heparitin-sulfate and arginyl-glycyl-aspartic-acid

Coxsackievirus A9 (CVA9) is an enterically transmitted enterovirus and one of the most pathogenic type among human enteroviruses. CVA9 isolates use a distinctive RGD (Arg-Gly-Asp) motif within VP1 capsid protein that defines its ability to bind to integrin receptor(s) for cellular entry. To investigate CVA9 evolution and pathogenicity, genetic relationships and recombination events were analyzed between 54 novel clinical isolates of CVA9, as well as 21 previously published full length CVA9 sequences from GenBank. Samples were investigated by partial sequencing of the novel VP1 and 3Dpol genes, as well as including the corresponding areas from GenBank sequences. Phylogenetic analyses were combined with clinical data in a further attempt to analyze whether sequence evolution reflects CVA9 pathogenicity in the phylogenies. Furthermore, VP1 gene was also analyzed for receptor binding sites including the RGD motif and the putative heparan sulfate (HS) site. Analysis of the 559-nucleotide-long VP1 sequences identified six clades. Although most of the strains within each clade showed geographical clustering, the grouping pattern of the isolates in the analysis of the VP1 gene was strikingly different from grouping of 3Dpol, which suggests that recombination events may have occurred in the region encoding the nonstructural proteins. Inclusion of clinical data did not provide any evidence of symptom based phylogenetic clustering of CVA9 isolates. Amino acid sequence analysis of the VP1 polypeptide demonstrated that the RGD motif was fully conserved among the isolates while the putative HS binding site was only found in one isolate. These data suggest that integrin binding is essential for virus tropism, but do not explain the symptom repertoire.

Topics: Amino Acid Motifs; Binding Sites; Capsid Proteins; Conserved Sequence; Coxsackievirus Infections; DNA-Directed RNA Polymerases; Enterovirus B, Human; Evolution, Molecular; Heparitin Sulfate; Humans; Oligopeptides; Phylogeny; Protein Binding; Receptors, Virus; Recombination, Genetic

Some cell-adapted strains of foot-and-mouth disease virus (FMDV) can utilize heparan sulfate (HS) as a receptor to facilitate viral infection in cultured cells. A number of independent sites on the capsid that might be involved in FMDV-HS interaction have been studied. However, the previously reported residues do not adequately explain HS-dependent infection of two cell-adapted PanAsia-1 strains (O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc) of FMDV serotype O. To identify the molecular determinant(s) for the interaction of O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc with HS receptor, several chimeric viruses and site-directed mutants were generated by using an infectious cDNA of a non-HS-utilizing rescued virus (Cathay topotype) as the genomic backbone. Phenotypic properties of these viruses were determined by plaque assays and virus adsorption and penetration assays in cultured cells.. Only two of the rescued viruses encoding VP0 of O/Tibet/CHA/6/99tc or VP1 of O/Fujian/CHA/9/99tc formed plaques on wild-type Chinese hamster ovary (WT-CHO; HS+) cells, but not on HS-negative pgsD-677 cells. The formation of plaques by these two chimeric viruses on WT-CHO cells could be abolished by the introduction of single amino acid mutations Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc and Lys-1083 → Glu in VP1 of O/Fujian/CHA/9/99tc, respectively. Nonetheless, the introduced mutation Leu-2080 → Gln in VP2 of O/Fujian/CHA/9/99tc for the construction of expectant recombinant plasmid led to non-infectious progeny virus in baby hamster kidney 21 (BHK-21) cells, and the site-directed mutant encoding Glu-1083 → Lys in VP1 of O/Tibet/CHA/6/99tc did not acquire the ability to produce plaques on WT-CHO cells. Significant differences in the inhibition of the infectivity of four HS-utilizing viruses by heparin and RGD-containing peptide were observed in BHK-21 cells. Interestingly, the chimeric virus encoding VP0 of O/Fujian/CHA/9/99tc, and the site-directed mutant encoding Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc could bind to HS, but there was no expression of the 3A protein of these two viruses in WT-CHO cells.. The results suggest that the cooperation of certain specific amino acid residues in the capsid proteins of these two cell-adapted PanAsia-1 strains is essential for viral infectivity, the heparin affinity and the capability on FMDV-HS interaction.

Topics: Amino Acid Substitution; Animals; Capsid Proteins; Cell Line; CHO Cells; Codon; Cricetinae; Cricetulus; Foot-and-Mouth Disease Virus; Heparin; Heparitin Sulfate; Models, Molecular; Oligopeptides; Open Reading Frames; Protein Binding; Protein Conformation; Receptors, Virus; Serogroup; Viral Plaque Assay; Virus Attachment; Virus Replication

Retargeting oncolytic adenoviruses from their systemic preeminent liver tropism to disseminated tumor foci would highly improve the efficacy of these agents at eradicating tumors. We have replaced the KKTK fiber shaft heparan sulfate glycosaminoglycan-binding domain with an RGDK motif in order to achieve simultaneously liver detargeting and tumor targeting. When inserted into a wild-type backbone, this mutation palliated liver transaminase elevation and hematological alterations in mice. Importantly, when tested in a backbone that redirects E1A transcription towards pRB pathway deregulation, RGD at this novel shaft location also improved significantly systemic antitumor therapy compared with the broadly used RGD location at the HI-loop of the fiber knob domain.

Topics: Adenoviridae; Animals; Binding Sites; Cell Line, Tumor; Gene Transfer Techniques; Genetic Vectors; Heparitin Sulfate; Mice; Mice, Inbred BALB C; Neoplasms; Oligopeptides; Oncolytic Virotherapy; Receptors, Cell Surface

Tissue transglutaminase (TG2) is a multifunctional Ca(2+)-activated protein cross-linking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease, and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a nontransamidating mechanism via its association with fibronectin, heparan sulfates (HS), and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modeling and mutagenesis, we have identified the HS-binding site of TG2 (202)KFLKNAGRDCSRRSSPVYVGR(222). We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS-binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate for the RGD-induced loss of cell adhesion on fibronectin via binding to syndecan-4, leading to activation of PKCα, pFAK-397, and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed.

Topics: Animals; Antigens, Surface; Catalytic Domain; Cell Adhesion; CHO Cells; Cricetinae; Extracellular Matrix; GTP-Binding Proteins; HEK293 Cells; Heparin; Heparitin Sulfate; Humans; Mice; Models, Molecular; Mutagenesis; NIH 3T3 Cells; Oligopeptides; Protein Glutamine gamma Glutamyltransferase 2; Protein Structure, Quaternary; Protein Structure, Tertiary; Protein Transport; Signal Transduction; Syndecan-4; Transglutaminases

Human adenovirus serotype 5 (HAdV5)-based vectors administered intravenously accumulate in the liver as the result of their direct binding to blood coagulation factor X (FX) and subsequent interaction of the FX-HAdV5 complex with heparan sulfate proteoglycan (HSPG) at the surface of liver cells. Intriguingly, the serotype 35 fiber-pseudotyped vector HAdV5F35 has liver transduction efficiencies 4-logs lower than HAdV5, even though both vectors carry the same hexon capsomeres. In order to reconcile this apparent paradox, we investigated the possible role of other viral capsid proteins on the FX/HSPG-mediated cellular uptake of HAdV5-based vectors. Using CAR- and CD46-negative CHO cells varying in HSPG expression, we confirmed that FX bound to serotype 5 hexon protein and to HAdV5 and HAdV5F35 virions via its Gla-domain, and enhanced the binding of both vectors to surface-immobilized hypersulfated heparin and cellular HSPG. Using penton mutants, we found that the positive effect of FX on HAdV5 binding to HSPG and cell transduction did not depend on the penton base RGD and fiber shaft KKTK motifs. However, we found that FX had no enhancing effect on the HAdV5F35-mediated cell transduction, but a negative effect which did not involve the cell attachment or endocytic step, but the intracellular trafficking and nuclear import of the FX-HAdV5F35 complex. By cellular imaging, HAdV5F35 particles were observed to accumulate in the late endosomal compartment, and were released in significant amounts into the extracellular medium via exocytosis. We showed that the stability of serotype 5 hexon:FX interaction was higher at low pH compared to neutral pH, which could account for the retention of FX-HAdV5F35 complexes in the late endosomes. Our results suggested that, despite the high affinity interaction of hexon capsomeres to FX and cell surface HSPG, the adenoviral fiber acted as the dominant determinant of the internalization and trafficking pathway of HAdV5-based vectors.

Topics: Adenoviruses, Human; Amino Acid Motifs; Animals; Capsid Proteins; Cell Adhesion; Cell Compartmentation; Cell Membrane; CHO Cells; Cricetinae; Cricetulus; Endocytosis; Factor X; Green Fluorescent Proteins; HEK293 Cells; Heparitin Sulfate; Humans; Intracellular Space; Mutation; Oligopeptides; Protein Binding; Protein Structure, Tertiary; Serotyping; Surface Plasmon Resonance; Transduction, Genetic; Virion; Virus Internalization

Fibronectin (FN) is a major component of the extracellular matrix and functions in cell adhesion, cell spreading and cell migration. In the retina, FN is transiently expressed and assembled on astrocytes (ACs), which guide sprouting tip cells and deposit a provisional matrix for sprouting angiogenesis. The precise function of FN in retinal angiogenesis is largely unknown. Using genetic tools, we show that astrocytes are the major source of cellular FN during angiogenesis in the mouse retina. Deletion of astrocytic FN reduces radial endothelial migration during vascular plexus formation in a gene dose-dependent manner. This effect correlates with reduced VEGF receptor 2 and PI3K/AKT signalling, and can be mimicked by selectively inhibiting VEGF-A binding to FN through intraocular injection of blocking peptides. By contrast, AC-specific replacement of the integrin-binding RGD sequence with FN-RGE or endothelial deletion of itga5 shows little effect on migration and PI3K/AKT signalling, but impairs filopodial alignment along AC processes, suggesting that FN-integrin α5β1 interaction is involved in filopodial adhesion to the astrocytic matrix. AC FN shares its VEGF-binding function and cell-surface distribution with heparan-sulfate (HS), and genetic deletion of both FN and HS together greatly enhances the migration defect, indicating a synergistic function of FN and HS in VEGF binding. We propose that in vivo the VEGF-binding properties of FN and HS promote directional tip cell migration, whereas FN integrin-binding functions to support filopodia adhesion to the astrocytic migration template.

Topics: Animals; Astrocytes; Cell Movement; Extracellular Matrix; Fibronectins; Heparitin Sulfate; Integrin alpha5beta1; Integrins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Neovascularization, Physiologic; Oligopeptides; Phosphatidylinositol 3-Kinases; Protein Interaction Domains and Motifs; Proto-Oncogene Proteins c-akt; Retinal Vessels; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Fibronectin (FN) deposition mediated by fibroblasts is an important process in matrix remodeling and wound healing. By monitoring the deposition of soluble biotinylated FN, we show that the stress-induced TG-FN matrix, a matrix complex of tissue transglutaminase (TG2) with its high affinity binding partner FN, can increase both exogenous and cellular FN deposition and also restore it when cell adhesion is interrupted via the presence of RGD-containing peptides. This mechanism does not require the transamidase activity of TG2 but is activated through an RGD-independent adhesion process requiring a heterocomplex of TG2 and FN and is mediated by a syndecan-4 and β1 integrin co-signaling pathway. By using α5 null cells, β1 integrin functional blocking antibody, and a α5β1 integrin targeting peptide A5-1, we demonstrate that the α5 and β1 integrins are essential for TG-FN to compensate RGD-induced loss of cell adhesion and FN deposition. The importance of syndecan-2 in this process was shown using targeting siRNAs, which abolished the compensation effect of TG-FN on the RGD-induced loss of cell adhesion, resulting in disruption of actin skeleton formation and FN deposition. Unlike syndecan-4, syndecan-2 does not interact directly with TG2 but acts as a downstream effector in regulating actin cytoskeleton organization through the ROCK pathway. We demonstrate that PKCα is likely to be the important link between syndecan-4 and syndecan-2 signaling and that TG2 is the functional component of the TG-FN heterocomplex in mediating cell adhesion via its direct interaction with heparan sulfate chains.

Topics: Actins; Animals; Cell Adhesion; CHO Cells; Cricetinae; Cricetulus; Cytoskeleton; Extracellular Matrix; Fibronectins; GTP-Binding Proteins; Heparitin Sulfate; Humans; Integrin alpha5beta1; Mice; Mice, Mutant Strains; Oligopeptides; Protein Glutamine gamma Glutamyltransferase 2; Protein Kinase C-alpha; rho-Associated Kinases; RNA, Small Interfering; Signal Transduction; Syndecan-2; Syndecan-4; Transglutaminases

Coxsackievirus and adenovirus receptor (CAR), alphav integrins, and heparan sulfate glycosaminoglycans (HSGs) are the tropism determinants of adenoviral (Ad) vectors in vivo. For the development of a targeted Ad vector, its broad tropism needs to be blocked (or reduced). We have previously developed Ad vectors with ablation of CAR, alphav integrin, and HSG binding by mutation of the FG loop in the fiber knob (deletion of T489, A490, Y491, and T492 of the fiber protein), deletion of the RGD motif of the penton base, and substitution of the fiber shaft domain for that derived from Ad type 35, respectively, and have shown that this triple-mutant Ad vector [Ad/deltaF(FG)deltaP-S35-L2] exhibits significantly lower transduction in mouse liver compared with the conventional Ad vector [Koizumi, N., Mizuguchi, H., Sakurai, F., Yamaguchi, T., Watanabe, Y., and Hayakawa, T. (2003). J. Virol. 77, 13062-13072]. In the present study, we optimized the fiber knob mutation for further reduced in vivo transduction and examined toxicity of the modified Ad vectors. Ad/deltaF(AB)deltaPS35- L2, a triple-mutant Ad vector containing a mutation of the AB loop in the fiber knob (R412S, A415G, E416G, and K417G), mediated approximately 15,000- and 500-fold lower mouse liver transduction by intravenous and intraperitoneal administration, respectively, than the conventional Ad vector, and mediated 10- fold lower mouse liver transduction than did Ad/deltaF(FG)deltaP-S35-L2. Ad/deltaF(AB)deltaP-S35-L2 also exhibited lower transduction of other organs compared with Ad/deltaF(FG)deltaP-S35-L2 and the conventional Ad vector. Levels of both liver serum enzymes (aspartate transferase [AST] and alanine transferase (ALT)] and interleukin (IL)-6 in mouse serum after intravenous administration of Ad/deltaF(AB)deltaP-S35-L2 were similar to those in the nontreatment mouse serum, whereas the conventional Ad vector led to high levels of AST, ALT, and IL-6. We therefore succeeded in further improving the mutant Ad vector, abolishing both viral natural tropism and toxicity. This new Ad vector appears to be a fundamental vector for targeted gene delivery.

Topics: Adenoviruses, Human; Alanine Transaminase; Animals; Aspartate Aminotransferases; Binding Sites; Coxsackie and Adenovirus Receptor-Like Membrane Protein; Gene Transfer Techniques; Genetic Vectors; Heparitin Sulfate; Humans; Integrin alphaV; Interleukin-6; Liver; Male; Mice; Mice, Inbred C57BL; Oligopeptides; Receptors, Virus; Sequence Deletion; Transduction, Genetic

Adaptation of field isolates of foot-and-mouth disease virus (FMDV) to grow in cells in culture can result in changes in viral properties that include acquisition of the ability to bind to cell surface heparan sulfate (HS). After 13 passages on BHK cells to produce a vaccine, a Cathay topotype isolate of FMDV serotype O from China (O/CHA/90) extended its cell culture host range and bound to heparin-Sepharose, although it did not require cell surface HS as a receptor molecule. To understand these phenomena, we constructed chimeric viruses by using a type A(12) infectious cDNA and the capsid protein-coding regions of O/CHA/90 and its cell culture-adapted derivative (vac-O/CHA/90). Using a set of viruses derived from these chimeras by exchanging portions of the capsid-coding regions, we discovered that a group of amino acid residues that surround the fivefold axis of the icosahedral virion determine host range in cell culture and influence pathogenicity in pigs. These residues included aromatic amino acids at positions 108 and 174 and positively charged residues at positions 83 and 172 in protein 1D. To test if these residues participated in non-integrin-dependent cell binding, the integrin-binding RGD sequence in protein 1D was changed to KGE in two different chimeras. Evaluation of these KGE viruses indicated that growth in cell culture was not dependent on HS. One of these viruses was tested in pigs, where it produced a mild disease and maintained its KGE sequence. These results are discussed in terms of receptor utilization and pathogenesis of this important pathogen.

Topics: Amino Acid Sequence; Animals; Binding Sites; Capsid Proteins; Cell Line; China; Cricetinae; Foot-and-Mouth Disease; Foot-and-Mouth Disease Virus; Genetic Engineering; Heparitin Sulfate; Models, Molecular; Molecular Sequence Data; Oligopeptides; Recombinant Fusion Proteins; Sepharose; Swine; Swine Diseases; Virion

Cell surface molecules that can act as virus receptors may exert an important selective pressure on RNA viral quasispecies. Large population passages of foot-and-mouth disease virus (FMDV) in cell culture select for mutant viruses that render dispensable a highly conserved Arg-Gly-Asp (RGD) motif responsible for integrin receptor recognition. Here, we provide evidence that viability of recombinant FMDVs including a Asp-143-->Gly change at the RGD motif was conditioned by a number of capsid substitutions selected upon FMDV evolution in cell culture. Multiply passaged FMDVs acquired the ability to infect human K-562 cells, which do not express integrin alpha(v)beta(3). In contrast to previously described cell culture-adapted FMDVs, the RGD-independent infection did not require binding to the surface glycosaminoglycan heparan sulfate (HS). Viruses which do not bind HS and lack the RGD integrin-binding motif replicate efficiently in BHK-21 cells. Interestingly, FMDV mutants selected from the quasispecies for the inability to bind heparin regained sensitivity to inhibition by a synthetic peptide that represents the G-H loop of VP1. Thus, a single amino acid replacement leading to loss of HS recognition can shift preferential receptor usage of FMDV from HS to integrin. These results indicate at least three different mechanisms for cell recognition by FMDV and suggest a potential for this virus to use multiple, alternative receptors for entry even into the same cell type.

Topics: Amino Acid Sequence; Animals; Aphthovirus; Binding Sites; Capsid; Capsid Proteins; Cell Line; CHO Cells; Cricetinae; Heparitin Sulfate; Humans; K562 Cells; Molecular Sequence Data; Oligopeptides; Receptors, Virus; Receptors, Vitronectin; Recombination, Genetic; Virus Replication

Lipoprotein lipase (LPL) is made by several cell types, including macrophages within the atherosclerotic lesion. LPL, a dimer of identical subunits, has high affinity for heparin and cell surface heparan sulfate proteoglycans (HSPGs). Several studies have shown that cell surface HSPGs can mediate cell binding to adhesion proteins. Here, we tested whether LPL, by virtue of its HSPG binding could mediate monocyte adhesion to surfaces. Monocyte binding to LPL-coated (1-25 micrograms/mL) tissue culture plates was 1.4- to 7-fold higher than that of albumin-treated plastic. Up to 3-fold more monocytes bound to the subendothelial matrix that had been pretreated with LPL. LPL also doubled the number of monocytes that bound to endothelial cells (ECs). Heparinase and heparitinase treatment of monocytes or incubation of monocytes with heparin decreased monocyte binding to LPL. Heparinase/heparitinase treatment of the matrix also abolished the LPL-mediated increase in monocyte binding. These results suggest that LPL dimers mediate monocyte binding by forming a "bridge" between matrix and monocyte surface HSPGs. Inhibition of LPL activity with tetrahydrolipstatin, a lipase active-site inhibitor, did not affect the LPL-mediated monocyte binding. To assess whether specific oligosaccharide sequences in HSPGs mediated monocyte binding to LPL, competition experiments were performed by using known HSPG binding proteins. Neither antithrombin nor thrombin inhibited monocyte binding to LPL. Next, we tested whether integrins were involved in monocyte binding to LPL. Surprisingly, monocyte binding to LPL-coated plastic and matrix was inhibited by approximately 35% via integrin-binding arginine-glycine-aspartic acid peptides. This result suggests that monocyte binding to LPL was mediated, in part, by monocyte cell surface integrins. In summary, our data show that LPL, which is present on ECs and in the subendothelial matrix, can augment monocyte adherence. This increase in monocyte-matrix interaction could promote macrophage accumulation within arteries.

Topics: Animals; Cattle; Cell Adhesion; Cell Adhesion Molecules; Heparan Sulfate Proteoglycans; Heparin; Heparin Lyase; Heparitin Sulfate; Integrins; Lipoprotein Lipase; Milk; Monocytes; Oligopeptides; Polysaccharide-Lyases; Proteoglycans

Cytotactin/tenascin is a multidomain extracellular matrix protein that inhibits both cell spreading and intracellular alkalinization. The protein has multiple different domains which are homologous to regions in epidermal growth factor, fibronectin, and fibrinogen. In previous studies, we produced nonoverlapping fusion proteins corresponding to these domains and examined their effects on cell attachment and spreading. Based on their ability either to promote or to inhibit cell attachment, two of these fusion proteins were shown to be adhesive and two were shown to be counteradhesive. To determine how the adhesive and counteradhesive activities of different cytotactin/tenascin domains alter intracellular pH (designated pHi), we have measured pHi, in NIH3T3 and U251MG cells in the presence of the cytotactin/tenascin fusion proteins and intact cytotactin/tenascin, as well as fibronectin. Cells incubated in the presence of intact cytotactin/tenascin or of the counteradhesive fusion proteins had a pHi lower than control cells. In contrast, the presence of the adhesive fusion proteins or of fibronectin caused cells to have higher pHi values than control cells. When two fragments were simultaneously presented, one of which alone increased pHi and the other of which alone decreased pHi, the predominant effect was that of lowered pHi. Incubation with an RGD-containing peptide derived from the cytotactin/tenascin sequence inhibited alkalinization promoted by the adhesive fragment containing the second through sixth fibronectin type III repeats that was known to bind to integrins. Incubation of the cells with heparinase I or III inhibited the intracellular alkalinization of cells plated in the presence of the other adhesive fusion protein containing the fibrinogen domain, suggesting that heparan sulfate proteoglycans were involved in these pHi changes. The activity of protein kinase C appeared to be important for the changes in pHi mediated by all of the proteins. The protein kinase C inhibitor Calphostin C blocked the rise in pHi elicited by the adhesive fusion proteins and by fibronectin. Moreover, activation of protein kinase C by the addition of phorbol esters increased the pHi in cells plated on cytotactin/tenascin or counteradhesive fusion proteins and reversed their effects. The results of this study support the hypothesis that cytotactin/tenascin can bind to multiple cell surface receptors and thereby elicit different physiological responses. Decreases in pH

Topics: 3T3 Cells; Animals; Cell Adhesion; Cell Adhesion Molecules, Neuronal; Cell Size; Extracellular Matrix Proteins; Heparitin Sulfate; Hydrogen-Ion Concentration; In Vitro Techniques; Mice; Oligopeptides; Protein Kinase C; Recombinant Fusion Proteins; Signal Transduction; Structure-Activity Relationship; Tenascin

We have previously demonstrated that chemically modified thrombin preparations induce endothelial cell (EC) adhesion, spreading and cytoskeletal reorganization via an Arg-Gly-Asp (RGD) sequence and the alpha v beta 3 integrin. Native thrombin, however, did not exhibit adhesive properties, consistent with crystal structure analysis, showing that Gly-Asp residues of the RGD epitope are buried within the molecule. We have now identified a possible physiological mean of converting thrombin to an adhesive protein. Plasmin, the major end product of the fibrinolytic system, converted thrombin to an adhesive protein for EC in a time and dose-dependent manner. EC adhesion and spreading was also induced by a low molecular weight (approximately 3,000 D) cleavage fragment generated upon incubation of thrombin with plasmin. Cell adhesion mediated by this fragment was completely inhibited by the synthetic peptide GRGDSP. Conversion of thrombin to an adhesive molecule was significantly enhanced in the presence of heparin or heparan sulfate, while other glycosaminoglycans (GAGs) (e.g., dermatan sulfate, keratan sulfate, chondroitin sulfate) had no effect. The role of cell surface heparan sulfate in thrombin conversion to EC adhesive protein was investigated using CHO cell mutants defective in various aspects of GAG synthesis. Incubation of both thrombin and a suboptimal amount of plasmin on the surface of formaldehyde fixed wild-type CHO-KI cells resulted in an efficient conversion of thrombin to an adhesive molecule, as indicated by subsequent induction of EC attachment. In contrast, there was no effect to incubation of thrombin and plasmin with fixed CHO mutant cells lacking both heparan sulfate and chondroitin sulfate, or with cells expressing no heparan sulfate and a three-fold increase in chondroitin sulfate. A similar gain of adhesive properties was obtained upon incubation of thrombin and plasmin in contact with native, but not heparinase-treated extracellular matrix (ECM) produced by cultured ECs. It appears that cell surface and ECM-associated heparan sulfate modulate thrombin adhesive properties through its heparin binding site in a manner that enables suboptimal amounts of plasmin to expose the RGD domain. Our results demonstrate, for the first time, a significant modulation of thrombin molecule by heparin, resulting in its conversion to a potent adhesive protein for ECs. This conversion is most effective in contact with cell surfaces, basement membranes and E

Topics: Animals; Cattle; Cell Adhesion Molecules; Cells, Cultured; Cricetinae; Endothelium; Extracellular Matrix; Fibrinolysin; Glycosaminoglycans; Heparitin Sulfate; Oligopeptides; Protein Conformation; Thrombin

Aortic endothelial cells adhere to the core protein of murine perlecan, a heparan sulfate proteoglycan present in endothelial basement membrane. We found that cell adhesion was partially inhibited by beta 1 integrin-specific mAb and almost completely blocked by a mixture of beta 1 and alpha v beta 3 antibodies. Furthermore, adhesion was partially inhibited by a synthetic peptide containing the perlecan domain III sequence LPASFRGDKVTSY (c-RGD) as well as by GRGDSP, but not by GRGESP. Both antibodies contributed to the inhibition of cell adhesion to immobilized c-RGD whereas only beta 1-specific antibody blocked residual cell adhesion to proteoglycan core in the presence of maximally inhibiting concentrations of soluble RGD peptide. A fraction of endothelial surface-labeled detergent lysate bound to a core affinity column and 147-, 116-, and 85-kD proteins were eluted with NaCl and EDTA. Polyclonal anti-beta 1 and anti-beta 3 integrin antibodies immunoprecipitated 116/147 and 85/147 kD surface-labeled complexes, respectively. Cell adhesion to perlecan was low compared to perlecan core, and cell adhesion to core, but not to immobilized c-RGD, was selectively inhibited by soluble heparin and heparan sulfates. This inhibition by heparin was also observed with laminin and fibronectin and, in the case of perlecan, was found to be independent of heparin binding to substrate. These data support the hypothesis that endothelial cells interact with the core protein of perlecan through beta 1 and beta 3 integrins, that this binding is partially RGD-independent, and that this interaction is selectively sensitive to a cell-mediated effect of heparin/heparan sulfates which may act as regulatory ligands.

Topics: Actinin; Amino Acid Sequence; Animals; Antibodies; Basement Membrane; Cattle; Cell Adhesion; Cells, Cultured; Endothelium, Vascular; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Integrin beta1; Integrin beta3; Integrins; Mice; Molecular Sequence Data; Oligopeptides; Peptide Fragments; Proteoglycans

We have examined the molecular interactions of avian neural crest cells with fibronectin and laminin in vitro during their initial migration from the neural tube. A 105-kDa proteolytic fragment of fibronectin encompassing the defined cell-binding domain (65 kDa) promoted migration of neural crest cells to the same extent as the intact molecule. Neural crest cell migration on both intact fibronectin and the 105-kDa fragment was reversibly inhibited by RGD-containing peptides. The 11.5-kDa fragment containing the RGDS cell attachment site was also able to support migration, whereas a 50-kDa fragment corresponding to the adjacent N-terminal portion of the defined cell-binding domain was unfavorable for neural crest cell movement. In addition to the putative "cell-binding domain," neural crest cells were able to migrate on a 31-kDa fragment corresponding to the C-terminal heparin-binding (II) region of fibronectin, and were inhibited in their migration by exogenous heparin, but not by RGDS peptides. Heparin potentiated the inhibitory effect of RGDS peptides on intact fibronectin, but not on the 105-kDa fragment. On substrates of purified laminin, the extent of avian neural crest cell migration was maximal at relatively low substrate concentrations and was reduced at higher concentrations. The efficiency of laminin as a migratory substrate was enhanced when the glycoprotein occurred complexed with nidogen. Moreover, coupling of the laminin-nidogen complex to collagen type IV or the low density heparan sulfate proteoglycan further increased cell dispersion, whereas isolated nidogen or the proteoglycan alone were unable to stimulate migration and collagen type IV was a significantly less efficient migratory substrate than laminin-nidogen. Neural crest cell migration on laminin-nidogen was not affected by RGDS nor by YIGSR-containing peptides, but was reduced by 35% after addition of heparin. The predominant motility-promoting activity of laminin was localized to the E8 domain, possessing heparin-binding activity distinct from that of the N-terminal E3 domain. Migration on the E8 fragment was reduced by greater than 70% after addition of heparin. The E1' fragment supported a minimal degree of migration that was RGD-sensitive and heparin-insensitive, whereas the primary heparin-binding E3 fragment and the cell-adhesive P1 fragment were entirely nonpermissive for cell movement.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Binding Sites; Cell Movement; Cells, Cultured; Chondroitin Sulfate Proteoglycans; Collagen; Fibronectins; Heparan Sulfate Proteoglycans; Heparin; Heparitin Sulfate; Laminin; Membrane Glycoproteins; Membrane Proteins; Neural Crest; Oligopeptides; Peptide Fragments; Platelet Factor 4; Quail