Antiangiogenic and antitumor activities of peptide inhibiting the vascular endothelial growth factor binding to neuropilin-1

Neuropilin-1 (NRP-1), a non-tyrosine kinase receptor of vascular endothelial growth factor-165 (VEGF165), was found expressed on endothelial and some tumor cells. Since its overexpression is correlated with tumor angiogenesis and progression, the targeting of NRP-1 could be a potential anti-cancer strategy. To explore this hypothesis, we identified a peptide inhibiting the VEGF165 binding to NRP-1 and we tested whether it was able to inhibit tumor growth and angiogenesis. To prove the target of peptide action, we assessed its effects on binding of radiolabeled VEGF165 to recombinant receptors and to cultured cells expressing only VEGFR-2 (KDR) or NRP-1. Antiangiogenic activity of the peptide was tested in vitro in tubulogenesis assays and in vivo in nude mice xenotransplanted in fat-pad with breast cancer MDA-MB-231 cells. Tumor volumes, vascularity and proliferation indices were determined. The selected peptide, ATWLPPR, inhibited the VEGF165 binding to NRP-1 but not to tyrosine kinase receptors, VEGFR-1 (flt-1) and KDR; nor did it bind to heparin. It diminished the VEGF-induced human umbilical vein endothelial cell proliferation and tubular formation on Matrigel and in co-culture with fibroblasts. Administration of ATWLPPR to nude mice inhibited the growth of MDA-MB-231 xenografts, and reduced blood vessel density and endothelial cell area but did not alter the proliferation indices of the tumor. In conclusion, ATWLPPR, a previously identified KDR-interacting peptide, was shown to inhibit the VEGF165 interactions with NRP-1 but not with KDR and to decrease the tumor angiogenesis and growth, thus validating, in vivo, NRP-1 as a possible target for antiangiogenic and antitumor agents.     

Analysis of the pivotal residues of the immunodominant and highly uveitogenic determinant of interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein (IRBP), a retinal-specific Ag, induces experimental autoimmune uveitis in a variety of animals. We have previously shown that sequence 1169-1191 of bovine IRBP is the immunodominant epitope of this protein in Lewis rats and is highly immunogenic and uveitogenic in these rats. The active site of peptide 1169-1191 was determined by testing its truncated forms. The shortest peptide to be immunologically active was found to be 1182-1190 (WEGVGVVPD). To determine the role of individual residues of this sequence, we have tested the immunologic activities of nine analogs of peptide 1181-1191, in which each of residues 1182-1190 was substituted with alanine (A). The tested activities included the capacity to induce experimental autoimmune uveitis and cellular responses in immunized rats, as well as the capability to stimulate lymphocytes sensitized against IRBP or the parent peptide 1181-1191. Analogs that did not stimulate these lymphocytes were also tested for their capacity to competitively inhibit the proliferative response to 1181-1191. Analogs A(1184), A(1186), and A(1187) resembled 1181-1191 in their activities, whereas the other analogs exhibited remarkably reduced activities, with several patterns being noticed. Analog A(1182) was inactive in all tests. Analog A(1190) was very weakly uveitogenic and non-immunogenic, but it stimulated lymphocytes sensitized against IRBP or 1181-1191 when added at exceedingly high concentrations. Analogs A(1183) and A(1185) resembled A(1190) in being weakly uveitogenic and A(1185) was also found to be poorly immunogenic. In addition, relatively high concentrations of A(1183) and A(1185) were needed to stimulate lymphocytes sensitized against IRBP or 1181-1191. However, a different pattern of activities was exhibited by analogs A(1188) and A(1189). These peptides were uveitogenic and immunogenic, but failed to stimulate lymphocytes sensitized to IRBP or 1181-1191. Furthermore, A(1188) and A(1189), but not A(1182), also inhibited the response to 1181-1191 of a cell line specific toward this parent peptide. The data are interpreted to show that residues 1188 and 1189 are involved in the interaction of the peptide with the TCR, whereas residues 1182 and 1190 and, perhaps, 1183 and 1185, are pivotal for the binding of peptide 1181-1190 to the MHC molecules on APC.     

CD4-derived synthetic peptide blocks the binding of HIV-1 GP120 to CD4-bearing cells and prevents HIV-1 infection

The T cell surface glycoprotein CD4 plays an important role in mediating cellular immunity and serves as the receptor for human immunodeficiency virus. In order to identify primary sequences within the CD4 molecule that may be involved in the binding of the HIV-I envelope, we synthesized various peptides corresponding to the V1, V2, V3, and V4 domains of CD4. We tested the ability of these peptides to block the binding of purified HIV-I gp120 to CD4+ human lymphoblastic leukemia cells (CEM) using fluorescence-activated cell sorting. One of these peptides, corresponding to CD4 amino acids (74-95), when preincubated with gp120, blocked its subsequent binding to CEM cells by 80%. A truncated form of this peptide (81-95), was found to be as efficient as the longer peptide (74-95) in inhibiting the binding of gp120 to CEM cells. The same peptide did not block the binding of OKT4A or Leu3A anti-CD4 monoclonal antibodies, which were previously shown to block HIV-I binding to CD4. The peptides were also tested for their ability to block HIV-I infection of a T cell line in vitro. Only CD4 peptide (74-95) and the shorter fragment (81-95) succeeded in protecting T cells against infection with different HIV-I strains. All the other peptides examined had no effect on gp120 binding to CEM cells and did not block syncytia formation. Goat polyclonal antibodies against the CD4 peptide (74-95) gave modest interference of gp120 binding to CEM cells. These data suggest that the CD4 region (74-95) participates in the CD4-mediated binding and/or internalization of HIV-I virion.     

Structural and functional characterization of the porcine proline-rich antifungal peptide SP-B isolated from salivary gland granules.

A 1905-Da cationic proline-rich peptide, named SP-B, was recently isolated by our group as the main component of salivary gland granules, and its primary sequence fully characterized by means of automated Edman sequencing and LC-MS/MS tools. In the present study SP-B is shown to possess antifungal activity when challenged with strains of Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus, while only negligible antibacterial activity was detected. Furthermore, SP-B was found to be non-cytotoxic when tested on fibroblast cell lines. To obtain information regarding its structure affinity, capillary electrophoresis (CE), circular dichroism (CD) and attenuated total reflection (ATR)-FT/IR experiments were performed. CE revealed a pH dependence of the hydrodynamic radial dimensions both in aqueous and 2,2,2-trifluoroethanol solutions. CD and ATR-FT/IR measurements confirmed the structure-pH relationship, revealing a secondary structure composed of mixed proportions of polyproline-II, unordered and turn motifs, the last being more evident in the zwitterionic form of the peptide. From these findings SP-B peptide could be classified as a new member of the proline-rich antimicrobial peptide family.     

Enhancing the immunogenicity of a permissive binding T cell epitope derived from the simian immunodeficiency virus-encoded negative regulatory factor.

Using the murine system we have analyzed an immunogenic T cell peptide epitope corresponding to amino acids 96-112 of the simian immunodeficiency virus-negative regulatory protein sequence. This epitope was unusual in that it was strongly immunogenic in mice of five of the six H-2 haplotypes tested. We generated a T cell hybridoma (SVNF) specific for this peptide in order to determine how manipulating the peptide might alter its immunogenicity. Substitution analysis showed that His 103, Pro 104, Val 106, and Pro 107 were important amino acids for stimulating SVNF because substitutions at these positions diminished the reactivity of SVNF. However, we also found that substituting an Ala for a Val at position 100 or a Val for an Ala at position 110 enhanced reactivity of SVNF. We were able to further enhance the immunogenicity of this epitope by extending the carboxyl terminus two amino acids and making the resulting carboxyl-terminal Lys an amide and by adding a Glu to the amino terminus. These modifications shifted the in vitro activity of SVNF at least two orders of magnitude. We also compared the ability of this modified peptide and the wild-type SIV nef 96-112 to prime a T cell response in vivo. We primed mice with various doses of either the wild-type or the modified peptide and looked at the ability of the draining lymph node cells to proliferate to wild-type peptide. We found that the modified peptide was 10- to 100-fold better at priming a T cell response than the wild-type peptide. Therefore, we were able to create a peptide that was more immunogenic than the wild-type peptide in vivo as well as in vitro. Manipulations such as these that enhance the immunogenicity of T cell epitopes must be considered in developing peptide vaccines against HIV or other infectious agents.     

Identification of downstream target genes of latent membrane protein 1 in nasopharyngeal carcinoma cells by suppression subtractive hybridization

Proteolytic digestion of bovine beta-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15-20), AASDISLLDAQSAPLR (residues 25-40), IPAVFK (residues 78-83) and VLVLDTDYK (residues 92-100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55-64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of beta-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of beta-lactoglobulin could be useful to increase its antimicrobial function.     

Inhibition of HIV-1 entry before gp41 folds into its fusion-active conformation

HIV-1 entry into its host cell is modulated by its transmembrane envelope glycoprotein (gp41). The core of the activated conformation of gp41 consists of a trimer of heterodimers comprising a leucine/isoleucine zipper sequence (represented here by the synthetic peptide N36 or by the longer N51 peptide) and a C-terminal highly conserved region (represented here by C34). A correlation was found between the action of DP178, which is a potent inhibitor of HIV-1 entry into its host cell, and its ability to interact with the leucine/isoleucine zipper sequence. This correlation was further tested and confirmed by circular dichroism spectroscopy. We found that whereas DP178 perturbs the partial alpha-helix nature of peptides corresponding to the leucine/isoleucine zipper sequence (N36 or N51), it cannot perturb the trimer of heterodimers conformation, modeled by the complex of N36 or N51 with C34. Therefore, we suggest that the already formed trimer of heterodimers is not the target of inhibition by DP178. Our results are consistent with a model in which DP178 acquires its inhibitory activity by binding to an earlier intermediate of gp41, in which the N and C peptide regions are not yet associated, thus allowing DP178 to bind to the leucine/isoleucine zipper sequence and consequently to inhibit transition to the fusion-active conformation.     

Uptake of cell-penetrating peptides is dependent on peptide-to-cell ratio rather than on peptide concentration

The influence of the peptide-to-cell ratio and energy depletion on uptake and degradation of the cell-penetrating peptides (CPPs) MAP (model amphipathic peptide) was investigated. The intracellular concentration of the CPPs, MAP and penetratin was monitored while varying the number of cells at fixed peptide concentration and incubation volume, or changing the concentration and incubation volume at fixed cell number. The uptake of CPPs was shown to be dependent on the peptide/cell ratio. At given peptide concentration and incubation volume, the intracellular concentration of peptide increased with lower cell number. At given cell number, doubling of the incubation volume increased intracellular peptide concentration to a similar extent as the doubling in incubation concentration. From a practical view, this means that the peptide/cell ratio has at least the same importance for the uptake of CPPs as the used peptide concentration. No influence of the peptide/cell ratio was found for the cellular uptake of peptide nucleic acid (PNA), or a non-amphipathic MAP analogue, investigated in parallel for comparison purposes.Energy depletion resulted in significantly reduced quantities of intracellular fluorescence label. Moreover, we show that this difference is mainly due to a membrane-impermeable fluorescent-labelled degradation product, which is lacking in energy-depleted cells. The mechanism of its generation is not likely to be endosomal degradation of endocytosed material, as it is not chloroquine- or brefeldin-sensitive.     

22R-Hydroxycholesterol protects neuronal cells from beta-amyloid-induced cytotoxicity by binding to beta-amyloid peptide

22R-hydroxycholesterol, a steroid intermediate in the pathway of pregnenolone formation from cholesterol, was found at lower levels in Alzheimer's disease (AD) hippocampus and frontal cortex tissue specimens compared to age-matched controls. beta-Amyloid (Abeta) peptide has been shown to be neurotoxic and its presence in brain has been linked to AD pathology. 22R-hydroxycholesterol was found to protect, in a dose-dependent manner, against Abeta-induced rat sympathetic nerve pheochromocytoma (PC12) and differentiated human Ntera2/D1 teratocarcinoma (NT2N) neuron cell death. Other steroids tested were either inactive or acted on rodent neurons only. The effect of 22R-hydroxycholesterol was found to be stereospecific because its enantiomer 22S-hydroxycholesterol failed to protect the neurons from Abeta-induced cell death. Moreover, the effect of 22R-hydroxycholesterol was specific for Abeta-induced cell death because it did not protect against glutamate-induced neurotoxicity. The neuroprotective effect of 22R-hydroxycholesterol was seen when using Abeta1-42 but not the Abeta25-35 peptide. To investigate the mechanism of action of 22R-hydroxycholesterol we examined the direct binding of this steroid to Abeta using a novel cholesterol-protein binding blot assay. Using this method the direct specific binding, under native conditions, of 22R-hydroxycholesterol to Abeta1-42 and Abeta17-40, but not Abeta25-35, was observed. These data suggest that 22R-hydroxycholesterol binds to Abeta and the formed 22R-hydroxycholesterol/Abeta complex is not toxic to rodent and human neurons. We propose that 22R-hydroxycholesterol offers a new means of neuroprotection against Abeta toxicity by inactivating the peptide.     

Uptake of cell-penetrating peptides is dependent on peptide-to-cell ratio rather than on peptide concentration

The influence of the peptide-to-cell ratio and energy depletion on uptake and degradation of the cell-penetrating peptides (CPPs) MAP (model amphipathic peptide) was investigated. The intracellular concentration of the CPPs, MAP and penetratin was monitored while varying the number of cells at fixed peptide concentration and incubation volume, or changing the concentration and incubation volume at fixed cell number. The uptake of CPPs was shown to be dependent on the peptide/cell ratio. At given peptide concentration and incubation volume, the intracellular concentration of peptide increased with lower cell number. At given cell number, doubling of the incubation volume increased intracellular peptide concentration to a similar extent as the doubling in incubation concentration. From a practical view, this means that the peptide/cell ratio has at least the same importance for the uptake of CPPs as the used peptide concentration. No influence of the peptide/cell ratio was found for the cellular uptake of peptide nucleic acid (PNA), or a non-amphipathic MAP analogue, investigated in parallel for comparison purposes. Energy depletion resulted in significantly reduced quantities of intracellular fluorescence label. Moreover, we show that this difference is mainly due to a membrane-impermeable fluorescent-labelled degradation product, which is lacking in energy-depleted cells. The mechanism of its generation is not likely to be endosomal degradation of endocytosed material, as it is not chloroquine- or brefeldin-sensitive.     

Isolation and characterization of four bactericidal domains in the bovine β-lactoglobulin

Proteolytic digestion of bovine β-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15–20), AASDISLLDAQSAPLR (residues 25–40), IPAVFK (residues 78–83) and VLVLDTDYK (residues 92–100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55–64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of β-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of β-lactoglobulin could be useful to increase its antimicrobial function.     

前列腺干细胞抗原(PSCA)的表达及其特异结合肽的筛选

通过反转录 PCR从人前列腺癌细胞中克隆了前列腺干细胞抗原 (PSCA)基因 ,在大肠杆菌中利用pQE30载体对截断型PSCA基因进行了可溶性表达。蛋白纯化后 ,利用噬菌体随机展示 12肽库筛选了PSCA蛋白的特异结合肽 ,通过与EGFP蛋白的耦联表达验证了结合肽的特异性。此特异结合肽的获得 ,为进一步研究针对PSCA的前列腺癌靶向免疫治疗奠定了基础     

A synthetic peptide containing the IKVAV sequence from the A chain of laminin mediates cell attachment, migration, and neurite outgrowth

Laminin is a basement membrane glycoprotein which consists of A, B1, and B2 chains. Laminin has diverse biological activities including promoting cell adhesion, migration, differentiation, growth, and neurite extension. Synthetic peptides from the active region of the A chain were prepared and tested for their biological activity. A 19-mer peptide (designated PA22-2), from just above the carboxyl globule on the long arm of the A chain, was found to promote cell adhesion, spreading, migration, and neurite outgrowth. By testing smaller sequences within the 19-mer peptide, a constituent pentapeptide, IKVAV (Ile-Lys-Val-Ala-Val), was identified as the active site for cell adhesion and neurite outgrowth. These data suggest that this sequence is one of the principle sites in laminin which regulate cellular behavior.     

In vitro and in silico studies on substrate recognition and acceptance of human PKMYT1, a Cdk1 inhibitory kinase

The human Myt1 kinase (PKMYT1) is an important regulator of the G2/M transition in the cell cycle. Presently, limited knowledge about its substrate recognition is available. Here, various potential substrates were investigated by different antibody based techniques including fluorescence polarization immunoassays and immunoblotting. Regarding both Thr and Tyr kinase activity, only protein substrates were found to be phosphorylated by Myt1, whereas any tested peptide was not recognized. In silico molecular dynamics studies were used to compare the stability of the Myt1 peptide complex with Wee1 peptide complex and support the biochemical findings. Furthermore, a Myt1 kinase binding assay suggests Myt1 being insensitive to staurosporine.     

Structure–activity relationship study of the plasma membrane translocating potential of a short peptide from HIV-1 Tat protein

Summary Tat, a 86-amino acid protein involved in the replication of Human Immunodeficiency Virus type 1 (HIV-1), is able to translocate efficiently through the plasma membrane and to reach the nucleus to transactivate the viral genome. The region 37–72 of the Tat protein, centered on a cluster of basic amino acids, has been assigned to this translocation activity. Recent data in our group have attributed this membrane translocating activity to a peptide extending from residues 48 to 60, which contains a cluster of eight basic amino acids within a linear sequence of nine residues. Internalization of this peptide into cells occurred within minutes at concentrations as low as 100 nM. In order to define more precisely the involvement of these basic amino acids in peptide translocation, several analogues carrying deletions or substitutions of one, or several, of the basic residues were synthesized and tested for their cellular uptake and nuclear translocation. A direct correlation between the overall charge of the peptide and its cell internalization was found. In addition, the covalent linkage of this short basic peptide allows the efficient translocation of a non-membrane permeant peptide.     

Human follicular gonadotropin releasing peptide analogs. Evaluation of biological (in vitro) and immunological activity

Human follicular gonadotropin releasing peptide (hF-GRP) has been shown to stimulate pituitary LH and FSH secretion in vitro. Six hF-GRP analogs have been synthesized and evaluated for gonadotropin releasing activity in a rat anterior pituitary primary cell culture system. A tyrosine analog of hF-GRP, [Tyr4]-hF-GRP, retained comparable biological activity in releasing gonadotropins. However, acetylation of hF-GRP in Ac-hF-GRP greatly reduced the in vitro activity. The shorter segments of hF-GRP, hF-GRP-(5-14), and hF-GRP-(10-14), were tested for LH and FSH releasing activity, and it was found that the decapeptide retained moderate activity while the activity of the pentapeptide was markedly lower than hF-GRP. The baboon alpha 1 antitrypsin-(27-40) peptide, b-alpha 1 AT-(27-40), is relatively less potent in releasing LH than hF-GRP. Interestingly, the baboon peptide is more potent (2.5-fold) in releasing FSH under identical conditions. The effect of hF-GRP in releasing LH and FSH was not affected by the presence of LHRH antagonists in cell culture systems. When these peptides were tested for immunological activity in a hF-GRP radioimmunoassay, it was found that hF-GRP and [Tyr4]-hF-GRP have comparable activities. The C-terminal decapeptide of hF-GRP is more active (1.5-fold) in the RIA, and the C-terminal pentapeptide had only one third of the immunoreactivity. The b-alpha 1-AT-(27-40) failed to cross-react in the RIA even at a concentration of 20 micrograms per tube.     

Amino acids and peptides. Part 39: a bivalent poly(ethylene glycol) hybrid containing an active site (RGD) and its synergistic site (PHSRN) of fibronectin

Fibronectin contains the active sequence Arg-Gly-Asp (RGD), along with its synergic site Pro-His-Ser-Arg-Asn (PHSRN). However, the PHSRN peptide does not show synergic activity when it is mixed with the RGD peptide, indicating that a spatial array between RGD and PHSRN in fibronectin may be necessary for synergic activity. Here, we have used an amino acid type poly(ethylene glycol) derivative (aaPEG) to design a bivalent PEG hybrid of fibronectin active peptides. We prepared the aaPEG hybrid peptides PHSRN-aaPEG, aaPEG-RGD, and PHSRN-aaPEG-RGD, and tested their biological activity. Whereas aaPEG-RGD promoted cell spreading activity, PHSRN-aaPEG had no activity. The PHSRN-aaPEG-RGD hybrid strongly promoted cell spreading compared with aaPEG-RGD. These results suggest that the PHSRN sequence in the PHSRN-aaPEG-RGD molecule synergistically enhances the cell spreading activity of the RGD sequence, and that the bivalent aaPEG hybrid method may be useful for conjugating functionally active peptides.     

A cell-permeable peptide inhibits activation of PKR and enhances cell proliferation

The double-stranded RNA dependent protein kinase (PKR) is a negative regulator of cell proliferation and thus itself a target for modulation. We show that a cell-permeable peptide (PRI), containing a conserved double-stranded RNA binding motif found in PKR, inhibits activation of the kinase and activity to phosphorylate its substrate. Further, the PRI-peptide localizes to the cytoplasm of murine embryonic fibroblasts and ablates cellular PKR activation. The PRI-peptide enhances cell proliferation compared to treatment with a variant control peptide, resulting in cultures with increased cell density. We conclude that peptides that interfere with PKR may be useful tools for regulating cell proliferation.     

Antiviral activity and conformational features of an octapeptide derived from the membrane-proximal ectodomain of the feline immunodeficiency virus transmembrane glycoprotein

Feline immunodeficiency virus (FIV) provides a valuable animal model by which criteria for lentivirus control strategies can be tested. Previous studies have shown that a 20-mer synthetic peptide of the membrane-proximal ectodomain of FIV transmembrane glycoprotein, designated peptide 59, potently inhibited the growth of tissue culture-adapted FIV in feline fibroblastoid CrFK cells. In the present report we describe the potential of this peptide to inhibit the replication of primary FIV isolates in lymphoid cells. Because antiviral activity of peptide 59 was found to map to a short segment containing three conserved Trp residues, further analyses focused on a derivative of eight amino acids ((770)W-I(777)), designated C8. Peptide C8 activity was found to be dependent on conservation of the Trp motif, to be removed from solution by FIV absorbed onto substrate cells, and to be blocked by a peptide derived from the N-terminal portion of FIV transmembrane glycoprotein. Structural studies showed that peptide C8 possesses a conformational propensity highly uncommon for peptides of its size, which may account for its considerable antiviral potency in spite of small size.     

Identification of a novel recognition sequence for the integrin alpha 4 beta 1 in the COOH-terminal heparin-binding domain of fibronectin.

The type III connecting segment of fibronectin contains two cell binding sites, represented by the peptides CS1 and CS5, that are recognized by the integrin receptor alpha 4 beta 1. Using assays measuring the spreading of A375-SM human melanoma cells, we now report that the adhesion promoting activity of a 29 kDa protease fragment of fibronectin containing the COOH-terminal heparin-binding domain (HepII), but lacking CS1 and CS5, is completely sensitive to anti-alpha 4 and anti-beta 1 antibodies, suggesting that HepII contains a third alpha 4 beta 1-binding sequence. Examination of the primary structure of HepII revealed a sequence with homology to CS1. A 19mer peptide spanning this region (designated H1) was found to support cell spreading to the same level as the 29 kDa fragment. H1-dependent adhesion was completely sensitive to anti-alpha 4 and anti-beta 1 antibodies. When soluble peptides were tested for their ability to block cell spreading on the 29 kDa fragment, a 13mer peptide comprising the central core of H1 was found to be completely inhibitory. The active region of H1 was localized to the pentapeptide IDAPS, which is homologous to LDVPS from the active site of CS1. Taken together, these results identify a novel peptide sequence in the HepII region of fibronectin that supports alpha 4 beta 1-dependent cell adhesion.