Melanoma Cell Adhesion and Spreading Activities of a Synthetic 124 ...

Vol. 268, No. 19. Issue of July 5, pp. 14153-14160, 1993 Printed in U.S.A.

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc

Melanoma Cell Adhesion and Spreading Activitiesof a Synthetic 124-Residue Triple-helical“Mini-collagen”* (Received for publication, January 4, 1993, and in revised form, February 23, 1993)

Cynthia G. Fields, Daniel J. Mickelson, Sandra L. Drake, JamesB. McCarthy, and GreggB. Fields$ From the DeDartment of Laboratow Medicine and Pathology and TheBiomedical Engineering Center, University of Minnesota, Minneapolis, Minnesota 55455

glycosylated collagen sequences in homotrimeric and A great variety of cells,such as melanomacells, fibroblasts,platelets,keratinocytes, and epithelial heterotrimeric forms. cells, adhere to and migrate on specific regions within the triple-helical domains of types I, 111, and IV collagen. The relative importance of collagen primary, secTumor cell invasion and metastasis involves the adhesion ondary, and tertiary structures on these cellular acand motility of tumor cells on extracellular matrix compotivities has not been ascertained, as no general synnents, such as fibronectin andcollagen. Collagens are distinthetic methodology exists to allow for the study of guished structurally from other extracellular matrix proteins peptides incorporatingbiologically active sequences in by their composition of three a chains of primarily repeating triple-helical conformation. We have thusdeveloped a Gly-X- Y triplets, which induces each a chain to adopta leftnovel, generally applicable solid-phase branching handedpoly-Pro I1 helix. Threeleft-handedchainsthen methodology for the synthesis of aligned, triple-helical intertwine to form a right-handed superhelix. Triple-helical collagen-model polypeptides (Le. “mini-collagens”). conformation is conducive for adhesion of a variety of cells to Three nascent peptide chains are carboxyl-terminally types linked through one Nu-amino and two Ilf-amino groups I and IV collagen (Rubin et al., 1981; Santoro, 1986; Aumailley and Timpl, 1986; Vandenberg et al., 1991; Goldof Lys, while repeating Gly-Pro-Hyp triplets induce triple helicity. A homotrimeric triple-helical polypep- man et al., 1992; Gullberg et al., 1992) and the migration of tide (THP)of 124 amino acids, incorporating residues keratinocytes on type I collagen (Scharffetter-Kochanek et 1263-1277 of al(IV) collagen, was synthesized. Highly al., 1992) and has been implicated as an essential feature for metastatic mouse melanoma cells showed a profound interstitial collagen catabolism (Fields,1991). Severalsepreference for adhesion to this THP as compared with quences within the triple-helical domains of types I, 111, and a single-stranded peptide(SSP)incorporating the same IV collagen have been identified as cell adhesion sites. The type IV collagen sequence or a branched peptide con- adhesion of Chinese hamster ovary cells to type I collagen is inhibited by the 757-791 sequence of therat a l ( I ) chain tainingeightrepeats of Gly-Pro-Hyp(designated GPP*). Specifically, 50% cell adhesion occurred at a (Kleinman et al., 1978). The cy2& integrin from human fibroTHP concentrationof 1.12FM, while comparable levels blasts and platelets adheres to a peptide incorporating resiof adhesion required [SSP]= 170 pM or [GPP*] > 100 dues 430-442 of the rat al(I)chain (Staatzet d . , 1991), while human platelet aggregation is inhibited by a peptide incorPM. Melanoma cells also spread on the THPa greater to porating residues 76-84 of the bovine al(III) chain (Legrand extent thanon the SSPor GPP*. These results are the first direct demonstrations of the significance of triple et al., 1980). Residues 531-543 of the human al(IV) chain promotehumankeratinocyteadhesionandrabbitcorneal helicity for cell adhesion to and spreading on a specific collagen sequence and support earlier conclusions of epithelial cell adhesionandmigration(WilkeandFurcht, 1990; Cameron et al., 1991). A peptide incorporating residues conformational dependency for cell adhesion to and 1263-1277 from the cyI chain of humantype IV collagen migration on types I and IV collagen. In addition, the motility of mouse and melanoma cell THP activities support the concept that supportsadhesion,spreading,and glioma and neuroblastomacell lines tumor cell adhesionand spreading on type IV collagen human melanoma and rat (Chelberg et al., 1990). involvesmultiple,distinctdomainsintriple-helical cell adhesiontotriple-helical collagen conformation. The triple-helical peptide synthetic pro- Priorstudieson sequences have utilized only single-stranded peptides, even tocol developed here will allow eventually for the study of both structure and biological activity of specific, though disruptions of collagen secondary and tertiary structures have been shown to be detrimental for adhesion. For * This work was supported by National Institutes of Health and example, the initial rateof rat hepatocyte adhesionwas more Diabetes andDigestive and Kidney Diseases Grants KD 44494 (to G. rapid to native, triple-helical collagen compared with denaB. F.) and CA 43924 and CA 54263 (to J. B. M.), Minnesota Medical tured collagen due to conformationally-dependent alp1inteFoundation Grant CRF-152-91, American Cancer Society Grant 1332-6, the Leukemia Task Force, University of Minnesota Grant-in- grin binding sites (Rubin et al., 1981; Gullberg et al., 1992), while platelets adhered effectively to native, but not denaAid of Research, Artistry, and Scholarship, and the Millipore Corp. The costs of publication of this article were defrayed in part by the tured, type I collagen (Santoro, 1986). Human fibrosarcoma payment of pagecharges. Thisarticlemust thereforebehereby ( H T 1080) cells adhered to the major triple-helical domain marked “aduertisement” in accordance with 18 U.S.C. Section 1734 and cyanogen bromide-derived CB3 fragment (residues 291solely to indicate this fact. 576) of type IV collagen, but not to the denatured domainor $ Recipient of a McKnight-Land Grant Professorship. To whom fragment (Aumailley andTimpl, 1986; Vandenberg et al., correspondence andreprintrequestsshould be addressed Dept. of Laboratory Medicine and Pathology, Box 107, 420 Delaware St. 1991). Cell migration and aggregation is also influenced by triple helicity, aschemotaxis of humankeratinocytes was S.E., University of Minnesota,Minneapolis, MN 55455. Tel.: 612-626-2446; Fax: 612-625-1121. twice asgreatonnative uersus denaturedtype I collagen

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14154

Melanoma-adhesive Mini-collagen

(Gly-Pro-Hyp)8-Gly-Val-Lys-Gly-Asp-Lys-Gly-Asn-Pro-Gly-Trp-Pro-Gly-Ala-Pro-AHA

1

(Gly-Pro-Hyp)8-Gly-Val-Lys-Gly-Asp-Lys-Gly-Asn-~o-Gly-Trp-Pro-Gly-Ala-Pro-AHA-Lys-L s-Tyr-Gly

1

(Gly-Pro-Hyp)8-Gly-Val-Lys-Gly-Asp-Lys-Gly-A~-~o-Gly-Trp-Pro-Gly-Ala-Pr~A~

(Gly-Pro-Hyp)s-AHA FIG. 1. Sequences of THP (top),SSP (middle),and GPP* (bottom).T h e T H Pis composed of a carboxyl-terminal branch generated from 2 Lys residues, 1 al(I) 1263-1277 sequence per chain, and 8 Gly-Pro-Hyp repeats per chain. The SSP is composed of the a1(I) 12631277 sequence. GPP* is composed of the carboxyl-terminal branch and8 Gly-Pro-Hyp repeats per chain.

(Scharffetter-Kochanek et al., 1992), while platelet aggregation was induced by only triple-helical, not denatured, cyanogen bromide-derived fragments of types I and I11 collagen (Zijenah andBarnes, 1990). Theseresults suggest that a general synthetic peptide methodology needs to be developed by which the importance of collagen triple-helical structure on cellular activities can be ascertained. Additionally, this unique methodology should allow for the incorporation of post-translationally modified amino acids, as both of the cell adhesion sequences within type IV collagen contain O-glycosylated Hyl residues (Babel and Glanville, 1984). Cell-surface galactosyltransferase has been shown to mediate fibroblast adhesion to type IV collagen via glycosylated residues (Babiarz and Cullen, 1992). We describe presently the threedimensional orthogonal solid-phase synthesis and characterization of a triple-helical polypeptide (THP)’ incorporating

residues 1263-1277 from the a1chain of type IV collagen (Fig. 1).A covalent branching scheme was developed for the synthesis of the THP, in similar fashion to branching schemes utilized for the syntheses of a-helical bundle proteins (Hahn et al., 1990; Mutter et al., 1992) and multiple antigen peptide systems (Tam, 1988). Also synthesized were a single-stranded peptide (SSP) incorporating residues 1263-1277 from the a1 chain of type IV collagen and a branched peptide containing eight repeats of Gly-Pro-Hyp (designated GPP*) (Fig. 1).The adhesion and spreading activities of highly metastatic melanoma cells were quantitated as a function of THP, SSP, or GPP* concentration. EXPERIMENTALPROCEDURES

Materials-All standard peptide synthesis chemicalswere analytical reagent grade or better and purchased from Applied Biosystems, The abbreviations used are: THP, triple-helical peptide; AHA, 6- Inc. (Foster City, CA) or Fisher. DBU, EDT, and (Ph3PLPd were aminohexanoic acid; AI, allyl; allyllinker, 4-trityloxy-Z-but-2-enylox- from Aldrich, ~ - H y pand FDAA fromSigma,Gly-Pro-Hypand yacetic acid dicyclohexylamine salt; Boc, tert-butoxycarbonyl; DBU, MBHA resin (substitution level = 0.80 mmol/g) from Bachem Biosciences (Philadelphia, PA), Fmoc-Gly-HMP resin (substitutionlevel 1,8-diazabicyclo[5.4.0]undec-7-ene; DCM,dichlorometbane;DIEA, = 0.43 mmol/g) from Millipore Corp., Fmoc-Tyr(A1) and allyl linker N,N-diisopropylethylamine;DIPCDI, N,N”diisopropylcarbodiimide; DMF, N,N-dimethylformamide; EDT, 1,2-etbanedithiol; FDAA, 1- from Propeptide (Vert-Le-Petit, France), HBTU fromRichelieu Biofluoro-2,4-dinitrophenyl-5-~-alaninamide; Fmoc, N-fluoren-g-ylme- technologies (St.-Hyacinthe,Quebec), Fmoc-AHA andFmoc-Nle from Advanced ChemTech (Louisville, KY), andFmoc-Hyp(tBu) thoxycarbonyl; GPP*, [N-tris[(Gly-Pro-Hyp),-AHA]-Lys-Lysl-Tyrfrom Novabiochem (La Jolla, CA).All other Fmoc-amino acids were Gly; HBTU, 2-(1H-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluoropbosphate; HMP, 4-hydroxymethylphenoxy; HOBt, l-hy- from Bachem Biosciences or Millipore. Amino acids are of the Ldroxybenzotriazole; HPLC, high performance liquid chromatography; configuration (except for Gly) except where noted. The synthesis, MBHA, 4-methylbenzhydrylamine;Nle, norleucine; NMP, l-methyl- purification, and characterization of the SSP have been described 2-pyrrolidinone; PBS, phosphate-buffered saline solution; (Ph,P),Pd, (Chelberg et al., 1990), while that of GPP* will he described elsetetrakis(triphenylphosphine)palladium(O);resin, copoly(styrene-1%- where.’ divinylbenzene); PAGE, polyacrylamide gel electrophoresis; SSP, sin* C. G. Fields and G. B. Fields, manuscript in preparation. gle-stranded peptide; TFA, trifluoroaceticacid.

Mini-collagen Melanoma-adhesive

14155

Preparation of Fmoc-Gly-Pro-Hyp-Fmoc-Gly-Pro-Hyp was syn- amino acids was by Fmoc solid-phase methodology on an Applied thesized from Gly-Pro-Hyp using an established protocol for Fmoc- Biosystems 431A Peptide Synthesizer using cycles described previamino acids (Fields et al., 1989). A typical preparation was as follows. ously (Fields et al., 1991; C. G. Fields et al., 1992). Incorporation of 3.0 g of Gly-Pro-Hyp (10.5 mmol) was dissolved in 54 ml of Na2C03/ Fmoc-Gly-Pro-Hyp tripeptides was performed manually in a shaker water (1:9) and stored at 4 "C. 4.05 g of N-fluoren-9-ylmethyl succi- as follows. The Fmoc-peptide-resin (0.10 mmol) was deprotected with nimidyl carbonate (12.0 mmol) was dissolved in 45 ml of dimethoxy- 10 ml of DBU/piperidine/DMF (1:1:48) for 0.5 h and washed three ethane and stirred at 4 "C. The aqueous Na2C03solution was added times with DMF. 0.202 g of Fmoc-Gly-Pro-Hyp (0.40 mmol) and M slowly to the dimethoxyethane solution, and the reaction proceeded 0.061 g of HOBt (0.40 mmol) were dissolved in 10 mlof0.035 for 2.5 h at 4 "C and 21 h at room temperature. The solution was HBTU/DMF. 0.131 mlof DIEA (0.75 mmol) was added, and the filtered, and 360 ml of water was added to the filtrate. The aqueous solution reacted with the resin for 2.5 h. The Fmoc-peptide-resin was layer was extracted with 300 ml of diethyl ether, acidified to pH 2 washed three times with DMF. Deprotection and coupling steps were with concentrated HC1, reduced to half-volume at 80 "C under re- repeated seven times. 0.196 g of the peptide-resin was side-chain-deprotected by treatduced pressure, and stored at 4 "C for 24 h. The aqueous layer was decanted from the oily precipitate, reduced to -30 ml at 81 "C under ment with TFA/EDT/water (92.5:2.5:5)for 1 h and washed with reduced pressure, and stored at 4 "C for 24 h. The aqueous layer was DCM. Polypeptide was liberated from the resin by treatment with decanted from the oily precipitate. Both oily precipitates were dis- 0.074 g of (Ph3P),Pd (0.065 mmol) as described (Lloyd-Williams et solved in a total of 20 ml of methanol, then 250 ml of ethyl acetate al., 1991) for 20 h. The resin was filtered and washed with piperidine was added. A white residue was recovered by evaporation at 73 "C for and DCM. The dried resin mass was 0.075 g, indicating a release of 1 h under reduced pressure; yield3.23 g (6.39 mmol, 60.6%). The 0.121 g of peptide (62% of theoretical). The combined filtrate and identity of the product as Fmoc-Gly-Pro-Hyp and its homogeneity washings were reduced under pressure to minimal volume (1.0-1.5 was verified by thin layer chromatography (chloroform/methanol/ ml), 2.0 ml of0.5 N aqueous HCl was added, and the solution was acetic acid (95:20:3)),scanning UV spectroscopy, and Edman degra- extracted with diethyl ether andDCM. The aqueous layer was applied to a Pharmacia LKB Biotechnology Inc. PD-10 G-25 size-exclusion dation sequence analysis. Preparation of Fmoc-Gly-Allyl Resin-0.848 g of Fmoc-Nle (2.4 column (5.0 X 1.5 cm, equilibrated with acetic acid/water (1:9)) and mmol) was coupled to 2.0 g of MBHA-resin (1.6 mmol) with 0.367 g eluted with acetic acid/water (1:9). The eluent was lyophilized to a powder, then dissolved in 2.0 mlof acetic acid/water (1:9) for semiof HOBt (2.4 mmol) and 0.373 ml of DIPCDI (2.4 mmol) in 20 ml of DCM/DMF (1:l)for 2.3 h. The resin was washed three times with preparative HPLC. Semipreparative HPLC was performed on a Rainin AutoPrep DMF, deprotected with 20 ml of piperidine/DMF (1:l) for 30 min, and washed three times with DMF. 1.82 g of allyl linker (3.2 mmol) System with a Dynamax C-18 column (12-pm particle size, was coupled to the resin with 0.489 g of HOBt (3.2 mmol) and 0.497 300-A pore size, 250 X 10 mm). The elution gradient was 0-60% B in ml of DIPCDI (3.2 mmol) in 20 ml of DMF for 18.5 h. The resin was 45 min at a flow rate of 2.0 ml/min, where A was water containing washed once with DMF andtwice with DCM, deprotected twice with 0.1% TFA and B was acetonitrile containing 0.08% TFA. Detection 20mlof TFA-DCM (9:1), first for 20 min, then for 10 min, washed was at 229 nm. Analytical HPLC was performed on a Hewlettthree times with DCM, neutralized with 20 ml of DIEA/DCM (1:9) Packard 1090 Liquid Chromatograph equipped with an ODS Hypersil for 15 min, and washed once with DCM and twice with DMF. 1.43 g C-18 column (5-pm particle size, 100 X 2.1 mm). 10 pl from semipreof Fmoc-Gly (4.8 mmol) was esterified to the allyl resin with 0.735 g parative HPLC fractions were loaded onto the column. The elution of HOBt (4.8 mmol), 0.746 ml of DIPCDI (4.8 mmol), and 0.059 g of gradient was 0-60% B in 60 min at a flow rate of 0.3 ml/min, where 4-(dimethy1amino)pyridine(0.48 mmol) in 20 ml of DMF for 6.5 h. A and B were the same as for semipreparative HPLC. Diode array The resin waswashed once with DMF and twice with DCM and detection was a t 220, 254, and 280 nm. stored under vacuum overnight. Peptide Analyses-Ninhydrin analysis (Fields et al., 1992b) was Preparation of [N-tris[Fmoc-AHA]-Lys-Lys]-Tyr(Al)-Gly-Allylused to monitor all manual coupling and deprotection steps. Edman Resin-Fmoc-Tyr(Al), Fmoc-Lys(Boc), and Fmoc-Lys(Boc) were degradation sequence analysis was performed on an Applied Biosyscoupled to Fmoc-Gly-allyl resin with 1.40 mmol of Fmoc-amino acid, tems 477A Protein Sequencer/l20A Analyzer as described previously 0.215 g of HOBt (1.40 mmol), and 0.218 ml of DIPCDI (1.40 mmol) (C. G. Fields et al., 1992, 1993). Fulvene-piperidine concentrations in 20mlof DMF for 2-4 h. Both Fmoc-Lys(Boc) residues were (301 nm) and scanning UV spectra (200-320 nm) were determined double-coupled. Fmoc removal was by 20 ml of piperidine/DMF (1:1) with a Beckman DU-70 spectrophotometer. Amino acid analyses were for 0.5 h. The peptide-resin was washed three times with DMF after performed on a Beckman 6300 Analyzer with a sulfated polystyrene each coupling and deprotection, then once with DCM prior to removal cation exchange column (0.4 X 25 cm). Peptides were hydrolyzed with of the Boc groups. The N-amino Boc groups wereremoved by 6 N aqueous HCI a t 110 "C for 18-48 h. CD spectroscopy was perresin with formed on a Jasco710 spectropolarimeter using a 100-pl,0.1-mm cell. treatment of Fmoc-Lys(Boc)-Lys(Boc)-Tyr(A1)-Gly-allyl 20mlof TFA/DCM (1:l) for 0.5 h. The peptide-resin was washed Reversibility of triple-helical melting was examined by recording a three times with DCM, neutralized with 20 ml of DIEA/DCM (1:9) CD spectrum at 14.6 "C, heating to 84.7 "C, and recording a spectrum, for 0.5 h, washed twice with DCM and once with DMF, and Fmoc- and cooling to 15.6 "C, equilibrating for 0.5 h, and recording a specdeprotected as described above. Fmoc-AHA was double-coupled for trum. The van't Hoff enthalpy (&) was calculated for the triple 2.5-3 h as described above using 4.10 mmol of Fmoc-AHA (1.45 g), helix ej coil transition using HOBt (0.630 g), and DIPCDI (0.638 ml). The substitution level was determined by fulvene-piperidine analysis (Fields et al., 199213) to be 0.181 mmol/g. Peptide Synthesis and Purification-Incorporation of individual where F is the fraction of triple helicity (Engel et al., 1977).The THP and GPP* were 3H-labeled by reductive methylation (Jentoft and Dearborn, 1979) with NaCNBH3 and [3H]formaldehydeand desalted over a Sephadex G-50 column (50 X 1.5 cm) in PBS, p H 7.4. [3H] THP was used for 6-15% SDS-PAGE and size-exclusion chromatography. -Lys -Gly linker Racemization of Hyp during Fmoc-Gly-Pro-Hyp Coupling-FmocGly-Pro-Hyp was coupled to Gly-HMP resin and deprotected under the same conditions as described in Peptide Synthesis and Purijication. Gly-Pro-Hyp-Gly was liberated from the resin with a 1-h treatment of TFA/water (95:5), precipitated with methyl tBu ether, and FIG. 2. General strategy for the solid-phase synthesis of hydrolyzed for 3, 6, 9, 20, and 46 h as described in Peptide Analyses. branched, triple-helical peptides. The "amino protecting group Two different Gly-Pro-Hyp-Gly concentrations were used. Racemi( B )must be stable to theconditions of the N"-amino protecting group zation studies were performed by derivatizing samples with FDAA as ( A )removal, while the 0-phenolprotecting group (C) and linker must described (Adamson et al., 1992) and eluting 2,4-dinitrophenyl-5-~be stable to theconditions of the N"- and N-aminoprotecting group alaninamide-amino acids by analytical HPLC (see Peptide Synthesis removal. Once the N"- and "amino groups are deprotected, simul- and Purification) with an elution gradient of 5-20% B in 70 min at a taneous peptide synthesis proceeds from all three termini. The spe- flow rate of 0.5 ml/min. Detection was at 340 nm. cific protecting group strategy used in this work is given under Melanoma Cell Adhesion and Spreading"K1735 M4 tumor cell "Results and Discussion." adhesion and spreading assays were as described previously (Chelberg

I

I

-2

+-"

14156


i

1800

1400 1200

FIG. 3. Analytical HPLC elution profile of the purified THP. The THP elution time was 30.9 min. Condi-

3

1000

tionsare given under“Experimental Procedures.”

+J 40

30 10

20

Time

(min.)

0

-10

-20

FIG. 4. Circular dichroism spectra of the THP in acetic acid/water (1:19), pH 2.4. Spectra were recorded at [THP] = 83.5 pM by accumulating 5 scans at 0.5 nm intervals (response of 1 s). The spectrum at14.6 “C is characteristic of an intact triple-helix, while the spectrum at 84.7 “C indicates no triplehelix is present (see “Results and Discussion”).

-30

et al., 1990) with minor alterations. Peptides were dissolved in PBS Immulon plates was determined as described (Chelberg et al., 1990) andadsorbed directly onto 96-well polystyreneImmulon 1 plates using 3H- or ‘2”I-labeledpeptide. (Dynatech Laboratories Inc., Chantilly, VA) overnight a t 37 “C. Nonspecific binding sites were blocked with 5 mg/ml bovine serum albuRESULTSANDDISCUSSION min in adhesion media (Dulbecco’s modified Eagle’s medium containDesign, Synthesis, and Characterization of the Triple-helical ing 20 mM HEPES) for 2 h a t 37 “C. Tumorcells were released from tissue culture flasks with 37 “C PBS containing 10 mM EDTA and Polypeptide-To ensure proper alignment of three peptide washed several times with adhesion media. Cells were labeled overstrands in a triple-helix, a branching protocol was developed night with 1 pCi/ml 13H]thymidine (Du Pont-New England Nuclear) based on the liquid-phase synthetic scheme of Heidemann for adhesion assays. Cells were added to the plate wells a t a density and co-workers (Roth et al., 1979). The branchwas introduced of 50,000 cells/ml in a total volume of 100 pl and adhered for 1 h a t at the carboxyl terminus of the synthetic peptide, consistent 37 “C. For adhesion assays, wells were washed several times with adhesion media, the remainingcells were lysed, and radioactivitywas with the natural nucleation of collagen triple-helices from the determined asdescribed (Chelberg et al., 1990). For spreading assays, carboxyl toaminoterminus(EngelandProckop, 1991). wells were fixed and stained using DiffQuik reagents (Baxter) and Branching of three peptide strands from one initial chain photographedwith a NikonMF-15cameramountedon a Nikon Diaphot inverted microscope at 2 0 0 ~magnification. Cell spreading required three different protecting group strategies (Fig. 2): (B), was quantitated by an Optomax System IV image analyzer equipped W-aminoprotection ( A ) , Lys side chainprotection which must be stable to the N”-amino protecting group rewith a Hitachi Monitor. The efficiency of peptide adsorption to the

14157


.o -

I I I I I I I

.o -

FIG. 5. Thermal transitioncurve of the THP inaceticacidlwater (1:19), pH 2.4. Molar ellipticities ([HI) were recorded at X = 225 nm while the temperature was increased from 15 "C to 85 "C. The triple-helix was somewhat destabilized by aggregation of the THP from 15-30 "C. The triple-helix was stable from 30-48 "C, then melted with T,,, = 58.5 "C.

I I 0-

.o -

-

, 20

I I I I I I I I I I I I I I I I I I I I I

1

1

I ,

I

1

70

60

50

40

30

A FIG. 6. Elution profile from Sephadex G-50-80 (100 X 1.0 cm) sizeexclusion chromatography of [3H] THP at 4 "C ( A ) or 35 "C ( B ) .Samples were runin PBS containing 1% Triton at a flow rate of 5.0 ml/h. The column, buffer, and samples were equilibrated at 4 "C or 35 "Cfor 12 h. Sample volumes were 750 pl, collected fractions 1.5 ml,and collection time 20 h. Fraction radioactivity was quantitated by a Beckman LS 3801 Liquid Scintillation Counter. THP estimated molecular mass = 11.6 kDa at 4 "C and 36.0 kDa at 35 "C.

0

2 0 4 0 6 0 8 0 1 0 0 0 2 0 4 0

&I 100 120

60

Fraction Number

moval conditions, and C"-carboxyl protection (linker), which must be stable to theN"-amino and Lys side chain protecting group removal conditions. As eventual incorporation of glycosylated Hyl is desired, the synthetic scheme could not use repetitive acidolysis for W-amino protecting group removal or strong acidolysis or alkali for C-carboxyl protecting group removal (cleavage of the peptide-linker). This desired synthetic scheme was achieved with amild three-dimensional orthogonal3 protecting group strategy. N"-Amino protection ~

~

~

.

~

~

~

~

.

~

~

An orthogonal system has been defined as a set of completely independent classes of protectinggroups,such that eachclass of

( A ) was the Fmoc group, which is base-labile and stable to acidolysis and (Ph3P)rPd-catalyzed nucleophilic transfer (Kunz and Dombo, 1988; Fields et al., 1992b). Lys side chain protection ( B ) was the Boc group, which is moderate acidlabile and stable to base and (Ph3P).,Pd-catalyzed nucleophilic transfer (Blankemeyer-Menge and Frank, 1988; Fields et al., 199213). (?"-Carboxyl protection was the allyl linker, which is labile to (Ph3P),Pd-catalyzed nucleophilic transfer and acidand base-stable (Guibi et al., 1989; Kunz et al., 1989; Lloyd~

_

_

_

_

_

_

_

.

~

group can be removed in any order and in the presence of all other classes (Barany and Merrifield, 1977; Barany and Albericio, 1985).

~

.

14158


Frc. 7. Adhesion of melanoma cells as a function of THP, SSP, or GPP* concentration. Cells were allowed to adhere to peptide-coated Immulon plates for 1h at 37 "C. Melanoma cell adhesion was most specific for the THP, where 50% adhesion occurred at a concentration of 1.12 p M , Half-maximal adhesion to SSP and GPP' occurred at concentrations of 170 and >lo0 p M , respectively. Conditions are given under "Experimental Procedures."

Lo9 [ P e P t w (PM) Williams et al., 1991). Allyl-based side chain protection was ratios were Gly40 (40 expected), Pro 30.2 (31), Hyp 25.8 (24), used for Tyr (C). Asx 6.4 (6), Ala 3.2 (3),Val 2.8 (3), Tyr 1.3 ( l ) , Lys 8.8 (8), Branching was achieved by synthesizing Fmoc-Lys(Boc)- and AHA 3.4 (3).The integrity of the Trp residues was Lys(Boc)-Tyr(A1)-Gly-allyl resin and deprotecting the Ne- established by scanning UV spectroscopy. and "amino groups. Fmoc-AHA was then incorporated onto The THP CD spectrum at 14.6 "C exhibited a large negative all three amino termini to provide a flexible spacer, The [@I200 and a positive [8]225(Fig. 4), distinguishing features of a specific sequence from the a1chain of type IV collagen (resi- coiled-coil triple-helix (Heidemann and Roth,1982). The CD dues 1263-1277)was assembled with Fmoc-amino acids. spectrum at 84.7 "C (Fig. 4) was indicative of a melted tripleFmoc-Gly-Pro-Hyp triplets were then incorporated following helix, as [8]225 was negative (HeidemannandRoth, 1982). the specific type IV collagen sequence, as repeating Gly-Pro- Melting o f the triple-helix was reversible. To determine the Hyp sequences form stable triple-helices at relatively short triple-helix melting temperature (triple-helix coil transichain lengths (Sakakibara et al., 1973; Engel et al., 1977). tion), [ 8 ] 2 2 5 was monitored as a function of temperature. Two Since the desired T H P was 124 residues, rapid coupling and transitions were seen, one with a midpoint at 23.5 "C and one deprotection reagents wereused to ensure high synthetic with a midpoint of 58.5 "C (Fig. 5 ) . Size-exclusion chromatogefficiency. Coupling of Fmoc-amino acids and Fmoc-Gly-Pro- raphy (Fig. 6) gave apparent T H P molecular masses of 11.6 Hyp was achieved with HBTU, which is highly reactive kDa at 4 "C and 36.0 kDa at 35 "C, indicating the first (Fields et al., 1991) and utilized with optimal peptide-resin transition was an aggregation phenomenon. Aggregation has solvation conditions (Fields and Fields, 1991). Fragment con- been observed previously by x-ray diffraction and electron (Glydensation of Fmoc-Gly-Pro-Hyp tripeptides proceeded microscopy studies of triple-helical (Pro-Pro-Gly)zo and smoothly, with less than 0.5% ~ - H y per p Fmoc-Gly-Pro-Hyp Pro-Hyp), polypeptides (Andreeva et al., 1963; Millionova et incorporated. Rapid removal of the Fmoc group from Fmoc- al., 1963; Olsen et al., 1971). Although the large number of Gly-Pro-Hyp was achieved with 2% DBU plus 2% piperidine Hyp residues in the Y position of the THP could promote (for scavenging dibenzofulvene) in DMF. DBU removal of the aggregation via intermolecular hydrogen bonding (RamachanFmoc group is rapid even in "difficult" sequences (Wade et dran et al., 1973; Nkmethy and Scheraga, 1986), aggregation al., 1991). Solid-phase sequence analysis gave the desired induced by increasing temperature from 15-30 "C is indicative of a hydrophobically driven process (Fields et al., 1992a). The sequence [ (Gly-Pro-Hyp),-Gly-Val-Lys-Gly-Asp-Lys-GlyAsn-Pro-Gly-Trp-Pro-Gly-Ala-Pro] and 3% cumulative pre- second T H P transition, a triple-helical melt at 58.5 "C, was view, indicating that the synthesis of the homotrimeric pep- of a similar value to T,,, = 57.5-61 "C for (Pro-Hyp-Gly),, in tide washighly efficient. Polypeptide-resin mass following 0.6-10% acetic acid/water (Sakakibara et al., 1973; Engel et synthesis was0.694 g (91% of theoretical). Following side al., 1977; Brodsky et al., 1992; Long et al., 1992). The van't chain deprotection and cleavage of the polypeptide-resin, Hoff enthalpy ( A P ) for the THP melt was -97 kcal/mol, removal of the majority of the (PhaP)rPd was achieved by similar to LW= -90 kcal/mol (Engel et al., 1977) and -124 dissolving the (Ph,P),Pd-polypeptide complex in 0.5 N HCl, kcal/mol (Long et al., 1992) for the triple-helix w coil tranextracting with diethyl ether and DCM, and chromatograph- sition of (Pro-Hyp-Cly)lo. Melanoma Cell Activities of the Triple-helical Polypeptideing on a G-25 column. Semipreparative HPLC purification yielded a homogeneous polypeptide, as determined by analyt- Melanoma cell adhesion was compared for the THP, SSP, ical HPLC (Fig. 3), SDS-PAGE, and size-exclusion chroma- and GPP* (sequences given in Fig. 1) over a coated peptide tography. The apparent molecular mass of the purified T H P concentration range of 0.1-300 pM. For the SSP concentrawas 11.6 kDa by size-exclusion chromatography at 4 "C (cal- tion, a molecular mass of 4797 Da was used, where 1 mol of culated molecular mass = 11,205 Da). Mass of the purified SSP accounted for three peptide chains.This conversion T H P was 6.7 mg, 4.7% of the overall theoretical yield. Non- allowed for 1 mol of THP, SSP, or GPP* to represent three covalent sequence analysis gave the sequence (Gly-Pro-Hyp)a- potential peptide active sites. The coating efficiencies for the Gly-Val-Lys. Aminoacid identification could not proceed three peptides were comparable, ensuring that cell adhesion after Lys-27 due to gradual peptide loss from the filter and/ results would not be a reflection of differential peptide ador inefficient amino acid yield per cycle. Amino acid analysis sorption to theplates. Half-maximal melanoma cell adhesion

-


FIG. 8. Comparison of spreading of melanoma cells on THP ( A ) ,SSP ( B ) ,and GPP* ( C ) . Photographs shown are representative examples of spreading on peptide-coatedImmulonplates at [THP] = 0.9 p ~ [SSP] , = 2.1 p ~and , [GPP*]= 1.4 p ~Cells . adhered and spreadfor 1 h a t 37 "C, then were fixedand stained withDiffQuik reagents. Conditions are given under "Experimental Procedures."

occurred at [THP] = 1.12 p ~ [SSP] , = 170 p ~ and , [GPP*] > 100 p M (Fig. 7). Thus, triple-helical conformation in combination with the aI(IV) 1263-1277 sequence resulted in a 100-foldincreasein melanoma cell adhesion activity compared with the aI(IV) 1263-1277 sequence alone. This result

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is the first direct demonstration of the significance of triple helicity for cell adhesion toa specific collagen sequence. Prior studies had concluded that alP1 and a& integrin-mediated cell adhesion to types I and IV collagen was conformationor no dependent,asdenaturing collagen resultedinlittle integrin-mediated adhesion (Santoro,1986; Vandenberg et al., 1991; Gullberg et al., 1992). The level of melanoma cell adhesion to GPP* was similar to that reported previously for rat hepatocyte adhesion to (Pro-Pro-Gly)lo and (Pro-Hyp-Gly), (Rubin et al., 1981) and platelet adhesion to (Pro-Gly-Pro), (ZijenahandBarnes, 1990). Intact triple-helical structure may, independent of sequence, serveas a low level recognition feature for cells, as platelet adhesion to types I or I11 collagen was not inhibited by (Pro-Gly-Pro), (Zijenah and Barnes, 1990). Structural elements other than triple-helices, such as polyproline, do not appear to direct collagen-mediated cell adhesion (Rubin et al., 1981). Melanoma cell spreading was compared for the THP, SSP, , [GPP*] and GPP* at [THP] = 0.9 pM, [SSP] = 2.1 p ~ and = 1.4 p ~ Cell . spreading was more extensive on the T H P (Fig. 8 A ) than on either theSSP (Fig. 8 B ) or GPP* (Fig. 8C). Cell areas averaged 0.048, 0.012, and 0.017 mm*/pM peptide in response to THP, SSP, and GPP*, respectively. As in the case of melanoma cell adhesion, cell spreading was most efficient when triple-helicity was combined with the al(IV) 1263-1277 sequence. This result is consistent with conclusions from prior studies that cell migration or motility on collagen or collagen sequences can be conformation-dependent.Humankeratinocyte migration was twice as great on triple-helical uersus denatured type I collagen (ScharffetterKochanek et al., 1992). A single-stranded peptide model of theal(IV) 1263-1277 sequenceshowed substantially decreased melanoma spreading activity when the three imino acids (Pro'*'', and Pro'?'') were replaced by Ala (Chelberg et al., 1990). This reduced activity was correlated by twodimensional'HNMRtothe absence of a double P-turn structure in the carboxyl-terminalregion of the peptide (Mayo et al., 1991). Although triple helicity per se was not a factor in these single-stranded peptides, the relative importance of secondary structure for melanoma cell spreading on a collagen-model sequence was clearly demonstrated. The combined melanomacell T H P adhesion and spreading activities support the concept that tumor cell adhesion and spreadingontype IV collagen involves multiple, distinct domains, as a t least two domains within type IV collagen in triple-helical conformation are tumorcell adhesion sites (Chelberg et al., 1989; Vandenberg et al., 1991). In addition, the enhancement of cellular activities due to triple helicity confirms the aI(IV)1263-1277 sequence as a specific melanoma cell adhesion and spreading site, as this sequence in its native conformation has greater activity than the isolated sequence and implies that basement membrane type IV collagen is a site for tumor cell invasion based on collagen primary, secondary, and tertiary structures. Other activities that arecollagen-mediated, such as cell adhesion to sites in types I, 111, and IV collagen and matrix metalloproteinase cleavage of types I-IV collagen at specific sites, are accessible to study by the synthetic method described here. We have demonstrated that homotrimeric mini-collagens can be synthesized without repetitive or strong acidolysis and used to study the relative influences of collagen primary, secondary, and tertiary on cellular behavior. By utilizing further dimensionsof chemical orthogonality, heterotrimeric mini-collagens will be synthesized, and thus all structural features of native collagens will be incorporated into studies of collagen-mediated biological activities. Acknowledgments-We thank Celeste Hymel for excellent techni-

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Guibe, F., Dangles, O., Balavoine, G., and Loffet, A. (1989) Tetrahedron Lett. cal assistance, Dr. M o r t e n Meldal for helpful suggestions regarding 3 0 , 2641-2644 Fmoc removal conditions, Marc F e r r e r for a s s i s t a n c e w i t h the CD Gullberg, D., Gehlsen, K. R., Turner, D. C., Ahlen, K., Zijenah, L. S., Barnes, studies, Dr. Sally Palm for assistance with cell spreading quantitation, M. J., and Rubin, K. (1992) E M B O J . 11,3865-3873 Hahn, K. W., Klis, W. A., and Stewart, J. M. (1990) Science 2 4 8 , 1544-1547 and Dr. Albert Loffet for the g e n e r o u s d o n a t i o nof Fmoc-Tyr(A1). Heidemann, E., and Roth, W. (1982) Adu. Polym. Sci. 43,143-203 Jentoft, N., and Dearborn, D. G. (1979) J. Biol. Chem. 254,4359-4365 REFERENCES Kleinman, H. K., McGoodwin, E. B., Martin, G. R., Klebe, R. J.. Fietzek, P. P., and Woolley, D. E. (1978) J. B i d . Chem. 253,5642-5646 Adamson, J. G., Hoang, T., Crivici, A,, and Lajoie, G. A. (1992) Anal. Biochem. Kunz, H., and Dombo, B. 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