Glycosylation Modulates Melanoma Cell α2β1 and α3β1 Integrin Interactions with Type IV Collagen

Background: The influence of collagen glycosylation on integrin binding has not been studied previously.

Results: Glycosylation affected α3β1 integrin binding more strongly than α2β1 integrin binding.

Conclusion: Glycosylation modulated integrin/collagen interactions.

Significance: If changes in collagen glycosylation occur in malignancy, then metastasis may be altered by these changes.

Abstract

Although type IV collagen is heavily glycosylated, the influence of this post-translational modification on integrin binding has not been investigated. In the present study, galactosylated and nongalactosylated triple-helical peptides have been constructed containing the α1(IV)382–393 and α1(IV)531–543 sequences, which are binding sites for the α2β1 and α3β1 integrins, respectively. All peptides had triple-helical stabilities of 37 °C or greater. The galactosylation of Hyl³⁹³ in α1(IV)382–393 and Hyl⁵⁴⁰ and Hyl⁵⁴³ in α1(IV)531–543 had a dose-dependent influence on melanoma cell adhesion that was much more pronounced in the case of α3β1 integrin binding. Molecular modeling indicated that galactosylation occurred on the periphery of α2β1 integrin interaction with α1(IV)382–393 but right in the middle of α3β1 integrin interaction with α1(IV)531–543. The possibility of extracellular deglycosylation of type IV collagen was investigated, but no β-galactosidase-like activity capable of collagen modification was found. Thus, glycosylation of collagen can modulate integrin binding, and levels of glycosylation could be altered by reduction in expression of glycosylation enzymes but most likely not by extracellular deglycosylation activity.

Introduction

Despite the continuous advances made, patients suffering from advanced stage melanoma still face a rather bleak prognosis. Melanoma remains unpredictable in its biological behavior, with a high risk of recurrence and a 50% chance to develop metastases in lymph nodes after recurrence (1). To support advances in treatment and detection, attention has turned to locating and identifying key elements that could be utilized either as possible therapeutic targets or as potential markers for the different stages of the disease. Metastasis occurs via a series of linked steps (2, 3). Tumor cells need to extravasate through the endothelium of the lymph node or blood vessel and become anchored in the local extracellular matrix (ECM)³ to initialize secondary growth formation. The principal proteins of the ECM are laminins and collagens, the latter of which serves as a lattice network to create the cellular microenvironment. Type IV collagen is the most important structural component of the basement membrane (BM), which is a specialized form of the ECM.

Metastasis requires a subtype of tumor cells capable of enduring release from the primary tumor site and traveling through the lymphatic or vasculatory system while evading killer cells and/or platelet aggregation. This process requires an altered phenotype, which allows cells to quickly adhere to and release from the BM to promote a faster migration. These phenotypic changes are most easily defined by changed expression profiles of transmembrane receptors, such as integrins, responsible for the rolling motion cells display during migration (4). Integrins are the foremost contributors in mediating cell-cell and cell-ECM adhesions. Interactions between integrins and ECM proteins, such as collagen, are crucial for adherence, migration, and invasion of tumor cells (5).

Integrins are heterodimers of noncovalently associated α and β subunits. In vertebrates, there are 18 α and 8 β subunits that can assemble into 24 different receptors with unique binding properties and tissue distributions (6, 7). Based on the structural characteristics of their α and β subunits, integrins are classified as either an I-domain or a non-I-domain, which signals a fundamentally different association mechanism between the two groups of receptor types and their respective ligands (6, 8–12). I-domain-containing integrins preferentially bind to ligands via their I-domain, which is located on the α subunit, providing a more approachable binding site and a more relaxed spatial arrangement, whereas non-I-domain integrins carry out binding partly by another portion of the α subunit and partly by the β subunit, which sterically places the ligand in a more confined space and makes the binding site less approachable and possibly less favorable (10, 12). The I-domain contains a conserved MIDAS that binds divalent metal cations. Ligand binding alters the coordination of the metal ion and shifts the I-domain from a closed, resting state to an open, active conformation, which results in increased ligand affinity and promotes subsequent integrin activation (13). Four I-domain α subunits (α1, α2, α10, and α11) associate with β1 and form a distinct collagen-binding subfamily. The structural basis of the interaction of these integrins with their ligand is a Glu residue within a collagenous Gly-Phe-Hyp-Gly-Glu-Arg motif, providing the cation coordination (9).

The β subunit plays an important role in ligand binding when α subunits lack the I-domain. Integrin β subunits contain an ion binding site homologous to MIDAS with a sequence motif of Asp-Xaa-Ser-Xaa-Ser. Mutation of any of these ion-coordinating residues within the β1, β2, β3, or β5 subunits ablated ligand binding to the respective integrins (14–16). In αβ integrin heterodimers, ligands bind to a crevice in the head domain between the αβ subunit interface. In many cases the ligand interacts with the metal ion-occupied MIDAS located within the β subunit and the propeller domain of the α subunit (17). The α3β1 integrin is a non-I-domain integrin that binds collagen and laminin (18–20). More specifically, the α3β1 integrin binds type IV collagen (21) and contributes to melanoma cell migration on this ligand (22, 23). Thus, collagen receptors include both α I-domain and non-I-domain integrins, implying differing binding mechanisms at play.

Tumor cell adhesion to the triple-helical domain of BM (type IV) collagen occurs at several different regions (24). Type IV collagen cyanogen bromide fragment 3 (CB3(IV)), which includes α1(IV) residues 388–551 and α2(IV) residues 407–570, was reported to contain the individual binding sites for the α1β1 and α2β1 integrins (25, 26). The α1β1 integrin simultaneously binds Asp⁴⁴¹ from two α1(IV) chains and Arg⁴⁵⁸ from the α2(IV) chain (27–31). The Gly-Phe-Hyp-Gly-Glu-Arg recognition motif for the α2β1 integrin is located within the α1(IV)382–391 sequence (32). A THP model of α1(IV)382–393 binds to melanoma cells (33).

The α1(IV)531–543 sequence promotes adhesion and spreading of melanoma and other cell types (34). The α3β1 integrin was identified as the receptor that binds to type IV collagen via this sequence (35). Interestingly, peptides promoted adhesion of melanoma cells in single-stranded and triple-helical conformation (34), thus providing the first evidence for existence of triple-helix-independent integrin binding sites within the collagenous domain.

An essential characteristic of native type IV collagen is the high level of Lys hydroxylation and subsequent Hyl glycosylation present in each α chain. These post-translational modifications are carried out on almost all Lys residues present in type IV collagen, compared with the relatively low level (∼10%) of modification that is present on types I and II collagen (36, 37). Prior research conducted in our laboratory indicated an altered affinity of a cell surface proteoglycan, CD44, toward binding sites in type IV collagen based on Hyl glycosylation (38). However, prior studies have not considered how Hyl glycosylation impacts integrin recognition of collagen. To specifically examine the possible modulation of integrin function by glycosylation, THPs with Lys substituted by glycosylated Hyl for Lys⁵⁴³ and Lys⁵⁴⁰ from the human α1(IV)531–543 gene sequence (α3β1 integrin-specific) and Lys³⁹³ from the human α1(IV)382–393 gene sequence (α2β1 integrin-specific) were synthesized. These ligands were utilized to compare the promotion of melanoma cell adhesion, to observe the effects of ligand glycosylation. Cellular integrin concentrations were quantified utilizing immunocytochemistry. Alternative receptors were examined for recognition of glycosylated collagen. We also tested the possibility of melanoma cell modulation of collagen glycosylation by examining extracellular β-galactosidase-like activity.

MATERIALS AND METHODS

All chemicals were molecular biology or peptide synthesis grade and purchased from ThermoFisher Scientific (Waltham, MA) or Sigma-Aldrich.

Synthesis of Fmoc-d,l-Hyl[(5-O-β-Gal(Ac₄))(N^ϵ-Cbz)]-OPfp Building Block

The synthesis of Fmoc-d,l-Hyl[(5-O-β-Gal(Ac₄))(N^ϵ-Cbz)]-OPfp was performed in six steps, starting from the racemate of d,l-5-Hyl (Sigma-Aldrich). The synthetic approach has been described previously (39), and analytical data (¹H NMR, ¹³C NMR, and mass spectra) of all intermediates and the desired product were in accordance with published ones (40). For example, MALDI-TOF MS of Fmoc-d,l-Hyl[(5-O-β-Gal(Ac₄))(N^ϵ-Cbz)]-OPfp yielded m/z = 1037.7535 (calculated for C₄₉H₄₇F₅N₂NaO₁₆⁺, m/z = 1037.2738). RP-HPLC retention time = 19.745 min using a Vydac C18 column (5 μm, 300 Å, 150 × 4.6 mm), analytical gradient of 2–98% B in 20 min (where A was 0.1% TFA in H₂O and B was 0.1% TFA in acetonitrile), with a flow rate of 1 ml/min and detection at λ = 220 and 280 nm.

Synthesis of (Glyco)peptides

The (glyco)peptide sequences were based on type IV collagen motifs possessing integrin recognition sites (see Table 1). (Glyco)peptides were synthesized by Fmoc solid phase chemistry using TentaGel S Ram resin (Advanced ChemTech, Louisville, KY) with a substitution level of 0.26 mmol/g. Peptide synthesis was carried out on the Liberty (CEM, Matthews, NC) automated microwave-assisted peptide synthesizer equipped with a Discover microwave module. Fmoc amino acids were coupled using 5 eq of each amino acid, 4.9 eq O-(¹H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, and 8 eq N-methylmorpholine (microwave power of 25 W at 50 °C, 300 s). Fmoc-d,l-Hyl[(5-O-β-Gal(Ac₄))(N^ϵ-Cbz)]-OPfp was incorporated manually using 3 eq of amino acid and 6 eq N,N-diisopropylethylamine with a reaction time of 17 h. The N termini of the peptides and glycopeptides were modified by coupling with n-dodecanoic acid.

TABLE 1.

Sequences, analytical data, and thermal stabilities of THPs used in this study

Peptide designation	Sequence	Integrin binding	[M+H]+ observed (calculated)	RP-HPLC RTa	Tm
				min	°C
α1(IV)382–393	C10- (Gly-Pro-Hyp)4-Gly-Ala-Hyp-Gly-Phe-Hyp-Gly-Glu-Arg-Gly-Glu-Lys- (Gly-Pro-Hyp)4-Tyr-NH2	α2β1	3687.4666 (3687.7796)	13.31	43
α1(IV)382–393(Gal)	C10-(Gly-Pro-Hyp)4-Gly-Ala-Hyp-Gly-Phe-Hyp-Gly-Glu-Arg-Gly-Glu-Hyl(Gal)-(Gly-Pro-Hyp)4-Tyr-NH2	α2β1	3865.1714 (3865.8273)	11.32	37
α1(IV)531–543	C10- (Gly-Pro-Hyp)5-Gly-Glu-Phe-Tyr-Phe-Asp-Leu-Arg-Leu-Lys-Gly-Asp-Lys- (Gly-Pro-Hyp)5-NH2	α3β1	4414.0239 (4414.1941)	12.38	45
α1(IV)531–543(Gal)(Gal)	C10-(Gly-Pro-Hyp)5-Gly-Glu-Phe-Tyr-Phe-Asp-Leu-Arg-Leu-Hyl(Gal)-Gly-Asp-Hyl(Gal)-(Gly-Pro-Hyp)5-NH2	α3β1	4770.0703 (4770.2896)	11.74	37

^a Using a gradient of 2–70% B in 20 min under conditions given under “Materials and Methods.”

In the case of glycopeptide α1(IV)382–393(Gal), part of the peptidyl-resin was N-terminally modified with biotin containing a 20-atom PEG spacer (instead of n-dodecanoic acid). N-Biotinyl-NH-(PEG)₂-COOH (EMD Millipore, San Diego, CA) was coupled manually using 3 eq molar excess along with 3 eq of O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and 6 eq of N-methylmorpholine in DMF for 90 min. Biotinylated α1(IV)382–393(Gal) THP was utilized for β-galactosidase studies because of its favorable RP-HPLC elution profile.

Removal of side chain protecting groups and peptide-resin cleavage were carried out as reported previously (41) for 3 h in an atmosphere of ambient gas (Ar) using 7 ml of cleavage mixture (5% H₂O, 5% thioanisole, 2.5% phenol, and 2.5% 1,2-ethanedithiol in TFA). Cleaved (glyco)peptides were precipitated in cold methyl tert-butyl ether, centrifuged, and lyophilized. Crude glycopeptides were subjected to Ac protecting group removal from sugar moieties using 0.1 m NaOH solution for 15 min. After this time the glycopeptide solution was neutralized with HCl and lyophilized.

Crude (glyco)peptides were purified using RP-HPLC on an Agilent 1260 Infinity series preparative HPLC equipped with a Vydac C18 column (15–20 μm, 300 Å, 250 × 22 mm). The elution gradient was 5–50% B in 60 min (where A was 0.1% TFA in H₂O, and B was 0.1% TFA in acetonitrile), with a flow rate of 10 ml/min and detection at λ = 220 and 280 nm. The HPLC fractions were combined, frozen, and lyophilized.

A portion of the biotinylated glycopeptide α1(IV)382–393(Gal) (2.5 mg) was subjected to selective N-acetylation of the Hyl residue. Briefly, the glycopeptide was dissolved in 1 ml of 50 mm ammonium carbonate solution, and 100 μl of acetic anhydride was added. The reaction progress was monitored by RP-HPLC and MALDI-TOF MS, and upon completion the reaction mixture was frozen and lyophilized.

(Glyco)peptide purity was evaluated on an Agilent 1260 Infinity analytical HPLC using a Vydac C18 column (5 μm, 300 Å, 150 × 4.6 mm), analytical gradient 2–98% B in 20 min, with a flow rate of 1 ml/min and detection at λ = 220 and 280 nm. MALDI-TOF MS analysis was performed using an Applied Biosystems Voyager DE-PRO Biospectrometry work station (Carlsbad, CA) with a α-cyano-4-hydroxy-cinnamic acid/2,5-dihydroxybenzoic acid matrix.

CD Spectroscopy

Peptides were dissolved in 0.5% acetic acid and equilibrated at 4 °C (>24 h) to facilitate triple-helix formation. Peptide concentrations were determined using a Thermo Scientific NanoDrop 1000 (Waltham, MA) via absorbance at λ = 280 nm, ϵ_Tyr = 1490 m⁻¹ cm⁻¹. Triple-helical structure was evaluated by near UV CD spectroscopy using a Jasco J-810 spectropolarimeter (Easton, MD) with a path length of 1 mm. Thermal transition curves were obtained by recording the molar ellipticity ([θ]) at λ = 225 nm with an increase in temperature of 20 °C/h in a range of 5–80 °C. Temperature was controlled by a JASCO PTC-348WI temperature control unit. The THP melting temperature (T_m) was defined as the inflection point in the transition region (first derivative). The spectra were normalized by designating the highest [θ]_{225 nm} as 100% folded and the lowest [θ]_{225 nm} as 0% folded.

Cell Culture

The M14#5 human metastatic melanoma cell line was generously provided by Dr. Barbara Mueller (Torrey Pines Institute for Molecular Studies, La Jolla, CA). The WM-115 (primary melanoma), WM-266-4 (metastatic melanoma), and SK-MEL-2 (metastatic melanoma) cell lines were obtained from American Type Culture Collection (Manassas, VA). For cell adhesion assays, cells were grown in EMEM with l-Gln (American Type Culture Collection) supplemented with 10% fetal bovine sera (HyClone), 50 units/ml penicillin, and 0.05 mg/ml streptomycin using 175-cm² flasks. At ∼80% confluency cells were subcultured (1:3 ratio for SK-MEL-2, 1:6 for the other cell lines). For cell detachment 0.25% trypsin-EDTA solution was used (Invitrogen). Flasks were kept in a humidified incubator containing 5% CO₂, and cells were passaged only eight times to avoid genetic drifts and other variations.

Immunocytochemistry

Biotinylated mouse antihuman integrin α3 subunit (CD49c, clone IA3, catalog number BAM1345) and biotinylated mouse anti-human integrin α2 subunit (CD49b, clone HAS3, catalog number BAM1233) mAbs were purchased from R&D Systems (Minneapolis, MN). Biotin-SP-conjugated ChromPure mouse IgG, whole molecule (product code 015-060-003), and mouse serum and the Cy3-conjugated (indocarbocyanine) streptavidin (product code 016-160-084) were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Immunocytochemistry experiments were carried out according to the direct detection method employing working concentrations based on the manufacturer's recommendation. The specificities of the integrin antibodies were previously established (42, 43). Briefly, cells were plated in a 96-well Costar plate (Corning No. 3632; Fisher Scientific) at 20,000–40,000 cells/100 μl growth medium and allowed to become 70–80% confluent overnight. Growth medium was removed, and cells were rinsed with 1× PBS. Cells were fixed by incubation with 4% paraformaldehyde/PBS for 20 min at room temperature. The plate was blocked against nonspecific binding with 1% mouse serum, 1% BSA/PBS for 1 h at room temperature. Biotinylated anti-integrin α3 or α2 mAbs were diluted in 1% mouse serum, 1% BSA/PBS to 25 μg/ml, added to the cells, and incubated for 1 h at room temperature. As a negative control, biotin-SP-conjugated mouse IgG was utilized, diluted to 25 μg/ml in 1% mouse serum, 1% BSA/PBS. Following incubation with the antibodies and/or mouse IgG, cells were rinsed three times with 1 × PBS and subsequently incubated for 1 h at room temperature in the dark with Cy3-conjugated streptavidin diluted to 2 μg/ml in 1% mouse serum, 1% BSA/PBS. Background fluorescence was established by incubating the cells with the Cy3-streptavidin solution for 1 h at room temperature in the dark. The plate was washed with 1× PBS, and bound Cy3 was detected using the red filter on an Olympus IX70 inverted fluorescence microscope camera. Semi-quantitative image analyses were carried out using the Quantity One® v.4.2.2 software (Bio-Rad) on quadruplets of 16 cell/image areas. Cells were counted in selected areas using photos taken at bright light at a visible range wavelength, and then, after switching on the red filter, the selected areas were photographed to determine fluorescence. Exposure times were 250, 400, or 666 ms. Because of either cellular accumulation or entrapped dye, some areas indicated artificially high levels of fluorescence. Those “bright spots” were not utilized for quantification purposes.

Adhesion Assays

The melanoma cell adhesion assay was performed as described previously (44). Peptide ligands (see Table 1) were dissolved in 40% ethanol in PBS and further diluted to desired concentrations in PBS. Pro-Bind^TM 96-well plates (BD Biosciences, San Jose, CA) were coated with 100 μl of desired peptide and incubated at 4 °C overnight. Nonspecific binding sites were blocked with 2 mg/ml BSA in PBS for 2 h at 37 °C (200 μl/well). Cells were split 1–2 days before the experiment and were washed with PBS (without Ca²⁺ and Mg²⁺) and then released with Accutase (Invitrogen). Cells were then washed and resuspended to 75,000 cells/well in adhesion medium (20 mm HEPES, 2 mg/ml albumin in RPMI 1640 medium). Cell suspension was added to the plate (100 μl/well), and plates were incubated for 60 min at 37 °C. All nonattached cells were removed by washing three times with warm adhesion medium. Adherent cells were counted using CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI) and quantitated with a Synergy H4 Hybrid multimode microplate reader (BioTek, Winooski, VT).

AlphaScreen Assay

The AlphaScreen assay was performed accordingly to recently published methodology (45). His-tagged galectin-3 (1.25 μl) and biotin-ASF (1.25 μl) were added to wells containing varying concentrations of α1(IV)382–393 and α1(IV)382–393(Gal) THPs (2.5 μl, 0–1 mm final concentration) in optimized assay buffer (25 mm HEPES, 100 mm NaCl, 0.05% Tween 20, pH 7.4). Because of the low solubility of the peptides at higher concentrations, final solutions contained 1% DMSO. The nonbiotinylated ASF was used as a control. The final concentration of His-tagged galectin-3 was 100 nm and biotin-ASF 5 nm. The reaction mixture was incubated for 1 h at room temperature, and then 5 μl of nickel-chelate Acceptor and 5 μl streptavidin-conjugate Donor beads were simultaneously added to a final concentration of 25 μg/ml. Incubation proceeded for 1 h in the dark at room temperature, and the assay plate was subsequently read at 22 °C in the AlphaScreen mode on a Synergy H4 Hybrid multimode microplate reader. AlphaScreen signal counts (cps) versus log [ligand] (M) were expressed as the mean of five replicate measurements. The IC₅₀ values were obtained by nonlinear regression analysis using the Graph Pad Prism 5.04 software.

Molecular Modeling

To generate a model of α1(IV)382–393(Gal) THP interacting with the α2β1 integrin, a homology modeling approach was utilized. Briefly, starting structure 1DZI was used as a template (9). Collagen-like peptide residues were mutated manually in PyMOL (46) and UCSF Chimera software (47). Residues were mutated using Dunbrack backbone-dependent rotamer library (48). Charges were added using AMBER ff12SB force field, and for unknown residues (Gal) were calculated using AM1-BCC model (49). Mutated residues were subjected to minimization using the antechamber program (50) included in Chimera.

The α3β1 integrin model was built using the α5β1 integrin x-ray crystallographic structure (51). The α5 subunit was replaced with α3 by homology modeling using the Modeler program (52) and subsequent minimization steps of α3/β1 interface residues using the antechamber module of the Chimera package. Next, docking of α1(IV)531–543 single-stranded peptide was performed using Autodock Vina (53). Because the geometry of the α1(IV)531–543 peptide backbone is unknown, we have selected three different combinations of φ/ψ torsion angles within the polyproline type II family, namely φ/ψ of −60°/150°, −70°/160°, and −75°/175°. Three separate docking runs were performed and compared. In each docking the peptide backbone was kept rigid, and side chains contained rotatable bonds. The docking site was chosen arbitrarily and contained the top of the α3β1 interface along with the MIDAS site in the β1 subunit.

β-Galactosidase Activity Assessment

Isolated β-Galactosidase with Synthetic Substrate

Escherichia coli β-galactosidase (EC 3.2.1.23, grade VIII) was purchased from Sigma-Aldrich. Enzymatic assays were performed in 100 mm phosphate buffer, pH 7.2, supplemented with 10 mm MgCl₂ and 5 mm 2-mercaptoethanol (added freshly before the assay). The enzyme activity was determined using the fluorogenic substrate MUG (Sigma-Aldrich) at λ_excitation = 365 nm and λ_emission = 445 nm. β-Galactosidase activity was measured using the Synergy H4 Hybrid Multi-Mode Microplate Reader over a period of 1 h, with occasional shaking to assure even substrate distribution.

Isolated β-Galactosidase with Peptide Substrates

α1(IV)382–393(Gal) THP was selected as a model putative substrate. To determine the influence of the Hyl ϵ-NH₂ group on β-galactosidase activity, an acetylated version of α1(IV)382–393(Gal) THP, α1(IV)382–393(Gal)-Ac THP, was prepared (see earlier description).

α1(IV)382–393(Gal) and α1(IV)382–393(Gal)-Ac THPs (46 μg each) were incubated at 37 °C with 100 U of β-galactosidase in 100 mm phosphate buffer, pH 7.2, supplemented with 10 mm MgCl₂ and 5 mm 2-mercaptoethanol. After 4 and 24 h, aliquots were taken and analyzed using RP-HPLC/MALDI-TOF MS.

Melanoma Cells with Synthetic Substrate

Extracellular β-galactosidase activity was assessed using a whole cell assay with primary and metastatic melanoma cells. The WM-115 and WM-266-4 cell lines were plated in 24-well plate format at 50,000 cells/well (Corning CellBIND, Corning) and cultured overnight (24 h) using four different media types: EMEM, EMEM supplemented with HI-FBS, OptiMEM, and OptiMEM with HI-FBS. The HI-FBS concentration was 5% (v/v), and the total volume of the media was 500 μl. After 24 or 48 h, MUG was added to a final concentration of 30 μm, and β-galactosidase activity was measured using the Synergy H4 Hybrid multimode microplate reader over a period of 1 h, with occasional shaking to assure even substrate distribution.

Melanoma Cells with Peptide Substrates

The WM-115 and WM-266-4 cell lines were plated in 24-well format at 50,000 cells/well and cultured overnight (24 h) using four different media types: EMEM, EMEM supplemented with HI-FBS, OptiMEM, and OptiMEM with HI-FBS. The HI-FBS concentration was 5% (v/v), and the total volume of the media was 500 μl. Cells were grown overnight, and THPs were added to a final concentration of 35 μm. The incubation was carried out for 24 and 48 h. Aliquots of media were taken, filtered through a 0.22-μm HPLC filter, and subjected to RP-HPLC analysis. All fractions were then collected and analyzed using MALDI-TOF MS.

Released Melanoma Cells with Synthetic and Peptide Substrates

CUG synthetic substrate (Invitrogen) was diluted to a 60 μm in 1 × PBS. To test cell suspensions, subconfluent cells were rinsed with 1 × PBS and released with 5 mm EDTA/PBS. Cells were washed and rediluted to 2 × 10⁻⁵–1 × 10⁻⁶ cells/ml in PBS containing 10 mm MgCl₂ and 5 mm 2-mercaptoethanol. 100 μl of cells were combined with 50 μl of CUG, and enzymatic activity was measured for 30–90 min, with recurring agitation to maintain the cells in suspension, at λ_excitation = 400 nm and λ_emission = 450 nm on a SpectraMAX GeminiEM 96-well plate spectrafluorometer and quantified by the SoftMax Pro 4.3LS software. As a positive control, 50 μl of β-galactosidase and 50 μl of CUG were added to 100 μl of PBS containing the activators and tested simultaneously.

RESULTS

Synthesis of Galactosylated Hyl Building Block

To prepare a galactosylated Hyl building block, we utilized a synthetic approach developed by Kihlberg and co-workers (39), in which 9-BBN simultaneously protects the carboxyl and amino functionalities of amino acids (54). 9-BBN was used for regioselective protection of the α-amino and α-carboxyl groups of d,l-5-Hyl. The resulting 9-BBN complex was then employed in transformations such as ϵ-amino group protection and O-glycosylation. Further manipulations led to preparation of the Fmoc-d,l-Hyl[(5-O-β-Gal(Ac₄))(N^ϵ-Cbz)]-OPfp building block, suitable for direct use in peptide synthesis under standard Fmoc chemistry conditions. The Cbz group was chosen instead of tert-butyloxycarbonyl for ϵ-amino group protection of Hyl because it is more acid stable during O-glycosylation conditions. Fmoc-d,l-Hyl[(5-O-β-Gal(Ac₄))(N^ϵ-Cbz)]-OPfp was obtained in six steps, with a yield comparable with previously published ones (39).

(Glyco)peptide Synthesis and Characterization

d,l-Hyl[(5-O-β-Gal(Ac₄))(N^ϵ-Cbz)] was incorporated into THPs possessing sequences from the α1(IV) collagen chain recognized by α2β1 and α3β1 integrins. We prepared two sets of peptides containing either the Hyl(O-Gal) residue or its Lys counterpart (Table 1). The Hyl(O-Gal) residue was incorporated manually, whereas other amino acids were incorporated using an automated synthesizer under microwave conditions. The N termini of all (glyco)peptides were modified with n-dodecanoic acid to ensure triple-helical character of the (glyco)peptides and to facilitate their attachment to plastic surfaces during the adhesion assay (55, 56).

All peptides were characterized by RP-HPLC and MALDI-TOF MS (Table 1), with appropriate purity and mass values observed. The triple-helical character of the peptides was analyzed by CD spectroscopy, in the range of λ = 250–180 nm (Fig. 1). All peptides had characteristic triple-helical spectra, with a positive peak at λ = 222 nm and a negative peak at λ = 205 nm. Thermal transition curves were obtained by recording molar ellipticity ([θ]) at λ = 225 nm as a function of increasing temperature (Fig. 2). The melting point (T_m) was defined as the inflection point in the transition region (Table 1). All peptides exhibited good triple-helix stability, with T_m values ranging from 37 to 45 °C. Galactosylation of Hyl had a destabilizing effect on the triple-helix, because the glycopeptides had T_m values 6–8 °C lower than the corresponding nonglycosylated peptides. This could be caused by the presence of racemic d,l-5-Hyl used in the present study.

FIGURE 1.

CD spectra of THPs. THPs were dissolved in 0.1% acetic acid to a final concentration of 50 μm. Scans were taken over the range of λ = 180–250 nm.

FIGURE 2.

Thermal transition curves of THPs obtained by recording the molar ellipticity ([θ]) at λ = 225 nm with a temperature change of 20 °C/h over the range of 5–80 °C and normalized to fraction folded. The THP melting temperature (T_m) was defined as the inflection point in the transition region (first derivative).

Immunocytochemistry

The cell surface concentrations of the α2 and α3 subunits of the α2β1 and α3β1 integrins were evaluated for all melanoma cell lines by immunocytochemistry. Image analysis for each cell line for both receptor subunits (Fig. 3A) provided semiquantitative numerical information on cellular integrin concentrations. Numerical values reflected relative fluorescence intensities of the same cell number, normalized by area measured, and by subtraction of the autofluorescence of the cells (Fig. 3B). All four cell lines showed abundant levels of both integrin subunits, with somewhat higher levels for the α2 subunit compared with the α3 subunit (Fig. 3B). The primary cell line (WM-115) had lower levels of the α2 and α3 subunits compared with the metastatic cell lines. Overall, integrin levels were sufficient to investigate melanoma-ligand interactions.

FIGURE 3.

The cell surface concentrations of the α2 and α3 subunits of the α2β1 and α3β1 integrins evaluated for melanoma cell lines by immunocytochemistry. A, image analysis for each cell line for both receptor subunits and IgG background. Bars indicate 100 μm. B, semiquantitative numerical values for cellular integrin concentrations, obtained by relative fluorescence intensities of the same number of cells, normalized by area measured and by subtraction of the autofluorescence of the cells. Conditions are given under “Materials and Methods.”

Melanoma Cell Adhesion

The influence of glycosylation on melanoma cell adhesion was examined over a THP concentration range of 0–50 μm (Fig. 4). All melanoma cell lines exhibited similar binding curves to each of the nonglycosylated peptides, α1(IV)382–393 THP and α1(IV)531–543 THP (Fig. 4, top left and bottom left). Adhesion to α1(IV)382–393 THP was observed at the lowest peptide concentration tested (0.1 μm), whereas adhesion to α1(IV)531–543 THP initiated at 1.0 μm and reached a maximum at 10 μm (Fig. 4, top left and bottom left). The α1(IV)531–543 THP dose dependence mirrors that reported previously (34). Although adhesion to α1(IV)382–393 THP has been reported previously (32, 33), the present results are the first dose dependences.

FIGURE 4.

Adhesion of melanoma cells as a function of α1(IV)382–393 THP (top left), α1(IV)382–393(Gal) THP (top right), α1(IV)531–543 THP (bottom left), and α1(IV)531–543(Gal)(Gal) THP (bottom right) concentration. Cells were allowed to adhere for 1 h at 37 °C. All assays were repeated in triplicate. Conditions are given under “Materials and Methods.”

In the case of α1(IV)382–393 THP, glycosylation of Hyl³⁹³ had modest influence on cell adhesion (Fig. 4, top right). Nonglycosylated α1(IV)382–393 THP promoted cell adhesion over the whole range of concentrations studied (0.1–50 μm). The glycosylated α1(IV)382–393(Gal) THP showed reduced adhesion levels in the lower THP concentration range (5 μm and below) with the most pronounced difference for the M14#5 metastatic cell line (Fig. 4, top right).

All cell lines were very sensitive toward the glycosylation of the α1(IV)531–543 sequence (Fig. 4, bottom right). Cells were adherent to α1(IV)531–543 THP in a range of 1–50 μm (Fig. 4, bottom left). The glycosylation of Hyl⁵⁴⁰ and Hyl⁵⁴³ dramatically decreased adhesion (Fig. 4, bottom right). Cells were adherent to α1(IV)531–543(Gal)(Gal) THP only at the highest concentration tested (50 μm).

Galectin-3 Interaction with THPs

Galectin-3 is known to mediate cell binding to galactosylated ligands (57). Thus, we examined the possibility that glycosylation may result in a switch of receptor binding, in that galectin-3 may mediate binding to glycosylated THPs. Binding was tested using a galectin-3 AlphaScreen assay (45) with α1(IV)382–393 and α1(IV)382–393(Gal) THPs along with ASF as a control (Fig. 5). Only nonspecific binding of the THPs with galectin-3 was observed (IC₅₀ in the millimolar range) with no difference between glycosylated and nonglycosylated ligands. Thus, galectin-3 does not appear to be involved in binding to these ligands.

FIGURE 5.

Results of AlphaScreen assay for determination of the binding of α1(IV)382–393 and α1(IV)382–393(Gal) THPs to galectin-3. ASF was used as positive control.

Molecular Modeling of integrin·THP Complexes

To further investigate the influence of glycosylation of α1(IV)382–393 and α1(IV)531–543 THPs on binding to their respective integrins, molecular modeling was performed. Models for both integrin·THP complexes were prepared (Figs. 6 and 7).

FIGURE 6.

Molecular modeling of α1(IV)382–393(Gal) THP interaction with the α2 integrin subunit I-domain.

FIGURE 7.

Molecular modeling studies of α3β1 integrin with α1(IV)531–543 peptide. A, a small deviation of φ/ψ angles (10°) caused by flexibility of this region results in strand separation. B, docking studies of α1(IV)531–543 peptide using three different sets of φ/ψ angles. Docked peptide (backbone in gray, Lys in blue, and Asp/Glu in yellow) binds across the α/β integrin interface (magenta/cyan, respectively). C, refined docking simulations where only Gly⁵⁴¹ and/or Gly⁵⁴⁴ were allowed to be flexible.

For the α2β1 integrin, a model was generated using the x-ray crystallographic structure of the α2 I-domain in complex with a THP (PDB: 1DZI) (9). The Hyl³⁹³ glycosylation site is located four residues away from the Glu³⁸⁹ responsible for binding to the MIDAS of the I-domain. It appears that the Hyl³⁹³ site is at the outer interface of the integrin interaction site, and thus mono-glycosylation does not impact binding significantly (Fig. 6). When the disaccharide-containing residue (Glc-Gal)Hyl is considered, glycosylation of Hyl³⁹³ will interfere with binding to the α2 integrin because the binding site is masked by the sugar moiety (data not shown). Thus, the effect of glycosylation on α2β1 integrin binding to type IV collagen may very well depend on whether monosaccharide or disaccharide glycosylation has occurred.

The recognition site for the α3β1 integrin is much different from α2β1 because there is no homology between the peptide ligand sequences. Also, the α1(IV)531–543 sequence does not have the classic -Gly-Xaa-Yaa- repeat required for stabilization of the triple-helix, but rather has a noncollagen-like insertion/break region of n = 7 (58). The break region within α1(IV)531–543 is anticipated to have some strand separation, based on prior studies of break regions within THPs (59, 60). Conversely, the α1(IV)531–543 break region does not possess either Pro or Hyp and nor do the flanking regions, and thus the break is not anticipated to significantly affect the triple-helical structure of the remainder of the THP (61, 62). In addition, the presence of hydrophobic residues within the break region further aids in the stability of the THP (62).

For molecular modeling studies, it was assumed that the α1(IV)531–543 region possesses a polyproline type II-like structure (Fig. 7A). Single-stranded α1(IV)531–543 was used for molecular docking purposes. Three different sets of polyproline type II-like torsion angles, φ/ψ = −60°/150°, −70°/160°, and −75°/175°, were considered, and the peptide backbone was kept rigid upon docking (Fig. 7B). Previous studies revealed the crucial role of both Asp residues of the α1(IV)531–543 sequence for binding to the α3β1 integrin (34). Taking this into account, another set of docking was performed where the Gly⁵⁴¹ and/or Gly⁵⁴⁴ residues were allowed flexibility around the Cα atom. In each docking result the lowest energy ligands were obtained by having the Asp⁵⁴² residue in close proximity of the MIDAS (Fig. 7C). The docking studies revealed that glycosylation of Hyl⁵⁴⁰ and Hyl⁵⁴³ occurred right in the middle of key electrostatic/metal binding interactions and thus would dramatically impact the binding of the α3β1 integrin to the α1(IV)531–543 THP.

β-Galactosidase Activity Evaluation

Knowing that glycosylation could negatively impact integrin binding to type IV collagen, we next examined whether melanoma cells could modulate O-glycosylation of the microenvironment. Initially, E. coli β-galactosidase was tested with the α1(IV)382–393(Gal) THP. Different enzyme concentrations (1–100 units) were compared using incubation at 37 °C for up to 72 h. At certain time points aliquots were taken and subjected to RP-HPLC/MALDI-TOF MS analysis. No hydrolysis of Gal by E. coli β-galactosidase was observed (data not shown). Activity of the β-galactosidase was confirmed with MUG fluorogenic substrate (data not shown).

The influence of the ϵ-amino group of Hyl on β-galactosidase activity was then tested. Prior studies indicated that β-galactosidase was effective in cleaving the Gal moiety from (Gal)Hyl only if the ϵ-amino group was acetylated (63–65). No cleavage of the sugar moiety was observed when α1(IV)382–393(Gal) THP, in either nonacetylated or acetylated (α1(IV)382–393(Gal)-Ac THP) form, was treated with β-galactosidase (data not shown). Although this result is in contrast to the results obtained by Spiro (63–65), the prior study tested the enzyme activity on isolated (Gal)Hyl moiety only.

Whole cell assays were next performed. Two cell lines were selected: primary (WM-115) and metastatic (WM-266–4) melanoma obtained from the same patient. Different cell culture media (EMEM and OptiMEM with or without HI-FBS) were also tested. In the first experiment, cells were grown for 24 or 48 h, and then MUG was added and activity monitored for 1 h (Fig. 8). There was no activity present in EMEM and OptiMEM media (Fig. 8, A and C). In contrast, media containing HI-FBS possessed some β-galactosidase activity (Fig. 8, B and D). In the case of OptiMEM + HI-FBS, the activity was associated with the presence of serum, because control (without cells) also exhibited this activity (Fig. 8D). However, both WM-115 and WM-266-4 exhibited some activity toward MUG above background in EMEM + HI-FBS (Fig. 8B). The results from 48 h were identical (data not shown).

FIGURE 8.

Evaluation of extracellular β-galactosidase activity using the fluorogenic substrate MUG with WM-115 and WM-266–4 melanoma cells. After initial 24 h growth, cells were incubated with MUG, and activity was monitored using λ_excitation = 365 nm and λ_emission = 445 nm. Cell culture media tested were EMEM (A), EMEM + HI-FBS (B), OptiMEM (C), and OptiMEM + HI-FBS (D). RFU, relative fluorescent units.

Next, WM-115 and WM-266-4 were incubated with α1(IV)382–393 THP, α1(IV)382–393(Gal) THP, α1(IV)531–543 THP, and α1(IV)531–543(Gal,Gal) THP. After 24 or 48 h of incubation, an aliquot of culture media was taken and subjected to HPLC/MALDI-TOF MS analysis. The nonglycosylated peptides served as internal controls for determination of the hydrolysis pattern. The RP-HPLC profiles of peptides incubated with media served as a control. Interestingly, RP-HPLC analysis showed that all peptides were stable in media containing HI-FBS (data not shown).

All peptides were clearly identified during RP-HPLC/MALDI-TOF MS analysis of aliquots (Fig. 9). Under the employed conditions, neither of the glycosylated THPs was deglycosylated, as confirmed by MS analyses (data not shown). It was also possible to perform MALDI-TOF MS analyses of crude aliquots (without HPLC separation), and these results confirmed that glycopeptides were unmodified (Fig. 10). The results with cells in suspension were identical, in that (a) β-galactosidase activity could be observed with CUG and inhibited by phenylethyl β-d-thiogalactopyranoside (a selective β-galactosidase inhibitor) and (b) no degalactosylation of the THPs was found (data not shown).

FIGURE 9.

Representative example of RP-HPLC profile of crude media filtrate after incubation of WM-115 cells with THPs for 24 h in EMEM + HI-FBS medium. α1(IV)382–393(Gal) THP (A), biotinylated α1(IV)382–393(Gal) THP (B), α1(IV)531–543 THP (C), and α1(IV)531–543(Gal)(Gal) THP (D) were identified by retention time (indicated with arrow) following MALDI-TOF MS analysis. Similar profiles were obtained for all other tested media.

FIGURE 10.

A, MALDI-TOF MS profile of α1(IV)531–543(Gal)(Gal) THP collected from the indicated peak in Fig. 9D. An arrow indicates the m/z corresponding to the glycopeptide, [M+H]⁺ = 4772.38 (theoretical 4770.29). B, MALDI-TOF MS profile of crude WM-115 cell filtrate after 24 h of incubation with α1(IV)382–393(Gal) THP in EMEM + HI-FBS medium. An arrow indicates the m/z corresponding to the biotinylated glycopeptide, [M+H]⁺ = 4255.30 (theoretical 4254.97).

DISCUSSION

Tumor cells interact with type IV collagen at the site of extravasation through distinct cellular receptors, including the α1β1, α2β1, and α3β1 integrins. Integrins contribute to the ability of melanoma cells to migrate, invade, and metastasize to secondary sites (6, 66). Because they play a pivotal role in both inside-out and outside-in signaling (67), integrins affect most aspects of cell behavior, including shape, motility, differentiation, proliferation, and survival. Thus, it is not surprising that these receptors are also known to be differentially expressed in tumors relative to normal cells, depending on tumor type and stage of progression (4, 6, 66, 68, 69).

The types and concentration of cellular receptors has long been a focus for finding indicators of disease progression. Testing of 10 different human melanoma cell lines found that the α2, α3, and β1 integrin subunits were expressed on all of them (70). It is interesting to note that the α3 subunit showed the highest expression profile, with subtle differences in regards to the invasive profile of a given cell line. The same variation was observed for the α2 and β1subunits, showing higher expression levels in more invasive tumor types, although the overall concentrations were somewhat lower than that of the α3 subunit. In light of these prior results, the role of the associative relationships between type IV collagen and α2β1 and α3β1 integrins with regards to melanoma progression was examined here.

Prior research conducted in our laboratory indicated an altered affinity of a cell surface proteoglycan, CD44, toward binding sites in type IV collagen based on glycosylation (38). This result led to us to consider whether hydroxylation/glycosylation of Lys residues modulates ligand binding by other receptors, such as integrins. Previous studies have not considered how Hyl glycosylation impacts on integrin recognition of collagen. To specifically examine the possible modulation of integrin function by glycosylation, THPs with Lys substituted by glycosylated Hyl for Lys³⁹³ from the human α1(IV)382–393 gene sequence (α2β1 integrin-specific), and Lys⁵⁴³ and Lys⁵⁴⁰ from the human α1(IV)531–543 gene sequence (α3β1 integrin-specific) were synthesized. These ligands were utilized to compare the promotion of cell adhesion.

Collagen glycosylation was found to modulate integrin binding. The integrins were affected differently, with only modest inhibition of α2β1 binding (with the primary melanoma cell line being least affected) and significant inhibition for α3β1 interaction.

Molecular modeling studies of the α2β1 integrin in complex with the glycosylated ligand α1(IV)382–393(Gal) THP indicated that (Gal)Hyl³⁹³ was at the outer interface of the integrin interaction site, and thus galactosylation only slightly diminished binding (Fig. 6). However, for the cell lines tested herein, the effects of ligand glycosylation on binding varied, with the smallest effect on the primary melanoma cell line (WM-115) and the largest effect on the highly metastatic M14#5 cell line (Fig. 4, top right). Because the relative amount of α2β1 integrin on each cell surface was similar (Fig. 3B), variations in activity could be due to interactions of the glycosylated ligand with different activation states of the α2β1 integrin or with different cell surface complexes that incorporate the α2β1 integrin.

The simulations of interactions of the α3β1 integrin with α1(IV)531–543 THP indicated that the relatively flexible 531–543 region is capable of binding across the α3β1 interface with Asp⁵⁴² binding to the MIDAS motif within the β1 subunit (Fig. 7). Because the MIDAS motif is located in the αβ interface groove on β subunit, complexation of the Mg²⁺ cation by the Asp⁵⁴² side chain is a primary driving force for binding the peptide to the receptor (Fig. 7C). This model is with agreement with previously published data identifying Asp⁵⁴² as a critical residue for α3β1 integrin binding to α1(IV)531–543 (34) and consistent with several integrin x-ray crystallographic structures including αIIbβ3 (71), α5β1 (51), and αvβ3 (17). Glycosylation within the α1(IV)531–543 sequence results in significant inhibition of integrin binding, mostly likely because of the proximity of the galactosylated residues (Hyl⁵⁴⁰ and Hyl⁵⁴³) to the key electrostatic/metal binding interactions via Asp⁵⁴². Although inhibition caused by glycosylation is an uncommon phenomenon, the presence of sialic acid on sialoglycoprotein P2B reduced the binding of tumor cells to type IV collagen (72, 73).

The reduced binding of integrins caused by ligand glycosylation presents a possible “cryptic sites” mechanism by which tumor cells may invade the BM (38). In the native, glycosylated state, regions within type IV collagen may have minimal interaction with receptors such as the α3β1 integrin and CD44. After tumor cells bind to type IV collagen (presumably via the α2β1 integrin), cell surface or secreted glycosidases could liberate the collagen-bound carbohydrates. This process would expose cryptic sites for interaction with the α3β1 integrin, CD44/CSPG, and/or other cell surface receptors.

Extracellular removal of carbohydrates could also occur under other circumstances. Numerous bacterial pathogens bind to collagen (74), with binding occurring at several sites within the triple helix (75). The collagen binding protein from Staphylococcus aureus has been identified as CNA, and its mode of binding has been determined (76). Upon binding to collagen, bacteria could secrete β-galactosidases that facilitate deglycosylation. Reduced glycosylation could impact integrin interactions, as well as other collagen-binding proteins. The endocytic collagen receptor urokinase plasminogen activator receptor-associated protein mediates glycosylated collagen turnover (77). DDR1 binds to type IV collagen (78), and this binding may be mediated by ligand glycosylation (79).

Ultimately, glycosylation could be modulated extracellularly in similar fashion to intracellular protein dynamic glycosylation/phosphorylation (80, 81). The post-translational modification of Hyl is catalyzed by two groups of collagen glycosyltransferases, Hyl galactosyltransferase (EC 2.4.1.50) and (Gal)Hyl glucosyltransferase (EC 2.4.1.66), resulting in the formation of (Gal)Hyl and (Glc-Gal)Hyl, respectively (82). The Hyl galactosyltransferase activity has been ascribed to the multifunctional enzyme LH3 (83, 84) and/or GLT25D1 and GLT25D2 (85). LH3 appears responsible for the (Gal)Hyl glucosyltransferase activity (84, 86–89). LH3 can function extracellularly, glycosylating native, triple-helical collagens (90, 91). In fact, extracellular glycosyltransferase activity of LH3 is vital for the cell growth and viability (92). A cell surface galactosyltransferase functions as a type IV collagen adhesion molecule and galactosylates type IV collagen (93). Platelets can supply sugar donor substrates for extracellular glycosylation (94). Collagen Hyl residues can also be phosphorylated (95), and phosphorylation of collagen can occur extracellularly (96).

For dynamic modification of collagen to occur, carbohydrates would need to be removed from type IV collagen extracellularly. An age-dependent increase in β-galactosidase activity (at pH 6) has been reported (97), and a cell surface inactive β-galactosidase functions as an elastin and laminin receptor (98). An α-glucosidase that removes glucose from (Glc-Gal)Hyl has been characterized (99). However, we found no evidence that deglycosylation could be performed extracellularly, because triple-helical glycopeptides were not substrates for purified β-galactosidase or melanoma cells. Thus, although a deglycosylation/cryptic site mechanism provides interesting speculation, it should also be noted that glycosylation is not 100% efficient; collagen O-glycosylation sites are found as mixtures of Lys, Hyl, (Gal)Hyl, and (Glc-Gal)Hyl (36, 89, 100, 101). Thus, receptor interaction may just occur with the subpopulation of type IV collagen that does not contain carbohydrate.

Alternatively, tumor cell binding may be mediated by differential glycosylation that is tissue-specific. For example, LH3 is found extracellularly in kidney, spleen, and muscle (91). Thus, α2β1 and α3β1 integrin binding may be regulated by different levels of type IV collagen glycosylation as determined by LH3 activity. It is also possible that Hyl glycosylation enzymes are decreased in cancer, in similar fashion to β3-N-acetylglucosaminyltransferase-1 (102), and this in turn could enhance receptor association with type IV collagen.

The present study has focused on cellular interactions with glycosylated collagen models. When one considers the BM in vivo, variations in collagen glycosylation are generally unknown. If the levels of glycosylation are indeed modulated, tumor interactions with and response to the BM may be altered or simply compensated for by adherence to other BM ligands (i.e. laminin). Future studies may consider the complexities of cell-BM interactions and the role of glycosylation within that microenvironment.