Interaction of Lp(a) with Plasminogen Binding Sites on Cells

1995 ◽  
Vol 73 (03) ◽  
pp. 458-465 ◽  
Author(s):  
Lindsey A Miles ◽  
Gunther M Fless ◽  
Angelo M Scanu ◽  
Patricia Baynham ◽  
Matthew T Sebald ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Binding Sites ◽  
Surface Proteins ◽  
Time Dependent ◽  
Temperature Sensitive ◽  
U937 Cells ◽  
Cell Surface Proteins ◽  
Sensitive Characteristics ◽  
Lipoprotein Binding

SummaryLp(a) competes with plasminogen for binding to cells but it is not known whether this competition is due to the ability of Lp(a) to interact directly with plasminogen receptors. In the present study, we demonstrate that Lp(a) can interact directly with plasminogen binding sites on monocytoid U937 cells and endothelial cells. The interaction of Lp(a) with these sites was time dependent, specific, saturable, divalent ion independent and temperature sensitive, characteristics of plasminogen binding to these sites. The affinity of plasminogen and Lp(a) for these sites also was similar (Kd = 1-3 μM), but Lp(a) bound to fewer sites (̴10-fold less). Both gangliosides and cell surface proteins with car- boxy-terminal lysyl residues, including enolase, a candidate plasminogen receptor, inhibited Lp(a) binding to U937 cells. Additionally, Lp(a) interacted with low affinity lipoprotein binding sites on these cells which also recognized LDL and HDL. The ability of Lp(a) to interact with sites on cells that recognize plasminogen may contribute to the pathogenetic consequences of high levels of circulating Lp(a).

scholarly journals Interaction of single-chain urokinase with its receptor induces the appearance and disappearance of binding epitopes within the resultant complex for other cell surface proteins

Blood ◽  
1996 ◽  
Vol 88 (2) ◽  
pp. 542-551 ◽  
Author(s):  
AA Higazi ◽  
RH Upson ◽  
RL Cohen ◽  
J Manuppello ◽  
J Bognacki ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cell Surface ◽  
Binding Sites ◽  
Ldl Receptor ◽  
Cell Types ◽  
Surface Proteins ◽  
Single Chain ◽  
Cell Surface Proteins ◽  
Functional Changes ◽  
And Migration

Binding of urokinase-type plasminogen activator (uPA) to its glycosylphosphatidylinositol-anchored receptor (uPAR) initiates signal transduction, adhesion, and migration in certain cell types. To determine whether some of these activities may be mediated by associations between the uPA/uPAR complex and other cell surface proteins, we studied the binding of complexes composed of recombinant, soluble uPA receptor (suPAR) and single chain uPA (scuPA) to a cell line (LM-TK- fibroblasts) that does not express glycosylphosphatidylinositol (GPI)-anchored proteins to eliminate potential competition by endogenous uPA receptors. scuPA induced the binding of suPAR to LM-TK- cells. Binding of labeled suPAR/scuPA was inhibited by unlabeled complex, but not by scuPA or suPAR added separately, indicating cellular binding sites had been formed that are not present in either component. Binding of the complex was inhibited by low molecular weight uPA (LMW-uPA) indicating exposure of an epitope found normally in the isolated B chain of two chain uPA (tcuPA), but hidden in soluble scuPA. Binding of LMW-uPA was independent of its catalytic site and was associated with retention of its enzymatic activity. Additional cell binding epitopes were generated within suPAR itself by the aminoterminal fragment of scuPA, which itself does not bind to LM-TK- cells. When scuPA bound to suPAR, a binding site for alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2 MR/LRP) was lost, while binding sites for cell-associated vitronectin and thrombospondin were induced. In accord with this, the internalization and degradation of cell-associated tcuPA and tcuPA-PAI- 1 complexes proceeded less efficiently in the presence of suPAR. Further, little degradation of suPAR was detected, suggesting that cell- bound complex dissociated during the initial stages of endocytosis. Thus, the interaction of scuPA with its receptor causes multiple functional changes within the complex including the dis-appearance of an epitope in scuPA involved in its clearance from the cell surface and the generation of novel epitopes that promote its binding to proteins involved in cell adhesion and signal transduction.

scholarly journals Identification of Cell-Surface Proteins Endocytosed by Human Brain Microvascular Endothelial Cells In Vitro

Pharmaceutics ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 579 ◽  
Author(s):  
Shingo Ito ◽  
Mariko Oishi ◽  
Seiryo Ogata ◽  
Tatsuki Uemura ◽  
Pierre-Olivier Couraud ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Human Brain ◽  
Targeted Delivery ◽  
Surface Proteins ◽  
Intercellular Adhesion Molecule ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Cell Surface Proteins ◽  
Microvascular Endothelial

Cell-surface proteins that can endocytose into brain microvascular endothelial cells serve as promising candidates for receptor-mediated transcytosis across the blood–brain barrier (BBB). Here, we comprehensively screened endocytic cell-surface proteins in hCMEC/D3 cells, a model of human brain microvascular endothelial cells, using surface biotinylation methodology and sequential window acquisition of all theoretical fragment-ion spectra-mass spectrometry (SWATH-MS)-based quantitative proteomics. Using this method, we identified 125 endocytic cell-surface proteins from hCMEC/D3 cells. Of these, 34 cell-surface proteins were selectively internalized into human brain microvascular endothelial cells, but not into human umbilical vein endothelial cells (HUVECs), a model of human peripheral microvascular endothelial cells. Two cell-surface proteins, intercellular adhesion molecule-1 (ICAM1) and podocalyxin (PODXL), were identified as BBB-localized endocytic cell-surface proteins in humans, using open mRNA and protein databases. Immunohistochemical evaluation confirmed PODXL expression in the plasma membrane of hCMEC/D3 cells and revealed that anti-PODXL antibody-labeled cell-surface PODXL internalized into hCMEC/D3 cells. Immunohistochemistry further revealed that PODXL is localized at the luminal side of human brain microvessels, supporting its potential suitability for translational applications. In conclusion, our findings highlight novel endocytic cell-surface proteins capable of internalizing into human brain microvascular endothelial cells. ICAM1 or PODXL targeted antibody or ligand-labeled biopharmaceuticals and nanocarriers may provide effective targeted delivery to the brain across the BBB for the treatment of central nervous system (CNS) diseases.

scholarly journals Transmission of pseudorabies virus from immune-masked blood monocytes to endothelial cells

2003 ◽  
Vol 84 (3) ◽  
pp. 629-637 ◽  
Author(s):  
Gerlinde R. Van de Walle ◽  
Herman W. Favoreel ◽  
Hans J. Nauwynck ◽  
Thomas C. Mettenleiter ◽  
Maurice B. Pensaert
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Neutralizing Antibodies ◽  
Pseudorabies Virus ◽  
Surface Proteins ◽  
Blood Monocytes ◽  
Cell Surface Proteins ◽  
Pregnant Uterus ◽  
Specific Antibodies

Pseudorabies virus (PRV) may cause abortion, even in the presence of vaccination-induced immunity. Blood monocytes are essential to transport the virus in these immune animals, including transport to the pregnant uterus. Infected monocytes express viral proteins on their cell surface. Specific antibodies recognize these proteins and should activate antibody-dependent cell lysis. Previous work showed that addition of PRV-specific polyclonal antibodies to PRV-infected monocytes induced internalization of viral cell surface proteins, protecting the cells from efficient antibody-dependent lysis in vitro (immune-masked monocytes). As a first step to reach the pregnant uterus, PRV has to cross the endothelial cell barrier of the maternal blood vessels. The current aim was to investigate in vitro whether immune-masked PRV-infected monocytes can transmit PRV in the presence of virus-neutralizing antibodies via adhesion and fusion of these monocytes with endothelial cells. Porcine blood monocytes, infected with a lacZ-carrying PRV strain, were incubated with PRV-specific antibodies to induce internalization. Then, cells were co-cultivated with endothelial cells for different periods of time. Only PRV-infected monocytes with internalized viral cell surface proteins adhered efficiently to endothelial cells. LacZ transmission to endothelial cells, as a measure for monocyte–endothelial cell fusion, could be detected after co-cultivation from 30 min onwards. Virus transmission was confirmed by the appearance of plaques. Adhesion of immune-masked PRV-infected monocytes to endothelial cells was mediated by cellular adhesion complex CD11b–CD18 and subsequent fusion was mediated by the virus. In conclusion, immune-masked PRV-infected monocytes can adhere and subsequently transmit virus to endothelial cells in the presence of PRV-neutralizing antibodies.

scholarly journals Export of major cell surface proteins is blocked in yeast secretory mutants

1983 ◽  
Vol 96 (2) ◽  
pp. 541-547 ◽  
Author(s):  
P Novick ◽  
R Schekman
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Secretory Pathway ◽  
Early Stage ◽  
Surface Proteins ◽  
Temperature Sensitive ◽  
Major Protein ◽  
Permissive Temperature ◽  
Trinitrobenzenesulfonic Acid ◽  
Cell Surface Proteins

The transport of newly synthesized proteins to the yeast cell surface has been analyzed by a modification of the technique developed by Kaplan et al. (Kaplan, G., C. Unkeless, and Z.A. Cohn, 1979, Proc. Natl. Acad. Sci. USA, 76:3824-3828). Cells metabolically labeled with (35)SO(4)(2-) are treated with trinitrobenzenesulfonic acid (TNBS) at 0 degrees C under conditions where cell-surface proteins are tagged with trinitrophenol (TNP) but cytoplasmic proteins are not. After fractionation of cells into cell wall, membrane and cytoplasmic samples, and solubilization with SDS, the tagged proteins are immunoprecipitated with anti-TNP antibody and fixed staphylococcus aureus cells. Analysis of the precipitates by SDS gel electrophoresis and fluorography reveals four major protein species in the cell wall (S(1)-S(4)), seven species in the membrane fraction (M(1)-M(7)), and no tagged proteins in the cytoplasmic fraction. Temperature-sensitive mutants defective in secretion of invertase and acid phosphatase (sec mutants; Novick, P., C. Field, and R. Schekman, 1980, Cell, 21:204-215) are also defective in transport of the 11 major cell surface proteins at the nonpermissive temperature (37 degrees C). Export of accumulated proteins is restored in an energy- dependent fashion when secl cells are returned to a permissive temperature (24 degrees C). In wild-type cells the transit time for different surface proteins varies from less than 8 min to about 30 min. The asynchrony is developed at an early stage in the secretory pathway. All of the major cell wall proteins and many of the externally exposed plasma membrane proteins bind to concanavalin A. Inhibition of asparagine-linked glycosylation with tunicamycin does not prevent transport of several surface proteins.

What do nanometer molecular trajectories tell us about heterogeneous cell surface domaines?

1995 ◽  
Vol 53 ◽  
pp. 786-787
Author(s):  
Watt W. Webb
Keyword(s):  
Cell Surface ◽  
Lipid Membranes ◽  
Surface Proteins ◽  
Protein Diffusion ◽  
Coated Pits ◽  
Cell Surface Proteins ◽  
Membrane Heterogeneity ◽  
Fluorescence Photobleaching Recovery ◽  
Photobleaching Recovery ◽  
Plasma Membrane Heterogeneity

Plasma membrane heterogeneity is implicit in the existence of specialized cell surface organelles which are necessary for cellular function; coated pits, post and pre-synaptic terminals, microvillae, caveolae, tight junctions, focal contacts and endothelial polarization are examples. The persistence of these discrete molecular aggregates depends on localized restraint of the constituent molecules within specific domaines in the cell surface by strong intermolecular bonds and/or anchorage to extended cytoskeleton. The observed plasticity of many of organelles and the dynamical modulation of domaines induced by cellular signaling evidence evanescent intermolecular interactions even in conspicuous aggregates. There is also strong evidence that universal restraints on the mobility of cell surface proteins persist virtually everywhere in cell surfaces, not only in the discrete organelles. Diffusion of cell surface proteins is slowed by several orders of magnitude relative to corresponding protein diffusion coefficients in isolated lipid membranes as has been determined by various ensemble average methods of measurement such as fluorescence photobleaching recovery(FPR).

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