Midkine, a cytokine that inhibits HIV infection by binding to the cell surface expressed nucleolin

The growth factor midkine （MK） is a cytokine that inhibits HIV infection in cell cultures in an autocrine and paracrine manner by blocking the attachment of HIV particles to permissive cells. MK mRNA is systematically expressed in adult peripheral blood lymphocytes from healthy donors, while its expression becomes markedly but transiently increased upon in vitro treatment of lymphocytes with IL-2 or IFN-7 and activation of T lymphocytes by PHA or through the engagement of the CD28 antigen. The binding of MK to cells occurs specifically at a high and a low affinity binding site. This low affinity-binding site is the cell-surface expressed nucleolin, which is implicated in the mechanism of the initial attachment of HIV particles to cells. Accordingly, the nucleolin-binding HB-19 pseudopeptide has no effect on the MK binding to the high affinity binding site, whereas it prevents the binding of MK to the low affinity binding site, thus suggesting the low affinity receptor of MK is the cell-surface-expressed nucleolin. Confocal immunofluorescence laser microscopy revealed the colocalization of MK and the cell-surface-expressed nucleolin at distinct spots. The use of various deletion constructs of nucleolin then indicates that the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif RGG, as the domain that binds MK. The specific binding of MK to the surface nucleolin is independent of heparan sulfate and chondroitin sulfate proteoglycans. After binding to cells, MK enters cells by an active process in which nucleolin and lipid rafts appear to be implicated. The potent and the distinct anti-HIV action of MK along with its enhanced expression in lymphocytes by various physiological stimuli, point out that MK is a cytokine that could be involved in HIV pathogenesis.     

The anti-HIV pseudopeptide HB-19 forms a complex with the cell-surface-expressed nucleolin independent of heparan sulfate proteoglycans.

The HB-19 pseudopeptide 5[Kpsi(CH(2)N)PR]-TASP, psi(CH(2)N) for reduced peptide bond, is a specific inhibitor of human immunodeficiency virus (HIV) infection in different CD4(+) cell lines and in primary T-lymphocytes and macrophages. Here, by using an experimental CD4(+) cell model to monitor HIV entry and infection, we demonstrate that HB-19 binds the cell surface and inhibits attachment of HIV particles to permissive cells. At concentrations that inhibit HIV attachment, HB-19 binds cells irreversibly, becomes complexed with the cell-surface-expressed nucleolin, and eventually results in its degradation. Accordingly, by confocal immunofluorescence microscopy, we demonstrate the drastic reduction of the cell-surface-expressed nucleolin following treatment of cells with HB-19. HIV particles can prevent the binding of HB-19 to cells and inhibit complex formation with nucleolin. Such a competition between viral particles and HB-19 is consistent with the implication of nucleolin in the process of HIV attachment to target cells. We show that another inhibitor of HIV infection, the fibroblast growth factor-2 (FGF-2) that uses cell-surface-expressed heparan sulfate proteoglycans as low affinity receptors, binds cells and blocks attachment of HIV to permissive cells. FGF-2 does not prevent the binding of HB-19 to cells and to nucleolin, and similarly HB-19 has no apparent effect on the binding of FGF-2 to the cell surface. The lack of competition between these two anti-HIV agents rules out the potential involvement of heparan sulfate proteoglycans in the mechanism of anti-HIV effect of HB-19, thus pointing out that nucleolin is its main target.     

Pleiotrophin inhibits HIV infection by binding the cell surface-expressed nucleolin

The growth factor pleiotrophin (PTN) has been reported to bind heparan sulfate and nucleolin, two components of the cell surface implicated in the attachment of HIV-1 particles to cells. Here we show that PTN inhibits HIV-1 infection by its capacity to inhibit HIV-1 particle attachment to the surface of permissive cells. The beta-sheet domains of PTN appear to be implicated in this inhibitory effect on the HIV infection, in particular the domain containing amino acids 60-110. PTN binding to the cell surface is mediated by high and low affinity binding sites. Other inhibitors of HIV attachment known to bind specifically surface expressed nucleolin, such as the pseudopeptide HB-19 and the cytokine midkine prevent the binding of PTN to its low affinity-binding site. Confocal immunofluorescence laser microscopy revealed that the cross-linking of surface-bound PTN with a specific antibody results in the clustering of cell surface-expressed nucleolin and the colocalization of both PTN and nucleolin signals. Following its binding to surface-nucleolin, PTN is internalized by a temperature sensitive mechanism, a process which is inhibited by HB-19 and is independent of heparan and chondroitin sulfate proteoglycans. Nevertheless, proteoglycans might play a role in the concentration of PTN on the cell surface for a more efficient interaction with nucleolin. Our results demonstrate for the first time that PTN inhibits HIV infection and suggest that the cell surface-expressed nucleolin is a low affinity receptor for PTN binding to cells and it is also implicated in PTN entry into cells by an active process.     

The anti-HIV pentameric pseudopeptide HB-19 binds the C-terminal end of nucleolin and prevents anchorage of virus particles in the plasma membrane of target cells

The multivalent pseudopeptide HB-19 that binds the cell-surface-expressed nucleolin is a potent inhibitor of human immunodeficiency virus (HIV) infection by blocking virus particle attachment and thus anchorage in the plasma membrane. We show that cross-linking of surface-bound HB-19A (like HB-19 but with a modified template) results in aggregation of HB-19A with surface nucleolin. Consistent with its specific action, HB-19A binding to different types of cells reaches saturation at concentrations that have been reported to result in inhibition of HIV infection. By using Chinese hamster ovary mutant cell lines, we confirm that the binding of HB-19A to surface nucleolin is independent of heparan and chondroitin sulfate proteoglycans. In vitro generated full-length nucleolin was found to bind HB-19A, whereas the N-terminal part containing the acidic amino acid stretches of nucleolin did not. The use of various deletion constructs of the C-terminal part of nucleolin then permitted the identification of the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif, RGG, as the domain that binds HB-19A. Finally, a synthetic peptide corresponding to the last C-terminal 63 amino acids was able to inhibit HIV infection at the stage of HIV attachment to cells, thus suggesting that this domain could be functional in the HIV anchorage process.     

Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cells.

Lactoferrin (Lf), a multifunctional molecule present in mammalian secretions and blood, plays important roles in host defense and cancer. Indeed, Lf has been reported to inhibit the proliferation of cancerous mammary gland epithelial cells and manifest a potent antiviral activity against human immunodeficiency virus and human cytomegalovirus. The Lf-binding sites on the cell surface appear to be proteoglycans and other as yet undefined protein(s). Here, we isolated a Lf-binding 105 kDa molecular mass protein from cell extracts and identified it as human nucleolin. Medium-affinity interactions ( approximately 240 nm) between Lf and purified nucleolin were further illustrated by surface plasmon resonance assays. The interaction of Lf with the cell surface-expressed nucleolin was then demonstrated through competitive binding studies between Lf and the anti-human immunodeficiency virus pseudopeptide, HB-19, which binds specifically surface-expressed nucleolin independently of proteoglycans. Interestingly, binding competition studies between HB-19 and various Lf derivatives in proteoglycan-deficient hamster cells suggested that the nucleolin-binding site is located in both the N- and C-terminal lobes of Lf, whereas the basic N-terminal region is dispensable. On intact cells, Lf co-localizes with surface nucleolin and together they become internalized through vesicles of the recycling/degradation pathway by an active process. Morever, a small proportion of Lf appears to translocate in the nucleus of cells. Finally, the observations that endocytosis of Lf is inhibited by the HB-19 pseudopeptide, and the lack of Lf endocytosis in proteoglycan-deficient cells despite Lf binding, point out that both nucleolin and proteoglycans are implicated in the mechanism of Lf endocytosis.     

Cell surface nucleolin on developing muscle is a potential ligand for the axonal receptor protein tyrosine phosphatase-sigma

Reversible tyrosine phosphorylation, catalyzed by receptor tyrosine kinases and receptor tyrosine phosphatases, plays an essential part in cell signaling during axonal development. Receptor protein tyrosine phosphatase-sigma has been implicated in the growth, guidance and repair of retinal axons. This phosphatase has also been implicated in motor axon growth and innervation. Insect orthologs of receptor protein tyrosine phosphatase-sigma are also implicated in the recognition of muscle target cells. A potential extracellular ligand for vertebrate receptor protein tyrosine phosphatase-sigma has been previously localized in developing skeletal muscle. The identity of this muscle ligand is currently unknown, but it appears to be unrelated to the heparan sulfate ligands of receptor protein tyrosine phosphatase-sigma. In this study, we have used affinity chromatography and tandem MS to identify nucleolin as a binding partner for receptor protein tyrosine phosphatase-sigma in skeletal muscle tissue. Nucleolin, both from tissue lysates and in purified form, binds to receptor protein tyrosine phosphatase-sigma ectodomains. Its expression pattern also overlaps with that of the receptor protein tyrosine phosphatase-sigma-binding partner previously localized in muscle, and nucleolin can also be found in retinal basement membranes. We demonstrate that a significant amount of muscle-associated nucleolin is present on the cell surface of developing myotubes, and that two nucleolin-binding components, lactoferrin and the HB-19 peptide, can block the interaction of receptor protein tyrosine phosphatase-sigma ectodomains with muscle and retinal basement membranes in tissue sections. These data suggest that muscle cell surface-associated nucleolin represents at least part of the muscle binding site for axonal receptor protein tyrosine phosphatase-sigma and that nucleolin may also be a necessary component of basement membrane binding sites of receptor protein tyrosine phosphatase-sigma.     

Identification of a 110-kDa nonintegrin cell surface laminin-binding protein which recognizes an A chain neurite-promoting peptide.

Laminin is a potent promoter of neurite outgrowth, and a synthetic peptide of 19 amino acids, PA22-2, from the A chain has been found to promote process formation. Using peptide affinity chromatography, we have identified a 110-kDa, cell surface ligand from both neural cells and brain which binds this sequence. This binding protein does not share immunological identity with the B1 chain of integrin, and reduction does not alter its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibody to the 110-kDa protein stained cellular processes in vivo. Sequence analysis of the first 18 amino acids from the amino terminus yielded almost exact sequence identity with nucleolin, a major 110-kDa nucleolar phosphoprotein. Antibody to nucleolin, however, does not interact with the neural-derived, laminin-peptide-binding 110-kDa protein. The 110-kDa protein appears to be a ligand for a specific site on laminin.     

Midkine and pleiotrophin: two related proteins involved in development,survival, inflammation and tumorigenesis

Midkine (MK) and pleiotrophin (PTN) are low molecular weight proteins with closely related structures. They are mainly composed of two domains held by disulfide bridges, and there are three antiparallel beta-sheets in each domain. MK and PTN promote the growth, survival, and migration of various cells, and play roles in neurogenesis and epithelial mesenchymal interactions during organogenesis. A chondroitin sulfate proteoglycan, protein-tyrosine phosphatase zeta (PTPzeta), is a receptor for MK and PTN. The downstream signaling system includes ERK and PI3 kinase. MK binds to the chondroitin sulfate portion of PTPzeta with high affinity. Among the various chondroitin sulfate structures, the E unit, which has 4,6-disulfated N-acetylgalactosamine, provides the strongest binding site. The expression of MK and PTN is increased in various human tumors, making them promising as tumor markers and as targets for tumor therapy. MK and PTN expression also increases upon ischemic injury. MK enhances the migration of inflammatory cells, and is involved in neointima formation and renal injury following ischemia. MK is also interesting from the viewpoints of the treatment of neurodegenerative diseases, increasing the efficiency of in vitro development, and the prevention of HIV infection.     

High and low affinity binding sites for basic fibroblast growth factor on cultured cells: absence of a role for low affinity binding in the stimulation of plasminogen activator production by bovine capillary endothelial cells

Scatchard analysis of binding of 125I-basic fibroblast growth factor (FGF) to baby hamster kidney (BHK) cells revealed the presence of two binding sites: a high affinity site with KD of 20 pM and 80,000 sites per cell and a low affinity site with KD of about 2 nM and 600,000 sites per cell. The binding to the two sites could be separated by first washing the cells with 2 M NaCl at pH 7.5 which released the low affinity binding and then extracting the cells with 0.5% Triton X-100 to recover the 125I-basic FGF bound to high affinity sites. The binding to the high affinity site was acid sensitive, suggesting that it represented binding to the receptor. Binding to the low affinity site could be competed strongly by heparin and less strongly by heparan sulfate but not by chondroitin sulfate, dermatan sulfate, or keratan sulfate. Treatment of BHK cells with heparinase abolished 62% of the low affinity binding, suggesting that the low affinity binding represented binding to cell-associated, heparin-like molecules. A variety of other cell types, including bovine capillary endothelial (BCE) cells, also demonstrated both low and high affinity binding sites. To test whether the low affinity binding might play a role in the basic FGF stimulation of plasminogen activator (PA) production by BCE cells, heparin was added to BCE cultures at concentrations which totally blocked binding of 125I-basic FGF to the low affinity sites. Addition of the heparin did not diminish the increased PA production induced by basic FGF. This suggests that the low affinity binding has no direct role in the stimulation of PA production in BCE cells.     

Cell-surface nucleolin is a signal transducing P-selectin binding protein for human colon carcinoma cells

We have previously shown that P-selectin binding to Colo-320 human colon carcinoma cells induces specific activation of the alpha(5)beta(1) integrin with a concomitant increase of cell adhesion and spreading on fibronectin substrates in a phosphatidylinositol 3-kinase (PI3-K) and p38 MAPK-dependent manner. Here, we identified by affinity chromatography and characterized nucleolin as a P-selectin receptor on Colo-320 cells. Nucleolin mAb D3 significantly decreases the Colo-320 cell adhesion to immobilized P-selectin-IgG-Fc. Moreover, nucleolin becomes clustered at the external side of the plasma membrane of living, intact cells when bound to cross-linked P-selectin-IgG-Fc chimeric protein. We have also found P-selectin binding to Colo-320 cells induces tyrosine phosphorylation specifically of cell-surface nucleolin and formation of a signaling complex containing cell-surface nucleolin, PI3-K and p38 MAPK. Using siRNA approaches, we have found that both P-selectin binding to Colo-320 cells and formation of the P-selectin-mediated p38 MAPK/PI3-K signaling complex require nucleolin expression. These results show that nucleolin (or a nucleolin-like protein) is a signaling receptor for P-selectin on Colo-320 cells and suggest a mechanism for linkage of nucleolin to P-selectin-induced signal transduction pathways that regulate the adhesion and the spreading of Colo-320 on fibronectin substrates.     

Characterization of the receptor for keratinocyte growth factor. Evidence for multiple fibroblast growth factor receptors

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. KGF exhibits potent mitogenic activity for a variety of epithelial cell types but is distinct from other known FGFs in that it is not mitogenic for fibroblasts or endothelial cells. We report saturable specific binding of 125I-KGF to surface receptors on intact Balb/MK mouse epidermal keratinocytes. 125I-KGF binding was completed efficiently by acidic FGF (aFGF) but with 20-fold lower efficiency by basic FGF (bFGF). The pattern of 125I-acidic FGF binding and competition on Balb/MK keratinocytes and NIH/3T3 fibroblasts suggests that these cell types possess related but distinct FGF receptors. Scatchard analysis of 125I-KGF binding suggested major and minor high affinity receptor components (KD = 400 and 25 pM, respectively) as well as a third high capacity/low affinity heparin-like component. Covalent affinity cross-linking of 125I-KGF to its receptor on Balb/MK cells revealed two species of 115 and 140 kDa. KGF also stimulated the rapid tyrosine phosphorylation of a 90-kDa protein in Balb/MK cells but not in NIH/3T3 fibroblasts. Together these results indicate that Balb/MK keratinocytes possess high affinity KGF receptors to which the FGFs may also bind. However, these receptors are distinct from the receptor(s) for aFGF and bFGF on NIH/3T3 fibroblasts, which fail to interact with KGF.     

Distribution of ecto-5'-nucleotidase on subsets of human T and B lymphocytes as detected by indirect immunofluorescence using goat antibodies

Human T and B lymphocyte subsets were characterized for ecto-5'-nucleotidase (ecto-5'-NT) expression by two-color immunofluorescence by using polyclonal goat antibodies to 5'-NT and murine monoclonal antibodies to T and B cell subsets. Anti-5'-NT antibodies were prepared by immunizing a goat with purified human placental 5'-NT. Lymphocyte surface 5'-NT was detected with F(ab')2 fragments of immune goat IgG followed by biotinylated F(ab')2 rabbit anti-goat IgG and fluorescein isothiocyanate-avidin. Lymphocyte cell surface antigens were detected with phycoerythrin (PE)-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD16, and anti-CD19. HB-4, an antigen present on a major subset of human peripheral blood B cells, was detected with murine monoclonal anti-HB-4 and PE-anti-mouse-kappa. Analysis showed that ecto-5'-NT was expressed on 32 +/- 7% of CD3+, 19 +/- 6% of CD4+, and 50 +/- 21% of CD8+ T cells, but not on CD16+ lymphocytes. Ecto-5'-NT was also expressed on 81 +/- 8% of adult peripheral blood B cells as defined by PE-anti-CD19; HB-4 was expressed on 84 +/- 7% of CD19+ cells. The two populations of B cells were not identical, however, because HB-4 was co-expressed on only 79 +/- 18% of ecto-5'-NT+ B cells. Two-color immunofluorescent staining of T cells from a patient with congenital agammaglobulinemia and low T cell ecto-5'-NT activity revealed reduced percentages of ecto-5'-NT+ cells in his CD3+, CD4+, and CD8+ populations. Thus, reduced ecto-5'-NT activity by enzyme assay was paralleled by reduced numbers of 5'-NT molecules on the cell surface. Two-color immunofluorescent staining of B cells from a patient with hypogammaglobulinemia and low B cell ecto-5'-NT activity also revealed markedly reduced expression of 5'-NT. HB-4 expression was normal, however, suggesting that the patient's B cells were blocked in maturation subsequent to the acquisition of HB-4 but prior to that of ecto-5'-NT. These results demonstrate that anti-5'-NT antibodies will be valuable tools for analyzing ecto-5'-NT expression and lymphocyte maturation in patients with immuno-deficiency diseases.     

Anti-viral activity of human recombinant heparin-binding proteins HBNF and MK.

Herpes simplex viruses bind to cell surface heparan sulfate proteoglycans, as a first step of viral infection. We report here that two recombinant heparin-binding proteins HBNF and MK inhibit infectivity of human herpes simplex viruses types 1 and 2 and human cytomegalovirus. Carboxymethylated HBNF and MK, which retain affinity for heparin-Sepharose, do not exhibit anti-viral activities. Arguments are presented that anti-viral effects of HBNF and MK are due to the competition for the specific binding to the cell surface heparan sulfate proteoglycans.     

Intracellular redistribution of nucleolin upon interaction with the CD3epsilon chain of the T cell receptor complex

T cell activation through the antigen receptor (TCR) involves the cytoplasmic tails of the CD3 subunits CD3gamma, CD3delta, CD3epsilon, and CD3zeta. Whereas the biological significance of the cytoplasmic tails of these molecules is suggested, in part, by their evolutionarily conserved sequences, their interactions with signal transduction molecules are not completely understood. We used affinity chromatography columns of glutathione S-transferase fused to the CD3epsilon cytoplasmic tail to isolate proteins that specifically interact with this subunit. In this way, we identified the shuttling protein nucleolin as a specific CD3epsilon-interacting molecule. Using competition studies and affinity chromatography on peptide columns, we were able to identify a central proline-rich sequence as the nucleolin-interacting sequence in CD3epsilon. Transfection in COS cells of wild type CD3epsilon, but not of nonbinding mutants of CD3epsilon, resulted in redistribution of nucleolin from the nucleus and nucleoli to the cytoplasm. This property was transferred to a CD8 protein chimera by appending the cytoplasmic tail of CD3epsilon. We also found that nucleolin associated with the TCR complex. This association was increased upon TCR engagement, suggesting that the CD3epsilon/nucleolin interaction may have a role in T cell activation.