Pg Binding Research Articles

Plasmin(ogen) carbohydrate chains mediate binding to dipeptidyl peptidase IV (CD 26) in rheumatoid arthritis human synovial fibroblasts

Summary Objective: To assess the reactivity between dipeptidyl peptidase IV (DPP IV) and the α2,3-linked sialic acid of the plasminogen (Pg)Thr 345 O-linked carbohydrate chain as the mechanism enabling plasmin (Pm) to induce intracellular Ca 2+ via DPP IV on rheumatoid synovial fibroblasts, and identify the DPP IV region responsible for this interaction. Methods: Cytosolic Ca 2+ mobilization in rheumatoid synovial fibroblasts was assayed by Digital Imaging Microscopy (DIM). DPP IV was purified by affinity chromatography on an immobilized polysaccharide structurally analogous to the last two residues of the Pg 2 carbohydrate chain. Binding of Pg to DPP IV and identification of the DPP IV reactive site were determined by an enzyme-linked immunosorbent assay (ELISA) and inhibition of Pm-induced intracellular Ca 2+ mobilization in these cells by peptides comprising three regions of DPP IV primary structure. Results: Cytosolic Ca 2+ mobilization induced by Pm on rheumatoid synovial fibroblasts is inhibited by L-lactose, a sugar that interferes with sialic acid binding to lectins. Asialo Pg which binds and can be converted into Pm on the surface of these cells is not able to induce intracellular Ca 2+ mobilization. A peptide comprising the DPP IV primary sequence L 313 QWLRRI inhibits both Pg binding to DPP IV and Pm-induced intracellular Ca 2+ mobilization on these cells. Conclusion: The intracellular Ca 2+ mobilization resulting from the reaction between Pg/Pm and DPP IV is mediated by a lectin-like region in DPP IV. This region is structurally analogous to the sequence (QxW) 3 , previously identified as a carbohydrate-binding region in several lectin families.

Kringle 2 Mediates High Affinity Binding of Plasminogen to an Internal Sequence in Streptococcal Surface Protein PAM

Many cells express receptors for plasminogen (Pg), although the responsible molecules in most cases are poorly defined. In contrast, the group A streptococcal surface protein PAM contains a domain with two 13-amino acid residue long repeated sequences (a1 and a2) responsible for Pg binding. Here we identify the region in Pg that interacts with PAM. A radiolabeled proteolytic plasminogen fragment containing the first three kringles (K1-K3) interacted with streptococci expressing PAM or a chimeric surface protein harboring the a1a2 sequence. In contrast, plasminogen fragments containing kringle 4 or kringle 5 and the activable serine proteinase domain failed to bind to PAM-expressing group A streptococci. A synthetic and a recombinant polypeptide containing the a1a2 sequence both bound to immobilized recombinant K2 (rK2) but not to rK1 or rK3. The interaction between the a repeat region and rK2 was reversible, and rK2 completely blocked the binding of Pg to the a1a2 region. The binding of the a repeat containing polypeptide to K2 occurred with an equilibrium association constant of 4.5 x 10(7) M-1, as determined by surface plasmon resonance, a value close to that (1.6 x 10(7) M-1) calculated for the a1a2-Pg interaction. Inhibition experiments suggested involvement of the lysine-binding site of K2 in the interaction. These data demonstrate that K2 contains the major Pg-binding site for PAM, providing the first well defined example of an interaction between an internal Pg-binding region in a protein and a single kringle domain.

Dipeptidyl peptidase IV (CD26) is a receptor for streptokinase on rheumatoid synovial fibroblasts

Plasminogen (Pg) binds to synovial fibroblasts from patients with rheumatoid arthritis with a very high affinity and in a dose-dependent manner. The Pg receptor in these cells is composed of a glycoprotein IIb/IIIa related protein (α IIb β 3 ) in association with dipeptidyl peptidase IV (DPP IV). Pg binds via its kringles to the β 3 subunit of α IIb β 3 , which is in the vicinity of receptor-bound urinary-type plasminogen activator (uPA). Activation of Pg to plasmin (Pm) by uPA induces a conformational change in the molecule, which facilitates the interaction of Pm-carbohydrate chains with DPP IV. This interaction induces a rise in cytosolic free Ca 2+ concentration. The streptococcal Pg activator streptokinase (SK) also binds to rheumatoid synovial fibroblasts with high affinity and in a dose-dependent manner. By contrast, normal synovial fibroblasts do not bind SK. Cell-bound SK can activate Pg and induces a cytosolic Ca 2+ mobilization which differs in nature from the response observed after activation by uPA. SK binding can be inhibited by fibronectin (FN), but not by Pg- or Pm-derived heavy chain. The receptor for SK was purified by affinity chromatography and identified as DPP IV. SK and FN bind to DPP IV via the amino acid sequence LTSRPA, common to both ligands. Our studies suggest that Pg/Pm binding to rheumatoid synovial fibroblasts can be regulated via two distinct types of binding sites on DPP IV. The first site involves direct binding via a lectin-like interaction. The second site functions in association with FN/SK. These findings suggest an important role of DPP IV in the regulation of Pg binding and activation on the surface of rheumatoid synovial fibroblasts.

Conserved thermochemistry of guanosine nucleophile binding for structurally distinct group I ribozymes.

We report thermodynamic values for binding of the guanosine nucleophile to the ribozyme derived from the Anabaena group I intron, and find that they are similar to those measured previously for the structurally distinct Tetrahymena ribozyme. The free energy of binding guanosine 5'-monophosphate (pG) at 30 degrees C is similar for the two ribozymes. The delta(H)degrees' and delta(S)degrees' for pG binding to the Anabaena ribozyme--RNA substrate complex (E x S) are 3.4 +/- 4 kcal/mol and 27 +/- 10 e.u., respectively. The negligible enthalpic contribution and positive entropy change were found previously for the Tetrahymena ribozyme, and are considered remarkable for a hydrogen-bonding interaction between a nucleotide and a nucleic acid. These thermodynamic values may reflect conformational changes or water release upon pG binding that are comparable for the two ribozymes. In addition, the apparent chemical steps of the two ribozyme reactions share similar activation energies and a positive deltaS++. It now appears that such thermochemical values for guanosine binding and activation may be intrinsic properties of the group I intron catalytic center.

A positive entropy change for guanosine binding and for the chemical step in the Tetrahymena ribozyme reaction.

The ribozyme derived from the group I intron of Tetrahymena thermophila binds an exogenous guanosine nucleotide, which acts as the nucleophile in the sequence-specific cleavage of oligonucleotides. By examining the temperature dependence of the reaction under conditions where Km = Kd, we conclude the following: (1) Guanosine 5'-monophosphate (pG) binds to the closed ribozyme-oligonucleotide substrate complex with a positive entropy change (delta S degree' = +23 eu) and an enthalpy change (delta H degree') close to zero. This is contrary to the expectation that binding would cause increased order (negative delta S degree) and be driven by a negative delta H degree. (2) Inosine and 2-aminopurine riboside, each lacking two hydrogen-bonding moieties relative to guanosine, also bind with a positive entropy value and an unfavorable (positive) delta H degree'. From this result, we suggest that the hydrogen-bonding moieties make an enthalpic contribution to guanosine binding overcoming an intrinsic unfavorable delta H. (3) At 0 degree C, there is equally tight binding of pG in the presence and absence of oligonucleotide substrate bound to the ribozyme. Thus, energetic interactions responsible for the thermodynamic coupling between pG and oligonucleotide substrate binding seen at higher temperatures are indirect. (4) The activation barrier of the chemical step is stabilized by a positive delta S++ (+31 to 39 eu). This stabilization is seen in four reactions using substrates with two different leaving groups in the presence and absence of pG, suggesting that the entropic contribution is inherent to the active site. The positive delta S values for the chemical step and for the binding of pG can be explained by a conformational change or release of water. Thus, although hydrogen bonding contributes to binding of nucleotides to this RNA enzyme as previously thought, it is these other events which produce a positive delta S that provide the energetic driving force for binding.

Interaction of high molecular weight kininogen with plasminogen inhibits binding of plasminogen to cell surfaces

Recent studies have shown that high molecular weight kininogen (HK) inhibits the binding of plasmin to platelets and thereby inhibits platelet activation by plasmin. We hypothesized that these results might be explained by HK binding to plasmin(ogen) in solution. Using ligand blotting, we showed that 125I-plasminogen (Pg) binds to immobilized HK and that 125I-HK binds to immobilized Pg. HK partially inhibited 125I-Pg binding to lysine-Sepharose. Non-covalent binding of HK to Pg in solution was demonstrated using the cross-linking agent, bis(sulfosuccinimidyl)suberate (BS 3) . The cross-linking agent stabilized a 180kDa complex as determined by SDS-PAGE. The 180kDa complex was not observed in the absence of BS 3 or when ϵ-amino caproic acid was added. In addition, excess unlabelled Pg or HK inhibited the binding of each radiolabelled protein to the other. The equilibrium dissociation constant (K D) for the binding of Pg to HK in solution was 0.8 μmol/L, as determined by BS 3 cross-linking. Binding of Pg to C6 glioma cells in culture was inhibited by HK in a concentration-dependent manner with an IC 50 of approximately 0.4 μmol/L. Similar inhibition was observed using human umbilical vein endothelial cells (IC 50≈0.5 μmol/L). Binding of 125I-HK to C6 glioma cells was apparently saturable, zinc-dependent and inhibited by Pg (IC 50≈0.9 μmol/L). The results presented here suggest that HK inhibits Pg binding to cell surfaces primarily by forming a complex with Pg in solution. Pg binding to HK may affect the localization of these proteins in the vascular compartment.

Characterization of the plasminogen receptors of normal and rheumatoid arthritis human synovial fibroblasts.

Plasminogen (Pg) activation on the surface of rheumatoid arthritis (RA) synovial fibroblasts by the urinary-type Pg activator induced a significant increase in cytosolic free Ca2+ concentration. This response was not observed in normal synovial fibroblasts, suggesting different Pg binding and activation mechanisms in these cell types. Pg receptors from both cell types were isolated by affinity chromatography using Pg covalently bound to Sepharose 4B. RA synovial fibroblasts express a Pg receptor complex composed of a glycoprotein IIb/IIIa-related protein in association with a 130-kDa protein that is antigenically related to the alpha 2-macroglobulin receptor-associated protein and dipeptidyl peptidase IV. This receptor complex appears to bind to both Pg and fibronectin. The Pg "receptor" in normal synovial fibroblasts is composed of a 97-kDa protein also antigenically related to the alpha 2-macroglobulin receptor-associated protein. Both cell types express the urinary-type Pg activator receptor on their surfaces. Our results suggest that RA synovial fibroblasts express novel proteins involved in Pg binding, activation, and signal transduction, which are absent in normal synovial fibroblasts.

Oxidative modification of Lp(a) causes changes in the structure and biological properties of apo(a)

We previously showed that oxidative modification converted Lp(a) particles to a form readily recognized by macrophage scavenger receptor(s). This was mediated in part by apo(a) changes indicated by a stronger negative charge protein fragmentation and increased immunoreactivity to monoclonal antibody K07, which also cross-reacted with plasminogen (Pg). The present study demonstrated that the expression of K07 and K09 epitopes of apo(a) increased markedly during time-dependent oxidative modification of Lp(a) by copper ions. Incubation of oxidized Lp(a) with the monocytoid U937 cell line showed that these particles competed more effectively with 125I-type 2 Pg binding to specific cell surface receptors than native Lp(a). This study suggests that oxidative Lp(a) modification causes significant changes in apo(a) conformation, resulting in the enhanced interaction of these particles with macrophage scavenger receptors and Pg binding sites.

Microglia-derived plasminogen enhances neurite outgrowth from explant cultures of rat brain

Recently, we reported the production and secretion of plasminogen (Pg) in cultured rat brain microglia [Nakajima et al., (1992) Fedn. Eur. Biochem. Socs Lett.308, 179–182]. To investigate the physiological significance of Pg, we determined the effect of Pg on neurite outgrowth of cultured neocortical explants of an embryonic rat brain in serum-free chemically defined medium. Pg markedly enhanced the neurite outgrowth. Although plasmin, which is derived from Pg by activation by urokinase (UK), had a similar effect in this explant culture system, UK itself did not show any effect. Furthermore, we studied the characteristics of Pg binding to cultured neocortical neurons dissociated from an embryonic 16-day-old rat brain by using 125I-Pg. Specific binding of Pg to neocortical neurons was detected and Scatchard plot analysis revealed high- and low-affinity binding sites on the neurons. The estimated dissociation constants of high- and low-affinity binding sites were approximately 16.1 and 124.2 nM, respectively. These results suggest that microglia-derived Pg plays certain roles in the regulation of neurite extension through binding to the surface of neocortical neurons.

Plasmin binding to the plasminogen receptor enhances catalytic efficiency and activates the receptor for subsequent ligand binding

Specific cell surface receptors for plasminogen (Pg) are expressed by a wide variety of cell types. The colocalization of receptors for Pg and its activators restricts plasmin (Pm) activity to specific sites and serves to promote fibrinolysis and local Pg activation. These studies show that both Pg and Pm bind to cellular receptors on monocytoid U937 cells. Limited Pm pretreatment of the cells enhances total Pg binding and alters the kinetics of Pm binding. Furthermore, surface-bound Pg is converted to Pm in the absence of exogenous activators. Cell-bound Pm exhibits a 12-fold increase in catalytic efficiency ( k cat K m ) relative to Pm free in solution. These studies demonstrate that Pg/Pm receptor occupancy can be regulated by Pm in the microenvironment and may play a significant regulatory role in fibrinolysis and extravascular proteolysis.

Binding and Activation of Plasminogen on Immobilized Immunoglobulin G: Identification of the plasmin-derived Fab as the plasminogen-binding fragment

We have found that tissue plasminogen activator catalyzes the binding of plasminogen (Pg) to immunoglobulin G (IgG) immobilized on a surface. This enhancement is due to the formation of plasmin, since plasmin treatment of immobilized IgG produced a 20-fold increase in Pg binding. Pg binding is lysine site dependent and reversible. The augmentation of Pg binding by plasmin is specific as other proteases produced significantly less or no effect. Immobilized plasmin-treated IgG also specifically binds Pg in plasma. IgG-immobilized Pg is activated by tissue plasminogen activator, and a significant portion of the plasmin formed remains bound to the IgG. The Pg reactive species in a plasmin-treated IgG digest was identified as the Fab fragment by chromatography utilizing the immobilized high affinity lysine-binding site of plasminogen. Specificity of the interaction was further demonstrated by immunoblot-ligand analysis which demonstrated that the plasmin-derived Fab fragment bound Pg whereas papain-derived Fab or plasmin-derived Fc fragments did not. These data suggest that Pg binds to the new COOH-terminal lysine residue of the plasmin-derived Fab. Pg also binds to an immobilized immune complex following plasmin treatment. These findings indicate that surface-bound IgG localizes plasminogen thus extending the spectrum of activity of the plasmin system to immunologic reactions.

Studies on type II progesterone receptor in MCF-7 cells

These experiments demonstrate for the first time the existence of a Type II progesterone receptor (RpII) in MCF-7 human breast cancer cells. RpII was shown to have a lower affinity for tritiated progesterone ([ 3H]Pg) (Kd ≥ 13 nM) than classical Rp (Kd ≤ 3 nM). RpII was detected by cytosolic, nuclear, and whole cell assays of MCF-7 cells. Scatchard analysis of [ 3H]Pg binding data revealed that classical Rp but not RpII could be recompartmentalized from the cytosolic to the nuclear pool by treating cells 1 h at 37°C with 1 μM Pg. RpII levels were shown to be increased more than two-fold by growing MCF-7 cells for 4 days in 10 nM estradiol (E 2) plus 100 nM Pg when compared to either untreated cells or to cells treated with only E 2.

Prostaglandin binding activity and myoblast fusion in aggregates of avian myoblasts

Myoblast aggregates provide a system for studying cell interactions which have several advantages over standard, stationary cultures. In gyrotory rotation, aggregate size can be controlled and is independent of cell migration. In muscle aggregates, fibroblasts are excluded, yet myoblast differentiation and fusion occur in a highly synchronous fashion. Specific PG binding occurs in chick or quail myoblast aggregates: in chick the peak of binding is at 35–36 hr. Aggregation is complete 16 hr before PG binding activity appears. This suggests either that gyrotory aggregation is not identical to myoblast recognition, or that PG binding activity occurs subsequent to myoblast recognition. Myoblast aggregates begin to release PG before 18 hr. The amount detected remains constant until binding begins at 34 hr when PG binding to the aggregates begins. Thus, both the release of PG and PG receptor activity are characteristics of the myoblasts and release of prostaglandin precedes appearance of the binding activity. As a first step in identifying the PG receptor and determining its appearance on the myoblast cell surface, we have prepared antisera against myoblast surfaces which blocks receptor-ligand interaction and have absorbed it against both peripheral and intrinsic membrane fractions. The results indicate that the PG receptor is a myoblast peripheral membrane macromolecule.

Loss of Prostaglandin E Receptors During Progression of Rat Mammary Tumors From Hormonal Dependence to Autonomy<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

Prostaglandin E (PGE) receptors and PGE-adenylate cyclase responsiveness were measured in tumor samples from a hormone-dependent subline of the transplantable MTW9 rat mammary tumor and from an autonomous subline derived from the hormone-dependent tumor. Scatchard analysis of the equilibrium binding data suggested that the hormone-dependent, slow-growing (MTW9A) tumors contain two major types of binding sites for PGE2: a high-affinity component (Kd less than 10(-9) M) and a low-affinity component [Kd greater than 10(-8) M]. The hormone-autonomous, fast-growing tumors (MTW9D), however, have lost more than 80% of the PG binding sites and exhibited mainly a predominant PGE lower affinity component (Kd greater than 10(-8) M). Loss of PGE receptors in autonomous tumors was not due to in vivo down-regulation of these receptors by excessive production of PGE, since both the hormone-dependent and autonomous tumors endogenously produce and release approximately the same amounts of PGE. Incubation of tumor tissues in vitro with PGE caused a significant stimulation of adenylate cyclase activity in the MTW9A tumors, whereas adenylate cyclase activity was not stimulated in the MTW9D tumors even in the presence of the nonhydrolyzable analogue of GTP, Gpp[NH]p. The results suggest that loss of PGE receptors and PGE-adenylate cyclase responsiveness occurs during progression of mammary tumors from hormonal dependence to autonomy and that the subsequent loss of cyclic AMP is associated with the uncontrolled growth characteristics of the autonomous tumors.

Distribution of prostaglandin E 2 binding sites in the porcine gastrointestinal tract

Binding of biologically active 3H-PGE 2 to particulate fractions of porcine gastrointestinal mucosa and muscle was investigated. Specific binding activity was detected in the 2500 xg and 30,000 xg sedimentation fractions of mucosa from esophagus, fundus, antrum, duodenum, ileum and colon, as well as in serosal muscle taken from the antrum, ileum, and colon. Optimal binding (> 40 fmol/mg protein) was observed in the 30,000 xg fraction of fundic mucosa incubated at pH 5.0. The characteristics of 3H-PGE 2 binding were variable in the remainder of the gastrointestinal tract although binding in these tissues was significantly less (0.2 to 15 fmol/mg protein) than that observed in the fundic mucosa. These data suggest that the cellular and/or subcellular site of PG binding is not uniform throughout the gastrointestinal tract. In fundic mucosa removal of the surface epithelial layer by scraping did not significantly alter the total binding activity for PGE. This result suggests that in gastric secretory mucosa optimal binding activity for PGE 2 occurs within the gastric pits deep to the surface epithelium.