Biological designer self-assembling peptide nanofiber scaffolds significantly enhance osteoblast proliferation, differentiation and 3-D migration

A class of self-assembling peptide nanofiber scaffolds has been shown to be an excellent biological material for 3-dimension cell culture and stimulating cell migration into the scaffold, as well as for repairing tissue defects in animals. We report here the development of several peptide nanofiber scaffolds designed specifically for osteoblasts. We designed one of the pure self-assembling peptide scaffolds RADA16-I through direct coupling to short biologically active motifs. The motifs included osteogenic growth peptide ALK (ALKRQGRTLYGF) bone-cell secreted-signal peptide, osteopontin cell adhesion motif DGR (DGRGDSVAYG) and 2-unit RGD binding sequence PGR (PRGDSGYRGDS). We made the new peptide scaffolds by mixing the pure RAD16 and designer-peptide solutions, and we examined the molecular integration of the mixed nanofiber scaffolds using AFM. Compared to pure RAD16 scaffold, we found that these designer peptide scaffolds significantly promoted mouse pre-osteoblast MC3T3-E1 cell proliferation. Moreover, alkaline phosphatase (ALP) activity and osteocalcin secretion, which are early and late markers for osteoblastic differentiation, were also significantly increased. We demonstrated that the designer, self-assembling peptide scaffolds promoted the proliferation and osteogenic differentiation of MC3T3-E1. Under the identical culture medium condition, confocal images unequivocally demonstrated that the designer PRG peptide scaffold stimulated cell migration into the 3-D scaffold. Our results suggest that these designer peptide scaffolds may be very useful for promoting bone tissue regeneration.     

Kinetic and functional characterisation of the heparin‐binding peptides from human transglutaminase 2

Transglutaminase 2 (TG2) is an autoantigen in celiac disease (CD) and it has multiple biologic functions including involvement in cell adhesion through interactions with integrins, fibronectin (FN), and heparan sulfate proteoglycans. We aimed to delineate the heparin‐binding regions of human TG2 by studying binding kinetics of the predicted heparin‐binding peptides using surface plasmon resonance method. In addition, we characterized immunogenicity of the TG2 peptides and their effect on cell adhesion. The high‐affinity binding of human TG2 to the immobilized heparin was observed, and two TG2 peptides, P1 (amino acids 202–215) and P2 (261–274), were found to bind heparin. The amino acid sequences corresponding to the heparin‐binding peptides were located close to each other on the surface of the TG2 molecule as part of the α‐helical structures. The heparin‐binding peptides displayed increased immunoreactivity against serum IgA of CD patients compared with other TG2 peptides. The cell adhesion reducing effect of the peptide P2 was revealed in Caco‐2 intestinal epithelial cell attachment to the FN and FN‐TG2 coated surfaces. We propose that TG2 amino acid sequences 202–215 and 261–274 could be involved in binding of TG2 to cell surface heparan sulfates. High immunoreactivity of the corresponding heparin‐binding peptides of TG2 with CD patient's IgA supports the previously described role of anti‐TG2 autoantibodies interfering with this interaction. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Fish bone peptide promotes osteogenic differentiation of MC3T3‐E1 pre‐osteoblasts through upregulation of MAPKs and Smad pathways activated BMP‐2 receptor

Fish bone, a by‐product of fishery processing, is composed of protein, calcium, and other minerals. The objective of this study was to investigate the effects of a bioactive peptide isolated from the bone of the marine fish, Johnius belengerii, on the osteoblastic differentiation of MC3T3‐E1 pre‐osteoblasts. Post consecutive purification by liquid chromatography, a potent osteogenic peptide, composed of 3 amino acids, Lys‐Ser‐Ala (KSA, MW: 304.17 Da), was identified. The purified peptide promoted cell proliferation, alkaline phosphatase activity, mineral deposition, and expression levels of phenotypic markers of osteoblastic differentiation in MC3T3‐E1 pre‐osteoblast. The purified peptide induced phosphorylation of mitogen‐activated protein kinases, including p38 mitogen‐activated protein kinase, extracellular regulated kinase, and c‐Jun N‐terminal kinase as well as Smads. As attested by molecular modelling study, the purified peptide interacted with the core interface residues in bone morphogenetic protein receptors with high affinity. Thus, the purified peptide could serve as a potential pharmacological substance for controlling bone metabolism.     

Mechanism of interaction of thrombospondin with human endothelium and inhibition of sickle erythrocyte adhesion to human endothelial cells by heparin

Thrombospondin (TSP) mediates sickle erythrocyte adhesion to endothelium, but the mechanism remains unknown. Since TSP is comprised of heterogeneously distinct domains, this adhesion may depend on the interaction of specific regions of TSP with different cell surface receptors. To examine the mechanisms of interaction of TSP with human umbilical vein endothelial cells (HUVEC), we performed binding studies using soluble [¹²⁵I]TSP. Our data showed that (i) monoclonal antibodies (MoAbs) against cell surface heparan sulfate (HS) or the heparin-binding domain of TSP, or cleavage of HS on HUVEC by heparitinase reduced TSP binding by 28–40%, (ii) the RGD peptide or MoAbs against integrin α_vβ₃ or the calcium binding region of TSP inhibited binding by 18–28%, and (iii) a MoAb against the cell-binding domain of TSP inhibited binding by 36%. Unmodified heparin inhibited the binding of TSP to endothelial cells by 70% and did so far more effectively than selectively desulfated heparins, HS or chondroitin sulfate. Heparin inhibited TSP binding to HUVEC at much lower concentrations than were required to inhibit TSP binding to sickle erythrocytes. Unmodified heparin effectively inhibited the TSP-mediated adhesion of sickle erythrocytes to HUVEC. These data imply that cell surface HS-mediated mechanisms play a key role in TSP-mediated sickle erythrocyte adhesion to endothelium, and heparin may be of use for inhibition of this adhesion.     

Enhancement of cell attachment and tissue integration by a IKVAV containing multi-domain peptide

Laminin contains a number of cell binding motifs including IKVAV and some that bind heparin. We developed a multi-domain synthetic peptide, LA2, which combines IKVAV sequences with a heparin-binding domain with the goal of improving cell attachment to otherwise non-adherent substrates. LA2 was used to coat polystyrene, ethyl vinyl acetate (EVA), expanded polytetrafluoroethylene (ePTFE), polycarbonate, titanium and stainless steel. In cell attachment studies, LA2 dramatically increased cell attachment to polystyrene and EVA compared to uncoated counterparts or those coated with SIKVAV. Similar increases were observed on ePTFE and titanium. On polystyrene, LA2 enhanced the attachment of endothelial cells, smooth muscle cells, epithelial cells, myoblasts, and osteoblast progenitor cells. Following adhesion, the cells underwent proliferation to form confluent monolayers with phenotypic morphologies. Using osteoblast progenitor cells (MC3T3 cells) grown on LA2/polystyrene, the cells exhibited an increased production of a differentiation marker, alkaline phosphatase. In vivo, LA2 improved tissue integration into ePTFE when implanted subcutaneously in rats. After 2 weeks, cells had penetrated deep into the LA2 coated ePTFE implant whereas little cell penetration was found in uncoated grafts. The implant sites exhibited little inflammation or other untoward effects. The results indicated that the LA2 peptide improved cell adhesion and tissue integration and might be useful in a number of tissue engineering applications.     

Cell surface phosphatidylinositol-anchored heparan sulfate proteoglycan initiates mouse melanoma cell adhesion to a fibronectin-derived, heparin-binding synthetic peptide

Cell surface heparan sulfate proteoglycan (HSPG) from metastatic mouse melanoma cells initiates cell adhesion to the synthetic peptide FN-C/H II, a heparin-binding peptide from the 33-kD A chain-derived fragment of fibronectin. Mouse melanoma cell adhesion to FN-C/H II was sensitive to soluble heparin and pretreatment of mouse melanoma cells with heparitinase. In contrast, cell adhesion to the fibronectin synthetic peptide CS1 is mediated through an alpha 4 beta 1 integrin and was resistant to heparin or heparitinase treatment. Mouse melanoma cell HSPG was metabolically labeled with [35S]sulfate and extracted with detergent. After HPLC-DEAE purification, 35S-HSPG eluted from a dissociative CL-4B column with a Kav approximately 0.45, while 35S-heparan sulfate (HS) chains eluted with a Kav approximately 0.62. The HSPG contained a major 63-kD core protein after heparitinase digestion. Polyclonal antibodies generated against HSPG purified from mouse melanoma cells grown in vivo also identified a 63-kD core protein. This HSPG is an integral plasma membrane component by virtue of its binding to Octyl Sepharose affinity columns and that anti-HSPG antibody staining exhibited a cell surface localization. The HSPG is anchored to the cell surface through phosphatidylinositol (PI) linkages, as evidenced in part by the ability of PI-specific phospholipase C to eliminate binding of the detergent-extracted HSPG to Octyl Sepharose. Furthermore, the mouse melanoma HSPG core protein could be metabolically labeled with 3H-ethanolamine. The involvement of mouse melanoma cell surface HSPG in cell adhesion to fibronectin was also demonstrated by the ability of anti-HSPG antibodies and anti-HSPG IgG Fab monomers to inhibit mouse melanoma cell adhesion to FN-C/H II. 35S-HSPG and 35S-HS bind to FN-C/H II affinity columns and require 0.25 M NaCl for elution. However, heparitinase-treated 125I-labeled HSPG failed to bind FN-C/H II, suggesting that HS, and not HSPG core protein, binds FN-C/H II. These data support the hypothesis that a phosphatidylinositol-anchored HSPG on mouse melanoma cells (MPIHP-63) initiates recognition to FN-C/H II, and implicate PI-associated signal transduction pathways in mediating melanoma cell adhesion to this defined ligand.     

RGD-independent cell adhesion to the carboxy-terminal heparin-binding fragment of fibronectin involves heparin-dependent and -independent activities

Cell adhesion to extracellular matrix components such as fibronectin has a complex basis, involving multiple determinants on the molecule that react with discrete cell surface macromolecules. Our previous results have demonstrated that normal and transformed cells adhere and spread on a 33-kD heparin binding fragment that originates from the carboxy-terminal end of particular isoforms (A-chains) of human fibronectin. This fragment promotes melanoma adhesion and spreading in an arginyl-glycyl-aspartyl-serine (RGDS) independent manner, suggesting that cell adhesion to this region of fibronectin is independent of the typical RGD/integrin-mediated binding. Two synthetic peptides from this region of fibronectin were recently identified that bound [3H]heparin in a solid-phase assay and promoted the adhesion and spreading of melanoma cells (McCarthy, J. B., M. K. Chelberg, D. J. Mickelson, and L. T. Furcht. 1988. Biochemistry. 27:1380-1388). The current studies further define the cell adhesion and heparin binding properties of one of these synthetic peptides. This peptide, termed peptide I, has the sequence YEKPGSP-PREVVPRPRPGV and represents residues 1906-1924 of human plasma fibronectin. In addition to promoting RGD-independent melanoma adhesion and spreading in a concentration-dependent manner, this peptide significantly inhibited cell adhesion to the 33-kD fragment or intact fibronectin. Polyclonal antibodies generated against peptide I also significantly inhibited cell adhesion to the peptide, to the 33-kD fragment, but had minimal effect on melanoma adhesion to fibronectin. Anti-peptide I antibodies also partially inhibited [3H]heparin binding to fibronectin, suggesting that peptide I represents a major heparin binding domain on the intact molecule. The cell adhesion activity of another peptide from the 33-kD fragment, termed CS1 (Humphries, M. J., A. Komoriya, S. K. Akiyama, K. Olden, and K. M. Yamada. 1987. J. Biol. Chem., 262:6886-6892) was contrasted with peptide I. Whereas both peptides promoted RGD-independent cell adhesion, peptide CS1 failed to bind heparin, and exogenous peptide CS1 failed to inhibit peptide I-mediated cell adhesion. The results demonstrate a role for distinct heparin-dependent and -independent cell adhesion determinants on the 33-kD fragment, neither of which are related to the RGD-dependent integrin interaction with fibronectin.     

Multiple domains contribute to heparin/heparan sulfate binding by human HIP/L29

Human heparin/heparan sulfate interacting protein/L29 (HIP/L29) is thought to be involved in the promotion of cell adhesion, the promotion of cell growth in the cancerous state, and the modulation of blood coagulation. These activities are consistent with the proposed function of HIP/L29 as a heparin/heparan sulfate (Hp/HS) binding growth factor that has a preference for anticoagulantly active Hp/HS. Previous studies showed that a peptide derived from the C terminus of human HIP/L29 (HIP peptide-1) can selectively bind anticoagulant Hp and support cell adhesion. However, a murine ortholog does not have an identical HIP peptide-1 sequence, yet still retains the ability to bind Hp, suggesting that there may be additional Hp/HS binding sites outside of the HIP peptide-1 domain. To test this hypothesis, a systematic study of the domains within human and murine HIP/L29 responsible for Hp/HS binding activity was undertaken. Using deletion mutants, proteolytic fragments, and protease protection of HIP/L29 by Hp, we demonstrate that multiple binding domains contribute to the overall Hp/HS binding activity of HIP/L29 proteins. Furthermore, a conformational change is induced in human HIP/L29 upon Hp binding as detected by circular dichroism spectroscopy. These studies demonstrate the multiplicity of Hp/HS binding sequences within human and murine HIP/L29.     

The SH3 domain of HS1 protein recognizes lysine-rich polyproline motifs

HS1 is a protein involved in erythroid proliferation and apoptotic cell death, containing several structurally significant motifs including a C-terminal SH3 domain. HPK1 is a member of the Ste20-related kinase family, which contains four proline-rich sequences and is constitutively associated with HS1 in hematopoietic cells. Recombinant fusion protein GST-SH3_HS1 was expressed to assess the binding properties of 16 peptides derived from the HPK1 proline-rich regions. The binding affinities were determined by non-immobilized ligand interaction assay by circular dichroism. Our results revealed that the classical PxxPxK class II binding motif is not sufficient to induce the interaction with the GST-SH3_HS1 domain, an event dependent on the presence of additional basic residue(s) located at the C-terminus of the PxxPxK motif: Lys₋₅ in P2 peptide and Lys₋₈ in P4c peptide. Lys replacement by Arg residues decreases the ligand binding affinity. The finding that both SH3_HS1 domain and full-length HS1 protein bind to P2 peptide with similar affinity demonstrates that the whole protein sequence does not affect the interaction properties of the domain. In silico models of SH3_HS1 as a complex with P2 or P4c highlight the domain residues that interact with the recognition determinants of the peptide ligand and that cooperate in the complex stabilization.     

The design of a peptide sequence to inhibit HIV replication: a search algorithm combining Monte Carlo and self-consistent mean field techniques

We developed a search algorithm combining Monte Carlo (MC) and self-consistent mean field techniques to evolve a peptide sequence that has good binding capability to the anticodon stem and loop (ASL) of human lysine tRNA species, tRNA^Lys3, with the ultimate purpose of breaking the replication cycle of human immunodeficiency virus-1. The starting point is the 15-amino-acid sequence, RVTHHAFLGAHRTVG, found experimentally by Agris and co-workers to bind selectively to hypermodified tRNA^Lys3. The peptide backbone conformation is determined via atomistic simulation of the peptide-ASL^Lys3 complex and then held fixed throughout the search. The proportion of amino acids of various types (hydrophobic, polar, charged, etc.) is varied to mimic different peptide hydration properties. Three different sets of hydration properties were examined in the search algorithm to see how this affects evolution to the best-binding peptide sequences. Certain amino acids are commonly found at fixed sites for all three hydration states, some necessary for binding affinity and some necessary for binding specificity. Analysis of the binding structure and the various contributions to the binding energy shows that: 1) two hydrophilic residues (asparagine at site 11 and the cysteine at site 12) “recognize” the ASL^Lys3 due to the VDW energy, and thereby contribute to its binding specificity and 2) the positively charged arginines at sites 4 and 13 preferentially attract the negatively charged sugar rings and the phosphate linkages, and thereby contribute to the binding affinity.     

Identification of a heparin binding domain of the neural cell adhesion molecule N-CAM using synthetic peptides

The neural cell adhesion molecule (N-CAM) plays an integral role in cell interactions during neural development, with the binding of heparan sulfate proteoglycan to the amino-terminal region of N-CAM being required for N-CAM function. In the present study we have used synthetic peptides (HBD-1 and HBD-2), derived from the primary amino acid sequence of rat N-CAM, to identify the region of N-CAM that binds heparan sulfate. The 28 amino acid HBD-1 synthetic peptide was shown to bind both [3H]heparin and dissociated retinal cells. Retinal cells also attach to a substratum of HBD-2 peptide, but fail to bind to a control peptide containing a scrambled amino acid sequence of HBD-2. The HBD-2 peptide also inhibits retinal cell adhesion to N-CAM, demonstrating the physiological importance of the amino acid sequence encoded by the HBD peptide. These data therefore permit the localization of a heparin binding domain to a 17 amino acid region of immunoglobulin-like loop 2.     

A comparative study on the heparin‐binding proteomes of Toxoplasma gondii and Plasmodium falciparum

Toxoplasma gondii is an obligatory intracellular apicomplexan parasite which exploits host cell surface components in cell invasion and intracellular parasitization. Sulfated glycans such as heparin and heparan sulfate have been reported to inhibit cell invasion by T. gondii and other apicomplexan parasites such as Plasmodium falciparum. The aim of this study was to investigate the heparin‐binding proteome of T. gondii. The parasite‐derived components were affinity‐purified on the heparin moiety followed by MS fingerprinting of the proteins. The heparin‐binding proteins of T. gondii and P. falciparum were compared based on functionality and affinity to heparin. Among the proteins identified, the invasion‐related parasite ligands derived from tachyzoite/merozoite surface and the secretory organelles were prominent. However, the profiles of the proteins were different in terms of affinity to heparin. In T. gondii, the proteins with highest affinity to heparin were the intracellular components with functions of parasite development contrasted to that of P. falciparum, of which the rhoptry‐derived proteins were prominently identified. The profiling of the heparin‐binding proteins of the two apicomplexan parasites not only explained the mechanism of heparin‐mediated host cell invasion inhibition, but also, to a certain extent, revealed that the action of heparin on the parasite extended after endocytosis.     

Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans: 2. Biochemical analyses

Platelet endothelial cell adhesion molecule 1 (PECAM-1) (CD31), a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules with six Ig-like domains, has a range of functions, notably its contributions to leukocyte extravasation during inflammation and in maintaining vascular endothelial integrity. Although PECAM-1 is known to mediate cell adhesion by homophilic binding via domain 1, a number of PECAM-1 heterophilic ligands have been proposed. Here, the possibility that heparin and heparan sulfate (HS) are ligands for PECAM-1 was reinvestigated. The extracellular domain of PECAM-1 was expressed first as a fusion protein with the Fc region of human IgG1 fused to domain 6 and second with an N-terminal Flag tag on domain 1 (Flag-PECAM-1). Both proteins bound heparin immobilized on a biosensor chip in surface plasmon resonance (SPR) binding experiments. Binding was pH-sensitive but is easily measured at slightly acidic pH. A series of PECAM-1 domain deletions, prepared in both expression systems, were tested for heparin binding. This revealed that the main heparin-binding site required both domains 2 and 3. Flag-PECAM-1 and a Flag protein containing domains 1-3 bound HS on melanoma cell surfaces, but a Flag protein containing domains 1-2 did not. Heparin oligosaccharides inhibited Flag-PECAM-1 from binding immobilized heparin, with certain structures having greater inhibitory activity than others. Molecular modeling similarly identified the junction of domains 2 and 3 as the heparin-binding site and further revealed the importance of the iduronic acid conformation for binding. PECAM-1 does bind heparin/HS but by a site that is distinct from that required for homophilic binding.     

Effects on in vitro and in vivo angiogenesis induced by small peptides carrying adhesion sequences

It is well known that tumor growth is strictly dependent on neo‐vessel formation inside the tumor mass and that cell adhesion is required to allow EC proliferation and migration inside the tumor. In this work, we have evaluated the in vitro and in vivo effects on angiogenesis of some peptides, originally designed to promote cell adhesion on biomaterials, containing RGD motif mediating cell adhesion via integrin receptors [RGD, GRGDSPK, and (GRGDSP)₄K] or the heparin‐binding sequence of human vitronectin that interacts with HSPGs [HVP(351–359)]. Cell adhesion, proliferation, migration, and capillary‐like tube formation in Matrigel were determined on HUVECs, whereas the effects on in vivo angiogenesis were evaluated using the CAM assay. (GRGDSP)₄K linear sequence inhibited cell adhesion, decreased cell proliferation, migration and morphogenesis in Matrigel, and induced anti‐angiogenic responses on CAM at higher degree than that determined after incubation with RGD or GRGDSPK. Moreover, it counteracted both in vitro and in vivo the pro‐angiogenic effects induced by the Fibroblast growth factor (FGF‐2). On the other hand, HVP was not able to affect cell adhesion and appeared less effective than (GRGDSP)₄K. Our data indicate that the activity of RGD‐containing peptides is related to their adhesive properties, and their effects are modulated by the number of cell adhesion motifs and the aminoacidic residues next to these sequences. The anti‐angiogenic properties of (GRGDSP)₄K seem to depend on its interaction with integrins, whereas the effects of HVP may be partially due to an impairment of HSPGs/FGF‐2. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of antilytic peptide antibody: application for the detection of lytic‐based hybrid peptide in serum samples

We previously reported that a novel targeted drug termed hybrid epidermal growth factor receptor (EGFR)‐lytic peptide, made by chemical conjugation of targeted binding peptide and cell‐killing, lytic‐peptide components, has selective cytotoxic activity that allows it to discriminate between normal and cancer cells. In addition, in vivo analysis revealed that this hybrid peptide displays significant antitumor activity in a xenograft model of human breast and pancreatic cancer in mice. Here, we characterized antilytic peptide antibody, which was raised from rabbit serum using the antigen of lytic peptide conjugated with keyhole limpet hemocyanin. It was found that antilytic peptide antibody is specific to the lytic peptide as assessed by both ELISA and surface plasmon resonance analysis and can also bind to EGFR‐lytic peptide. Epitope mapping analysis using Biacore showed that two successive lysine regions in the lytic‐peptide sequence are significant for recognition by this antibody. In addition, it was shown that this antibody can detect lytic‐based hybrid peptide in serum samples from mouse blood and also in cultured breast cancer MDA‐MB‐231 cell samples by immunocytochemical staining experiments. It was found that the maximum concentrations of this peptide in serum were reached within 15–30 min of i.v. administration of EGFR‐lytic peptide to mice. These results indicate that this antibody will be a useful tool for the detection of lytic‐based peptides to investigate their in vivo stability and pharmacokinetics. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

A novel peptide sequence in perlecan domain IV supports cell adhesion, spreading and FAK activation.

Perlecan/HSPG2 is a large, multi-domain, multifunctional heparan sulfate proteoglycan with a wide tissue distribution. With the exception of its unique domain I, each of perlecan's other four domains shares sequence similarity to other protein families including low density lipoprotein (LDL) receptor, laminin alpha chain, neural cell adhesion molecule (NCAM), immunoglobulin (Ig) superfamily members, and epidermal growth factor (EGF). Previous studies demonstrated that glycosaminoglycan-bearing perlecan domain I supports early chondrogenesis and growth factor delivery. Other sites in the core protein interact with other matrix molecules and support cell adhesion, although the peptide sequences involved remain unidentified. To identify novel functional motifs within perlecan, we used a bioinformatics approach to predict regions likely to be on the exterior of the folded protein. Unique hydrophilic sequences of about 18 amino acids were selected for testing in cell adhesion assays. A novel peptide sequence (TWSKVGGHLRPGIVQSG) from an immunoglobulin (Ig) repeat in domain IV supported rapid cell adhesion, spreading and focal adhesion kinase (FAK) activation when compared to other peptides, a randomly scrambled sequence of the domain IV peptide or a negative control protein. MG-63 human osteosarcoma cells, epithelial cells and multipotent C(3)H10T1/2 cells, but not bone marrow cells, rapidly, i.e., within 30 min, formed focal adhesions and assembled an actin cytoskeleton on domain IV peptide. Cell lines differentially adhered to the domain IV peptide, suggesting adhesion is receptor specific. Adhesion was divalent cation independent and heparin sensitive, a finding that may explain some previously poorly understood observations obtained with intact perlecan. Collectively, these studies demonstrate the feasibility of using bioinformatics-based strategies to identify novel functional motifs in matrix proteins such as perlecan.     

Facile and selective covalent grafting of an RGD‐peptide to electrospun scaffolds improves HUVEC adhesion

The development of a biomimetic surface able to promote endothelialization is fundamental in the search for blood vessel substitutes that prevent the formation of thrombi or hyperplasia. This study aims at investigating the effect of functionalization of poly‐ε‐caprolactone or poly(L‐lactic acid‐co‐?‐caprolactone) electrospun scaffolds with a photoreactive adhesive peptide. The designed peptide sequence contains four Gly‐Arg‐Gly‐Asp‐Ser‐Pro motifs per chain and a p‐azido‐Phe residue at each terminus. Different peptide densities on the scaffold surface were obtained by simply modifying the peptide concentration used in pretreatment of the scaffold before UV irradiation. Scaffolds of poly‐ε‐caprolactone embedded with adhesive peptides were produced to assess the importance of peptide covalent grafting. Our results show that the scaffolds functionalized with photoreactive peptides enhance adhesion at 24 h with a dose‐dependent effect and control the proliferation of human umbilical vein endothelial cells, whereas the inclusion of adhesive peptide in the electrospun matrices by embedding does not give satisfactory results. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

The aggregated form of an AAMP derived peptide behaves as a heparin sensitive cell binding agent

A peptide termed P189, derived from the sequence of a newly discovered protein, AAMP (angio-associated migratory cell protein), contains a motif that is predicted to a bind heparin. It occurs near the amino terminal end of AAMP. Previous studies have shown that in its solubilized form P189 (RRLRRMESESES) binds heparin and melanoma cells. The peptide is bipolar in that it contains positive charges at its amino end and negative charges at its carboxyl end. It forms strongly aggregated particles that require exposure to 50% DMSO and 100 degrees C for solubilization to occur. Now heparin and cell binding (heparin sensitive) are also demonstrated for the peptide in its particular form. Cell binding/clustering to the peptide particles is strong and resists exposure to various reagents (sugars and inhibitors of glycolysis and protein synthesis) except heparin. Tumor cell migration is partially inhibited by the presence of the peptide. On electron photomicrographs the peptide is seen in close apposition to cell membranes. Heparin sensitivity of the cell binding indicates that cell surface glycosaminoglycans are involved. The aggregated peptide binds heparin in a saturable manner with a dissociation constant, K(d), of 306 pmol. Cell binding/clustering studies using peptide variants of P189, which have substitutions in either the charged and/or nonpolar residues, show that the specific sequence of P189 optimizes heparin-sensitive cell aggregation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 365-372, 1997.     

Binding and internalization of an ICAM-1 peptide by the surface receptors of T cells.

The objective of this work was to evaluate the binding characteristics of a cyclic peptide, cyclo (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-V al9-Thr10-Gly11-Cys12-OH (cIBR), to Molt-3 T cells. This cIBR peptide is derived from sequence numbers 11-20 of intercellular adhesion molecule-1 (ICAM-1). Binding studies were performed using a fluorescence-labeled peptide (FITC-cIBR) in which the fluorescence marker fluorescein 5-isothiocyanate (FITC) was conjugated to the N-terminal of the cIBR peptide. The binding affinity of the FITC-cIBR peptide to Molt-3 T cells was evaluated using a FACScan flow cytometer. The binding specificity of the FITC-cIBR peptide was also confirmed by inhibition of binding using unlabeled peptide (cIBR). The results show that FITC-cIBR binds to two populations of T cells with different affinities; population 1 has high cell numbers (75%) but low affinity, and population 2 has high binding affinity but low cell numbers (25%). Binding to both populations was saturable and could be inhibited by the unlabeled peptide (cIBR), suggesting a receptor-mediated binding process. In addition to binding, receptor-mediated internalization was also observed for population 2; this was confirmed by confocal microscopy and temperature-dependence studies at 37 degrees C and 4 degrees C. The binding and internalization of this peptide may be carried out by surface receptors on Molt-3 T cells such as LFA-1. In the future, the binding and internalization of cIBR peptide can be utilized as a method of targeted drug delivery to leukocytes for the treatment of leukocyte-related diseases.