as a ``bridge'' for ovarian
cell binding to the 1(I)757-791 sequence(17) . MMP-1
cleavage of the 775-776 bonds or mutation of 1(I)
Gln
and Ala
to Pro dramatically alters
fibronectin binding to type I collagen(17, 60) . The
can mediate chondrosarcoma cell
binding to denatured type II collagen via a fibronectin
bridge(21) , and thus an integrin may serve a similar function
for cell binding via a fibronectin bridge to the 1(I)772-786
region of type I collagen. Further investigations are ongoing to
definitively determine the integrin(s) utilized for cellular
recognition of the 1(I)772-786 THP.1(I)772-786 THP to promote cell adhesion
in a concentration-dependent fashion, but not inhibit cell adhesion to
type I collagen. In retrospect, it would have been somewhat surprising
if the 1(I)772-786 THP did inhibit NHDF binding to type I
collagen due to the multiple integrin binding sites within type I
collagen. It is also possible that there are interactions between the
THP and type I collagen. We have previously demonstrated aggregation of
THPs(35) , but have not examined the association of THPs and
collagen. inhibits
phagocytosis and enhances pro-MMP-1 release, while transforming growth
factor- has the opposite effect(4) . There is evidence
that suggests that the 1(I)772-786 sequence mediates both
proposed mechanisms of collagen turnover. We have demonstrated that
fibroblasts bind to the triple-helical 1(I)772-786 sequence.
Internalization of type I collagen by fibroblasts is reduced after
collagen is cleaved at the 775-776 bonds(3) . Thus,
fibroblast phagocytosis of type I collagen appears to include at least
part of the 772-786 region. Several members of the MMP family
(MMP-1, MMP-2, and MMP-8) hydrolyze the triple-helical region of type I
collagen at position 775 in the collagen
chains(27, 63) . Thus, extracellular degradation of
type I collagen occurs within the 772-786 region. This region may
also regulate MMP production. Prior studies have shown that a SSP
incorporating residues 1(I)769-783 supports human fibroblast
adhesion and induces the production of
MMP-1(18, 19, 64) . Although the induction
mechanism is unknown, it may be related to
-mediated binding to type I collagen
which results in tyrosine phosphorylation of pp125
(65) and induction of MMP-1 mRNA levels(66) . If the
integrin is indeed the cell surface
adhesion molecule that binds 1(I)772-786 THP, we would be
able to study a discreet cell signaling mechanism that influences
collagen metabolism. Also, our 1(I)772-786 THP may have even
greater activity for promoting cell signaling and MMP production than
the 1(I)769-783 SSP, as cell adhesion to triple-helical
collagen results in considerable up-regulation of protein synthesis
compared with denatured collagen(67) .
)
-Ahx)-Lys-Lys)-Tyr-Gly; HBTU,
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; mAb, monoclonal
antibody; MMP, matrix metalloproteinase; NHDF, normal human dermal
fibroblasts; PBS, phosphate-buffered saline solution; RP-HPLC,
reversed-phase high performance liquid chromatography; SSP,
single-stranded peptide; tBu, tert-butyl; TFA, trifluoroacetic
acid; THP, triple-helical peptide.
We thank Cynthia G. Fields for Edman degradation
sequence analyses and RP-HPLC assistance.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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