scholarly journals Immunotargeting of Nanocrystals by SpyCatcher Conjugation of Engineered Antibodies

2021 ◽  
Author(s):  
Cassio Pedroso ◽  
Victor Mann ◽  
Kathrin Zuberbühler ◽  
Markus-Frederik Bohn ◽  
Jessica Yu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Specific Binding ◽  
Time Lapse ◽  
Live Cell ◽  
Confocal Imaging ◽  
Cellular Targets ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Isopeptide Bonds ◽  
Time Lapse Imaging

Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quanti-tative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibod-ies (Ab) are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrys-tal labeling methods may orient Ab incorrectly, be reversible or denaturing, or lead to Ab-NP complexes too large for some applications. Here, we show that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered Ab to nanoparticle surfaces with control over stability, orientation, and stoichiometry. Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment Ab (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human can-cers. Confocal imaging of anti-uPAR scFv-QD conjugates shows the Ab mediates specific binding and internalization by breast cancer cells expressing uPAR. Time-lapse imaging of photostable scFv-UCNP conjugates show that Ab binding caus-es uPAR internalization with a ∼20-minute half-life on the cell surface, and uPAR is internalized to endolysosomal com-partments distinct from general membrane stains and without significant recycling to the cell surface. The controlled and stable conjugation of engineered Ab to NPs enables targeting of diverse receptors for live-cell study of their distribution, trafficking, and physiology.

scholarly journals Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells – Tools for analyzing dynamics of cell cycle, migration, and lineage selection

Methods ◽  
2018 ◽  
Vol 133 ◽  
pp. 81-90 ◽  
Author(s):  
Katja M. Piltti ◽  
Brian J. Cummings ◽  
Krystal Carta ◽  
Ayla Manughian-Peter ◽  
Colleen L. Worne ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Neural Stem Cells ◽  
Single Cell ◽  
Cell Tracking ◽  
Time Lapse ◽  
Live Cell ◽  
Time Lapse Imaging

Apolipoprotein(a)-Dependent Inhibition of Pericellular Plasminogen Activation Is Mediated by Specific Cellular Receptors

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2236-2236
Author(s):  
Rocco Romagnuolo ◽  
Michael B Boffa ◽  
Marlys L Koschinsky
Keyword(s):  
Cell Surface ◽  
Plasminogen Activator ◽  
Conflicts Of Interest ◽  
Cell Surface Receptor ◽  
Dependent Manner ◽  
Plasminogen Activation ◽  
Apolipoprotein A ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Abstract Abstract 2236 Lipoprotein(a) [Lp(a)] has been identified as an independent risk factor for cardiovascular diseases such as coronary heart disease. Lp(a) levels vary over 1000-fold within the human population and Lp(a) possesses both proatherogenic and prothrombotic properties due to the LDL-like moiety and apolipoprotein(a) [apo(a)] components, respectively. Apo(a) is highly homologous to plasminogen and thus can potentially interfere with plasminogen activation. Plasmin generated in the context of fibrin mediates the breakdown of blood clots, which are the causative factors in heart attacks and strokes. Plasmin generated on the surface of vascular cells plays a role in cell migration and proliferation, two of the fibroproliferative inflammatory events that underlie atherosclerosis. Previous studies have suggested that apo(a) may inhibit pericellular plasminogen activation on the basis of observations that apo(a) decreases plasminogen binding to cells. We have undertaken analysis of the mechanism by which apo(a) may interfere with pericellular plasminogen activation to allow for a more definitive description of the role of Lp(a) within the vasculature. Plasminogen activation was found to be markedly inhibited by the recombinant apo(a) variant 17K, in a dose dependent manner, on human umbilical vein endothelial cells (HUVECs), human monocytic leukemia cells (THP-1), THP-1 macrophages, and smooth muscle cells. The strong lysine binding site in kringle IV type 10, as well as kringle V appear to be required for this effect since apo(a) variants lacking these elements (17KΔAsp and 17KΔV, respectively) failed to inhibit activation. However, the role of lysine-dependent binding of apo(a) itself to the cells is not clear. Carboxypeptidase treatment of cells did not decrease apo(a) binding, and apo(a) does not compete directly for plasminogen binding to the cells. Rather, apo(a) and plasminogen may bind to the cells as a complex. We next attempted to identify the cell-surface receptor(s) that mediate plasminogen activation on the cell surface as well as its inhibition by apo(a). Urokinase-type plasminogen activator receptor (uPAR) has been previously shown to bind to urokinase-type plasminogen activator (uPA), vitronectin, and β3 integrins. uPAR is involved in the remodeling of the extracellular matrix (ECM) through regulation of plasminogen activation. We found evidence that uPAR is a potential receptor for both plasminogen and apo(a). Knockdown of uPAR in HUVECs results in decreased binding of plasminogen, 17K and, to a lesser extent, 17KΔAsp and 17KΔV. Similar experiments in SMCs revealed no changes in binding. A decrease in tPA-mediated plasminogen activation following uPAR knockdown occurred in HUVECs, and addition of 17K did not result in any further decrease. Overexpression of uPAR in THP-1 macrophages leads to greater than a two fold increase in 17K and plasminogen binding. Plasminogen activation increases over two-fold as a result of overexpression of uPAR, while 17K blunts the effect of uPAR overexpression. These results indicate that uPAR plays a crucial role in both plasminogen and apo(a) binding to the cell surface of specific cells and inhibition by apo(a) of plasminogen activation. Macrophage-1-antigen (Mac-1) receptor consists of CD11b (αM) and CD18 (β2) integrin and has been previously shown to recognize uPA and control migration and adhesion. Furthermore, αVβ3 has been previously shown to bind to vitronectin and the uPA-uPAR complex which promotes cell adhesion through binding of both vitronectin and αVβ3 integrins. We found that blocking the αM, β2, or αVβ3 receptors with monoclonal antibodies in THP-1 cells leads to a decrease in plasminogen activation, as well as a blunting of the inhibitory effects of apo(a) on plasminogen activation. These results indicate a role for Mac-1 and αVβ3 in apo(a) binding and inhibition of plasminogen activation. In conclusion, we have demonstrated, for the first time, the role of specific receptors in binding of apo(a) to vascular cell surfaces and in mediating the inhibitory effect of apo(a) on pericellular plasminogen activation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cross-talk of anosmin-1, the protein implicated in X-linked Kallmann's syndrome, with heparan sulphate and urokinase-type plasminogen activator

2004 ◽  
Vol 384 (3) ◽  
pp. 495-505 ◽  
Author(s):  
Youli HU ◽  
David GONZÁLEZ-MARTÍNEZ ◽  
Soo-Hyun KIM ◽  
Pierre Marc Gilles BOULOUX
Keyword(s):  
Cell Surface ◽  
Plasminogen Activator ◽  
Molecular Mechanisms ◽  
Expression System ◽  
Heparan Sulphate ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Kallmann's Syndrome ◽  
Kallmann’S Syndrome

Defective function of anosmin-1, the protein encoded by KAL-1, underlies X-linked Kallmann's syndrome (X-KS), a human hereditary developmental disorder. Anosmin-1 appears to play a role in neurite outgrowth and axon branching, although molecular mechanisms of its action are still unknown. Anosmin-1 contains a WAP (whey acidic protein-like) domain and four contiguous FnIII (fibronectin-like type III) repeats; its WAP domain shows similarity to known serine protease inhibitors, whereas the FnIII domains contain HS (heparan sulphate)-binding sequences. To investigate the functional role of these domains, we have generated both wild-type and mutant recombinant anosmin-1 proteins using a Drosophila S2 cell expression system. Here we present the first biochemical evidence demonstrating the high-binding affinity between HS and anosmin-1, as measured by SPR (surface plasmon resonance) (Kd=2 nM). The FnIII domains, particularly the first, are essential for dose-dependent HS binding and HS-mediated cell surface association. Furthermore, we have identified uPA (urokinase-type plasminogen activator) as an anosmin-1 interactant. Anosmin-1 significantly enhances the amidolytic activity of uPA in vitro; and anosmin-1–HS–uPA co-operation induces cell proliferation in the PC-3 prostate carcinoma cell line. Both the HS interaction and an intact WAP domain are required for the mitogenic activity of anosmin-1. These effects appear to be mediated by a direct protein interaction between anosmin-1 and uPA, since anosmin-1–uPA could be co-immunoprecipitated from PC-3 cell lysates, and their direct binding with high affinity (Kd=6.91 nM) was demonstrated by SPR. We thus propose that anosmin-1 may modulate the catalytic activity of uPA and its signalling pathway, whereas HS determines cell surface localization of the anosmin-1–uPA complex.

scholarly journals The receptor for urokinase-type plasminogen activator is deficient on peripheral blood leukocytes in patients with paroxysmal nocturnal hemoglobinuria

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1447-1455 ◽  
Author(s):  
M Ploug ◽  
T Plesner ◽  
E Ronne ◽  
V Ellis ◽  
G Hoyer-Hansen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Venous Thrombosis ◽  
Peripheral Blood ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Blood Monocytes ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal defect in bone marrow-derived cells and is clinically associated with intravascular hemolysis, hemoglobinuria, and an increased frequency of venous thrombosis. The common denominator of PNH-affected blood cells appears to be a defect in the membrane attachment of proteins normally anchored by glycosyl-phosphatidylinositol (GPI). We report here that the cellular receptor for urokinase-type plasminogen activator (u-PAR) is deficient on affected peripheral blood monocytes and granulocytes from four individuals with PNH as evidenced by chemical cross-linking analysis as well as by immunofluorescence flow cytometry using a monoclonal anti-u-PAR antibody. In contrast, on normal blood monocytes and granulocytes we find significant amounts of u-PAR, which is attached to the plasma membrane by a GPI-anchor as defined by its sensitivity towards a specific phospholipase treatment. By two-color flow cytometry it was shown that deficiency of u-PAR expression paralleled that of another GPI-anchored protein. As u-PAR is involved in the initiation of pericellular proteolysis, the reduced expression of u-PAR on PNH-affected leukocytes led to an overall reduction in the capacity for plasminogen activation by cell-surface-bound urokinase. Whereas the abnormal susceptibility of PNH-affected erythrocytes to lysis by autologous complement has been related to the low expression of three GPI-anchored complement regulatory proteins on the cell surface, we now propose that lack of u-PAR expression on the surface of peripheral blood leukocytes may be causally related to the high incidence of venous thrombosis observed in PNH patients.

scholarly journals Mycoplasma fermentans Binds to and Invades HeLa Cells: Involvement of Plasminogen and Urokinase

2004 ◽  
Vol 72 (9) ◽  
pp. 5004-5011 ◽  
Author(s):  
Amichai Yavlovich ◽  
Avigail Katzenell ◽  
Mark Tarshis ◽  
Abd A.-R. Higazi ◽  
Shlomo Rottem
Keyword(s):  
Cell Surface ◽  
Hela Cells ◽  
Surface Protein ◽  
Cholesterol Depletion ◽  
Proteolytic Digestion ◽  
Mycoplasma Fermentans ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Saturation Kinetics ◽  
Choline Phosphate

ABSTRACT Adherence of Mycoplasma fermentans to HeLa cells followed saturation kinetics, required a divalent cation, and was enhanced by preincubation of the organism at 37°C for 1 h in a low-osmolarity solution. Proteolytic digestion, choline phosphate, or anti-choline phosphate antibodies partially inhibited the adherence, supporting the notion that M. fermentans utilizes at least two surface components for adhesion, a protease-sensitive surface protein and a phosphocholine-containing glycolipid. Plasminogen binding to M. fermentans greatly increased the maximal adherence of the organism to HeLa cells. Anti-plasminogen antibodies and free plasminogen inhibited this increase. These observations suggest that in the presence of plasminogen the organism adheres to novel sites on the HeLa cell surface, which are apparently plasminogen receptors. Plasminogen-bound M. fermentans was detected exclusively on the cell surface of the infected HeLa cells. Nevertheless, plasminogen binding in the presence of the urokinase-type plasminogen activator (uPA) promoted the invasion of HeLa cells by M. fermentans. The latter finding indicates that the invasiveness of M. fermentans does not result from binding plasminogen but from activation of the bound plasminogen to plasmin. Cholesterol depletion and sequestration with β-cyclodextrin and filipin, respectively, did not affect the capacity of M. fermentans to adhere, but invasion of HeLa cells by uPA-activated plasminogen-bound M. fermentans was impaired, suggesting that lipid rafts are implicated in M. fermentans entry.

The ALG-2 Binding Site in Sec31A Influences the Retention Kinetics of Sec31A at the Endoplasmic Reticulum Exit Sites as Revealed by Live-Cell Time-Lapse Imaging

2010 ◽  
Vol 74 (9) ◽  
pp. 1819-1826 ◽  
Author(s):  
Hideki SHIBATA ◽  
Tatsutoshi INUZUKA ◽  
Haruna YOSHIDA ◽  
Hirofumi SUGIURA ◽  
Ikuo WADA ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Binding Site ◽  
Time Lapse ◽  
Live Cell ◽  
Time Lapse Imaging ◽  
Kinetics Of

scholarly journals The receptor for urokinase type plasminogen activator polarizes expression of the protease to the leading edge of migrating monocytes and promotes degradation of enzyme inhibitor complexes.

1990 ◽  
Vol 111 (2) ◽  
pp. 783-792 ◽  
Author(s):  
A Estreicher ◽  
J Mühlhauser ◽  
J L Carpentier ◽  
L Orci ◽  
J D Vassalli
Keyword(s):  
Cell Surface ◽  
Plasminogen Activator ◽  
Enzyme Inhibitor ◽  
Leading Edge ◽  
Plasminogen Activator Inhibitor ◽  
Upa Receptor ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Monocyte Cell ◽  
Extracellular Proteolysis

Receptor-bound urokinase-type plasminogen activator (uPA) remains associated to the surface of human monocytes for many hours. Monocytes induced to migrate in a chemotactic gradient of f-Met-Leu-Phe rapidly polarize their uPA receptors to the leading front of the cells. Receptor-bound enzyme can be inhibited by plasminogen activator inhibitor 2 (PAI-2), with a kinetics comparable to that determined for the free enzyme, and uPA/PAI-2 complexes can bind to the uPA receptor. In contrast to the active enzyme, the uPA/PAI-2 complex is rapidly cleared from the monocyte cell surface; this involves an initial cleavage of the complex at the cell surface, followed by endocytosis and degradation. These results indicate that the uPA receptor can function both to focus plasmin-mediated extracellular matrix degradation in front of migrating cells, and to target uPA/PAI-2 enzyme/inhibitor complexes for degradation; they suggest that this receptor is a key determinant in the control of uPA-catalyzed extracellular proteolysis.

FIBRIN-BINDING STUDIES OF PRO-UROKINASE (PRO-UK) USING SOLID PHASE FIBRIN PLATES

1987 ◽  
Author(s):  
E Angles-Cano ◽  
R Pannell ◽  
V Gurewich
Keyword(s):  
Solid Phase ◽  
Specific Binding ◽  
Chain Molecule ◽  
Single Chain ◽  
Binding Studies ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Sds Page ◽  
Fibrin Network ◽  
Photometric Measurements

Pro-UK is a single chain urokinase-type plasminogen activator (scu-PA) which has fibrin selective thrombolytic properties. However, quantitative data on pro-UK binding to fibrin and on the mechanism of its fibrin enhanced activation of plasminogen have been difficult to obtain. In the present study, a well defined fibrin network constructed on glutaraldehyde-activated PVC plates (Anal. Biochem. 153 : 201-210, 1986) and highly purified pro-UK (99 % scu-PA) were used. Binding was investigated as follows : varying dilutions of pro-UK in the presence of a trace amount of I-labeled pro-UK in buffer without or with glu-plasminogen, plasmin or oC -thrombin and in urine, plasma or serum, were incubated overnight at 4°C and then 2 h at 37°C in the fibrin plates. After washing, the wells were cut out and counted in a gamma-counter. The labeled pro-UK and the effect of enzymes on scu-PA were investigated by SDS-PAGE and autoradiography. In parallel experiments, the activity of the fibrin bound and unbound products was investigated spectrophotometrically by adding glu-plasminogen and a synthetic substrate selective for plasmin. The binding of pro-UK to fibrin was 1.7 ± 0.1 % in buffer and 0.2 ± 0.08 % in plasma, as determined from isotopic and spectro-photometric measurements. This binding is similar to that of (0.13 ± 0.05%) two-chain urokinase (plasmin-transformed scu-PA), but is extremely low compared to the specific binding of tPA (68 - 4%). By contrast, in urine, 11.2 ± 4.47 % binding of pro-UK to fibrin was observed. Thrombin did not modify the binding but transformed scu-PA into a two-chain molecule which had lost activity. These data indicate that pro-UK has little affinity for fibrin under these conditions but that some binding may be induced by a co-factor which is present in urine. Confirmation that thrombin degrades scu-PA was obtained and it is suggested that this effect may help to regulate fibrinolysis.

The receptor for urokinase-type plasminogen activator is deficient on peripheral blood leukocytes in patients with paroxysmal nocturnal hemoglobinuria

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1447-1455 ◽  
Author(s):  
M Ploug ◽  
T Plesner ◽  
E Ronne ◽  
V Ellis ◽  
G Hoyer-Hansen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Venous Thrombosis ◽  
Peripheral Blood ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Blood Monocytes ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal defect in bone marrow-derived cells and is clinically associated with intravascular hemolysis, hemoglobinuria, and an increased frequency of venous thrombosis. The common denominator of PNH-affected blood cells appears to be a defect in the membrane attachment of proteins normally anchored by glycosyl-phosphatidylinositol (GPI). We report here that the cellular receptor for urokinase-type plasminogen activator (u-PAR) is deficient on affected peripheral blood monocytes and granulocytes from four individuals with PNH as evidenced by chemical cross-linking analysis as well as by immunofluorescence flow cytometry using a monoclonal anti-u-PAR antibody. In contrast, on normal blood monocytes and granulocytes we find significant amounts of u-PAR, which is attached to the plasma membrane by a GPI-anchor as defined by its sensitivity towards a specific phospholipase treatment. By two-color flow cytometry it was shown that deficiency of u-PAR expression paralleled that of another GPI-anchored protein. As u-PAR is involved in the initiation of pericellular proteolysis, the reduced expression of u-PAR on PNH-affected leukocytes led to an overall reduction in the capacity for plasminogen activation by cell-surface-bound urokinase. Whereas the abnormal susceptibility of PNH-affected erythrocytes to lysis by autologous complement has been related to the low expression of three GPI-anchored complement regulatory proteins on the cell surface, we now propose that lack of u-PAR expression on the surface of peripheral blood leukocytes may be causally related to the high incidence of venous thrombosis observed in PNH patients.

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