T-cell activation requires clustering of a threshold number of T-cell receptors (TCRs) at the site of antigen presentation, a number that is reduced by CD28 co-receptor recruitment of signalling proteins to TCRs. Here we demonstrate that a deficiency in beta1,6 N-acetylglucosaminyltransferase V (Mgat5), an enzyme in the N-glycosylation pathway, lowers T-cell activation thresholds by directly enhancing TCR clustering. Mgat5-deficient mice showed kidney autoimmune disease, enhanced delayed-type hypersensitivity, and increased susceptibility to experimental autoimmune encephalomyelitis. Recruitment of TCRs to agonist-coated beads, TCR signalling, actin microfilament re-organization, and agonist-induced proliferation were all enhanced in Mgat5-/- T cells. Mgat5 initiates GlcNAc beta1,6 branching on N-glycans, thereby increasing N-acetyllactosamine, the ligand for galectins, which are proteins known to modulate T-cell proliferation and apoptosis. Indeed, galectin-3 was associated with the TCR complex at the cell surface, an interaction dependent on Mgat5. Pre-treatment of wild-type T cells with lactose to compete for galectin binding produced a phenocopy of Mgat5-/- TCR clustering. These data indicate that a galectin-glycoprotein lattice strengthened by Mgat5-modified glycans restricts TCR recruitment to the site of antigen presentation. Dysregulation of Mgat5 in humans may increase susceptibility to autoimmune diseases, such as multiple sclerosis.

Abstract. Malignant transformation of fibroblast and epithelial cells is accompanied by increased 131-6 N-acetylglucosaminyltransferase V (GIcNAc-TV) activity, a Golgi N-linked oligosaccharide processing enzyme. Herein, we report that expression of GIcNAc-TV in MvlLu cells, an immortalized lung epithelial cell line results in loss of contact-inhibition of cell growth, an effect that was blocked by swainsonine, an inhibitor of Golgi processing enzyme et-mannosidase II. In serumdeprived and high density monolayer cultures, the GlcNAc-TV transfectants formed loci, maintained microfilaments characteristic of proliferating cells, and also experienced accelerated cell death by apoptosis. Injection of the GIcNAc-TV transfectants into nude mice produced a 50% incidence of benign tumors, and progressively growing tumors in 2:12 mice with a latency of 6 mo, while no growth was observed in mice injected with control cells. In short term adhesion assays, the GIcNAc-TV expressing cells were less adhesive on surfaces coated with fibronectin and collagen type IV, but no changes were observed in levels of cell surface a5131 or otv133 integrins. The larger apparent molecular weights of the LAMP-2 glycoprotein and integrin glycoproteins o~5, O~v and 131 in the transfected cells indicates that their oligosaccharide chains are substrates for GlcNAc-TV. The results suggest that 131--6GIcNAc branching of N-linked oligosaccharides contributes directly to relaxed growth controls and reduce substratum adhesion in premalignant epithelial cells. NLINI~D glycosylation of proteins begins in the lumen of the rough endoplasmic reticulum where a subset of Asn-X-Ser/Thr residues on newly synthesized proteins are subjected to substitution with Glc3-MangGlcNAc2. The oligosaccharides are then remodeled or processed as the newly synthesized glycoproteins are transported through the Golgi compartments en route to the cell surface (Kornfeld and Kornfeld, 1985;Schachter, 1986). Many of the glycosyltransferases and glycosidases that constitute the Golgi oligosaccharide processing pathways are regulated in a tissue-specific manner (Paulson et al., 1989). Their patterns of expression and acceptor specificity appear to be the basis for the plethora of oligosaccharide structures observed in secreted and membrane glycoproteins. For the complex-type N-linked oligosaccharides, structural diversity begins with GlcNAc-branching of the trimannosyl core and continues with a variety of glycosyltransferases that complete these antennae. The [31-6GlcNAc-linked antenna which is initiated by the action of 131--6 N-acetylglucosaminyltransferase V (GlcNAc-TV) 1

The serum glycoprotein fetuin is expressed during embryogenesis in multiple tissues including limb buds and has been shown to promote bone remodeling and stimulate cell proliferation in vitro. In this report, we demonstrate that fetuin antagonizes the antiproliferative action of transforming growth factor-␤1 (TGF-␤1) in cell cultures. Surface plasmon resonance measurements show that fetuin binds directly to TGF-␤1 and TGF-␤2 and with greater affinity to the TGF-␤-related bone morphogenetic proteins (BMP-2, BMP-4, and BMP-6). In a competitive enzyme-linked immunosorbent assay, fetuin blocked binding of TGF-␤1 to the extracellular domain of TGF-␤ receptor type II (T␤RII), one of the primary TGF-␤-binding receptors. A comparison of fetuin and T␤RII shows homology in an 18 -19-amino acid sequence, which we have designated TGF-␤ receptor II homology 1 domain (TRH1). Since the TRH1 sequence is known to form a disulfide loop in fetuin, cyclized TRH1 peptides from both fetuin and T␤RII were chemically synthesized and tested for cytokine binding activity. Cyclized TRH1 peptide from T␤RII bound to TGF-␤1 with greater affinity than to BMP-2, while the cyclized TRH1 peptide from fetuin bound preferentially to BMP-2. Finally, fetuin or neutralizing anti-TGF-␤ antibodies blocked osteogenesis and deposition of calciumcontaining matrix in cultures of dexamethasone-treated rat bone marrow cells. In summary, these experiments define the TRH1 peptide loop as a cytokine-binding domain in both T␤RII and fetuin and suggest that fetuin is a natural antagonist of TGF-␤ and BMP activities.