Significance of heparin binding to basic residues in homologous to the amino terminus of hepatoma-derived growth factor and related proteins

Hepatoma-derived growth factor (HDGF) recognizes cell surface heparan sulfate to promote its internalization though binding to its N-terminal HATH (homologous to amino terminus of HDGF) domain. HDGF-related proteins (HRPs) all have the HATH domain in their N terminus. In this study, we report on the commonality of heparin binding in all HRPs with a broad range of heparin-binding affinity: HRP-4 is the strongest binder, and the lens epithelium-derived growth factor shows a relatively weak binding, with binding affinities (K(D)) showing 30-fold difference in magnitude. With the HDGF HATH domain used as a model, residue K19 was the most critical basic residue in molecular recognition and protein internalization, and with its proximal proline-tryptophan-tryptophan-proline motif, coordinated a conformational change when binding to the heparin fragment. Other basic residues, K21, K61, K70, K72 and R79, confer added contribution in binding that the total ionic interaction from these residues represents more than 70% of the binding energy. Because the positive-charged residues are conserved in all HRP HATH domains, heparin binding outside of cells might be of equal importance for all HRPs in mediating downstream signaling; however, distinct effects and/or distribution might be associated with the varying affinities to heparin.     

Hepatoma-derived growth factor. Significance of amino acid residues 81-100 in cell surface interaction and proliferative activity

Hepatoma-derived growth factor (HDGF) has proliferative, angiogenic, and neurotrophic activity. It plays a putative role in the development and progression of cancer. When expressed in cells, the mitogenic activity of HDGF depends on its nuclear localization, but it also stimulates proliferation when added to the cell culture medium. A cell surface receptor for HDGF has not been identified so far. We investigated the interaction of various purified recombinant HDGF fusion proteins with the cell surface of NIH 3T3 fibroblasts. We showed that binding of a HDGF-beta-galactosidase fusion protein to the cell surface of NIH 3T3 fibroblasts was saturable, occurred with high affinity (K(D) = 14 nm), and had a proliferative effect. We identified a peptide comprising amino acid residues 81-100 within the amino-terminal part of HDGF that bound to the cell surface of NIH 3T3 cells with saturation and affinity values similar to those of HDGF. When added to primary human fibroblasts, this peptide stimulated proliferation. Substitution of a single amino acid (K96A) within this peptide was sufficient to abolish its binding to the cell surface and its proliferative activity. In contrast, when expressed transiently in NIH 3T3 cells, a HDGF-beta-galactosidase fusion protein in which amino acid residues 81-100 were deleted still had proliferative activity, whereas a fusion protein containing only the 81-100 peptide did not. Our results suggest the existence of a plasma membrane-located HDGF receptor for which signaling depends on amino acid residues 81-100 of HDGF. This region differs from the one that has been recently identified to be essential for mitogenic activity depending on the nuclear localization of HDGF. Thus, HDGF exerts its proliferative activity via two different pathways.     

N- and 6-O-sulfated heparan sulfates mediate internalization of coxsackievirus B3 variant PD into CHO-K1 cells

Recently, it was demonstrated that the coxsackievirus B3 variant PD (CVB3 PD) is able to infect coxsackievirus-adenovirus receptor (CAR)-lacking cells by using heparan sulfates (HS) as additional receptors (A. E. Zautner, U. Korner, A. Henke, C. Badorff, and M. Schmidtke, J. Virol. 77:10071-10077, 2003). For this study, competition experiments with growth factors binding to known HS sequences as well as with specifically desulfated heparins were performed with Chinese hamster ovary cells (CHO-K1) to determine the structural requirements of HS for interaction with CVB3. Hepatocyte growth factor interacting with HS sequences containing [IdUA-GlcNSO(3)(6OSO(3))](n), but not basic fibroblast growth factor binding to [HexUA-GlcNSO(3)-HexUA-GlcNSO(3)-IdUA(2OSO(3))](n), was shown to compete effectively with CVB3 PD for cell surface HS. Whereas unmodified heparin and 2-O-desulfated heparin strongly inhibited the CVB3 PD-induced cytopathic effect, the antiviral activity was markedly reduced after N-, O- and 6-O-desulfation of heparin. Taken together, these results indicate that 6-O- and N-sulfation of GlcNAc of HS is crucial for HS interaction with CVB3 PD and that the disaccharide [IdUA-GlcNSO(3)(6OSO(3))](n) is involved in viral binding. Results from experiments with various inhibitors of endocytic pathways suggest that HS-mediated virus internalization is pH dependent. Despite the fact that CVB3 PD initiates infection about four times slower by making use of HS as a receptor than by using CAR, the time required for a complete viral life cycle in Chinese hamster ovary cells was independent of the utilized receptor.     

Heparin-binding EGF-like growth factor stimulation of smooth muscle cell migration: dependence on interactions with cell surface heparan sulfate

Heparin-binding EGF-like growth factor (HB-EGF), but not EGF, binds to cell surface heparan sulfate proteoglycan (HSPG). This was demonstrated in (a) the binding of 125I-HB-EGF to mutant CHO cells deficient in HS production was diminished by 70% compared to wild-type CHO cells, (b) the binding of 125I-HB-EGF to CHO cells and bovine aortic smooth muscle cells (BASMC) was diminished 80% by heparitinase or chlorate treatment, and (c) 125I-EGF did not bind to CHO cells and its binding to BASMC was not diminished at all by heparitinase and only slightly by chlorate treatment. Accordingly, the role of HB-EGF interactions with HSPG in modulating bioactivity was examined. Heparitinase or chlorate treatment of BASMC diminished the ability of HB-EGF to stimulate BASMC migration by 60-80%. A similar inhibition of migration occurred when BASMC were treated with a synthetic peptide (P21) corresponding to the sequence of the putative heparin-binding domain of HB-EGF. As a control for BASMC viability, and for specificity, it was found that heparitinase and P21 did not inhibit at all and chlorate inhibited only slightly the stimulation of BASMC migration by PDGF AB. Since heparitinase, chlorate, and P21 treatment also diminished by 70-80% the cross-linking of 125I-HB-EGF to the EGF receptor, it was concluded that the interaction of HB-EGF, via its heparin-binding domain, with cell surface HSPG was essential for its optimal binding to the EGF receptor on BASMC and hence for its optimal ability to stimulate migration.     

Hepatoma-derived growth factor stimulates podosome rosettes formation in NIH/3T3 cells through the activation of phosphatidylinositol 3-kinase/Akt pathway

Hepatoma-derived growth factor (HDGF) stimulates the migration, invasion and metastasis in several types of cancer cells. However, the mechanism underlying HDGF-stimulated migration remains unclear. In this study, we investigated the influence of HDGF on cytoskeleton remodeling and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in non-transformed NIH/3T3 cells. Exogenous HDGF promoted the migration and the formation of dorsal ruffles and podosome rosettes. Besides, HDGF supply increased the PI3K expression and Akt phosphorylation in dose- and time-dependent manners. Application of LY294002, a PI3K inhibitor, attenuated the HDGF-induced migration, dorsal ruffles and podosome rosettes formation. Consistently, the HDGF-overexpressing NIH/3T3 transfectants exhibited significantly increased motility and elevated PI3K/Akt activities, which were repressed by LY294002 or adenovirus-mediated overexpression of endogenous PI3K antagonist, PTEN. In summary, HDGF elicits the activation of PI3K/Akt signaling cascade, thereby promoting cytoskeleton remodeling to stimulate cellular migration.     

Integrin-dependent and -independent functions of astrocytic fibronectin in retinal angiogenesis

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.     

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.     

Beyond attachment: Roles of DC‐SIGN in dengue virus infection

Dendritic cell‐specific intercellular adhesion molecule‐3‐grabbing non‐integrin (DC‐SIGN), a C‐type lectin expressed on the plasma membrane by human immature dendritic cells, is a receptor for numerous viruses including Ebola, SARS and dengue. A controversial question has been whether DC‐SIGN functions as a complete receptor for both binding and internalization of dengue virus (DENV) or whether it is solely a cell surface attachment factor, requiring either hand‐off to another receptor or a co‐receptor for internalization. To examine this question, we used 4 cell types: human immature dendritic cells and NIH3T3 cells expressing either wild‐type DC‐SIGN or 2 internalization‐deficient DC‐SIGN mutants, in which either the 3 cytoplasmic internalization motifs are silenced by alanine substitutions or the cytoplasmic region is truncated. Using confocal and super‐resolution imaging and high content single particle tracking, we investigated DENV binding, DC‐SIGN surface transport, endocytosis, as well as cell infectivity. DC‐SIGN was found colocalized with DENV inside cells suggesting hand‐off at the plasma membrane to another receptor did not occur. Moreover, all 3 DC‐SIGN molecules on NIH3T3 cells supported cell infection. These results imply the involvement of a co‐receptor because cells expressing the internalization‐deficient mutants could still be infected.      

Surface-exposed amino acid residues of HPV16 L1 protein mediating interaction with cell surface heparan sulfate

Efficient infection of cells by human papillomaviruses (HPVs) and pseudovirions requires primary interaction with cell surface proteoglycans with apparent preference for species carrying heparan sulfate (HS) side chains. To identify residues contributing to virus/cell interaction, we performed point mutational analysis of the HPV16 major capsid protein, L1, targeting surface-exposed amino acid residues. Replacement of lysine residues 278, 356, or 361 for alanine reduced cell binding and infectivity of pseudovirions. Various combinations of these amino acid exchanges further decreased cell attachment and infectivity with residual infectivity of less than 5% for the triple mutant, suggesting that these lysine residues cooperate in HS binding. Single, double, or triple exchanges for arginine did not impair infectivity, demonstrating that interaction is dependent on charge distribution rather than sequence-specific. The lysine residues are located within a pocket on the capsomere surface, which was previously proposed as the putative receptor binding site. Fab fragments of binding-neutralizing antibody H16.56E that recognize an epitope directly adjacent to lysine residues strongly reduced HS-mediated cell binding, further corroborating our findings. In contrast, mutation of basic surface residues located in the cleft between capsomeres outside this pocket did not significantly reduce interaction with HS or resulted in assembly-deficient proteins. Computer-simulated heparin docking suggested that all three lysine residues can form hydrogen bonds with 2-O-, 6-O-, and N-sulfate groups of a single HS molecule with a minimal saccharide domain length of eight monomer units. This prediction was experimentally confirmed in binding experiments using capsid protein, heparin molecules of defined length, and sulfate group modifications.     

Roles of the heparin and low density lipid receptor-related protein-binding sites of protease nexin 1 (PN1) in urokinase-PN1 complex catabolism. The PN1 heparin-binding site mediates complex retention and degradation but not cell surface binding or internalization

We have previously described thrombin (Th)-protease nexin 1 (PN1) inhibitory complex binding to cell surface heparins and subsequent low density lipid receptor-related protein (LRP)-mediated internalization. Our present studies examine the catabolism of urinary plasminogen activator (uPA)-PN1 inhibitory complexes, which, unlike Th.PN1 complexes, bind almost exclusively through the uPA receptor. In addition, the binding site in PN1 required for the LRP-mediated internalization of Th.PN1 complexes is not required for the LRP-mediated internalization of uPA.PN1 complexes. Thus, the protease moiety of the complex partially determines the mechanistic route of entry. Because cell surface heparins are only minimally involved in the binding and internalization of uPA.PN1 complexes, we then predicted that complexes between uPA and the heparin binding-deficient PN1 variant, PN1(K7E), should be catabolized at the same rate as complexes formed with native PN1. Surprisingly, the uPA.PN1(K7E) complexes were degraded at only a fraction of the rate of native complexes. Internalization studies revealed that both uPA. PN1(K7E) and native uPA.PN1 complexes were initially internalized at the same rate, but uPA.PN1(K7E) complexes were rapidly retro-endocytosed in an intact form. By examining the pH dependence of complex binding in the range of 4.0-7.0, it was determined that the uPA.PN1 inhibitory complexes must specifically bind to endosomal heparins at pH 5.5 to be retained and sorted to lysosomes. These studies are the first to document a role for heparins in the catabolism of SERPIN-protease complexes at a point further in the pathway than cell surface binding, and this role may extend to other heparin-binding LRP-internalized ligands.     

Separate endocytic pathways of kinase-defective and -active EGF receptor mutants expressed in same cells

Ligand binding to the membrane receptor for EGF induces its clustering and internalization. Both receptor and ligand are then degraded by lysosomal enzymes. A kinase defective point mutant (K721A) of EGF receptor undergoes internalization similarly to the wild-type receptor. However, while internalized EGF molecules bound to either the wild-type or mutant receptors are degraded, the K721A mutant receptor molecules recycle to the cell surface for reutilization. To investigate the mechanism of receptor trafficking, we have established transfected NIH-3T3 cells coexpressing the kinase-negative mutant (K721A) together with a mutant EGF receptor (CD63) with active kinase. CD63 was chosen because it behaves like wild-type EGF receptor with respect to biological responsiveness and cellular routing but afforded immunological distinction between kinase active and inactive mutants. Although expressed in the same cells, the two receptor mutants followed their separate endocytic itineraries. Like wild-type receptor, the CD63 mutant was downregulated and degraded in response to EFG while the kinase-negative mutant K721A returned to the cell surface for reutilization. Intracellular trafficking of EGF receptor must be determined by a sorting mechanism that specifically recognizes EGF receptor molecules according to their intrinsic kinase activity.     

Characterization of heparin-binding growth-associated factor receptor on NIH 3T3 cells.

Scatchard plot analysis of the binding of 125I-labeled heparin binding cell growth-associated factor (125I-HBGAF) to NIH 3T3 cells revealed a single class of high affinity receptors (-5000/cell) with kd of -0.6 nM. 125I-HBGAF was covalently cross-linked to the cell surface receptor on NIH 3T3 cells with disuccinimidyl suberate (DSS). Two 125I-HBGAF-cross-linked complexes of 170 kDa and 142 kDa were observed on SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The 125I-HBGAF-cross-linked complex formation was completely abolished in the presence of greater than or equal to 100-fold excess of unlabeled HBGAF but not PDGF, EGF, aFGF, bFGF, or insulin. 125I-HBGAF appeared to undergo rapid internalization and relatively slow degradation following binding to the HBGAF receptor on NIH 3T3 cells. These results suggest that NIH 3T3 cells express a high affinity HBGAF receptor which shows two different estimated molecular masses of -155 kDa and -127 kDa. This high affinity HBGAF receptor was also found to express in other cell types.     

IL-8-mediated cell migration in endothelial cells depends on cathepsin B activity and transactivation of the epidermal growth factor receptor

Microvascular endothelial cells (HMECs) express both the CXCR1 and the CXCR2, but cell migration is almost entirely mediated by the CXCR2. Similarly, NIH 3T3 cells transfected with the CXCR2 migrated toward IL-8, whereas CXCR1-transfected cells failed to do so. This situation differs from that seen in leukocytes, where chemotaxis is primarily a function of the CXCR1. To define signal transduction pathways that explain this difference in behavior, various inhibitors were used to block cell migration. Apart from inhibitors of phosphatidylinositol 3-kinase, which blocked migration in all cases, inhibition of the epidermal growth factor (EGF) receptor blocked IL-8-mediated cell migration in HMECs and in CXCR2-transfected NIH 3T3 cells, but not in RBL2H3 cells, which do not express an EGFR. Blocking Abs against the EGFR or against heparin-binding EGF-like growth factor similarly blocked IL-8-mediated cell migration and in vitro tubulogenesis in HMECs. Furthermore, inhibition of the EGFR also attenuated focus formation in NIH 3T3 expressing the CXCR2. Immunoprecipitations of the EGFR in HMECs and in NIH 3T3 cells expressing the CXCR2 confirmed that the EGFR was phosphorylated following stimulation with IL-8. However, in contrast to previous reports, e.g., for the thrombin receptor, inhibition of matrix metalloproteases blocked IL-8-mediated cell migration only partially, whereas it was ablated by inhibition of cathepsin B. These results indicate that IL-8-induced transactivation of the EGFR is mediated by the CXCR2 and involves cathepsin B, and that this pathway is important for the migratory and tumorigenic effects of IL-8.     

Cell surface retention sequence binding protein-1 interacts with the v-sis gene product and platelet-derived growth factor beta-type receptor in simian sarcoma virus-transformed cells

The cell surface retention sequence (CRS) binding protein-1 (CRSBP-1) is a newly identified membrane glycoprotein which is hypothesized to be responsible for cell surface retention of the oncogene v-sis and c-sis gene products and other secretory proteins containing CRSs. In simian sarcoma virus-transformed NIH 3T3 cells (SSV-NIH 3T3 cells), a fraction of CRSBP-1 was demonstrated at the cell surface and underwent internalization/recycling as revealed by cell surface 125I labeling and its resistance/sensitivity to trypsin digestion. However, the majority of CRSBP-1 was localized in intracellular compartments as evidenced by the resistance of most of the 35S-metabolically labeled CRSBP-1 to trypsin digestion, and by indirect immunofluorescent staining. CRSBP-1 appeared to form complexes with proteolytically processed forms (generated at and/or after the trans-Golgi network) of the v-sis gene product and with a approximately 140-kDa proteolytically cleaved form of the platelet-derived growth factor (PDGF) beta-type receptor, as demonstrated by metabolic labeling and co-immunoprecipitation. CRSBP-1, like the v-sis gene product and PDGF beta-type receptor, underwent rapid turnover which was blocked in the presence of 100 microM suramin. In normal and other transformed NIH 3T3 cells, CRSBP-1 was relatively stable and did not undergo rapid turnover and internalization/recycling at the cell surface. These results suggest that in SSV-NIH 3T3 cells, CRSBP-1 interacts with and forms ternary and binary complexes with the newly synthesized v-sis gene product and PDGF beta-type receptor at the trans-Golgi network and that the stable binary (CRSBP-1.v-sis gene product) complex is transported to the cell surface where it presents the v-sis gene product to unoccupied PDGF beta-type receptors during internalization/recycling.     

Anti-tumor antibody BR96 blocks cell migration and binds to a lysosomal membrane glycoprotein on cell surface microspikes and ruffled membranes

BR 96 is an internalizing antibody that binds to Lewis Y (Le(y)), a carbohydrate determinant expressed at high levels on many human carcinomas (Hellstrom, I., H. J. Garrigues, U. Garrigues, and K. E. Hellstrom. 1990. Cancer Res. 50:2183-2190). Breast carcinoma cell lines grown to confluence bind less BR96 than subconfluent cultures (Garrigues, J., U. Garrigues, I. Hellstrom, and K. E. Hellstrom. 1993. Am. J. Path. 142:607-622). However, when the confluent cells are induced to migrate by scratch wounding, they again bind BR96 suggesting that antigens bearing the Le(y) determinant may promote cell migration. In the present study, BR96 was found to be highly enriched on microspikes and ruffled membranes, cell surface structures involved in cell migration. In addition, BR96 was a potent inhibitor of cell migration in vitro. When stationary BR96 treated cells were exposed to fresh culture media, membrane ruffles and microspikes developed at the cell margin and migration resumed. Immunogold microscopy showed that BR96 antigens were enriched on these membrane protrusions. BR96 cell surface immunoprecipitation analysis of 3H-glucosamine labeled breast carcinoma cells identified antigens with approximate molecular weights of 135 kd (upper antigen) and 85 kd (lower antigen). A short amino terminal sequence (8 residues) of the upper antigen matched that of human lysosomal membrane glycoprotein 1 (LAMP-1). In addition, the upper antigen was detected on immunoblots probed with anti-LAMP-1, and within the intracellular compartment BR96 was found predominantly in endosomes and lysosomes. A soluble LAMP-1/immunoglobulin fusion protein (LAMP-1/Ig) was transiently expressed in both BR96 binding and nonbinding cell lines. Immunoblot analysis of LAMP-1/Ig's from the various cell lines showed that (a) acquisition of the BR96 epitope is probably controlled at the level of polylactosamine modification (e.g., fucosylation) rather than LAMP-1 gene expression; (b) alternate forms of LAMP-1/Ig comigrate with the lower BR96 antigen raising the possibility that it may be a degradation product of the upper antigen; and (c) LAMP-1/Ig expressed in 3396 breast carcinoma cells has approximately 30-fold more BR96 epitopes than LAMP-1/Ig from non- tumorigenic mammary epithelial cells. Together these data indicate that a major BR96 antigen, LAMP-1, is present on unique cell surface domains involved in cell locomotion as well as membranes of the endocytic compartment. Altered glycosylation of LAMP-1 expressed in transformed cells may contribute to their ability to disseminate.     

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.     

The anti-angiogenic His/Pro-rich fragment of histidine-rich glycoprotein binds to endothelial cell heparan sulfate in a Zn2+-dependent manner

The plasma protein histidine-rich glycoprotein (HRGP), which has been identified as an angiogenesis inhibitor, binds to heparan sulfate (HS) in a Zn(2+)-dependent manner. We wished to test whether this interaction is mechanistically important in mediation of the anti-angiogenic effect of HRGP. Inhibition of angiogenesis by HRGP is exerted through its central His/Pro-rich domain, which is proteolytically released. A 35-amino-acid residue synthetic peptide, HRGP330, derived from the His/Pro-rich domain retains the inhibitory effect on blood vessel formation in vitro and in vivo, an effect dependent on the presence of Zn(2+). We now show that HRGP330 binds heparin/HS with the same capacity as full-length HRGP, and the binding is Zn(2+)-dependent. Peptides derived from the His/Pro-rich domain of HRGP downstream of HRGP330 fail to inhibit endothelial cell migration and display a significantly reduced heparin-binding capacity. An even shorter peptide, HRGP335, covering a 26-amino-acid sequence within HRGP330 retains full heparin/HS-binding capacity. Characterization of the HS interaction shows that there is a tissue-specific HS pattern recognized by HRGP335 and that the minimal length of heparin/HS required for binding to HRGP335 is an 8-mer oligosaccharide. Saturation of the HS binding sites in HRGP330 by pre-incubation with heparin abrogates the HRGP330-induced rearrangement of endothelial cell focal adhesions, suggesting that interaction with cell surface HS is needed for HRGP330 to exert its anti-angiogenic effect.     

Electric field-directed fibroblast locomotion involves cell surface molecular reorganization and is calcium independent

Directional cellular locomotion is thought to involve localized intracellular calcium changes and the lateral transport of cell surface molecules. We have examined the roles of both calcium and cell surface glycoprotein redistribution in the directional migration of two murine fibroblastic cell lines, NIH 3T3 and SV101. These cell types exhibit persistent, cathode directed motility when exposed to direct current electric fields. Using time lapse phase contrast microscopy and image analysis, we have determined that electric field-directed locomotion in each cell type is a calcium independent process. Both exhibit cathode directed motility in the absence of extracellular calcium, and electric fields cause no detectable elevations or gradients of cytosolic free calcium. We find evidence suggesting that galvanotaxis in these cells involves the lateral redistribution of plasma membrane glycoproteins. Electric fields cause the lateral migration of plasma membrane concanavalin A receptors toward the cathode in both NIH 3T3 and SV101 fibroblasts. Exposure of directionally migrating cells to Con A inhibits the normal change of cell direction following a reversal of electric field polarity. Additionally, when cells are plated on Con A- coated substrata so that Con A receptors mediate cell-substratum adhesion, cathode-directed locomotion and a cathodal accumulation of Con A receptors are observed. Immunofluorescent labeling of the fibronectin receptor in NIH 3T3 fibroblasts suggests the recruitment of integrins from large clusters to form a more diffuse distribution toward the cathode in field-treated cells. Our results indicate that the mechanism of electric field directed locomotion in NIH 3T3 and SV101 fibroblasts involves the lateral redistribution of plasma membrane glycoproteins involved in cell-substratum adhesion.