Isolation and characterization of the potential receptor for wheat germ agglutinin from human neutrophils

Abstract

Neutrophils participate in host protection and central to this process is the regulation of oxidative mechanisms. We purified by affinity chromatography the receptor for the GlcNAc-specific WGA from CD14+ CD16+ cell lysates (WGAr). The receptor is a 141 kDa glycoprotein constituted by two subunits of 78 and 63 kDa. It is mainly composed of Ser, Asx, and Gly, and, in a minor proportion, His, Cys, and Pro. Its glycan portion contains GlcNAc, Gal, and Man; NeuAc and GalNAc were identified in a minor proportion. The amino acid sequence of the WGA receptor was predicted from tryptic peptides by MALDI-TOF, both subunits showed homology with cytokeratin type II (26 and 29% for the 78 and 63 kDa subunits, respectively); the 78 kDa subunit showed also homology with the human transferrin receptor (24%). Antibodies against WGAr induce higher oxidative burst than WGA, determined by NBT reduction; however, this effect was inhibited (p < 0.05) with GlcNAc suggesting that WGAr participates as mediator in signal transduction in neutrophils.

New β-Lactam Derivatives Modulate Cell Adhesion and Signaling Mediated by RGD-Binding and Leukocyte Integrins

“…The α 4 β 1 integrin was extracted and purified from HEK293 cells stably expressing the α 4 integrin subunit (these cells endogenously express the β1 integrin subunit: data not shown). The cells were collected with a cell scraper, then membrane proteins were extracted and α 4 β 1 integrin was purified by affinity chromatography, as described . The binding buffer contained Tris–HCl 25 mM pH 7.5, CaCl 2 1 mM, MgCl 2 1 mM, MnCl 2 1 mM, BSA 2% (w/v), phenylmethanesulfonyl fluoride (PMSF) 1 mM, aprotinin 1 mg/mL, and leupeptin 50 mM.…”

“…The cells were collected with a cell scraper, then membrane proteins were extracted and α 4 β 1 integrin was purified by affinity chromatography, as described. 68 The binding buffer contained Tris−HCl 25 mM pH 7.5, CaCl 2 1 mM, MgCl 2 1 mM, MnCl 2 1 mM, BSA 2% (w/v), phenylmethanesulfonyl fluoride (PMSF) 1 mM, aprotinin 1 mg/mL, and leupeptin 50 mM. First, we allowed for the slow interaction between the α 4 integrin protein and the rabbit antihuman α 4 integrin antibody by incubating them together for 1 h at 4 °C.…”

Synthesis and assay of retro-α4β1 integrin-targeting motifs

“…The binding of the peptidomimetics 1e6 to a4b1 integrin was measured by (SPA), using Jurkat cells stably expressing human a4 integrin, and 125 I-FN as the specific radioligand [46,47]. Western blot analysis of the a4b1 integrin, extracted from cell lysate and purified by chromatography, confirmed that both a4 and b1 integrin subunits were present in the eluate employed for the test (S.M.).…”

“…Specific binding of 125 I-FN to an antibody-captured a4 and b1 integrin was time-dependent; the signal reached a plateau in 10 h and remained constant for the rest of the 24 h incubation (S.M.). The relatively slow kinetics of the SPA may require the establishment of equilibrium between the different components [46,47]. 125 I-FN specific binding to the SPA bead-associated a4b1 integrin, measured after overnight incubation, was inhibited in a concentration-dependent manner by BIO1211, MPUMP-Leu-Asp-Val-Pro-OH (1), with IC 50 and Ki values of 4.8 and 1.5 nM, respectively (Table 1).…”

Classification of Cells in CTC-Enriched Samples by Advanced Image Analysis

“…We also explored an alternative LED excitation light source to improve the excitation efficiency of the APC fluorochrome, as opposed to the mercury arc lamp which is used as a light source in the CellTracks [ 12 ]. In addition, wheat germ agglutinin (wga), a lectin that binds to sialic acid and N-acetylglucosamine residues, was explored because it is present on cellular plasma membranes [ 13 , 14 , 15 , 16 , 17 , 18 ]. We examined the presence of a cell membrane by using wga conjugated to AlexaFluor-488 and adding it to the CellSearch immunostaining cocktail.…”