Abstract
Intercellular adhesion molecule-1 (ICAM-1) occurs as both a membrane and a soluble, secreted glycoprotein (sICAM-1). ICAM-1 on endothelial cells mediates leukocyte adhesion by binding to leukocyte function associated antigen-1 (LFA-1) and macrophage antigen-1 (Mac-1). Recombinant mouse sICAM-1 induces the production of macrophage inflammatory protein-2 (MIP-2) in mouse astrocytes by a novel LFA-1- and Mac-1-independent mechanism. Here we showed that N-glycan structures of sICAM-1 influence its ability to induce MIP-2 production. sICAM-1 expressed in Chinese hamster ovary (CHO) cells was a more potent inducer of MIP-2 production than sICAM-1 expressed in HEK 293 cells, suggesting that posttranslational modification of sICAM-1 could influence its signaling activity. To explore the roles of glycosylation in sICAM-1 activity, we expressed sICAM-1 in mutant CHO cell lines differing in glycosylation, including Lec2, Lec8, and Lec1 as well as in CHO cells cultured in the presence of the alpha-mannosidase-I inhibitor kifunensine. Signaling activity of sICAM-1 lacking sialic acid was reduced 3-fold compared with sICAM-1 from CHO cells. The activity of sICAM-1 lacking both sialic acid and galactose was reduced 12-fold, whereas the activity of sICAM-1 carrying only high mannose-type N-glycans was reduced 12-26-fold. sICAM-1 glycoforms carrying truncated glycans retained full ability to bind to LFA-1 on leukocytes. Thus, sialylated and galactosylated complex-type N-glycans strongly enhanced the ability of sICAM-1 to induce MIP-2 production in astrocytes but did not alter its binding to LFA-1 on leukocytes. Glycosylation could therefore serve as a means to regulate specifically the signaling function of sICAM-1 in vivo.
Highlights
We showed that N-glycan structures of sICAM-1 influence its ability to induce macrophage inflammatory protein-2 (MIP-2) production. sICAM-1 expressed in Chinese hamster ovary (CHO) cells was a more potent inducer of MIP-2 production than sICAM-1 expressed in HEK 293 cells, suggesting that posttranslational modification of sICAM-1 could influence its signaling activity
The recombinant glycoproteins in culture supernatants were immunoaffinity-purified by chromatography on immobilized HPC4 monoclonal antibody, and their ability to induce MIP-2 production was assessed in primary mouse astrocytes
SICAM-1 is strongly elevated in the cerebrospinal fluid of patients with severe brain trauma [6], and recombinant mouse sICAM-1 induces production of the CXC chemokine MIP-2 in mouse astrocytes [9]
Summary
Materials—The pcDNA3.1(ϩ) plasmid was from Invitrogen and FuGENE 6 from Roche Applied Science. Blots were blocked with 5% nonfat dried milk in 20 mM Tris-HCl, pH 7.5, 0.3 M NaCl (TBS) for 90 min or overnight and incubated with HPC4 antibody (10 g/ml in TBS with 2 mM CaCl2 (TBS-CaCl2) and 1% bovine serum albumin). The samples were denatured with 5% -mercaptoethanol, 9.5 M urea, 2% Triton X-100 and focused at 100 V for 1 h, at 200 V for 1 h, and at 500 V overnight on ice. The gel was washed five times in 50% methanol, 1% SDS, 5 mM Tris-HCl, pH 8.0, for 10 min. The streptavidin-coated beads (11 l) were incubated with biotinylated HPC4 antibody (67 g/ml) in 0.5 ml of coupling buffer (20 mM Tris-HCl, pH 7.4, 0.9% NaCl, 2 mM CaCl2, 0.5% bovine serum albumin, 0.02% NaN3) at 37 °C for 2 h. Adhesion of the beads to HSB-2 cells was assessed by flow cytometry using a FACSCalibur (BD Biosciences)
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