From the Department of Chemistry and
Biochemistry, Florida Atlantic University, Boca Raton, Florida
33431-0991, ¶ Degussa/Rexim SA, 33 Rue de Verdun, F-80400 HAM,
France, and
Degussa AG, Rodenbacher Chaussee 4, D-63457 Hanau-Wolfgang, Germany
Received for publication, December 2, 2002, and in revised form, February 5, 2003
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ABSTRACT |
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Invasion of the basement membrane is believed to
be a critical step in the metastatic process. Melanoma cells have been
shown previously to bind distinct triple-helical regions within
basement membrane (type IV) collagen. Additionally, tumor cell binding sites within type IV collagen contain glycosylated hydroxylysine residues. In the present study, we have utilized triple-helical models
of the type IV collagen Tumor cell invasion, a key step in the metastatic process,
involves a complex series of correlated macromolecular interactions. These include interaction with, and movement through, collagen, most
often type I and/or basement membrane (type IV) collagen. In general,
invasion of the basement membrane is believed to be a critical step in
the metastatic process. Human melanoma cells have been shown to bind
distinct triple-helical regions within type IV collagen (1-5).
Melanoma receptors for triple-helical collagen fall into one of two
categories: members of the integrin heterodimeric protein family
( In addition to the integrin binding sites, the The IV-H1 sequence contains a glycosylated hydroxylysine (Hyl) residue
in position 1265 (28). Hyl is the major glycosylation site within
mammalian collagens. The 5-hydroxyl group may be posttranslationally modified by the monosaccharide galactose
( Prior studies have focused on the carbohydrate structures expressed on
the tumor cell surface (34, 41, 42). Cell surface oligosaccharides may
play a critical role in tumor cell angiogenesis, growth, and metastasis
(43-45). It is presently unknown, but of great interest, as to what
effect ligand glycosylation has on melanoma cell binding and signaling.
The effects of glycosylated triple-helical structure on cellular
systems have not been addressed.
In the present study, we sought to (a) determine the
melanoma cell receptor for triple-helical IV-H1 and (b)
analyze the results of IV-H1 single-site glycosylation on
triple-helical stability and melanoma cell recognition of this ligand.
We have used collagen-model triple-helical peptides of the general
sequence (Gly-Pro-Hyp)n-IV-H1-(Gly-Pro-Hyp)m (where
n = 4 or 6 and m = 4 or 0) for both
goals. Affinity chromatography and melanoma cell adhesion/inhibition
assays were utilized to determine the cellular receptor for
triple-helical IV-H1. To evaluate the effects of glycosylation, the
desired peptides and peptide-amphiphiles were assembled, and
biophysical comparisons were first performed using CD spectroscopy to
determine the conformational effects of single site glycosylation.
Melanoma cell adhesion and spreading on the respective
peptide-amphiphile ligands were then quantitated to evalulate the
biological consequences of glycosylation.
General--
All standard peptide synthesis chemicals
were peptide synthesis grade or better and purchased from
FisherBiotech. HOAt and HATU were purchased from PerkinElmer Life
Sciences, and N,N-diisopropyl-ethylamine was
purchased from Fisher Scientific.
Fmoc-4-((2',4'-dimethoxyphenyl)aminomethyl)phenoxy resin (substitution
level = 0.55 mmol/g), and Fmoc-amino acid derivatives were
purchased from Novabiochem/Calbiochem. Amino acids are of the
L-configuration (except for Gly). Palmitic acid (CH3-(CH2)14-CO2H,
designated C16) was purchased from Fisher.
Purification of 5-Hydroxy-L-lysine--
Hyl was
isolated from porcine gelatin by hydrolysis followed by multiple
passages over an ion-exchange column (46). The aqueous Hyl solution was
acidified with HCl and evaporated to obtain crystals of Hyl. The Hyl
was recrystallized when necessary. Analysis was performed using a
Crownpak CR+ column (150 × 4 mm; Daicel Chemical
Industries, Ltd.) at 0 °C at a flow rate of 0.4 ml/min. The mobile
phase was 0.13 M aqueous perchloric acid. Detection was at
Peptide Synthesis, Purification, and
Characterization--
Fmoc-Hyl(
RP-HPLC purification was performed on a Rainin AutoPrep system.
Peptides were purified with a Vydac 218TP152022 C18
column (15-20-µm particle size, 300-Å pore size, 250 × 25 mm)
at a flow rate of 5.0 ml/min. The elution gradient was 0-85% B in 85 min where A was 0.1% trifluoroacetic acid in water and B was 0.1% trifluoroacetic acid in acetonitrile. Detection was at
MALDI-MS was performed on a Hewlett-Packard G2025A LD-TOF mass
spectrometer using either a sinapinic acid or 2,5-dihydroxybenzoic acid/2-hydroxy-5-methoxy-benzoic acid (9:1, v/v) matrix (54). Peptide
mass values were as follows:
(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2, [M+H]+ 3573.3 Da (theoretical 3574.9 Da);
Hyl(Gal)1265-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2,
[M+H]+ 3752.4 Da (theoretical 3751.1 Da);
C16-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2, [M+H]+ 3811.5 Da (theoretical 3813.3 Da);
C16-Hyl(Gal)1265-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2,
[M+H]+ 3992.0 Da (theoretical 3989.1 Da); and
C16-Abu1267-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2,
[M+H]+ 3781.9 Da (theoretical 3783.4 Da).
Carbazole Test--
A sample of peptide was dissolved in 85%
H2SO4 and heated at 80 °C for 35 min. After
cooling, carbazole (1 mg/ml in ethanol) was added and incubated at room
temperature for 2 h. The presence of carbohydrate was monitored by
an increase in absorbance at CD Spectroscopy--
CD spectra were recorded over the range
Cells--
SK-Mel2, M14P, M14#5, and M14#11 human
melanoma cells were propagated as described previously (2, 4, 11).
Briefly, melanoma cells were cultured in Eagle's minimum essential
medium or RPMI 1640 supplemented with 10% fetal bovine sera, 1 mM sodium pyruvate, 0.1 mg/ml gentamycin (Roche Molecular
Biochemicals), 50 units/ml penicillin, and 0.05 mg/ml streptomycin.
Cells were passaged eight times and then replaced from frozen stocks of
early passage cells to minimize phenotypic drift. All cells were
maintained at 37 °C in a humidified incubator containing 5%
CO2. All media reagents were purchased from Fisher Scientific.
ELISA Analysis of CD44 or Cell Adhesion Assays--
Melanoma cell adhesion to
substrate-coated Pro-BindTM 96-well plates (BD Biosciences)
was performed as described previously (4). Peptide-amphiphiles
dissolved in PBS were diluted in 70% ethanol and added to the 96-well
plate and allowed to adsorb overnight at room temperature with mixing.
Nonspecific binding sites were blocked with 2 mg/ml BSA in PBS for
2 h at 37 °C. Cells were released with 5 mM EDTA in
PBS, washed 2× with adhesion medium (20 mM HEPES, 2 mg/ml
albumin in Eagle's minimum essential medium or RPMI 1640), and labeled
with 5- or 6-carboxyfluorescein diacetate. Unincorporated fluorophore
was removed by repeated washings with adhesion medium. Cells were then
resuspended in adhesion medium and added to the plate. The plate was
incubated for 60 min at 37 °C. Non-adherent cells were removed by
washing three times with adhesion medium. Adherent cells were lysed
with 0.2% SDS and quantitated with a SpectraMAX Gemini, 96-well plate
spectraflurometer (Molecular Devices, Sunnyvale, CA).
Inhibition of Cell Adhesion Assays--
Cells were labeled with
5- or 6-carboxyfluorescein diacetate, and Pro-BindTM 96-well
plates were coated with peptides or proteins as in the adhesion assay.
The cells were preincubated with various concentrations of mAbs
(Chemicon, Temecula, CA), GAGs, or chondroitinase ACII in the presence
of 10 µg/ml aprotinin, leupeptin, pepstatin A, and phenyl methyl
sulfonyl fluoride for 60 min at 37 °C after they had been harvested.
The cells were added to the wells to evaluate the adhesion to coated
peptides or proteins in the continued presence of mAbs or GAGs. Cells
were allowed to adhere for 30 min at 37 °C, and cell adhesion was
quantified as in the adhesion assay.
Cell Spreading Assays--
These assays are performed exactly as
the adhesion assays with the exception of the last step, cell lysis.
After washing unbound cells, the remaining cells are fixed with 2.5%
glutaraldehyde dissolved in formalin and stained with R-250 Coomassie
Blue. Digital photos of each well are taken, and the area of the cells
is quantitated with the assistance of Quantity One Software
(Bio-Rad).
Affinity Chromatography and Immunoprecipitation and Blotting
Analysis--
Branched Affinity Chromatography and GAG Analysis--
Branched
Construction and Characterization of Ligands
Design of Potential Ligands--
To determine the receptor binding
to the Construction and Characterization of Glycosylated
Peptides--
Pure L-Hyl was prepared from porcine gelatin
by hydrolysis and ion-exchange chromatography. The
Fmoc-Hyl( Biophysical Characterization of Potential Ligands--
CD spectra
characteristic of triple helices exhibit a positive molar ellipticity
at
To assess the biological effects of glycosylation, we prepared the
peptide-amphiphile models of
(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2, Abu1267-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2,
and
Hyl(Gal)1265-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2.
Prior work has shown that construction of peptide-amphiphiles,
whereby an alkyl chain is incorporated onto the N terminus of a
peptide, results in enhanced thermal stability of peptide conformation
and improved binding to hydrophobic surfaces (3, 14, 18, 19, 57).
The melting temperatures of the peptide-amphiphiles were 45.0 and
48.5 °C for
C16-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
and
C16-Hyl(Gal)1265-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2,
respectively (Table I). These Tm values are
sufficient for analysis of cellular activities. The
Tm of 45.0 °C for
C16-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2 is considerably lower than the Tm value reported
previously for this peptide-amphiphile (19). However, the
peptide-amphiphile concentration for the earlier study was 0.5 mM (19), which causes more extensive aggregation and a
correspondingly higher Tm value (57). The
peptide-amphiphile concentration used for the CD analysis described
herein (14 µM) approximates the concentration range
required for biological studies (see below).
Evaluation of the Melanoma Receptor for Triple-helical
Melanoma receptors for triple-helical collagen include members of
the integrin heterodimeric protein family
(1(IV)1263-1277 sequence to (a) determine the melanoma cell receptor for this ligand and
(b) analyze the results of single-site glycosylation on
melanoma cell recognition. Receptor identification was achieved by a
combination of methods, including (a) cell adhesion and
spreading assays using triple-helical
1(IV)1263-1277 and an
Asp1266Abu variant, (b) inhibition of cell
adhesion and spreading assays, and (c) triple-helical
1(IV)1263-1277 affinity chromatography with whole cell lysates and
glycosaminoglycans. Triple-helical
1(IV)1263-1277 was bound by
melanoma cell CD44/chondroitin sulfate proteoglycan receptors
and not by the collagen-binding integrins or melanoma-associated
proteoglycan. Melanoma cell adhesion to and spreading on the
triple-helical
1(IV)1263-1277 sequence was then compared for
glycosylated (replacement of Lys1265 with
Hyl(O-
-D-galactopyranosyl))
versus non-glycosylated ligand. Glycosylation was found to
strongly modulate both activities, as adhesion and spreading were
dramatically decreased due to the presence of galactose.
CD44/chondroitin sulfate proteoglycan did not bind to glycosylated
1(IV)1263-1277. Overall, this study (a) is the first
demonstration of the prophylactic effects of glycosylation on tumor
cell interaction with the basement membrane, (b) provides a
rare example of an apparent unfavorable interaction between
carbohydrates, and (c) suggests that sugars may mask
"cryptic sites" accessible to tumor cells with cell surface
or secreted glycosidase activities.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1
1,
2
1,
and
3
1 integrins) or cell surface
proteoglycans (such as CD44 and melanoma-associated proteoglycan/melanoma chondroitin sulfate proteoglycan
(MPG/MCSP/NG2)).1 Specific
ligands from type IV collagen have been described for the
1
1,
2
1, and
3
1 integrins. The
1
1 integrin simultaneously binds
Asp441 from two
1(IV) chains and Arg458 from
the
2(IV) chain (6, 7). The Gly-Phe-Hyp-Gly-Glu-Arg motif, in
triple-helical conformation, has been shown to bind to the
2
1 integrin (8-10). This motif is found
within type IV collagen at
1(IV)405-410; a triple-helical model of
1(IV)402-413 is bound by melanoma
cells.2 The melanoma cell
3
1 integrin binds to
1(IV)531-543 (2, 4, 11).
1(IV)1263-1277
region from type IV collagen (gene-derived sequence
Gly-Val-Lys-Gly-Asp-Lys-Gly-Asn-Pro-Gly-Trp-Pro-Gly-Ala-Pro, designated
IV-H1), promotes melanoma cell adhesion, spreading, and signaling (1,
3, 12-14). Affinity chromatography studies with a single-stranded
IV-H1 peptide resulted in the isolation of melanoma cell CD44
receptors, in the chondroitin sulfate proteoglycan (CSPG) form (15,
16). Subsequently, several triple-helical constructs incorporating the
IV-H1 sequence have been described (1, 14, 17). One of these, a
"peptide-amphiphile" of general structure
Cn-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2,
has undergone extensive biophysical characterization by CD and one- and
two-dimensional NMR spectroscopies (18-20). The IV-H1 region within
the peptide-amphiphile forms a continuous triple helix (19, 20). Loss
of triple-helical structure dramatically reduces melanoma cell
adhesion, spreading, and signaling modulated by this ligand (1, 14,
17). However, whether the triple-helical IV-H1 ligand is bound by CD44,
in analogous fashion to the linear version, has not been addressed.
Cells may engage different receptors depending upon the conformational
state of collagen (21-27).
-D-galactopyranosyl) or the disaccharide
glucosegalactose
(
-D-glucopyranosyl-(1
2)-
-D-galactopyranosyl) (29, 30). Interest in glycosylation of collagen stems from the recent
reports of activation of specific receptor tyrosine kinases by
glycosylated type I collagen (31), T-cell recognition of a glycosylated
sequence within type II collagen (32), and the identification of
melanoma and breast carcinoma binding sites within type IV collagen
that contain glycosylated Hyl residues (1, 2, 4, 11, 12, 14, 33). Most
secreted and cell surface eukaryotic proteins are found glycosylated
in vivo. Glycosylation is believed to have three important
biological roles (34-38). First, glycosylation can serve as a
recognition marker for a cell, both in the context of cell-cell and
cell-extracellular-matrix interactions. A second role concerns the
alteration glycosylation has on the physical properties of the protein.
Frequently, glycosylation will render a protein resistant to
hydrolysis, significantly increase solubility of a protein, or even
drastically affect the overall folding and/or physical bulk of a
protein. In the case of collagen-like triple helices, the addition of
-D-galactose to Thr in the Yyy position of either
(Gly-Pro-Yyy)10 or (Gly-Hyp-Yyy)10 greatly enhances triple-helical stability compared with Thr alone (39, 40). A
third role for glycosylation is in signal transduction and may be
analogous to or compete with protein phosphorylation (37). For example,
addition of O-linked
-N-acetylglucosamine to
insulin receptor substrate-1 and -2 apparently decreases
phosphorylation and affects insulin-mediated homeostatsis.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
= 200 nm.
-Boc,O-(2,3,4,6-tetra-O-acetyl-
-D-galactopyranosyl)),
branched
1(IV)1263-1277 THP, and branched
Hyl(Gal)1265-
1(IV)1263-1277 THP were prepared as
described previously (47, 48). Branched THP compositions were confirmed
by MALDI-MS analysis of the branch and Edman degradation sequence
analysis of the intact THP (47, 49). All other peptides were
synthesized as C-terminal amides to prevent diketopiperazine formation
(50). Peptide-resin assembly was performed by Fmoc solid-phase
methodology on a PerkinElmer Life Sciences/ABD 433A peptide
synthesizer by methods described previously in our laboratory (18, 19).
For incorporation of the glycosylated amino acid, the
H2N-peptidyl-resin was removed from the instrument.
Fmoc-Hyl(
-Boc,O-(2,3,4,6-tetra-O-acetyl-
-D-galactopyranosyl)) was coupled manually in an orbital shaker using 3-fold molar excesses of Fmocamino acid and HOAt, a 2.7-fold molar excess of HATU, and a
6-fold molar excess of N,N-diisopropyl-ethylamine in 10 ml
of N,N-dimethylformamide for 18 h.
Subsequent amino acids were coupled on the instrument. Peptide-resins
were characterized by Edman degradation sequence analysis as described
previously for "embedded" (non-covalent) sequencing (51) on an
Applied Biosystems 477A protein sequencer/120A analyzer. Peptide-resins
were then either (a) cleaved or (b) acylated with
the C16 alkyl tail (19) and then cleaved. Cleavage and
side-chain deprotection of the peptide-resin proceeded for 2 h
using ethanedithiol/thioanisole/phenol/water/trifluoroacetic acid
(2.5:5:5:5:82.5) as described (52). The cleavage solution was extracted
with methyl tert-butyl ether prior to purification. The
glycosylated peptide was deacetylated with methanolic sodium methoxide
(2 M) for 1 h at 20 °C (53).
= 229 nm.
Analytical RP-HPLC was performed on a Hewlett-Packard 1100 liquid
chromatograph equipped with an ODS Hypersil C18 RP column (5-µm particle size, 120-Å pore size, 100 × 2.1 mm). Eluants
were the same as for peptide purification. The elution gradient was 0-100% B in 30 min with a flow of 0.3 ml/min. Diode array detection was at
= 220, 254, and 280 nm.
= 490 nm (55).
= 190-250 nm on a JASCO J-600 using a 10-mm path length
quartz cell. The peptide concentration (14 µM in water)
was kept constant for all the experiments. Thermal transition curves
were obtained by recording the molar ellipticity ([
]) at
= 225 nm, whereas the temperature was continuously increased in the
range of 5-80 °C at a rate of 12 °C/h. Temperature was
controlled using a JASCO PTC-348WI temperature control unit. For
samples exhibiting sigmoidal melting curves, the reflection point in
the transition region (first derivative) is defined as the melting
temperature (Tm). Alternatively,
Tm was evaluated from the midpoint of the transition.
1 Integrin Subunit
Concentration--
The cell surface CD44 or
1 integrin
subunit concentration was evaluated for M14P, M14#5, and M14#11 human
melanoma cells by ELISA. Briefly, cells were diluted in PBS and plated
at various concentrations on a 96-well plate. The plate was incubated
at 4 °C for 2 h, and the PBS was removed. The cells were fixed
with methanol, and the plate was blocked with BSA at 4 °C.
Anti-CD44 mAb, anti
1-integrin subunit mAb, or an
equivalent concentration of IgG was diluted in PBST (PBS with 0.05%
Tween 20) containing 2 mg/ml BSA and incubated at 4 °C. The plate
was washed with PBST and subsequently incubated with goat anti-mouse
IgG conjugated to horseradish peroxidase in PBST and 2 mg/ml BSA. The
plate was washed and horseradish peroxidase detected using
3,3',5,5'-tetramethylbenzidine (Pierce).
1(IV)1263-1277 THP or branched
Hyl(Gal)1265-
1(IV)1263-1277 THP was coupled to
activated CH-Sepharose according to the instructions of the supplier
(Amersham Biosciences). In addition, a mock-coupled column was made
without the peptide. M14 and SK-Mel2 melanoma cells were extracted in
OGS lysis buffer (50 mM Tris·HCl, 15 mM NaCl,
pH 7.2, 0.5 mM CaCl2, 0.5 mM
MnCl2, 1 µM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml pepstatin A, 10 µg/ml leupeptin, and 50 mM OGS) by shaking 30 min at
4 °C. The lysates were cleared by centrifugation at 36,500 × g for 60 min at 4 °C. Cell lysates were shaken with the
mock beads for 4 h at 4 °C. The unbound materials were
collected and incubated with the peptide-Sepharose beads by rocking
overnight at 4 °C. The beads were washed with 3 volumes of OGS lysis
buffer, and the
1(IV)1263-1277 THP-bound proteins were eluted with
IP lysis buffer (0.25% Triton X-100, 75 mM NaCl, 25 mM Tris·HCl, 0.5 mM vanadate, 2.5 mM EDTA) supplemented with 50 mM EDTA and 1 M NaCl. The eluate was concentrated, and the buffer was
exchanged with IP lysis buffer using Microsep centrifugal concentrators. The samples were then immunoprecipitated with 5 µg/ml
anti-CD44 (Zymed Laboratories Inc., South San
Francisco, CA) or anti-
1 integrin subunit (Chemicon)
mAbs or mouse IgG (Chemicon). The CD44 samples were digested with 2.5 units/ml chondroitinase ACII for 3 h at 37 °C. All samples
were electrophoresed on a 4-20% gradient polyacrylamide gel (Bio-Rad)
and transferred to nitrocellulose (Micron Separations, Inc, Westboro,
MA). The nitrocellulose was incubated in TBST (10 mM
Tris·HCl, pH 7.6, 200 mM NaCl, 0.5% Tween 20) with 2%
BSA for at least 4 h and incubated in anti-CD44 or anti-
1 mAb or mouse IgG diluted in TBST with 2% BSA
overnight at 4 °C. The membrane was washed with TBST and incubated
with fluorescein-conjugated secondary antibody diluted in TBST with 2%
BSA for 1 h and washed with TBST. Fluorescence was monitored using
a FluorS Multi-imager (Bio-Rad).
1(IV)1263-1277 THP was coupled to activated CH-Sepharose according
to the instructions of the supplier (Amersham Biosciences). In
addition, a mock-coupled column was made without the peptide.
Chondroitin-4-sulfate, chondroitin-6-sulfate, or dermatan sulfate (all
from Calbiochem) were dissolved in carbonate buffer, pH 9.0. A 4-fold
molar excess of fluoroscein isothiocyanate dissolved in the same buffer
was added to each GAG and incubated overnight at 4 °C with mixing.
The goal was to achieve a 10-15% labeling of amino groups. Unreacted
fluoroscein isothiocyanate was removed by quenching with a 10-fold
excess of Gly followed by performing repeated buffer exchanges with
Microsep centrifugal concentrators. The final product was dissolved in
an OGS lysis buffer in which the OGS was omitted (
OGS lysis buffer).
The columns were also equilibrated in this buffer. The desired GAG
(2.5 µmoles) was incubated with the mock-coupled column for 2 h
at 4 °C. The unbound portion was then added to the peptide column,
and the column was mixed overnight at 4 °C. The column was washed
with 4 volumes of
OGS lysis buffer, and bound GAGs were removed by washing sequentially with 3 volumes of 0.25, 0.5, 0.75, 1.0, and 2.0 M NaCl while saving 2-3-ml fractions. The fluorescence of 200 µl from each fraction was measured at
excitation = 485 nm and
emission = 538 nm.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1(IV)1263-1277 region and evaluate the role of glycosylation
on melanoma activities, triple-helical models incorporating collagen
sequences of interest needed to be constructed. In addition, to
properly evaluate biological effects, the triple helices of these
"mini-collagens" needed to be stable to assay conditions.
Previously, we have described two methods for assembling THPs of
desirable thermal stabilities. One method uses a C-terminal covalent
branch (1, 47), whereas the other uses self-assembly driven by
pseudo-lipids (18-20). Both approaches were used in
the present study to create either the branched
1(IV)1263-1277 THP
(Fig. 1, top) or the
1(IV)1263-1277 peptide-amphiphile (Fig. 1, bottom). In
addition, one variant of the branched
1(IV)1263-1277 THP was
created in which Lys1265 was replaced with Hyl(Gal), and
two variants of the
1(IV)1263-1277 peptide-amphiphile were created
in which Asp1267 was replaced with Abu or
Lys1265 was replaced with Hyl(Gal).
View larger version (13K):
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Fig. 1.
Structures of the branched
1(IV)1263-1277 THP (top) and
C16-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
peptide-amphiphile (bottom). Ahx is
6-aminohexanoic acid.
-Boc,O-(2,3,4,6-tetra-O-acetyl-
-D-galactopyranosyl)) derivative was used for the synthesis of the collagen-model sequence (Gly-Pro-Hyp)4-Gly-Val-Hyl(Gal)-Gly-Asp-Lys-Gly-Asn-Pro-Gly-Trp-Pro-Gly-Ala-Pro-(Gly-Pro-Hyp)4-NH2, which is designated
Hyl(Gal)1265-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2.
The analogous sequence containing Lys1265 instead of
Hyl(Gal)1265 has been synthesized previously and
structurally characterized by CD and NMR spectroscopies (18-20).
Synthesis of the peptide proceeded without difficulty, and Edman
degradation sequence analysis of the peptide-resin indicated a highly
efficient assembly. The peptides were subsequently purified by RP-HPLC
and characterized by MALDI-MS. The
Fmoc-Hyl(
-Boc,O-(2,3,4,6-tetra-O-acetyl-
-D-galactopyranosyl)) derivative was also used for the synthesis of the branched
1(IV)1263-1277 THP, where Lys1265 was replaced with
Hyl(Gal)1265. Due to difficulties in obtaining mass spectra
for large branched peptides, branched
Hyl(Gal)1265-
1(IV)1263-1277 THP was characterized by
MALDI-MS analysis of the branch, sequence analysis of the intact THP
following RP-HPLC purification, and reaction with carbazole-sulfuric
acid to confirm the presence of carbohydrate.
= 222-227 nm and a negative molar ellipticity at
= 195-200 nm (56). Also, a triple-helical assembly can be
distinguished from a simple, non-intercoiled poly-L-proline II structure by its thermal denaturation behavior. A triple helix is
relatively sensitive to temperature, and thus triple-helical melts are
highly cooperative (56). The CD spectra for
(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2 and
Hyl(Gal)1265-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
at 5 °C were compared (Fig. 2). The CD
spectra for
(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2 and
Hyl(Gal)1265-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
are indicative of triple-helical structure. To examine the thermal
stability of the two peptides, the molar ellipticity at
= 225 nm was monitored as a function of increasing temperature. Both
(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2 and
Hyl(Gal)1265-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
exhibited sigmoidal transitions, consistent with the melting of a
triple-helical to single-stranded structure (Fig.
3). The Tm values for
(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2 and
Hyl(Gal)1265-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
were 39 and 42 °C, respectively (Table
I). Thus, glycosylation appeared to
slightly increase triple-helical stability.
View larger version (18K):
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Fig. 2.
Circular dichroism spectra of
(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
(A) and
Hyl(Gal)1265-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
(B) at 5 °C. Peptide concentrations were 14 µM in H2O.
View larger version (19K):
[in a new window]
Fig. 3.
Temperature dependence of molar ellipticity
at = 225 nm for
(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
(A) and
Hyl(Gal)1265-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
(B). Peptide concentrations were 14 µM in H2O.
Tm values for triple helix coil transitions
1(IV)1263-1277
1
1,
2
1,
and
3
1 integrins) and cell surface
proteoglycans. To examine the involvement of integrins for mediating
melanoma cell adhesion to
1(IV)1263-1277, a peptide-amphiphile
analog was prepared in which Asp1267 was replaced with Abu.
The collagen-binding integrins require either a Glu or Asp residue for
ligand binding (2, 9-11, 58, 59). Replacement of the only Asp/Glu
residue within
1(IV)1263-1277 by a sterically similar, but
uncharged, residue (Abu) allowed us to specifically examine the
possible role of integrin interaction with this sequence. The
Tm value for
C16-Abu1267-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
was 27 °C (Table I), and thus cell adhesion assays were performed at
20 °C to ensure that this ligand was primarily in triple-helical
conformation. Melanoma cell adhesion to
C16-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2 and
C16-Abu1267-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
was virtually identical over the concentration range of 0.1-10
µM (Fig. 4). Both
peptide-amphiphiles exhibited EC50 values of ~0.5
µM. Thus, the lack of a negatively charged residue had no
effect on melanoma cell binding to
1(IV)1263-1277, suggesting a
lack of integrin involvement.
View larger version (16K):
[in a new window]
Fig. 4.
Human melanoma cell adhesion to
C16-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
(closed triangles, solid line),
C16-Abu1267-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
(open triangles, dashed line), or BSA
(dashed line), at 20 °C. Peptide-amphiphile
concentrations were 0.01-10 µM.
Inhibition of melanoma cell adhesion and spreading assays was designed
to discriminate between integrin and proteoglycan involvement. Melanoma
cells were treated with 10 µg/ml anti-2 or 20 µg/ml anti-
1 integrin subunit mAbs prior to adhesion to
C16-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2. Neither mAb inhibited melanoma adhesion (data not shown). Treatment of
melanoma cells with GAGs prior to adhesion to
C16-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2 resulted in substantial inhibition of melanoma activity by
chondroitin-4-sulfate or chondroitin-6-sulfate (Fig.
5). Consistent with these results, treatment of melanoma cells with chondroitinase ACII inhibited cell
adhesion to and spreading on
C16-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2 in similar fashion as chondroitin-4-sulfate or chondroitin-6-sulfate (Fig. 5).
|
Since the adhesion and inhibition assays indicated that a CSPG was
potentially involved in melanoma cell interaction with 1(IV)1263-1277, we needed to determine whether CD44/CSPG or
MPG/MCSP/NG2 was involved. Two clones from the M14 parental cell line
have been created (M14#5 and M14#11) based on repeated cell sorting using the 9.2.27 MPG/MCSP/NG2
mAb.3 M14#5 expresses
CD44/CSPG but does not express MPG/MCSP (60). ELISA studies were
performed to determine the relative cell surface concentrations of CD44
and
1 integrins. M14#5 cells had higher levels of CD44
than M14P, whereas M14#11 cells had lower levels of CD44 than M14P
(Fig. 6). In contrast, the levels of the
1 integrin subunit were similar for M14P, M14#5, and
M14#11 (Fig. 6). We then examined the relative levels of melanoma
adhesion to and spreading on
C16-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2 by M14, M14#5, and M14#11 (Fig. 7). The
highest levels of adhesion and spreading were achieved by M14#5,
followed by M14P and lastly M14#11. Thus, melanoma adhesion and
spreading activities were not due to MPG/MCSP/NG2 and were most
likely mediated by CD44/CSPG.
|
|
Results from the cell adhesion/inhibition assays suggested that
CD44/CSPG was responsible for melanoma cell adhesion to triple-helical 1(IV)1263-1277. Affinity chromatography was performed to further characterize the receptor for triple-helical
1(IV)1263-1277. Branched
1(IV)1263-1277 THP was immobilized to CH-Sepharose, and
precleared human melanoma cell lysates were added to the beads. Following application of the cell lysates, the column was washed with 3 volumes of OGS lysis buffer, and then bound materials were eluted with
IP lysis buffer. Eluants were incubated with mAbs against either CD44
or the
1 integrin subunit followed by
immunoprecipitation and immunoblotting with the respective mAb. A
protein of ~85-90 kDa was immunoprecipitated by the anti-CD44 mAb
(Fig. 8). This apparent molecular mass
corresponded to melanoma CD44s core protein following chondroitinase
treatment (60, 61). No corresponding proteins were observed
using an anti-
1 integrin subunit mAb immunoprecipitation (data not shown; see below). Immunoprecipitation analysis of whole cell
lysates showed the presence of both CD44 and the
1
integrin subunit (Figs. 8 and 13), consistent with prior studies (60). Incubation of the column-bound materials or whole cell lysates with IgG
resulted in the detection of only IgG proteins (data not shown).
|
To further examine the role of chondroitin sulfate in the
binding of melanoma cells to 1(IV)1263-1277, affinity
chromatography was performed using branched
1(IV)1263-1277 THP and
chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate.
Both chondroitin-4-sulfate and chondroitin-6-sulfate were found to
specifically bind to
1(IV)1263-1277 THP, whereas dermatan sulfate
did not (Fig. 9). The relative elution profiles of chondroitin-4-sulfate and chondroitin-6-sulfate make it
appear that chondroitin-4-sulfate has a greater ability to bind
branched
1(IV)1263-1277 THP, but a significant amount (>4000 relative fluorescence units) of chondroitin-6-sulfate remains bound to
the THP and elutes only with successive washes with acetate buffer, pH
4.0, and Tris·HCl buffer, pH 8.0 (data not shown).
|
Effects of Glycosylation on Melanoma Activities
Human melanoma cell adhesion was examined for
C16-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
and
C16-Hyl(Gal)1265-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
at 37 °C (Fig. 10). The C16-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
peptide-amphiphile promoted significant adhesion of melanoma cells,
with an EC50 value of ~2.5 µM. The
glycosylated peptide-amphiphile promoted very low levels of adhesion of
melanoma cells at all concentrations tested. Neither the IV-H1 peptide
nor the C16 tail alone produced significant adhesion over
the concentration range studied (14). Prior studies had shown that the
single-stranded IV-H1 peptide promotes adhesion at concentrations
greater than 50 µM (EC50 ~170 µM) (1, 14).
|
The ability of
C16-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
and
C16-Hyl(Gal)1265-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
to promote cell spreading was next studied. Spreading was quantitated
over a ligand concentration range of 0.01-50 µM (Fig.
11). Melanoma cell spreading was more extensive on
C16-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
as compared with
C16-Hyl(Gal)1265-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2.
Representative microscopic images of melanoma cell spreading on 10 µM
C16-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2 and 10 µM
C16-Hyl(Gal)1265-(Gly-Pro-Hyp)4-IV-H1-(Gly-Pro-Hyp)4-NH2
(Fig. 12) illustrate the modulation of
cell activity based on glycosylation.
|
|
The dramatic decrease in cellular activities upon ligand glycosylation
could be due to decreased binding by CD44/CSPG. To address this
possibility, affinity chromatography experiments were repeated, this
time using the branched, glycosylated triple-helical ligand. Branched
Hyl(Gal)1265-1(IV)1263-1277 THP was immobilized to
CH-Sepharose, and precleared human melanoma cell lysates were added to
the beads. Following application of the cell lysates, the column was
washed with 3 volumes of OGS lysis buffer, and then bound materials
were eluted with IP lysis buffer. Eluants were incubated with mAbs
against either CD44 or the
1 integrin subunit followed
by immunoprecipitation and immunoblotting with the respective mAb. No
proteins were immunoprecipitated by either the anti-CD44 or the
anti-
1 integrin subunit mAb (Fig. 13). The result for the
1 integrin subunit is identical to that observed when
using the non-glycosylated ligand (see earlier discussion). Immunoprecipitation analysis of whole cell lysates showed the presence
of both CD44 and the
1 integrin subunit (Fig. 13).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The development of model triple-helical peptide ligands has led to
a better understanding of the role of the triple helix as a modulator
of biological function. In the present study, triple-helical models of
1(IV)1263-1277 have been used to define the roles of both
triple-helicity and glycosylation on tumor cell interactions with
basement membrane (type IV) collagen. Prior studies had shown that
CD44/CSPG from melanoma cells binds directly to single-stranded
1(IV)1263-1277 (15, 16) and that CD44/CSPG binds to type IV
collagen (60). However, cells may engage different receptors depending
upon the conformational state of collagen (21-27), and thus we needed
to determine the receptor for triple-helical
1(IV)1263-1277. A
variant of
1(IV)1263-1277 was constructed in which the single Asp
residue was replaced with Abu. The three collagen-binding melanoma
integrins,
1
1,
2
1, and
3
1,
require a negatively charged residue (Asp or Glu) for binding (2,
9-11, 58, 59). Replacement of the one negatively charged residue in
1(IV)1263-1277 had no effect on melanoma cell binding, indicating
that melanoma cell interaction with this triple-helical ligand is not
integrin-mediated. Inhibition of adhesion and spreading assays showed
that (a) anti-integrin mAbs had no effect on melanoma cell
activities, (b) chondroitin-4-sulfate and
chondroitin-6-sulfate interfered with melanoma cell activities, and
(c) removal of chondroitin and chondroitin sulfate GAG
chains by chondroitinase ACII inhibited melanoma cell activities.
MGP/MCSP was ruled out as a potential receptor since M14#5
melanoma cells, which do not express MGP/MCSP, efficiently adhered to
and spread on triple-helical
1(IV)1263-1277.
Affinity chromatography with the branched 1(IV)1263-1277 THP
indicated that melanoma cell CD44 bound directly to this ligand, whereas the
1 integrin subunit did not. Melanoma cell
binding to type IV collagen uses integrins of only the
1
family (62, 63), so interaction to the
1(IV)1263-1277 region of
this collagen does not appear to be integrin mediated. In addition,
CD44 is believed to be in the chondroitin sulfate form based on
(a) removal of the chondroitin sulfate glycosaminoglycan
chains from the receptor by chondroitinase ACII, (b) binding
of chondroitin-4 sulfate and chondroitin-6-sulfate to triple-helical
1(IV)1263-1277, (c) binding of melanoma CSPGs to type IV
collagen (60) and the linear form of
1(IV)1263-1277 (5, 15, 16),
and (d) virtually all of the melanocyte CD44 proteoglycans
being CSPGs (64).
Triple-helical 1(IV)1263-1277 represents the second distinct
extracellular matrix ligand described for CD44. CD44 has long been
recognized for the ability to bind hyaluronic acid (HA). HA
binds to the CD44 N-terminal globular domain (65). The HA binding motif consists of two basic amino acids separated by seven non-acidic amino acids (B(X7)B) (65). In CD44, HA binding
motifs are found within residues 21-45, with Arg41 being
of particular importance (65). Several distal residues also
contribute to HA binding, such as Lys158 and
Arg162 (65). Since chondroitin sulfate is required for CD44
binding to
1(IV)1263-1277 but interferes with CD44 binding to HA
(65), it appears that
1(IV)1263-1277 and HA bind to different
regions of CD44.
Position 1265 of the 1(IV) collagen chain can be glycosylated (28).
The effects of this glycosylation on either triple-helical structure or
CD44 binding are unknown. CD spectroscopic studies have shown that
glycosylation at residue 1265 of either the triple-helical peptide or
peptide-amphiphile increased the melting temperature by 3.0-3.5 °C
as compared with the non-glycosylated ligands. Prior work had
demonstrated that
-D-galactose glycosylation of Thr in
the Yyy position of (Gly-Hyp-Yyy)10 enhanced triple-helical stability by 32 °C as compared with Thr (40). This corresponds to
3.2 °C per glycosylated residue. Thus, both studies have come to
similar conclusions as to the role of glycosylation in stabilizing the
triple helix. However, in the case of Hyl glycosylation, this stabilization effect is most likely localized to a specific sequence and is not a general mechanism by which collagen thermal stability is
enhanced. Type II collagen, whether in fully glycosylated (10 residues/1016 total) or lowly glycosylated (2 residues/1016 total) form, has the same Tm value (66).
The role of Hyl glycosylation in the CD44 recognition processes was
first studied by comparing melanoma cell adhesion with the glycosylated
and non-glycosylated ligands. A dramatic reduction in cell adhesion was
observed due to the presence of the single galactose residue,
suggesting significant biological consequences of even subtle changes
in collagen carbohydrate content. Promotion of melanoma cell spreading
had similar, although not identical, trends, as seen for cell adhesion.
Subsequent affinity chromatography experiments indicated that CD44 no
longer bound to the 1(IV)1263-1277 sequence once carbohydrate was
present. The exquisite sensitivity of cell interaction with
glycosylated ligand has only rarely been observed. T cell hybridoma
response to type II collagen fragments has been shown to depend upon
contacts from a single glycosylated Hyl with the CD3 loops of the T
cell receptor (32).
We have found that glycosylation inhibits CD44 interaction with the
1(IV)1263-1277 region derived from basement membrane collagen. This
result is unexpected as prior studies had shown that melanoma cell
binding to
1(IV)1263-1277 is primarily via electrostatic
interactions with Lys1265 and Lys1268 (67).
Although it is possible that the glycosylation may mask the side-chain
charge of residue 1265, such behavior seems unlikely given the small
size of the carbohydrate. It is more likely that we have observed a
specific, unfavorable carbohydrate-carbohydrate interaction between the
CD44 chondroitin sulfate and the
1(IV)1263-1277 galactose residue.
Overall, little is known about how carbohydrates interact with cell
surface receptors, particularly in the case of unfavorable associations
(68, 69). More often, such interactions are favorable, as when
carcinoma cell surface mucins associate with platelet P-selectin,
creating a platelet "cloak" surrounding the tumor cells that aid in
the metastatic process (45). Although CD44 does bind certain
carbohydrates (HA), this interaction requires a minimum of six sugar
residues (three repeating disaccharide units) with affinity increasing
for longer HA molecules (Kd ~0.3 nM)
(65). The present study suggests that glycosylation can be used for
modulating tumor cell behaviors based on carbohydrate structure and
chain length.
The reduced binding of CD44/CSPG due to ligand glycosylation presents a
possible cryptic site mechanism by which tumor cells may invade the
basement membrane. In the native, glycosylated state, regions within
type IV collagen may have minimal interaction with receptors such as
CD44/CSPG. After tumor cells bind to type IV collagen (presumably via
integrins such as 2
1), cell surface or
secreted glycosidases could liberate the collagen-bound carbohydrates. This process would expose cryptic sites for interaction with CD44/CSPG and/or other cell surface receptors (such as the
3
1 integrin, which also binds to a
glycosylated region within type IV collagen (2, 11, 28)).
Galactosylation has been shown previously to mask Lewis X antigens
(70). Specific enzymes have been characterized for (a)
removal of glucose from disaccharide-modified Hyl
(2-O-
-D-glucopyranosyl-O-
-D-galactopyranosyl-Hyl glucohydrolase (86, 87)) and (b) transfer of
galactose to Hyl (UDP-galactose:hydroxylysine-collagen (basement
membrane) galactosyltransferase (88)). In addition, a cell surface
galactosyltransferase that binds to type IV collagen has been described
(71). Although a deglycosylation/cryptic site mechanism provides
interesting speculation, it should also be noted that not all Lys
residues in type IV collagen are fully hydroxylated and glycosylated
(28, 72), and thus receptor interaction may just occur with the
subpopulation of type IV collagen that does not contain carbohydrate.
CD44/CSPG interaction with 1(IV)1263-1277 and subsequent promotion
of signaling and spreading activities are dependent upon triple-helical
conformation and level of glycosylation. However, the role of CD44 in
tumor cell invasion is just beginning to be unraveled (65, 73). CD44
and several isoforms have been characterized on a variety of tumor cell
surfaces (74-77) and have been suggested to be prognostic indicators
of malignant melanoma (78, 79). Although CD44 binds to types I, IV, VI,
and XIV collagen, it is not a primary receptor for cell adhesion to
collagen (60, 80-82). The CD44 cytoplasmic domain binds to ankyrin and
members of the ezrin-radixin-moesin family of cytoskeletal proteins
(83). CD44 is also directly linked to two Tyr kinases,
p185HER2 and c-Src kinase (83). We have found that
signaling via the CD44/
1(IV)1263-1277 interaction results in
autophosphorylation of p125FAK (14), whereas others
have shown that CD44 mediates phosphorylation of ZAP-70 and the
activation of phospholipase C
, Ras, protein kinase C
(PKC
),
and NF-
B binding activity (84). One result of CD44 "outside-in"
signaling is up-regulation and activation of integrins (85) and the
expression of matrix metalloproteinases (61).2 It is
possible that CD44 works in concert with another receptor, such as the
2
1 integrin, to efficiently bind to type
IV collagen and subsequently up-regulate cell signaling pathways. Among
the products of these pathways are proteases and growth factors that aid in compromising the basement membrane. Such a mechanism is consistent with our "collagen structural modulation" model proposed previously for tumor cell invasion (4) and will be explored in future studies.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Barbara Mueller for providing the M14, M14#5, and M14#11 melanoma cell lines.
![]() |
FOOTNOTES |
---|
* This work was supported by the National Institutes of Health Grant CA 77402 (to G. B. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Nobex Corporation, P. O. Box 13940, Research Triangle Park, NC 27709.
** To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, Florida Atlantic University, 777 Glades Rd., Boca Raton, FL 33431-0991. Tel.: 561-297-2093; Fax: 561-297-2759; E-mail: fieldsg@fau.edu.
Published, JBC Papers in Press, February 6, 2003, DOI 10.1074/jbc.M212246200
2 J. A. Borgia, J. L. Lauer-Fields, and G. B. Fields, manuscript in preparation.
3 B. Mueller, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MPG/MCSP/NG2, melanoma-associated proteoglycan/melanoma chondroitin sulfate
proteoglycan;
Boc, tertiary-butyloxycarbonyl;
BSA, bovine
serum albumin;
CSPG, chondroitin sulfate proteoglycan;
Fmoc, 9-fluorenylmethoxy-carbonyl;
GAG, glycosaminoglycan;
HA, hyaluronic
acid;
HATU, O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate;
HOAt, 1-hydroxy-7-azabenzotriazole;
Abu, 2-aminobutyric acid;
Hyl, 5-hydroxy-L-lysine;
Hyp, 4-hydroxy-L-proline;
IV-H1, 1(IV)1263-1277 collagen
sequence Gly-Val-Lys-Gly-Asp-Lys-Gly-Asn-Pro-Gly-Trp-Pro-Gly-Ala-Pro;
THP, triple-helical peptide;
PBS, phosphate-buffered saline;
MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry;
RP-HPLC, reversed-phase high-performance liquid chromatography;
ELISA, enzyme-linked immunosorbent assay;
mAb, monoclonal antibody;
IP, immunoprecipitation;
OGS, octyl-
-glucoside.
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