ARTICLE

Screening for novel cell adhesive regions in bovine Achillestendon collagen peptidesPradipta Banerjee, Alka Mehta, and Shanthi C.

Abstract: Collagen, a major structural protein of the ECM, is known for its high cell adherence capacity. This study wasconducted to identify regions in collagen that harbour such bioactivity. Collagen from tendon was hydrolysed and the peptidesfractionated using ion-exchange chromatography (IEC). Isolated peptide fractions were coated onto disposable dishes andscreened for cell adherence and proliferative abilities. Active IEC fractions were further purified by chromatography, and twopeptides, C2 and E1 with cell adhesion ability, were isolated. A cell adhesion assay done with different amounts of C2 coated ontodisposable dishes revealed the maximum adhesion to be 94.6%, compared with 80% for collagen coated dishes and an optimumpeptide coating density of 0.507 nmoles per cm2 area of the dish. Growth of cells on C2, collagen, and E1 revealed a similarpattern and a reduction in the doubling time compared with cells grown on uncoated dishes. C2 had a mass of 2.046 kDa with22 residues, and sequence analysis revealed a higher percentage occurrence of hydrophilic residues compared with other regionsin collagen. Docking studies revealed GDDGEA in C2 as the probable site of interaction with integrins �2�1 and �1�1, andstability studies proved C2 to be mostly protease-resistant.

Key words: collagen peptides, chromatography, cryptic peptides, cell adhesion.

Résumé : Le collagène, une protéine structurale importante de la matrice extracellulaire, est connue pour sa capacitéd’adhérence cellulaire élevée. Cette étude a été réalisée afin d’identifier les régions du collagène qui présentent une telle activitébiologique. Du collagène de tendon a été hydrolysé et les peptides ont été fractionnés a l’aide d’une chromatographie échan-geuse d’ions (CEI). Les fractions peptidiques isolées ont été adsorbées sur des plaques jetables et criblées au plan de leur capacitéa promouvoir l’adhérence et la prolifération cellulaires. Les fractions de la CEI actives ont été davantage purifiées par chro-matographie et deux peptides, C2 et E1, possédant des capacités d’adhérence cellulaire ont été isolés. Des mesures d’adhésioncellulaire réalisées avec différentes quantités de C2 adsorbées sur des plaques jetables ont révélé que l’adhésion maximale étaitde 94.6 % comparativement a 80 % pour les plaques enrobées de collagène et une densité optimale de peptide de 0.507 nmoles par cm2

de surface de plaque. L’analyse de la croissance cellulaire sur C2, collagène et E1 a révélé des patrons similaires et une réductiondu temps de doublement comparativement aux cellules cultivées sur des plaques non enrobées. C2 possédait une masse de 2.046kDa et 22 résidus, et l’analyse de séquence a révélé un pourcentage d’occurrence de résidus hydrophiles plus élevé compara-tivement aux autres régions du collagène. Des études d’ancrage ont révélé que la séquence GDDGEA de C2 était probablementle site d’interaction avec les intégrines �2�1 et �1�1, et des études de stabilité ont prouvé que C2 était pratiquement résistant auxproteases. [Traduit par la Rédaction]

Mots-clés : peptides du collagène, chromatographie, peptides cryptiques, adhésion cellulaire.

IntroductionThe ECM is an intricate three-dimensional network composed

of fibrous proteins and proteoglycans surrounding the cells in atissue (Huxley-Jones et al. 2007). Collagen, a major component ofthe ECM, is a structural protein accounting for approximately 30%(dry weight) of all vertebrate body protein. More than 90% of theextracellular protein in the tendon and bone and more than 50%in the skin consist of collagen (Friess 1998).

Collagen, once considered only an architectural support forcells, has been recently found to possess regions that can displayphysiological activities including antioxidative and ACE-inhibition(Li et al. 2007; Cheng et al. 2009). Such bioactive regions comprisesmall portions, which generally remain “masked” until structuralchanges in the molecule expose them or when they are proteo-lytically cleaved. More than one such fragment can be derivedfrom a single protein precursor, and biological activities displayedby these peptides are often different from the parent molecule.Such peptides have been termed “cryptic” bioactive peptides

(Schenk and Quaranta 2003; Autelitano et al. 2006; Gómez-Guillénet al. 2011).

Cell adhesion is a complex process that plays a major role in thedevelopment of multicellular organisms. It consists of two majorsteps: the interaction of an ECM binding site with a cell adhesionreceptor protein leading to cell attachment followed by remodel-ling of the cytoskeletal filaments supporting the cell shape andspreading of the cell on the substratum (Abedin and King 2010).The interactions between collagen type I and cells are facilitatedby integrins, a large family of cell–ECM adhesion receptors in-volved in anchorage and bidirectional signal transfer. Bindinginitiates the formation of protein aggregates, termed focal adhe-sion sites, that link integrins to the cytoskeletons as well as to acascade of other cellular events involved in development, growth,apoptosis, and the response of cells to physical and chemicalstress (Heino 2007; Takada et al. 2007). It has already been estab-lished that collagen type I is highly efficient in cell adhesion,possibly by virtue of the sequence GFOGER and other yet uniden-tified sequences (Knight et al. 2000).

Received 16 March 2013. Revision received 5 July 2013. Accepted 14 August 2013.

P. Banerjee, A. Mehta, and S. C. School of Bio Science and Technology, VIT University, Vellore 632014, Tamil Nadu, India.Corresponding author: Shanti C. (e-mail: cshanthi@vit.ac.in).

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

1

Biochem. Cell Biol. 91: 1–15 (2013) dx.doi.org/10.1139/bcb-2013-0026 Published at www.nrcresearchpress.com/bcb on 21 August 2013.

rich2/bcb-bcb/bcb-bcb/bcb99914/bcb0076d14z xppws S�3 11/19/13 10:17 4/Color Fig: F3,F5,F7-F8 Art: bcb-2013-0026 Input-1st disk, 2nd ??

This study was undertaken to isolate such novel cell adhesiveregions from collagen type I, a common constituent of vertebratetendons, bones, skin, and scar tissue. Hydrolyzing the collagenenzymatically and screening the resultant peptides for bioactivityis a plausible way of identifying these cryptic cell adhesive re-gions. Use of bacterial collagenase for hydrolysis results in in-crease of low molecular weight peptides in the hydrolysate,leading to the “release” of the maximum possible peptides forsubmission to the screening process (Lecroisey & Keil 1979). Col-lagen type I was isolated from bovine Achilles tendon collagen, aslaughter house waste and a good source of soluble collagen, sub-jected to proteolysis, and the resultant peptides screened for celladhesive activity.

Materials and methodsChromatographic matrices (CM sepharose, sephadex G10, G25,

and G100) and molecular weight markers were obtained fromSigma-Aldrich (St. Louis, Mo., USA). Superdex 30 prep grade wasprocured from GE Healthcare (Little Chalfont, UK). All otherchemicals used were of analytical grade.

Bovine Achilles tendon was collected from slaughterhousesaround Vellore, Tamil Nadu, and pure collagen was isolated fromthe tendons through an acid extraction/salt precipitation tech-nique (Radhika and Sehgal 1997).

T-flasks (Nunclon surface) were procured from Nunc, Roskilde,Denmark and disposable culture dishes (35 × 10 mm) were ob-tained from Fischer Scientific, Hanover Park, Ill., USA. PowderedDulbecco’s modified Eagle’s Medium (DMEM) supplemented with2 mmol/L glutamine and 10× antibiotic-antimycotic solution wereobtained from Himedia, India. Foetal bovine serum and 10× sterilefiltered trypsin-EDTA solution was obtained from Sigma-Aldrich.Vero and HeLa cell lines were procured from National Centre forCell Science, Pune, India. All bench work associated with celllines, including peptide coating, were carried out inside a class IIbio-safety cabinet (Clean Air Systems, Chennai, India) to maintainsanitized conditions.

Initial separation of the peptidesCollagen hydrolysis was done according to the protocol de-

scribed in an earlier work (Banerjee et al. 2012). Briefly, crudeprotease was obtained from Alcaligenes odorans and incubated for24 h with bovine tendon collagen suspended in slightly alkalinebuffer at 37 °C. Separation of the peptides was performed in twostages. In the first stage, the hydrolysate was subjected to ion-exchange chromatography (IEC) and the fractions obtained screenedfor adhesive properties. Active fractions were further separatedby gel-permeation chromatography.

Hydrolysate (1.5 mL) was mixed with 0.5 mL of binding buffer(acetate buffer, 0.1 mol/L, pH 4.5) under aseptic conditions. Theresultant solution was heated in a water bath to 55 °C for 3 minand centrifuged at 2000 rpm for 1 min to remove the insolubledebris. The supernatant was applied to a 1.4cm × 15 cm column ofCM sepharose equilibrated in binding buffer. Elution was doneusing an increasing NaCl gradient varying from 0.025 to 0.5 mol/L.The flow rate was maintained at 1.2 mL·min−1, absorbance wasmonitored at 214 nm, and 3 mL fractions were collected. Fractionsshowing clear peaks were individually pooled and desalted byrunning through a 1.4cm × 10 cm column of sephadex G10. Thedesalted fractions were pooled individually and lyophilized in aMicromodulyo freeze-drier (Thermo Scientific, USA). The processwas repeated until sufficient amount of peptide fractions could becollected.

Effect of IEC fractions upon cell adhesion and cellproliferation

Peptide coating of disposable cell culture dishesCoating was done according to standard procedures (García

et al. 1999). The lyophilized test peptide fractions were dissolvedin 3.4 mmol/L acetic acid and filtered through a 0.45 �m celluloseacetate filter. The stock solution was diluted with de-ionized wa-ter according to the final coating amount desired (3.2 × 10−4 to 1 ×103 �g per dish) for the various fractions. One mL of each solutionwas dispensed into a 35 mm polystyrene dish and kept for 3 h inthe bio-safety cabinet under UV light. After 3 h, the excess solu-tion was aspirated and the dishes were air-dried in the hood. Oncedry, the dishes were washed with a sterilized solution of Dulbecco’sCa2+- and Mg2+- free phosphate buffered saline (PBS) and left to dryin the hood (Knight et al. 2000). Uncoated dishes were used asnegative control.

The coating of peptides and collagen to the dishes was con-firmed in a two-step process (Jacquemart et al. 2004). First, knownamounts of test peptides and collagen (confirmed by absorbancemeasurement at 210 nm) were used to coat dishes in the abovementioned method. After 3 h, the leftover solution was aspiratedout, and protein concentration rechecked, with suitably adjustedblanks, to determine the amount of uncoated collagen. Theamount was reconfirmed by leaching out the coated collagen us-ing a 10-fold stronger solution of acetic acid (34 mmol/L). Theaspirated solution was freeze-dried and checked for protein con-tent by absorbance measurement with suitably adjusted blanks.However, for concentrations lower than 8 × 10−1 �g per dish, theabsorbance measured was too low to determine the concentrationaccurately.

Cell maintenanceVero and HeLa cell lines were maintained in accordance with

standard protocols (Ammerman et al. 2008). Cells were cultured inDMEM, supplemented with 10% foetal bovine serum, and incu-bated at 37 °C in a 5% CO2 humidified incubator. For experiments,cells reaching 80% confluence were detached from T-flasks withtrypsin-EDTA, centrifuged, and cell number enumerated by Neu-ber’s chamber. The viability was tested by trypan blue exclusionassay.

Adhesion and proliferation assay on coated dishesIntact collagen and the IEC fractions were coated on the dishes

in amounts ranging from 3.2 × 10−3 to 1 × 103 �g. Cells at a concen-tration 3 × 106 mL−1 for adhesion studies and 5 × 105 mL−1 forproliferation studies were seeded and the dishes incubated in a 5%CO2 chamber for 6 h and 48 h, respectively. After incubation, themedium was removed and cells washed with Dulbecco’s Ca2+- andMg2+-free PBS to remove the un-adhered cells. The adherent cellswere harvested by trypsinization. The viability and cell numberenumeration was done as described in the above section Cellmaintenance. The IEC fractions responsible for maximum celladhesion in both cell lines within 6 h and proliferation in 48 hwere noted and subjected to further chromatographic separation.

Chromatographic separation of bioactive peptidesPeptides were purified by automated column chromatography

attached to an AKTAprime plus unit (GE Healthcare). For separa-tion, known amounts of IEC fractions C and E were dissolved in2 mL 50 mmol/L acetate buffer, pH 4.5, and incubated at 37 °C forcomplete dissolution. IEC fraction E was applied to a 1.4cm × 21 cmG100 sephadex bed with a column effluent flow rate of 1.5 mL·min−1.Fraction C was applied to a 1.4 cm × 20 cm superdex P30 bed witha flow rate of 0.8 mL·min−1. Elution was done with 50 mmol/Lacetate buffer, absorbance measured at 214 nm with a zinc lamp,and fractions of 2 mL were collected. Peaks obtained in the chro-matograms were individually pooled and ran in a 1.4 cm × 5 cmG10 column at a flow rate of 2 mL·min−1 for desalting. The desalted

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

2 Biochem. Cell Biol. Vol. 91, 2013

Published by NRC Research Press

rich2/bcb-bcb/bcb-bcb/bcb99914/bcb0076d14z xppws S�3 11/19/13 10:17 4/Color Fig: F3,F5,F7-F8 Art: bcb-2013-0026 Input-1st disk, 2nd ??

products were lyophilized and stored at −70 °C. For adhesionscreening, the dried peptide fractions were weighed and a knownquantity used to coat disposable dishes, as described in the earliersection on peptide coating. Peptide fractions found to support celladhesions were further run in a 1.4 cm × 30 cm G25 matrix andfinally in a 1.4 cm × 15 cm G10 matrix for desalting.

Bioactivity of purified peptidesPurified peptides E1 and C2 were coated on dishes in the range

of 3.2 × 10−3 to 1 × 103 �g per dish. Cell adhesion and cell prolifer-ation studies were performed with Vero cell lines as detailed inthe section above on adhesion and proliferation. A competitivebinding assay was also performed to confirm the results obtainedby coating. Peptides E1 and C2 were dissolved in DMEM media atdifferent concentrations (0.024−204 �g·mL−1) and incubated with3 × 106 cells·mL−1 for 30 min followed by adding the suspension tocollagen coated dishes (coating amount 40 �g per dish). The ad-hered cell count was checked after 6 h.

Growth studies of cells in the presence of E1 and C2The proliferation rate in the presence of purified peptides was

carried out for Vero and HeLa cell lines. Cells at a concentration of1.4 × 104 mL−1 were seeded onto a series of dishes coated withoptimum amount of test peptides, and growth was studied up to72 h of incubation. Collagen coated and untreated dishes wereused as positive and negative controls, respectively. After speci-fied time periods of 6, 12, 24, 36, 48, 60, and 72 h, cells werephotographed, trypsinized, and cell number counted. Doublingtime of the cells grown on different surfaces was calculated byfitting the curves to the exponential function given below by theleast-squares method.

(1) Y � Y0 × e(k×x)

where Y represents the changing cell count with time, Y0 repre-sents the cell count at time zero taken as a constant in this equa-tion, x represents the time in hours and k is the rate constant to bedetermined, expressed in h−1. The doubling time (t) was calculatedas:

(2) t �ln 2

k

Characterization of C2 peptide

Molecular weight determinationThe molecular weight of C2 was determined by automated gel-

permeation chromatography using an AKTAprime plus unit. A30 cm sephadex G25 bed was prepared in a C10/40 column andcalibrated with low molecular weight marker peptides accordingto the standard protocol given by Sigma. Void volume was calcu-lated using blue dextran. A known quantity of the purified pep-tide C2 was dissolved in 50 mmol/L acetate buffer, pH 4.5, andapplied to the calibrated columns with a similar flow rate as themarkers. From the (elution volume/void volume) values obtained,the molecular weight of the sample was calculated.

Tricine-SDS-PAGE of C2The lyophilized purified peptides were run in a 16%/6 mol/L

urea tricine-SDS-PAGE according to Schägger’s protocol (Schägger2006). Ultra-low range molecular weight markers were also rununder similar conditions. The purified peptide C2 and the hy-drolysate were dissolved in a reducing sample buffer consisting of0.4% SDS, 10% glycerol, along with 0.002% Coomassie blue G-250,and 50 mmol/L Tris buffer (pH 7). The samples were run in aminiVE vertical electrophoresis system (GE Healthcare) with anEPS 301 power pack for 3 h. After running, the gel was immersed

in 1% glutaraldehyde for 5 min followed by immersion in Coomas-sie blue staining solution.

The molecular weight of C2 was determined graphically by plot-ting the logarithms of the molecular weight of markers versus therespective retention factors of the marker bands.

Mass spectroscopy of C2

Trypsin digestionFive mg of the peptide C2 was dissolved in 50 mmol/L ammonium

bicarbonate buffer, pH 8. Mass spectroscopy grade trypsin wasadded to an enzyme: substrate ratio of 1:50 and the sample wasdigested for 15 h at 37 °C. The digestion was stopped by freeze-drying the sample.

MALDI-TOF mass spectroscopy of tryptic peptidesFor matrix-assisted laser desorption/ionization time-of-flight

mass spectrometry (MALDI-TOF MS), the Bruker Ultraflex TOF/TOFinstrument was run in reflective mode with delayed extractionand an acceleration voltage of 25 kvA to improve signal-to-noiseratio. Fity to 100 spectra were summed. Flex Analysis 2.0 softwarewas used to analyze the mass spectra.

Trypsin autolysis products were removed from the final spectra,and the data was exported to Mascot Peptide Mass Fingerprint(http://www.matrixscience.com/). The mass values were matchedto the UniProtKB/Swiss-Prot, a curated protein sequence database(http://expasy.org/sprot/). For the search, peptides were assumedmono-isotopic and oxidized at methionine residues. An “othermammalia” taxonomy restriction was used, a maximum of threemissed cleavages were allowed, and a peptide mass tolerance of0.16 kDa was used for peptide mass fingerprinting.

N-terminal sequencing of C2Purified C2 (100 �g) was electrophoresed in tricine-SDS-PAGE

and transferred overnight to a PVDF membrane (0.2 �m, Bio-Rad)using a Trans-Blot SD apparatus (Bio-Rad) at a constant voltage of14 V. The peptide band was visualized by staining the PVDF mem-brane with 0.1% Coomasie blue. The portion of the membranedisplaying the band was cut and loaded onto the protein sequencer.N-terminal sequencing was performed using a Procise proteinsequencing system (Applied Bioscience) equipped with a 120A on-line phenylthiohydantoin-amino acid analyzer.

Docking studies on C2 and I-domain of integrinsrecognizing collagen

The molecular structure of C2 was modelled using the polypep-tide builder function in ArgusLab 4.0.1 (Planaria Software LLC,Seattle, Wash., USA, http://www.arguslab.com) to be used as the“ligand”. The crystal structure of the I-domain of human integrin�1�1; PDB ID: IQCY and �2�1; PDB ID:1AOX was downloaded fromthe protein data bank. Two different software tools were used forthe docking analysis. PatchDock, a docking tool based on localshape feature matching (http://bioinfo3d.cs.tau.ac.il/PatchDock/)was used for docking the peptides to the integrin. The resultantstructures were refined through FireDock (http://bioinfo3d.cs.tau.ac.il/FireDock/) and observed through visualization software(Schneidman-Duhovny et al. 2011). ArgusDock, a docking tool ofArgusLab was also used to dock the peptide to the integrinI-domain (Yanamala et al. 2008) with a grid resolution of 0.4 Å.

Stability of C2The stability of C2 against common digestive proteases was

assayed by subjecting each peptide to three digestive conditions.A known amount of C2 was dissolved in 20 mmol/L Tris, pH 8.2,and incubated individually with (i) trypsin, (ii) chymotrypsin, and(iii) with an enzyme cocktail of trypsin and chymotrypsin. After anincubation period of 3 h, the reaction was terminated by boilingthe digest followed by extraction with 3.4 mmol/L acetic acid. Theacidic extract was dialyzed against de-ionized water for 3 h, with a

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

Banerjee et al. 3

Published by NRC Research Press

rich2/bcb-bcb/bcb-bcb/bcb99914/bcb0076d14z xppws S�3 11/19/13 10:17 4/Color Fig: F3,F5,F7-F8 Art: bcb-2013-0026 Input-1st disk, 2nd ??

change of water every hour and the resultant solution lyophilized.One hundred �g of the product was coated onto disposable dishesand cell adhesion assay performed with 3 × 106 cells·mL−1. Theresults obtained were subjected to statistical analysis to deter-mine the significance of the digestive enzymes on the peptides.

The peptide sequence was also uploaded to the virtual peptidecutter (http://web.expasy.org/peptide_cutter/) and cleaved usingvirtual enzymes to predict the number of products and cleavagesites.

Statistical analysisTriplicate samples were used for every cell adhesion assay per-

formed, and the cell counts are reported as mean ± SD. The resultsof the assays were checked for statistical significance by ANOVAand by t test. p values less than 0.05 were considered significant.

Results and discussion

Separation of the peptide fractions by IECCM sepharose, a weak cationic exchanger, was used for the

primary separation of the peptides. A binding as well as runningbuffer pH of 4.5 was chosen due to its closeness to the pKa of theCM, thus ensuring effective ionization. For the lack of a substan-tial number of tyrosine and tryptophan residues in collagen, awavelength specific for the peptide bond was used for absorbancemeasurement during elution (Hanson and Eyre 1996). Upon elu-tion with a salt step-gradient, six separate peaks were observedand labelled as A, B, C, D, E, and F, as displayed in Fig. 1. Theincreasing elution buffer strength led to elution of peptide frac-tions with increasing charge density, with the lowest being peak Aand the highest, peak F. In fact, peak A eluted out with bindingbuffer and possibly contained neutral or negatively charged pep-tides.

Bioactivity assessment of collagen and IEC fractionsCoated surfaces provide an effective binding site for cell adhe-

sion molecules, facilitating a faster and more firm cell attachmentwhen compared to uncoated surfaces. Historically, collagen iso-lated from diverse sources has been used successfully as an effec-tive coating material (Jokinen et al. 2004; Douglas et al. 2008). Inthe present study, the number of cells adhering to coated bovinetendon collagen has been used as a positive control against that ofthe peptides. Vero cells, fibroblastic in nature, and HeLa cells,epithelial-like, are known for robust growth along with anchor-age dependence (Oller et al. 1989; Freshney 2005) and have beenutilized to screen for adhesive activity.

Table 1 shows the concentration-dependent attachment of cellsto coated bovine tendon collagen and IEC fractions A, B, C, D, E,and F. A Gaussian-type pattern was observed with maximum ad-hesion constrained within a range of 0.8–250 �g coated materialper dish. The optimum coated amount of collagen registeringmaximum adhesion (80%) was found to be 40 �g per dish (a coat-ing density of 410 ng/cm2 area of the dish), and it reduces drasti-cally above 500 �g. IEC fractions C and E displayed 83% and 74%cell adhesion in the case of Vero, along with 62.3% and 63% celladhesion for the HeLa cell line, respectively. The range for maxi-mum adhesion for both cell lines was found to be 40–100 �gpeptide fraction coated per dish. The difference in adhered cellnumber was found to be significant with the change in amount ofthe coated IEC fraction (p < 0.001, by ANOVA). However, no signif-icant difference was observed between the adhesive capabilitiesof fractions C, E, and collagen type I (p > 0.05, by ANOVA).

Although the cell adhesion capacity of the coated peptideschanged significantly over a wide concentration range, the differ-ences between the extremities were found to be insignificant(p > 0.05). Lower amounts of peptide coating exhibited low celladhesion. As the amount of coated material increased, adheredcell count also increased until it reached a maximum, but atcoated amounts >100 �g per dish, the cell adhesion declined.

Higher coating amounts may “mask” the adhesion receptor rec-ognition sites, lowering the adhered cell count and resulting in asignificant similarity to the cell count on lower amounts of coatedpeptides.

The fraction F was found to be irresponsive to adhesion due tocomparatively higher charge density, as determined by its IECelution properties. Overall it could be inferred that the adhesioncapability was dependent mostly on the amount and the type ofpeptide coated.

A cell proliferation study was carried out on similarly coateddishes and as shown in Table 2, all four IEC fractions except Fdisplayed cell proliferative abilities. Fractions C and E were foundto be proficient at cell proliferation. The change in cell count wasfound to be significantly dependent on the type of IEC fractioncoated (p < 0.001, by ANOVA) and the amount of the peptide in thecoating (p < 0.001, by ANOVA). Higher cell proliferation was ob-served in the span of 40–250 �g with maxima around 100 �g.Overall, the observations were similar to the cell adhesion study.

Further screening was continued only with Vero cell lines be-cause of its higher adherent cell count.

Purification of the active cryptic peptidesIEC fractions E and C displayed better cell adhesive ability and

were separated further. The fraction E was resolved as reportedearlier (Banerjee et al. 2012) by running through sephadex G100column into three peptide fractions, E1, E2, and E2b, as displayedin Fig. 2a. The fraction C could not be resolved with sephadex gels,and hence superdex 30, a matrix specific for small peptides lessthan 5 kDa (Hellberg et al. 1996), was used. As shown in Fig. 2b, IECfraction C could be resolved into three fractions C1, C2, and C3.The peak portions of the fractions from E and C were individuallypooled, desalted, lyophilized, and were coated onto disposabledishes at an amount of 40 �g per dish and assayed for cell adher-ence. As shown in Figs. 2c and 2d, E1 and C2 were found to becomparatively more active and were further run in sephadex G25and G10 columns to ensure purity.

Bioactivity assessment of purified E1 and C2The adhesion and proliferation profiles of Vero cells were de-

termined with different amounts of purified E1 and C2. The pho-tomicrographs in Figs. 3a and 3b display adhered Vero cells after6 h of incubation. The adhered cell count as seen in Fig. 3c, part (i)displayed the same Gaussian-type adhesion pattern but with anoverall enhancement, particularly for the lower amounts coated.The dishes coated with 100 �g of purified peptides E1 and C2displayed a maximum cell adhesion of 98.3% and 94.6%, respec-tively, which was higher than that of collagen coated dishes (80%in 40 �g coated dishes). Although the amount of peptide required

Fig. 1. CM sepharose elution profile of hydrolyzed collagen.A nonlinear gradient of NaCl ranging from 0 to 0.3 mol/L in acetatebuffer (dashed line) was used as the eluant. The hydrolysate (solidline) was separated into 5 different fractions (from left to right, A, B,C, D, E, and F, as indicated), which were dialyzed, lyophilized, andsubjected to cell adhesive activity screening.

0

0.1

0.2

0.3

0.4

0.5

0.6

0 20 40 60 80 100Fraction number

Abs

orba

nce

at 2

14 n

m

0

0.1

0.2

0.3

0.4

0.5

0.6

NaC

l con

cent

ratio

n in

mol

/LA

BC D

E

F

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

4 Biochem. Cell Biol. Vol. 91, 2013

Published by NRC Research Press

F1

T1

T2

F2

F3

rich2/bcb-bcb/bcb-bcb/bcb99914/bcb0076d14z xppws S�3 11/19/13 10:17 4/Color Fig: F3,F5,F7-F8 Art: bcb-2013-0026 Input-1st disk, 2nd ??

for maximum cell adhesion was 2.5 times more than that requiredby collagen, the peptides could increase the maximum cell ad-hered by an average factor of 1.2 times. Peptide coated dishes alsoincreased the average number of cells adhered per unit area. Forboth peptides, half (1.5 × 106) of the total cells seeded were foundto be adherent in amounts ranging from 1.6 × 10−2 to 2.5 × 102 �g.In comparison, for the IEC fractions (sectionon bioactivity assess-ment above), half of the cells seeded could be adhered by a smalleramount, ranging from 0.8 to 1 × 102 �g, indicating a 125 timesincrease in cell adhesive capability after purification. As expected,the cell adhesion was found to be significantly dependent onamount of coated peptide (p < 0.001). However, the differencebetween the activities of E1 and C2 were insignificant (p > 0.05).Mann and West (2002) have reported that dishes coated withRGDS and synthetic peptides KQAGDV and VAPG display bettercell adherence at a coating density of 0.2 nmole/cm2 than at ahigher density of 2 nmole/cm2. This confirms the present study inwhich maximum cell adhesion was obtained at a coating densityof 0.507 nmoles/cm2 when compared with higher amounts ofcoated peptide.

The density of coated ligands and ligand spacing can modulateintegrin localization and clustering, resulting in changes in adhe-sion kinetics of the adhering cell (Cluzel et al. 2005). Coated sur-faces with purified peptides exhibit higher number of bindingsites per cm2 when compared to collagen coated dishes and thuspresent a more homogeneous binding site to the receptors (Maet al. 2005). This results in integrin clustering, leading to betteradhesion in peptide coated dishes when compared to collagen andIEC fractions-coated dishes.

In general, the hydrophobic nature of polystyrene surfacerenders it incapable of cell adhesion. Altering the surface bio-chemically via application of a protein coating can reduce thehydrophobicity and provide multiple nuclei for cell adhesion(García et al. 1999; Yang et al. 2007). Once coated, protein orienta-tion and conformation are influenced by the characteristics of thesurface, the amino acid composition, and native structure of theprotein. The adsorption of collagen onto the polystyrene surfacehas been found to result in a net-like pattern upon slow drying(Engel et al. 2004), resulting in collagen molecules “thrusting out”in a variety of orientations relative to the surface, in effect, pre-

Table 1. Vero and HeLa cell adhesion profiles on surfaces coated with IEC fractions A−F.

Amount coated (�g)

1 × 103 5 × 102 2.5 × 102 1 × 102 4 × 101 8 × 10−1 1.6 × 10−2 3.2 × 10−4

Coated sample Cell count (×105) ± SDVeroCollagen 4.8±0.7 9.64±0.7 10.4±1.4 14.2±0.8 20±2.1 16.4±1.8 10.4±1.7 11.0±1.2A 5.1±0.3 10.4±1.5 13.2±0.3 14.8±1.7 11.4±0.7 9.2±0.52 7.4±1.2 4.7±0.7B 2.40±0.7 3.0±0.5 5.8±1.9 12.4±0.5 20.0±1.9 15.4±0.9 7.6±1.4 7.1±2.3C 6.2±1.1 10.4±2.9 18.6±1.8 24.4±2.2 24.4±1.2 16.8±1.1 9.6±1.7 8.4±1.3D 4.4±0.6 7.2±0.3 7.0±0.7 15.2±0.9 18.2±1.4 16.8±0.5 9.8±0.6 2.4±0.8E 9.0±0.7 10.8±0.2 16.4±0.8 20.4±1.7 18.6±0.2 17.0±1.3 9.6±1.2 4.4±0.6F — — — — — — — —HeLaCollagen 2.5±0.8 7.8±0.9 11.0±1.5 18.8±1.7 15.2±1.3 14.1±1.3 11.8±0.6 9.2±0.8A 0 4.8±0.8 6.2±0.9 8.8±1.5 8.2±1.0 7.0±0.7 6.6±0.5 3.4±0.6B 0 1.3±0.7 5.6±0.5 10.6±2.1 13.2±1.3 8.8±1.5 6.2±0.8 5.8±0.8C 5.2±0.5 10.2±1.1 14.5±1.3 18.7±2.1 15.4±1.5 13.9±2.1 8.2±2.1 4.7±1.3D 0 4.5±1.3 7.2±0.8 11.5±1.3 9.8±0.8 8.4±0.9 7.9±0.7 6.2±1.2E 5.3±0.3 10.2±1.5 15.9±1.1 19.0±0.8 15.7±1.2 13.9±0.5 10.0±2.0 8.2±1.5F — — — — — — — —

Note: The amount of each fraction coated per dish ranged from 3.2 × 10−3 to 1 × 103 �g. Type I collagen coated inthe same range served as positive control. Concentration of cells seeded was 3 × 106 ml−1 and adherent cells wereenumerated after 6 h of incubation. The cell counts (×105) are given as mean of triplicates ± SD. Negative controlsfailed to display any adhesion within the observed time period.

Table 2. Vero and HeLa cell proliferation profiles on surfaces coated with IEC fractions A−F.

Amount coated (�g)

1 × 103 5 × 102 2.5 × 102 1 × 102 4 × 101 8 × 10−1 1.6 × 10−2 3.2 × 10−4

Coated sample Cell count (×105) ± SDVeroA 2.1±0.7 9.4±0.9 10.4±0.9 12.4±1.0 15.4±1.5 9.8±2.7 5.4±1.2 3.2±0.4B 5.3±1.3 8.0±1.0 10.2±0.8 13.7±2.3 14.3±1.5 11.0±1.0 8.2±1.1 4.6±1.9C 6.8±1.3 14.8±1.4 17.7±0.7 20.3±1.1 17.0±2.0 15.4±0.5 11.7±1.5 8.5±0.5D 3.8±1.3 9.1±1.9 12.8±2.0 18.0±1.0 15.9±0.8 12.6±1.5 8.5±1.8 4.5±2.1E 4.0±1.2 6.5±0.3 10.9±0.5 22.0±1.5 18.9±3.2 17.1±0.3 11.9±0.8 8.7±1.4F 2.5±1.9 4.9±0.8 9.3±02 8.8±1.5 9.2±0.35 8.3±0.8 5.9±1.7 2.5±0.5HeLaA 2.4±0.9 5.1±1.5 7.2±1.1 11.8±0.8 9.9±1.0 7.4±1.6 3.4±1.5 2.0±0.4B 3.7±1.1 5.2±0.8 5.9±0.9 7.5±0.5 11.4±0.8 13.8±1.0 8.2±1.7 5.1±0.5C 7.1±2.4 14.6±2.1 16.4±0.7 17.5±0.4 15.6±2.1 12.1±1.7 10.1±2.1 7.8±1.4D 0 5.6±1.4 4.0±0.2 5.5±0.8 9.4±0.6 7.0±0.5 7.9±1.9 6.2±0.4E 5.5±0.9 9.0±1.7 14.9±1.3 14.1±1.0 13.2±0.8 11.9±1.5 10.8±1.8 8.9±1.8F — — — — — — — —

Note: Concentration of cells seeded was 5 × 105 mL−1 and adherent cells were enumerated after 48 h of incuba-tion. The cell counts (×105) are given as mean of triplicates ± SD.

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

Banerjee et al. 5

Published by NRC Research Press

rich2/bcb-bcb/bcb-bcb/bcb99914/bcb0076d14z xppws S�3 11/19/13 10:17 4/Color Fig: F3,F5,F7-F8 Art: bcb-2013-0026 Input-1st disk, 2nd ??

senting a non-homogeneous binding site to the cell receptors(Lhoest et al. 1998). In comparison, the small peptides exhibit amore ordered repetitive orientation, providing a more uniformbinding domain resulting in tight localization of integrin recep-tors and consequent increase in FAK activity.

A similar result was obtained for cell proliferation on the pep-tides, as displayed in Fig. 3c, part (ii). Coated dishes with optimumamounts of peptide would result in an increased number of ad-hered cells compared to the cell count in dishes with sub-optimalor minimal amount of peptide. Higher cell adhesion facilitatesfaster cell proliferation, resulting in a re-occurrence of the Gaussian-type pattern, even after 48 h.

Figure 3d displays the effect of competitive binding of the dis-solved peptides and coated collagen. Both the peptides displayedsignificantly similar results (p > 0.05). As the peptide amount wasincreased in the medium, the amount of cells coated onto colla-gen subsequently decreased, hinting at the potential cell adhesivenature of the peptides. However, at concentrations higher than2 �g·mL−1, peptide binding decreased and cells were observedto adhere to collagen, an apparently unusual effect that arisesdue to the recoiling tendency of collagen-like peptides. At higheramounts, most collagen peptides recoil from each other formingtransient helices (Saeidi et al. 2009), and mostly remain unavail-able for binding with cellular receptors. Free binding sites of thecellular receptors attach to collagen instead, increasing the ob-served cell count. It could be summarized that in the lower con-centration ranges (0.024–2 �g·mL−1), the peptides provided a

consistent concentration-dependent cell binding activity, analo-gous to their cell adhesion activity in the coated form.

Growth curve in the presence of E1 and C2Cells adhere to and spread on surfaces by localizing integrin

receptors, which stimulate large protein complexes called focaladhesions (FA). Signals arising downstream from FA can ulti-mately affect cell survival, differentiation, proliferation, and var-ious aspects of cell division (Streuli 2009).

As seen in Figs. 4a and 4b, the cell count mostly increased tillhour 60. E1 and C2 supported growth patterns (p = 0.41) similar tothat of collagen. Vero cells were unable to adhere to uncoateddishes within 6 h, but registered adhered cells after a long incu-bation time of 24 h, possibly due to secretion of some minimalECM components required for attachment and proliferation.HeLa cells did not show any growth on uncoated dishes, and theoverall growth rate on peptides and collagen was shorter than forVero cells. For both cell lines, the growth rate observed in E1 andC2 along with collagen was found to be distinctly superior(p < 0.001) to that in the control uncoated dishes. After 72 h, Verocell count in the coated dishes was observed to be higher by afactor of 4.5 than the cell count in untreated dishes.

As displayed in Table 3, the doubling time of the cells in coatedplates was found to be drastically reduced from that of the un-coated ones (about 3 times less for Vero cells). The doubling timeof Vero and HeLa cells proliferated on E1 and C2 were found tosimilar to that grown on collagen. In fact for HeLa, the doubling

Fig. 2. Purification of peptides based on their bioactivity. (a) Elution profile of IEC fraction E resolving into 3 peptides E1, E2, and E2b afterrunning in sephadex G100. (b) Elution profile of IEC fraction C resolving into three peptides C1, C2, and C3 after running in Superdex P30.(c) Vero cell adhesive activity of fractions E1, E2, and E2b. (d) Vero cell adhesive activity of C1, C2, and C3. Dishes were coated with 40 �g ofpeptide fractions. Concentration of Vero cells seeded was 3 × 106 mL−1, and observations were taken after 6 h of incubation. The values aregiven as mean of triplicates ± SD.

a. b.

0

20

40

6080

100

120

140

160

0 10 20 30 40Fraction Number

mA

bsor

banc

e at

214

nm

0

5

10

15

20

25

0 20 40

Fraction number

mA

bsor

banc

e at

214

nm

c. d.

0

5

10

15

20

25

E1 E2 E2bFractions from E

Cel

l cou

nt×1

05

0

5

10

15

20

25

C1 C2 C3Fractions from C

Cel

l cou

nt×1

05

C1

C2

C3

E1

E2

E2b

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

6 Biochem. Cell Biol. Vol. 91, 2013

Published by NRC Research Press

F4

T3

rich2/bcb-bcb/bcb-bcb/bcb99914/bcb0076d14z xppws S�3 11/19/13 10:17 4/Color Fig: F3,F5,F7-F8 Art: bcb-2013-0026 Input-1st disk, 2nd ??

Fig. 3. Bioactivity assessment of purified peptides C2 and E1. (a) Photomicrographs of Vero cells adhered to C2 coated dishes after 6 h.(b) Photomicrographs of Vero cells adhered to E1 coated dishes after 6 h. The notations i, ii, iii, and iv depict amounts of peptide coated on thedishes; 3.2 × 10−4, 0.8, 1 × 102, and 2.5 × 102 �g per dish respectively. The bar represents a length of 0.1 mm. (c) (i) Cell adhesive activity of C2and E1; concentration of cell seeded was 3 × 106 mL−1, and observations were taken after 6 h of incubation. (ii) Cell proliferative activity of C2and E1; concentration of cells seeded was 5 × 105 mL−1, and observations were taken after 48 h of incubation. The cell counts are given asmean of triplicates ± SD. The notations I–VII represent the different coating amounts; 5 × 102, 2.5 × 102, 1 × 102, 4 × 101, 8 × 10−1, 1.6 × 10−2, and3.2 × 10−4 �g per dish respectively. (d) Competitive cell binding assay with dissolved peptides and coated collagen. Concentration of cellsseeded was 5 × 105 mL−1, and observations noted after 6 h. Values given are the mean of triplicates ± SD.

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

Banerjee et al. 7

Published by NRC Research Press

rich2/bcb-bcb/bcb-bcb/bcb99914/bcb0076d14z xppws S�3 11/19/13 10:17 4/Color Fig: F3,F5,F7-F8 Art: bcb-2013-0026 Input-1st disk, 2nd ??

time was shorter for cells grown on E1. The cell proliferation ratedepends on various factors, including signalling from ECM andcell-to-cell adhesion. A correct sequence of information transferacross cells, originating from ligand sequence recognition by in-tegrins, could influence proliferation rates and bring down thedoubling time (Maxová et al. 2010). E1 and C2 possibly mimic thecollagen signaling pathways responsible for controlling cell divi-sion events, thus leading to a shortened doubling time.

An E1 coated cell surface exhibits a higher density of the RGDsequence per cm2 area than the collagen coated plates, and a

greater cell proliferation was expected, but as observed, thegrowth rate of collagen and E1 (along with C2) were similar.This could probably stem from the fact that cell adhesion tocollagen must be synergistic and dependent on a number ofdifferent short sequences. In fact, mixed ligand coated surfaceshave been reported to significantly enhance cell adhesion andfocal adhesion assembly of adherent cells compared to surfacescoated with a single ligand (Reyes et al. 2008). The overall syn-ergistic effect of all such sequences in intact collagen resultsa higher cell proliferation similar to that on peptide coateddishes.

Fig. 3 (concluded).

c i.

05

10152025303540

I II III IV V VI VIIAmount of peptide coated per dish (µg)

Cel

l cou

nt×1

05 C2E1

ii.

05

101520253035

I II III IV V VI VIIAmount of peptide coated per dish (µg)

Cel

l cou

nt×1

05 C2E1

d

0.05.0

10.015.020.025.030.0

0 0.024 0.204 2.04 20.4 204Concentration of peptide in solution (µg mL-1)

Adh

ered

cel

l cou

nt

×105

C2E1

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

8 Biochem. Cell Biol. Vol. 91, 2013

Published by NRC Research Press

rich2/bcb-bcb/bcb-bcb/bcb99914/bcb0076d14z xppws S�3 11/19/13 10:17 4/Color Fig: F3,F5,F7-F8 Art: bcb-2013-0026 Input-1st disk, 2nd ??

Characterization and sequencing of C2Bioactive cryptic peptides are generally known for their small

size, which allows them to evade digestive proteolytic attack andincrease bioavailability (Segura-Campos et al. 2011). The peptide E1has been characterized in an earlier work (Banerjee et al. 2012) andhas been used as a test control in this study, as it harbours the celladhesive sequence RGD. E1 registers a mass of 3.2 kDa and thefollowing amino acid sequence: GETGPAGPAGPIGPVGARGPAGPQGPRGDKGETGEQ.

The mass of C2 obtained from column chromatography wasfound to be 2 ± 0.8 kDa. Figure 5 shows the electrophoretic patternof the hydrolysate along with the purified peptide C2. The hy-drolysate appears as a long dark streak with multiple bands visi-ble, and the peptide appears as a distinct single low molecularweight band between the 1.4 and 3.8 kDa marker bands. The mo-lecular weight obtained was found to be near 2 kDa and matchedthe value obtained from column chromatography.

MALDI-TOF peaks of the tryptic peptides of C2, as displayed inFig. 6, were obtained within a mass/charge range of 2045.055 to592.496 after peak smoothening and baseline subtraction. As thevalues did not match significantly with any portion of the bovinecollagen triple helical region through peptide mass-search soft-ware, N-terminal sequencing was carried out.

N-terminal sequencing done by Edman degradation methodcould identify a seven residue stretch beginning with a G residue:G-P-X-G-P-X-G-K, where the 3rd and 6th residues, represented by X,were most probably hydroxyproline, represented hereafter, by O.

A BLAST search performed with P in 3rd and 6th residue of C2with the bovine collagen type I triple helical sequence, fromresidue 178 to 1191, (http://www.uniprot.org/uniprot/P02453) wasfound to match best with residue 220 to 227 (Query ID 5249).

The sequence of collagen from residue 220 to 241, GPPGPPGKNGDDGEAGKPGRPG, gives a molecular weight of 2.0141 kDa.Replacement of two P by two O would increase the molecularweight to 2.0461 kDa and provide an almost accurate match withthe mass obtained from MALDI-TOF.

The final sequence was therefore taken to be GPOGPOGKNGDDGEAGKPGRPG; from collagen type I �1 chain, residue 220 to 241.

The peptide C2 did not possess the well-known RGD or REDVYor other reported sequences capable of cell adhesion, althoughits activity matched significantly with RGD-containing E1 andcollagen.

Bovine collagen type I (http://www.uniprot.org/uniprot/P02453)typically display a unique amino acid composition constituting32.8% G with 22.9% P and 11.6% A, followed by R, E, D, K, and S(3%–5%), along with other amino acids in very smaller quantities.The peptide C2 registered a lower percentage occurrence of A;from 11% to nil. An increase in percentage occurrence was ob-served for N (4.5%), D (9.1% from 3.2%), and K (9% from 3.6%). Thepercentage occurrences of R, E, G, and P were comparable to thatof collagen.

Overall, both C2 and E1 displayed a higher percentage occur-rence of negatively charged residues; 13.6% and 11.1% for C2 and E1,respectively, compared with 7.8% for collagen triple helical re-gion. The percentage occurrence of positively charged residueswas higher in C2, 18% when compared with 8.4% in E1 and 9.7% incollagen. Overall, 31.8% of the residues in C2 were hydrophilicwhen compared with 17.5% in collagen. Moreover, the negativelycharged residues D and E are confined to a short stretch of 11–14,almost in the middle of the sequence.

In silico interactions of C2 and I-domain of integrinreceptors

Human cells have 18 � and 8 � subunits and can be assembledtogether to result in 24 distinct heterodimers. The integrin familyresponsible for collagen recognition constitutes four members:�1�1, �2�1, �10�1, and �11�1. All four types are known to recog-nize GFOGER. �1�1 integrin can bind collagen I-VI and type XIII,

Fig. 4. Growth profile of (a) Vero and (b) HeLa cells on purifiedpeptides C2 and E1 compared with collagen; observations weremade for 72 h. (�), uncoated dishes, (�), collagen coated, (Œ), E1coated, and (�), C2 coated. The values are given as mean oftriplicates ± SD.

a.

0

5

10

15

20

25

30

0 20 40 60 80Incubation time (h)

Vero

cel

l cou

nt×1

04

CollE1UCC2

b.

02468

1012141618

0 20 40 60 80Incubation time (h)

HeL

a ce

ll co

unt×

104

CollE1UCC2

Table 3. Doubling times of Vero and HeLa cells grown onC2 coated dishes.

Coatedsample

Doubling time (h)for Vero cells

Doubling time (h)for HeLa cells

1 Collagen 10.25 18.392 C2 11.17 18.113 E1 10.29 16.874 Uncoated 32.96 —

Note: Collagen, positive control; E1, test control; uncoateddishes, negative control. The doubling time of HeLa cells oncoated dishes could not be measured and is left blank.

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

Banerjee et al. 9

Published by NRC Research Press

F5

F6

rich2/bcb-bcb/bcb-bcb/bcb99914/bcb0076d14z xppws S�3 11/19/13 10:17 4/Color Fig: F3,F5,F7-F8 Art: bcb-2013-0026 Input-1st disk, 2nd ??

whereas �2�1 is the major receptor for collagen type I and otherfibril-forming collagen. Ligand recognition occurs through an in-dependently folding domain, the inserted or I-domain located in �subunit, in juxtaposition to the N-terminal blades of the � propel-ler, also in the � subunit. The domain adopts a Rossman fold and

harbours a metal ion-dependent adhesion site or MIDAS (Knightet al. 2000; Douglas et al. 2008). Although the metal is held byoxygen atoms, one empty coordination site remains available forbond formation by a ligand carboxylate anion; which is why E orD residues present in ligand play a crucial role in ligand recogni-tion and signal transfer.

Figures 7a and 7b display the high-scored docked structures ofthe peptide and the �2�1 MIDAS sub-unit obtained from Patch-dock/Firedock; none allow the ligand to bind near the metal ion.Figures 7c and 7d display the best ranked poses of C2 docked nearthe metal ion residing site in MIDAS. As displayed, C2 is foundto interact with helix V, sheet C, D, and loops L1, L6, and L11. In thesecond pose (Fig. 7d), the 2nd D residue from the sequenceGDDGEA lies within the hydrogen bonding radii of the metal.This aspect has been enlarged and displayed in Fig. 7e. Almostsimilar results were obtained for �1�1-C2 interactions. The high-est ranked structure in Patchdock, displayed in Fig. 8a, allows thepeptide to interact with helix VI, sheet E, and loops L6 and L10,while rank 2 (Fig. 8b) shows the peptide interaction with helix V,VI, and loops L6 and L11. This interaction brings the N-terminalarea of the peptide within the hydrogen bonding distance of themetal ion and may be important. Poses 1 (Fig. 8c) and 2 (Fig. 8d) inArgusdock display the peptide to be in interaction with the heli-ces III and V; sheets A, B, and C along with loops L1, L6, and L11.Figure 8e displays a enlarged shot of the previous Fig. 8b, where anE residue from the peptide sequence GDDGEA lies within properhydrogen bonding distance of the metal.

Activation of integrin I-domain through ligand binding hasbeen understood as a two-state allosteric model ultimately lead-ing to the conformational changes necessary to pass the signaldown the chain. This model predicts the I-domain to have twomutually exclusive conformational states: one being the inactive“T” state, which is low affinity, more stable in the absence ofligands and the active “R” state, having a higher affinity for theligand. In the R state, the metal ion remains chelated throughdirect coordination by two serine residues (S), one threonine (T),and indirectly by two aspartate residues (D), via water molecules.The absence of a direct bond between the metal and a negatively

Fig. 5. Electrophoretic pattern of C2 obtained by tricine-SDS-PAGE. From the left: lane 1: ultra low range molecular weight markers in therange 1.060–26.600 kDa; lane 2: bovine tendon collagen hydrolysate; lane 3: purified C2.

Lane 1 Lane 2 Lane 3

26.6 kDa

17 kDa

14.2 kDa

6.5kDa

3.49 kDa

1.06 kDa

Fig. 6. MALDI-TOF spectrum of tryptic peptides of C2. The massvalues were transported to Mascot search engine and searched forpeptides. Protein mass scores greater than the critical value(p < 0.01) were used to identify the peptides.

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

10 Biochem. Cell Biol. Vol. 91, 2013

Published by NRC Research Press

F7

F8

rich2/bcb-bcb/bcb-bcb/bcb99914/bcb0076d14z xppws S�3 11/19/13 10:17 4/Color Fig: F3,F5,F7-F8 Art: bcb-2013-0026 Input-1st disk, 2nd ??

charged ligand (like D) enhances the electrophilicity of the metal,making it available for ligand binding. In the unliganded state,bonds with two S are maintained, but the bond to T is replaced bya direct interaction with asp, resulting in reduced electrophilicityof the metal. Thus, a strong tendency to bind a negatively chargedligand is lost, leading to a low-affinity state. Control over ligandaffinity is also probably maintained by differing surface chargedensity in the R and T states (Lee et al. 1995; Emsley et al. 2000;Takagi 2007). Peptides that can harbour acidic amino acids capa-ble of filing the 6th coordination position of the metal ion canchange the receptor inactive state to the active R state. In thisrespect, acidic amino acid containing sequences like GDDGEA inC2, as hinted in the present docking studies, may possibly be acryptic integrin “activator” site.

Sequence-activity correlation and comparison of C2 withdocumented cell adhesion peptides

Although each peptide would probably bind to a different fam-ily of integrin receptors, a comparative analysis of some of themajor adhesion peptides could bring into light common featuresof these peptides and point to their uniqueness. Sequence analysisof several peptides done by Kato et al (2006) and Okochi et al.(2008) has revealed a number of inherent characteristics. (i) Thepresence of a higher percentage of strictly hydrophobic residues I,V, L, and F has been found to reduce cell adhesion. An excess ofcharged residues, with an occurrence of greater than 50% of theresidues (in a tetra- or penta-peptide) have also been reported tohave a negative effect on adhesion. In effect, for a candidate pep-tide to display cell adhesion, a delicate balance between hy-drophobicity and hydrophilicity has to be achieved. (ii) N-terminalK, Q, H, and P have been reported to tilt the peptide towardsincreased cell adhesion. (iii) Small hydrophilic residues along withthe residue G were observed to be favoured. Top 20 cell adhesivepeptides described in Kato’s study displayed an average hy-drophobicity value of –4.0, which inclined more towards hy-drophilicity. (iv) Residues such as K and R, present in loweramounts, preferably around 5%–20% and 5%–10% respectively havebeen reported to increase cell adhesion. (v) More than 25% occur-rence of negatively charged residues disfavour cell adhesion.

These empirical rules obtained from Kato and Okochi’s studywere applied to some well-known cell adhesion peptides alongwith C2 to analyse their underlying common characteristics. Celladhesion peptides have been mostly identified in fibronectin, butless information exists on such cryptic peptides from collagentype I. A specific collagen sequence, GFOGER, has been identifiedto be recognized by the I-domain of several collagen-binding in-tegrins in the triple helical form (Emsley et al. 2000). Some otherdocumented cell adhesive peptides include: GVKGDKGNPGWPGAPY (Mayo et al. 1991) from human collagen type IV, ALNGR(Okochi et al. 2008), REDVY (Veiseh et al. 2007), LDV (Wayner andKovach 1992), and synergy peptide PHSRN (Feng and Mrksich

2004) from fibronectin, along with YIGSR from laminin (Mannet al. 1999).

A brief comparative analysis of these seven peptides along withRGD and C2 reveals some interesting correlations: (i) The mostcommon residue to be present ubiquitously is R, present in 7 out

Fig. 7. Probable interactions between C2 and the I-domain of the�2�1 integrin receptor obtained from docking studies. (a) Highestscored structure from PatchDock. (b) Second highest scoredstructure from PatchDock. The peptide is represented as a greenribbon, and the integrin subunits are the red (the I-domain) andblue ribbons (the � propeller). (c) Pose 1 (highest ranked) fromArgusDock. (d) Pose 2 from ArgusDock. The peptide is rendered inCPK mode, and the subunits are in red and blue ribbon mode.(e) The figure is an enlarged version of Fig. 7d depicting the Dresidue from GDDGEA sequence of C2 to lie well within the hy-drogen bonding distance of the metal ion. The peptide is renderedas cylinders and the integrin subunit in ribbon mode. The green linerepresents the distance between the keto group and the metal ion,represented in wire frame. Please see the online version for colourcontent.

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

Banerjee et al. 11

Published by NRC Research Press

rich2/bcb-bcb/bcb-bcb/bcb99914/bcb0076d14z xppws S�3 11/19/13 10:17 4/Color Fig: F3,F5,F7-F8 Art: bcb-2013-0026 Input-1st disk, 2nd ??

of 9 peptides (78%) followed by G (68% of the peptides), D (56% ofthe peptides), and N along with P (each 45%). The residues Y, E, K,L, A, and V were present in the range 15%–35%, while W, I, and Hhad abundance below 15%. However, close scrutiny reveals thatstrict hydrophobic residues are surrounded mostly by hydrophilicresidues, thus lowering the overall hydrophobicity. Also, thenumber of strict hydrophobic residues in most of the peptides(except LDV) was restricted to only one per peptide. (ii) The overallhydrophobicity of the majority of the peptides was found to below. The hydrophobicity values were calculated from Kyte andDoolittle’s reported scale from ExPASy (http://web.expasy.org/protscale/pscale/Hphob.Doolittle.html), and the average of8 peptides, excluding C2, was found to be –6.5, indicating a tilttowards overall hydrophilicity. Even though C2 had a calculatedhydrophobic index of –26.9, the sub-sequence GDDGEA had avalue of –9.5, close to the average hydrophobic index of otherpeptides. (iii) As reported in the earlier section, 13.6% of the resi-dues of C2 were hydrophilic, and the relative percentage occur-rence of K (9.1%) and R (4.5%) in C2 matched with the acceptablelimit set by Kato and Okochi’s work. To summarise, the majorityof the inferences reached here seemed to match with the empir-ical rules provided by Kato’s work.

Cell adhesion activity of E1 was probably due to the presence ofRGD sequence. However, the data that the RGD sequences in col-lagen type I can engage integrins actively is yet scanty, probablybecause the motif remains masked in the native form of collagen(Davis et al. 2000). However, the results of this study suggest thatpossibly an RGD-motif containing peptide in the coated form canbe recognized by integrins as a potential ligand.

C2 stability and abundanceFigure 9 displays the cell count of C2 treated with different

enzymes. Chymotrypsin did not have any effect on C2 activity.Trypsin and the enzyme cocktail was able to reduce the activity ofC2 by 10% and 7%, respectively; however, the decrease was notsignificant (p > 0.1, at 95% confidence level by t-test). Overall, theactivity of C2 remained mostly unaffected even after proteasedigestion.

Virtual cleavage conducted with C2 gave some possible expla-nations. The Peptide Cutter tool at ExPASy revealed three lowprobability (<6%) cleavage sites for chymotrpsin. The predictedtrypsin cleavage sites were after residues 8, 17, and 20, all with acleavage probability greater than 50% with the highest (90%) beingat the 8th position. However in both scenarios, the sub-sequenceGDDGEA remained intact, and this could be a possible reason forC2 to retain its cell adhesion activity even after protease treat-ment.

A BLAST search (query ID 63488) of the peptide C2 with otherprotein sequences (Altschul et al. 1997) in SwissProt revealed thesequence to be present in collagen type I and II of most species(5 out of 16 listed) with 100% identity. Interestingly, the peptide

Fig. 8. Probable interactions between C2 and the I-domain of the�1�1 integrin receptor obtained from docking studies. (a) Highestscored structure from PatchDock. (b) Second highest scoredstructure from PatchDock. It displays the peptide interacting withthe metal ion, represented by the sphere. The peptide is representedas a blue ribbon and the integrin subunit as a red ribbon. (c) Pose 1from ArgusDock. (d) Pose 2 from ArgusDock. Both poses displaypeptide residues in interaction with the metal ion. The peptide isrendered in CPK mode and the subunit in ribbon mode. (e) Thefigure is an enlarged version of Fig. 8b depicting E residue fromGDDGEA sequence of C2 to lie well within the hydrogen bondingdistance of the metal ion. The peptide and the integrin subunit arerendered in ribbon mode with functional groups in wire frame. Thegreen line represents the distance between the keto group and themetal ion, represented in green wire frame. Please see the onlineversion for colour content.

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

12 Biochem. Cell Biol. Vol. 91, 2013

Published by NRC Research Press

F9

rich2/bcb-bcb/bcb-bcb/bcb99914/bcb0076d14z xppws S�3 11/19/13 10:17 4/Color Fig: F3,F5,F7-F8 Art: bcb-2013-0026 Input-1st disk, 2nd ??

was present, with minor variations (86% identical), in the �2 chainof bovine collagen, with the GDDGEA sequence intact. In fact, thesub-sequence GDDGEA was found intact in 11 out of the 16 sequences,as displayed in Table 4. Some more intriguing results emergedwhen a second BLAST search was conducted with only the sub-sequence GDDGEA compared to nonredundant entries in PDB,SwissProt, PIR, and PRF (query ID 56336). The sub-sequence wasfound to be present intact in the collagen of a huge number ofspecies, including primates like rhesus macaques, galagos, gib-bons, gorillas, and humans along with other mammals like bats,horses, and boars, and even in several fish species. In fact, it waseven found to be present in a type II collagen homolog partialprotein isolated from an approximately 25 000 year old fossil car-tilage. Although the sub-sequence was identified in collagen frommost species, it was absent from other ECM sequences, leading tothe conclusion that unlike RGD, the sequence GDDGEA was acollagen-specific sequence, much like GFOGER.

It remains yet unclear whether the sequence can act as adhe-sion peptide in intact collagen fibrils; it lies quite close to theN-terminal telopeptide region, thus maximizing its chance to be“exposed” and be cleaved in vivo during tissue remodelling. Mam-malian collagenases cleave at a certain G-I/L sequence and wouldlead to destabilization of the triple helix, allowing other proteasesto act on collagen. The �1 chain of collagen displays cleavage sitesfor neutrophil elastase and proteinase 3 at the area 220–228and near 245–255, residues flanking peptide C2, maximizing thechances of C2 being excised in vivo. As displayed in Fig. 9, C2 itselfharbours neutrophil elastase cleavage sites flanking the sub-sequence GDDGEA. These facts, when combined with data fromstability study and in silico docking studies (section above on C2characterization), suggest that the sub-sequence GDDGEA couldindeed be crucial for integrin recognition that precedes celladhesion.

Potential applications of cell adhesive peptidesBiocompatibility has been a key issue in the integration of syn-

thetic implants into living tissue. A large number of syntheticpolymeric materials are available for medical applications, suchas prostheses, implants, and tissue engineering matrices. Most ofthese materials are stable and nontoxic but lead to inadequateinteraction between the polymer and the cells, leading to rejec-tion of the transplant along with inflammations, infections, localtissue waste, and thrombosis (Kantlehner et al. 2000).

A safe approach to improve cell-synthetic material interactionincludes coating the material with natural polymers, such as pro-teins, thus facilitating a controlled interaction between the celland the coated substrate. However, coating transplants with a

protein has its own set of disadvantages. Proteins, if not purifiedproperly, can elicit undesirable responses and increase the risk ofinfection. Furthermore, proteins may be subject to proteolytic deg-radation, thus limiting long-term application. Also, the texture ofthe surface may influence the conformation and orientation ofthe protein, which may cause a heterogeneous presentation of cellbinding motifs. And lastly, the large length of a protein may alsolead to variation of the segments that interact with the surface,resulting in a disarrayed coating (Hozumi et al. 2010).

Peptides do not face such limitations. Due to their small size,they can escape proteolytic digestion; collagen peptides are moresuitable in this respect as the increased proline content helpsthese peptides evade proteolysis by serine protease class of diges-tive enzymes, ultimately increasing its bioavailability. This leadsto a controlled reaction between the peptides and the surface,ensuring a more homogeneous binding surface being presentedto the adhering cells. As seen in this study, a bound peptide witha large number of uniform and effective binding sites wouldmimic an extracellular environment and promote greater cellattachment.

AcknowledgementsThis study was supported by research grant from DST, Govern-

ment of India (SERC SL No. 1328) and VIT University, Vellore. Wewould like to thank Dr. Suseela G. Chennai, Microbiology labora-tory, Central Leather Research Institute, for kindly providing theorganism. We would also like to thank Dr. S. Raghavan’s researchgroup, Biochemistry unit and Mrs. Sunita, Proteomics division,Molecular biophysics unit, Indian Institute of Science, Bangalore

Fig. 9. Effect of protease treatment on cell adhesion activity of C2.The notations C2/tr, C2/ch and C2/tr/ch represent C2 treated withtrypsin, chymotrypsin, and a mixture of trypsin and chymotrypsinrespectively. The cell counts are given as mean of 3 dishes ± SD.

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

C2 C2/tr C2/ch C2/tr/ch

Cel

l cou

nt×1

05Table 4. Comparative sequence analysis of C2 with closely matchingregions from other species.

Species Type Sequence

Bos taurus I 220 241�1 GPPGPPGKNGDDGEAGKPGRPG

1 Mammut americanum I 60 81 100�1 GPPGPPGKNGDDGEAGKPGRPG

2 Rattus norvegicus I 210 231 100�1 GPPGPPGKNGDDGEAGKPGRPG

3 Mus musculus I 210 231 100�1 GPPGPPGKNGDDGEAGKPGRPG

4 Canis lupus familiaris I 217 238 100�1 GPPGPPGKNGDDGEAGKPGRPG

5 Homo sapiens I 221 242 100�1 GPPGPPGKNGDDGEAGKPGRPG

6 Gallus gallus I 210 231 95�1 GPPGPPGKNGDDGEAGKPGRPG

7 Bos taurus II 243 264 86�1 GPPGPPGKPGDDGEAGKPGKSG

8 Homo sapiens II 243 264 86�1 GPPGPPGKPGDDGEAGKPGKAG

9 Gallus gallus I 132 153 86�2 GPPGPPGKAGEDGHPGKPGRPG

10 Cynops pyrrhogaster I 207 228 84�1 GLPGPPGKNGDDGESGKPGRPG

11 Bos taurus I 131 152 82�2 GPPGPPGKAGEDGHPGKPGRPG

12 Canis lupus familiaris I 133 154 82�2 GPPGPPGKAGEDGHPGKPGRPG

13 Rattus norvegicus II 175 196 82�1 GPPGPAGKPGDDGEAGKPGKAG

14 Xenopus laevis II 245 266 82�1 GPPGPSGKPGDDGEAGKPGKSG

15 Mus musculus II 243 264 82�1 GPPGPAGKPGDDGEAGKPGKSG

16 Xenopus (Silurana)tropicalis

II 248 269 82�1 GPPGPAGKPGDDGEAGKPGKSG

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

Banerjee et al. 13

Published by NRC Research Press

T4

rich2/bcb-bcb/bcb-bcb/bcb99914/bcb0076d14z xppws S�3 11/19/13 10:17 4/Color Fig: F3,F5,F7-F8 Art: bcb-2013-0026 Input-1st disk, 2nd ??

who have helped enormously in determining the peptide se-quence.

ReferencesAbedin, M., and King, N. 2010. Diverse evolutionary paths to cell adhesion.

Trends Cell Biol. 20(12): 734–742. doi:10.1016/j.tcb.2010.08.002. PMID:20817460.

Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., andLipman, D.J. 1997. Gapped BLAST and PSI-BLAST: a new generation of proteindatabase search programs. Nucleic Acids Res. 25(17): 3389–3402. doi:10.1093/nar/25.17.3389. PMID:9254694.

Ammerman, N.C., Beier-Sexton, M., and Azad, A.F. 2008. Growth and Mainte-nance of Vero Cell Lines. Curr. Protoc. Microbiol. APPENDIX, Appendix–4E.doi:10.1002/9780471729259.mca04es11. PMID:19016439.

Autelitano, D.J., Rajic, A., Smith, A.I., Berndt, M.C., Ilag, L.L., and Vadas, M. 2006.The cryptome: a subset of the proteome, comprising cryptic peptides withdistinctbioactivities.DrugDiscov.Today,11(7–8):306–314.doi:10.1016/j.drudis.2006.02.003. PMID:16580972.

Banerjee, P., Suseela, G., and Shanthi, C. 2012. Isolation and identification ofcryptic bioactive regions in bovine Achilles tendon collagen. Protein J. 31(5):374–386. doi:10.1007/s10930-012-9415-8. PMID:22562127.

Cheng, F.-Y., Wan, T.-C., Liu, Y.-T., Chen, C.-M., Lin, L.-C., and Sakata, R. 2009.Determination of angiotensin-I converting enzyme inhibitory peptides inchicken leg bone protein hydrolysate with alcalase. Anim. Sci. J. 80(1): 91–97.doi:10.1111/j.1740-0929.2008.00601.x. PMID:20163474.

Cluzel, C., Saltel, F., Lussi, J., Paulhe, F., Imhof, B.A., and Wehrle-Haller, B. 2005.The mechanisms and dynamics of �v�3 integrin clustering in living cells.J. Cell Biol. 171(2): 383–392. doi:10.1083/jcb.200503017. PMID:16247034.

Davis, G.E., Bayless, K.J., Davis, M.J., and Meininger, G.A. 2000. Regulation oftissue injury Responses by the exposure of matricryptic sites within Extra-cellular Matrix Molecules. Am. J. Pathol. 156(5): 1489–1498. doi:10.1016/S0002-9440(10)65020-1. PMID:10793060.

Douglas, T., Heinemann, S., Hempel, U., Mietrach, C., Knieb, C., Bierbaum, S.,Scharnweber, D., and Worch, H. 2008. Characterization of collagen II fibrilscontaining biglycan and their effect as a coating on osteoblast adhesion andproliferation. J. Mater. Sci. Mater. Med. 19(4): 1653–1660. doi:10.1007/s10856-007-3250-z. PMID:17851735.

Emsley, J., Knight, C.G., Farndale, R.W., Barnes, M.J., and Liddington, R.C. 2000.Structural Basis of Collagen Recognition by Integrin �2�1. Cell, 101(1): 47–56.doi:10.1016/S0092-8674(00)80622-4. PMID:10778855.

Engel, M.F.M., Visser, A.J.W.G., and van Mierlo, C.P.M. 2004. Conformationand orientation of a protein folding intermediate trapped by adsorption.Proc. Natl. Acad. Sci. U.S.A. 101(31): 11316–11321. doi:10.1073/pnas.0401603101.PMID:15263072.

Feng, Y., and Mrksich, M. 2004. The synergy peptide PHSRN and the adhesionpeptide RGD mediate cell adhesion through a common mechanism. Bio-chemistry, 43(50): 15811–15821. doi:10.1021/bi049174+. PMID:15595836.

Freshney, R.I. 2005. Culture of animal cells: a manual of basic technique. JohnWiley & Sons.

Friess, W. 1998. Collagen-biomaterial for drug delivery. Eur. J. Pharm. Biopharm.45: 113–136. doi:10.1016/S0939-6411(98)00017-4. PMID:9704909.

García, A.J., Vega, M.D., and Boettiger, D. 1999. Modulation of cell proliferationand differentiation through substrate-dependent changes in fibronectin con-formation. Mol. Biol. Cell, 10(3): 785–798. doi:10.1091/mbc.10.3.785. PMID:10069818.

Gómez-Guillén, M.C., Giménez, B., López-Caballero, M.E., and Montero, M.P.2011. Functional and bioactive properties of collagen and gelatin from alter-native sources: A review. Food Hydrocolloids, 25(8): 1813–1827. doi:10.1016/j.foodhyd.2011.02.007.

Hanson, D.A., and Eyre, D.R. 1996. Molecular site specificity of pyridinoline andpyrrole cross-links in type I collagen of human bone. J. Biol. Chem. 271(43):26508–26516. doi:10.1074/jbc.271.43.26508. PMID:8900119.

Heino, J. 2007. The collagen family members as cell adhesion proteins. J. Biol.Chem. 29(10): 1001–1010. doi:10.1002/bies.20636. PMID:17876790.

Hellberg, U., Ivarsson, J.-P., and Johansson, B.-L. 1996. Characteristics of Su-perdex® prep grade media for gel filtration chromatography of proteinsand peptides. Process Biochem. 31(2): 163–172. doi:10.1016/0032-9592(95)00044-5.

Huxley-Jones, J., Robertson, D.L., and Boot-Handford, R.P. 2007. On the origins ofthe extracellular matrix in vertebrates. Matrix Biol. 26(1): 2–11. doi:10.1016/j.matbio.2006.09.008. PMID:17055232.

Jacquemart, I., Pamuła, E., De Cupere, V.M., Rouxhet, P.G., and Dupont-Gillain, C.C. 2004. Nanostructured collagen layers obtained by adsorptionand drying. J. Colloid Interface Sci. 278(1): 63–70. doi:10.1016/j.jcis.2004.05.040. PMID:15313638.

Jokinen, J., Dadu, E., Nykvist, P., Käpylä, J., White, D.J., Ivaska, J., Vehviläinen, P.,Reunanen, H., Larjava, H., Häkkinen, L., and Heino, J. 2004. Integrin-mediated cell adhesion to type I collagen fibrils. J. Biol. Chem. 279(30): 31956–31963. doi:10.1074/jbc.M401409200. PMID:15145957.

Kantlehner, M., Schaffner, P., Finsinger, D., Meyer, J., Jonczyk, A., Diefenbach, B.,Nies, B., Hölzemann, G., Goodman, S.L., and Kessler, H. 2000. Surface coatingwith cyclic RGD peptides stimulates osteoblast adhesion and proliferation as

well as bone formation. ChemBioChem, 1(2): 107–114. doi:10.1002/1439-7633(20000818)1:2<107::AID-CBIC107>3.3.CO;2-W. PMID:11828404.

Kato, R., Kaga, C., Kunimatsu, M., Kobayashi, T., and Honda, H. 2006. Peptidearray-based interaction assay of solid-bound peptides and anchorage-dependant cells and its effectiveness in cell-adhesive peptide design. J. Biosci.Bioeng. 101(6): 485–495. doi:10.1263/jbb.101.485. PMID:16935250.

Knight, C.G., Morton, L.F., Peachey, A.R., Tuckwell, D.S., Farndale, R.W., andBarnes, M.J. 2000. The Collagen-binding A-domains of Integrins �1�1 and�2�1recognize the same specific amino acid sequence, GFOGER, in native(triple-helical) collagens. J. Biol. Chem. 275(1): 35–40. doi:10.1074/jbc.275.1.35.PMID:10617582.

Lee, J.-O., Bankston, L.A., Robert, C, and Liddington, M.A.A. 1995. Two conforma-tions of the integrin A-domain (I-domain): a pathway for activation? Struc-ture, 3(12): 1333–1340. doi:10.1016/S0969-2126(01)00271-4. PMID:8747460.

Lhoest, J.B., Detrait, E., Van den Bosch de Aguilar, P., and Bertrand, P. 1998.Fibronectin adsorption, conformation, and orientation on polystyrenesubstrates studied by radiolabeling, XPS, and ToF SIMS. J. Biomed. Mater.Res. 41(1): 95–103. doi:10.1002/(SICI)1097-4636(199807)41:1<95::AID-JBM12>3.3.CO;2-K. PMID:9641629.

Li, B., Chen, F., Wang, X., Ji, B., and Wu, Y. 2007. Isolation and identification ofantioxidative peptides from porcine collagen hydrolysate by consecutivechromatography and electrospray ionization–mass spectrometry. Food Chem.102(4): 1135–1143. doi:10.1016/j.foodchem.2006.07.002.

Ma, Z., He, W., Yong, T., and Ramakrishna, S. 2005. Grafting of gelatin on elec-trospun poly(caprolactone) nanofibers to improve endothelial cell spreadingand proliferation and to control cell Orientation. Tissue Eng. 11(7–8): 1149–1158. doi:10.1089/ten.2005.11.1149. PMID:16144451.

Mann, B.K., and West, J.L. 2002. Cell adhesion peptides alter smooth muscle celladhesion, proliferation, migration, and matrix protein synthesis on modi-fied surfaces and in polymer scaffolds. J. Biomed. Mater. Res. 60(1): 86–93.doi:10.1002/jbm.10042. PMID:11835163.

Mann, B.K., Tsai, A.T., Scott-Burden, T., and West, J.L. 1999. Modification ofsurfaces with cell adhesion peptides alters extracellular matrix deposition.Biomaterials, 20(23–24): 2281–2286. doi:10.1016/S0142-9612(99)00158-1. PMID:10614934.

Maxová, H., Bacáková, L., Eckhardt, A., Miksík, I., Lisá, V., Novotná, J., andHerget, J. 2010. Growth of vascular smooth muscle cells on collagen I exposedto RBL-2H3 mastocytoma cells. Cell. Physiol. Biochem. 25(6): 615–622. doi:10.1159/000315080. PMID:20511706.

Mayo, K.H., Parra-Diaz, D., McCarthy, J.B., and Chelberg, M. 1991. Cell adhesionpromoting peptide GVKGDKGNPGWPGAP from the collagen type IV triplehelix: cis/trans proline-induced multiple proton NMR conformations andevidence for a KG/PG multiple turn repeat motif in the all-trans prolinestate. Biochemistry, 30(33): 8251–8267. doi:10.1021/bi00247a022. PMID:1868097.

Okochi, M., Nomura, S., Kaga, C., and Honda, H. 2008. Peptide array-basedscreening of human mesenchymal stem cell-adhesive peptides derived fromfibronectin type III domain. Biochem. Biophys. Res. Commun. 371(1): 85–89.doi:10.1016/j.bbrc.2008.04.019. PMID:18413142.

Oller, A.R., Buser, C.W., Tyo, M.A., and Thilly, W.G. 1989. Growth of mammaliancells at high oxygen concentrations. J. Cell Sci. 94(1): 43–49. PMID:2613768.

Reyes, C.D., Petrie, T.A., and García, A.J. 2008. Mixed extracellular matrix ligandssynergistically modulate integrin adhesion and signaling. J. Cell. Physiol.217(2): 450–458. doi:10.1002/jcp.21512. PMID:18613064.

Saeidi, N., Sander, E.A., and Ruberti, J.W. 2009. Dynamic shear-influenced colla-gen self-assembly. Biomaterials, 30(34): 6581–6592. doi:10.1016/j.biomaterials.2009.07.070. PMID:19765820.

Schägger, H. 2006. Tricine-SDS-PAGE. Nat. Protoc. 1: 16–22. doi:10.1038/nprot.2006.4. PMID:17406207.

Schenk, S., and Quaranta, V. 2003. Tales from the crypt[ic] sites of the extracel-lular matrix. Trends Cell Biol. 13(7): 366–375. doi:10.1016/S0962-8924(03)00129-6. PMID:12837607.

Schneidman-Duhovny, D., Hammel, M., and Sali, A. 2011. Macromolecular dock-ing restrained by a small angle X-ray scattering profile. J. Struct. Biol. 173(3):461–471. doi:10.1016/j.jsb.2010.09.023. PMID:20920583.

Segura-Campos, M., Chel-Guerrero, L., Betancur-Ancona, D., andHernandez-Escalante, V.M. 2011. Bioavailability of bioactive peptides. Food Rev.Int. 27(3): 213–226. doi:10.1080/87559129.2011.563395.

Shibue, T., and Weinberg, R.A. 2009. Integrin �1-focal adhesion kinase signalingdirects the proliferation of metastatic cancer cells disseminated in thelungs. Proc. Natl. Acad. Sci. U.S.A. 106(25): 10290–10295. doi:10.1073/pnas.0904227106. PMID:19502425.

Streuli, C.H. 2009. Integrins and cell-fate determination. J. Cell Sci. 122(2): 171–177. doi:10.1242/jcs.018945. PMID:19118209.

Takada, Y., Ye, X., and Simon, S. 2007. The integrins. Genome Biol. 8(5): 215.doi:10.1186/gb-2007-8-5-215. PMID:17543136.

Takagi, J. 2007. Structural basis for ligand recognition by integrins. Curr. Opin.Cell Biol. 19(5): 557–564. doi:10.1016/j.ceb.2007.09.002. PMID:17942298.

Veiseh, M., Veiseh, O., Martin, M.C., Asphahani, F., and Zhang, M. 2007. Shortpeptides enhance single cell adhesion and viability on microarrays. Lang-muir, 23(8): 4472–4479. doi:10.1021/la062849k. PMID:17371055.

Wayner, E.A., and Kovach, N.L. 1992. Activation-dependent recognition by

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

14 Biochem. Cell Biol. Vol. 91, 2013

Published by NRC Research Press

rich2/bcb-bcb/bcb-bcb/bcb99914/bcb0076d14z xppws S�3 11/19/13 10:17 4/Color Fig: F3,F5,F7-F8 Art: bcb-2013-0026 Input-1st disk, 2nd ??

hematopoietic cells of the LDV sequence in the V region of fibronectin. J. CellBiol. 116(2): 489–497. doi:10.1083/jcb.116.2.489. PMID:1530947.

Yanamala, N., Tirupula, K.C., and Klein-Seetharaman, J. 2008. Preferential bind-ing of allosteric modulators to active and inactive conformational states ofmetabotropic glutamate receptors. BMC Bioinformatics, 9(Suppl. 1): S16. doi:10.1186/1471-2105-9-S1-S16. PMID:18315847.

Yang, H., Fung, S.-Y., Pritzker, M., and Chen, P. 2007. Modification of hydrophilic

and hydrophobic surfaces using an ionic-complementary peptide. PLoS One,2(12): e1325. doi:10.1371/journal.pone.0001325. PMID:18091996.

Yashiki, S., Umegaki, R., Kino-Oka, M., and Taya, M. 2001. Evaluation of attach-ment and growth of anchorage-dependent cells on culture surfaces with typeI collagen coating. J. Biosci. Bioeng. 92(4): 385–388. doi:10.1263/jbb.92.385.PMID:16233115.

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

Banerjee et al. 15

Published by NRC Research Press

rich2/bcb-bcb/bcb-bcb/bcb99914/bcb0076d14z xppws S�3 11/19/13 10:17 4/Color Fig: F3,F5,F7-F8 Art: bcb-2013-0026 Input-1st disk, 2nd ??