An E-Selectin Binding Assay Based on a Polyacrylamide-Type Glycoconjugate

Weitz‐Schmidt, Gabriele; Stokmaier, Daniela; Scheel, Günther; Nifant'ev, Nikolay E.; Tuzikov, Alexander; Bovin, Nicolai V.

doi:10.1006/abio.1996.0273

Cited by 62 publications

(47 citation statements)

References 5 publications

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“…Similarly, sialyl Lewis x oligosaccharides attached to polyacrylamide gel containing 30% or 10% mole of sialyl Lewis x (GlycoTech, Williamsport, PA; ref. 27) was added at 50 M to the cytotoxic assay mixture. To determine which NK cell receptors bind to sialyl Lewis x, anti-L-selectin antibody (PharMingen; ref.…”

Section: Isolation Of Iellqar Peptide That Mimics Sialyl Lewis Xmentioning

confidence: 99%

Natural killer cells attack tumor cells expressing high levels of sialyl Lewis x oligosaccharides

Οhyama

Kanto

Kato

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Section: Isolation Of Iellqar Peptide That Mimics Sialyl Lewis Xmentioning

confidence: 99%

Natural killer cells attack tumor cells expressing high levels of sialyl Lewis x oligosaccharides

Οhyama

Kanto

Kato

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…The specificity of GA was further assessed in a competition assay based on a biotinylated sulfo-Lewis A derivatized polyacrylamide (ie, biotin-PAA-Le a -SO 3 H, an established selectin ligand) 23 because TM11-PO does not bind to E-selectin. Although GA potently inhibited human P-selectin (85 mol/L), it was ineffective against E-selectin binding (Յ20% at a concentration of 1 mmol/L; Figure 1B), and it was a moderate inhibitor of L-selectin (IC 50 , 241 mol/L).…”

Section: Resultsmentioning

confidence: 99%

“…17 This led to the presumption that GA by itself may specifically interact with P-selectin. Indeed, the polyphenol was found to inhibit the binding of 2 different selectin ligands, TM11-PO 22 and biotin-PAA-Le a -SO 3 H, 23 to P-selectin at micromolar affinity. Conceivably, the TM11-based assay is most reflective of the functional activity of GA, which is corroborated by the finding that static and dynamic interactions between PSGL-1 displaying HL60 cells and CHO-P monolayers were inhibited by GA.…”

Section: Discussionmentioning

confidence: 99%

Gallic Acid Antagonizes P-Selectin–Mediated Platelet–Leukocyte Interactions

et al. 2005

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Background-Current paradigm attributes the low incidence of cardiovascular disorders in Mediterranean countries despite a high saturated fat intake, the "French paradox," to the antioxidant capacity of red wine polyphenols.Conceivably, other antiinflammatory pathways may contribute to at least a similar extent to the atheroprotective activity of these polyphenols. We have investigated whether gallic acid (GA), an abundant red wine polyphenol, modulates the activity of P-selectin, an adhesion molecule that is critically involved in the recruitment of inflammatory cells to the vessel wall and thus in atherosclerosis. Methods and Results-GA potently inhibited the binding of a peptide antagonist (IC 50 , 7.2 mol/L) and biotin-PAA-Le a -SO 3 H, an established high-affinity ligand, to P-selectin (IC 50 , 85 mol/L). Under dynamic flow conditions, GA markedly and dose dependently attenuated the rolling of monocytic HL60 cells over P-selectin-transfected Chinese hamster ovary cells (EC 50 , 14.5 mol/L) while increasing the velocity of P-selectin-dependent rolling of human blood leukocytes over a platelet monolayer. In vivo tests established that GA administration to normolipidemic C57/Bl6 and aged atherosclerotic apolipoprotein E-deficient mice impaired the baseline rolling of conjugates between activated platelets and circulating monocytes over femoral vein endothelium, as judged by online video microscopy (ED 50 , 1.7Ϯ0.3 and 1.5Ϯ0.4 mg · kg Ϫ1 · h Ϫ1 , respectively). Conclusions-Our findings provide a solid mechanistic foundation through which GA intervenes in major inflammatory pathobiologies by binding and antagonizing P-selectin.

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“…Competition Assay with TM11-PO or Biotin-PAA-Le a -SO 3 -The peptides were assayed for their ability to inhibit TM11-PO binding to human P-selectin (16) or biotin-PAA-Le a -SO 3 H binding to human and mouse P-selectin and human E-and L-selectin (17). TM11-PO, a tetrameric TM11/streptavidin peroxidase complex, were freshly prepared by incubating streptavidin peroxidase (8.4 l, 2.0 M) and TM11-biotin (biotin-CDVEWVDVSSLEWDLPC, 1.5 l, 190 mM) for 2 h at room temperature in assay buffer.…”

Section: Methodsmentioning

confidence: 99%

“…5). This PAA-based conjugate of sulfated Le a was reported to bind with low nanomolar affinity to all selectins (17).…”

Section: Table I Peptides and Their Affinities For Human P-selectin Amentioning

confidence: 99%

Rational Optimization of a Short Human P-selectin-binding Peptide Leads to Nanomolar Affinity Antagonists

Appeldoorn

Molenaar

Bonnefoy

et al. 2003

Journal of Biological Chemistry

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P-selectin plays an important role in the development of various diseases, including atherosclerosis and thrombosis. In our laboratory we recently identified a number of specific human P-selectin-binding peptides containing a Glu-Trp-Val-Asp-Val consensus motif, displaying a low micromolar affinity for P-selectin (IC 50 ‫؍‬ 2 M). In search of more potent antagonists for P-selectin, we have optimized the EWVDV pentapeptide core motif via a two-step combinatorial chemistry approach. A dedicated library of peptide derivatives was generated by introducing seven substituents at the N and C termini of the motif. In particular, pentapeptides with gallic acid or 1,3,5-benzenetricarboxylic acid substituents at the N terminus proved to be considerably more potent inhibitors of P-selectin binding than the parental peptide. After removal of the N-terminal glutamic acid from the core sequence, which appeared to be replaceable by a carboxamide function without loss of affinity, a second library was synthesized to map the chemical moieties within the gallic acid or 1,3,5-benzenetricarboxyl acid groups responsible for the enhanced P-selectin binding. Moreover, by varying the length and rigidity of the connective spacer, we have further optimized the spatial orientation of the N-terminal substituent. The combined use of phage display and subsequent combinatorial chemistry led to the design of a number of gallic acidcontaining peptides with low nanomolar affinity for P-selectin both under static and dynamic conditions (IC 50 ‫؍‬ 15.4 nM). These small synthetic antagonists, which are equally as potent as the natural ligand Pselectin glycoprotein ligand-1, are promising leads in anti-atherothrombotic therapy.P-selectin, a cell adhesion molecule involved in the initial attachment and "rolling" of leukocytes across the inflamed vessel wall (1-4), plays a key role in atherosclerosis. In fact, P-selectin deficiency in mice has been shown to reduce atherosclerotic lesion formation (5). Also, P-selectin activation induces hypercoagulance of platelets and mediates plateletmonocyte aggregation and has thus been associated with thrombosis (6 -9). Therefore, intervention in P-selectin-mediated processes is an attractive therapeutic entry.The endogenous ligand for P-selectin is P-selectin glycoprotein ligand-1 (PSGL-1), 1 a 220-kDa disulfide-linked homodimeric sialomucin (10). P-selectin binding to PSGL-1 proceeds via a short N-terminal amino acid sequence of the latter, containing three sulfated tyrosines and a sialyl Lewis X (sLeX, Neu5Ac␣2-3Gal␤1-4[Fuc␣1-3]GlcNAc) moiety (11-14). Most P-selectin antagonists are carbohydrate derivatives of the sLeX structure (15). However, as compared with PSGL-1, these glycosides are relatively poor and unselective P-selectin inhibitors, because binding to the other selectin family members (Eand L-selectin) is equally affected.In our lab we recently identified, through the use of phage display, a number of human P-selectin-binding peptides containing an EWVDV pentapeptide consensus motif (16). Binding of t...

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An E-Selectin Binding Assay Based on a Polyacrylamide-Type Glycoconjugate

Cited by 62 publications

References 5 publications

Natural killer cells attack tumor cells expressing high levels of sialyl Lewis x oligosaccharides

Natural killer cells attack tumor cells expressing high levels of sialyl Lewis x oligosaccharides

Gallic Acid Antagonizes P-Selectin–Mediated Platelet–Leukocyte Interactions

Rational Optimization of a Short Human P-selectin-binding Peptide Leads to Nanomolar Affinity Antagonists

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