Abstract
Sialic acids decorating blood and cell surface proteins can play important roles in various biological processes. The inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1, as well as bacterial lipopolysaccharide, can activate vascular endothelium, increasing expression of several surface glycoproteins. Here we show that treatment of cultured human endothelial cells (HEC) with TNF-alpha, interleukin-1, or lipopolysaccharide causes increased expression of the enzyme beta-galactoside alpha-2,6-sialytransferase (alpha 2-6STN). TNF-alpha was most effective, inducing a 3.5-fold enhancement of cell-associated sialytransferase activity by 72 h. In addition, activated HEC secreted a large portion of the induced sialyltransferase activity into the medium. Analysis of labeled HEC showed both a relative and an absolute increase of alpha 2,6-linked sialic acid on N-linked oligosaccharides after TNF-alpha stimulation. This coincided with increased expression of endothelial glycoproteins bearing N-linked glycans with alpha 2,6-linked sialic acid detected by the lectin Sambucus nigra agglutinin. The cytokine-inducible endothelial cell adhesion molecules E-selectin, ICAM-1, and VCAM-1 are among these glycoprotein substrates for alpha 2-6STN. These changes also correlated with a substantial increase in binding sites for CD22 beta, a mammalian lectin known to recognize oligosaccharides carrying multiple copies of alpha 2,6-linked sialic acid. Northern analysis revealed increased levels of mRNA encoding alpha 2-6STN. Thus, activation of endothelial cells during inflammatory and immunological processes may induce alpha 2-6STN, which can participate in sialylation of other activation-dependent molecules.
Highlights
Sialic acids decorating blood and cell surface proteins sialic acid and appears to serve asa biosynthetic precursor of can play important roles in various biological processes. the otherfamily members
Northern domain from its membrane anchor (17,18I)n. addition, soluble analysis revealed increased levels of mRNA encoding a2-6STN is one of a subset of serum glycoproteins known as a2-6STN.activation of endothelial cells during in- acute phase reactants that are elevated during a variety of flammatory and immunological processes may induce &-6STN, which can participate in sialylation of other activation-dependent molecules
The TNF-a-induced sialyltransferase activity was detected using as acceptor proteins asialofetuin, which contains both N- and 0-linked glycans [37], and asialoAGP, which contains only N-linked glycans [38].Treatments with linkage-specific sialidases and binding to the lectin S. nigra agglutinin demonstrated thmaotst, if not all, of the sialic acid incorporated into asialo-AGP by sialyltransferase was in a2,6-linkage
Summary
Endothelial Cell Cultureand Stimulation-Primary culturesof HEC from Clonetics Corp.(SanDiego, CA) were grown in dishes coated with 0.1% gelatin (FisherP, ittsburgh,PA) using Medium 199 (Life Technologies, Inc.) with 20% fetal calf serum (FCS; HyLcalobnoeratories,Logan, UT), 50 pg/ml endothelial cell growth supplement, and 100 pg/ml porcine heparin (Sigma), and subcultured utsriynpgsidversene The sis of human AGP (Sigma)(25))as anacceptor Under these conditions, ratio of a2,3 anda linkage of sialic acids on the same preparations of incorporation was linear with respect to incubation time as well as to 3H-labeled N-linked oligosaccharideswas determined by digestions the amount of cell lysate protein or pure a2-6STN After blockingwith TBS, 3% BSA, the membrane was incubated a2,8-linked sialyl residues, whereas the Newcastle disease virus siali- with 10 milliunitdml of sialidase (Clostridium perfringens, type X, dase hydrolyzes predominantly a2,3- and a2,8-linked sialyl residues Sigma) in sodium acetate buffer, pH 6.5, for 14 h at room temperature [27,28] These specificities werevalidated by digestingAGP, which had or with 10 unitdml of recombinant peptidew-glycosidaseF (Fluuobacbeen resialylated fromasialo-AGP with CMP-[14ClNeu5Acineither terium meningosepticurn,Boehringer Mannheim) in 100 m TridHC1, a2,3 or a2,6 linkages by purified sialyltransferases The membrane was washed twice with 1x SSC containing 0.5% SDS at room temperature, rinsed twice with 0.1 x SSC containing 0.1% SDSat 50 "C, and studied by autoradiography
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
