GM2 synthase is a homodimer in which the subunits are joined by lumenal domain disulfide bond(s). To define the disulfide bond pattern of this enzyme, we analyzed a soluble form by chemical fragmentation, enzymatic digestion, and mass spectrometry and a fulllength form by site-directed mutagenesis. Ganglioside synthesis is regulated during differentiation, development, and malignant transformation (1-3) and occurs in the Golgi apparatus by the stepwise addition of monosaccharides to glycolipid acceptors by membrane bound glycosyltransferases. The simple gangliosides GM3, 1 GD3, and GT3 are the precursors of the a-, b-, and c-series of gangliosides, respectively, and are synthesized by the addition of 1, 2, or 3 molecules of sialic acid to lactosylceramide (LacCer). Complex gangliosides are formed by the addition of GalNAc to simple gangliosides by the action of UDP-GalNAc:lactosylceramide/ GM3/GD3 ␤-1,4-N-acetylgalactosaminyltransferase (GM2 synthase) followed by the attachment of Gal and additional sialic acid residues (1). Thus, GM2 synthase is a key enzyme in ganglioside biosynthesis, controlling the balance between the expression of simple and complex gangliosides (4). Genetic ablation of GM2 synthase in mice resulted in male sterility (5) as well as decreased myelination and axonal degeneration of the central and peripheral nervous system (6).Previously we showed that GM2 synthase is a homodimer formed by disulfide bond(s) in the lumenal domain (7). The importance of Cys residues of glycosyltransferases has been shown in several functional and structural studies (8, 9) and also by the fact that the Cys residues of each glycosyltransferase family are conserved in spacing (10, 11). In addition a few glycosyltransferases have been shown to be disulfide bonded dimers although the majority are monomeric (see "Discussion").In this report we have utilized protein chemistry experiments, coupled with mass spectrometric analyses, to determine: 1) that all Cys of the soluble form of GM2 synthase are involved in disulfide bonds; and 2) which disulfides are responsible for dimer formation. These results demonstrate that in the dimer the NH 2 terminus of one subunit is close to the COOH terminus of the other subunit in space. EXPERIMENTAL PROCEDURESGM2 Synthase-CHO cell clone GTm1 which stably expresses a soluble form of myc-tagged GM2 synthase was described previously (12). Large scale cell culture was achieved in roller bottles using the serum-free medium CHO-S-SFM II (Life Technologies) according to Kolhekar et al. (13) and in bioreactors at the National Cell Culture Center, Minneapolis, MN. The soluble form of GM2 synthase was partially purified from culture supernatants by SP-Sepharose chromatography using a 0 -0.25 M NaCl gradient in 50 mM Hepes, pH 7.6, 5 mM MnCl 2 .

C12H16N4O10, monoclinic, C2/c (no. 15), a = 21.953(3) Å, b = 7.0292(9) Å, c = 13.5984(19) Å, β = 127.569(4)°, V = 1663.23(40) Å3, Z = 4, Rgt(F) = 0.0555, wRref(F2) = 0.1224, T = 122(2) K.