Complex gangliosides are essential in spermatogenesis of mice: Possible roles in the transport of testosterone

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108

We generated double knock-out mice lacking the GM2/ GD2 and the GD3 synthase gene by mating single gene mutants, and we analyzed the abnormal phenotypes of the mutant mice expressing only the GM3 ganglioside. We observed a refractory skin lesion that appeared primarily on the face of the mutant mice at 25 weeks after birth or later. Frequent scratching of the wound sites was observed in mutant mice with the skin injury, suggesting that it is a triggering factor that exacerbates the injury. This was confirmed by isolating mice in special cages for metabolic study in which the skin injury developed more rapidly. Characteristic proliferation of nerve fibers was found in the epidermis and subepidermis at the injured sites of the mutants, probably a result of continuous skin injury. Peripheral nerve degeneration was observed in young mutant mice, suggesting that reduced sensory function induced over-scratching and the resulting skin lesion. The fact that sensory response to mechanical stimuli decreased while that to hot stimuli increased in the mutant mice supports this interpretation. Thus, only GM3-expressing mice displayed the important role of gangliosides in maintaining skin integrity via regulation of the peripheral nerves.Acidic glycosphingolipids, gangliosides, are ubiquitously expressed in the various tissues of vertebrates and play important roles in the regulation of highly organized multicellular systems (1). In particular, they are very much enriched in the nervous system and have been considered to control development, proliferation, differentiation, and maintenance of the neural tissues and cells (2, 3). Expression patterns of various ganglioside species in the nervous system vary during development and are strictly regulated in a spatio-temporal manner (4), suggesting that individual structures of gangliosides contain significant implications for individual situations.A number of studies have been performed to demonstrate the biological function of gangliosides primarily by modifying their structures with enzymes or using inhibitors to block some steps of synthesis (5). Otherwise, the effects of addition of gangliosides to the culture medium or injection into the conditioned animals have been the primary approaches in addressing the significance of gangliosides. However, recent success in the molecular cloning of glycosyltransferase genes brought about an evolutional change in the experimental approaches for carbohydrate function analysis. Availability of glycosyltransferase genes responsible for the synthesis of gangliosides enabled us to remodel ganglioside compositions of cells and tissues in vivo (6, 7) and in vitro (8).Because we isolated GM2/GD2 synthase (EC 2.4.1.92) cDNA (9) and GD3 synthase (EC 2.4.99.8) cDNA (10), we established gene knock-out mice lines of the individual glycosyltransferases. GM2/GD2 synthase gene knock-out mice lacking all complex gangliosides showed almost normal morphogenesis of the nervous system and no definite abnormal behaviors (11), although they showed nerve degene...

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Refractory Skin Injury in Complex Knock-out Mice Expressing Only the GM3 Ganglioside

Inoue

Fujii

Furukawa

et al. 2002

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108

“…sialic acid-containing GSLs, resulting from Galgt1 deficiency (also known as Galgt1 encoding Gg3Cer/GM2/GD2-synthase (EC 2.4.1.92) are infertile ( Fig. 1) (1,2). In these mice, spermatogenesis proceeds only until the stage of round spermatids, and then the string of beads composed of spermatids is thought to lose Sertoli cell contact and round up to multinuclear giant cells (1).…”

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confidence: 99%

“…Glycosphingolipids (GSLs) 1 are amphipathic molecules present on cell membranes of the endocytotic and exocytotic pathway in mammalian cells where they influence membrane function. They also appear to play a role in cytological germ cell transformations because male mice lacking complex gangliosides, i.e.…”

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confidence: 99%

Novel Class of Glycosphingolipids Involved in Male Fertility

Sandhoff¹,

Geyer

Jennemann³

et al. 2005

Mice require testicular glycosphingolipids (GSLs) for proper spermatogenesis. Mutant mice strains deficient in specific genes encoding biosynthetic enzymes of the GSL pathway including Galgt1 (encoding GM2 synthase) and Siat9 (encoding GM3 synthase) have been established lacking various overlapping subsets of GSLs. Although male Galgt1؊/؊ mice are infertile, male Siat9؊/؊ mice are fertile. Interestingly, GSLs thought to be essential for male spermatogenesis are not synthesized in either of these mice strains. Hence, these GSLs cannot account for the different phenotypes. A novel class of GSLs was observed composed of eight fucosylated molecules present in fertile but not in infertile mutant mice. These GSLs contain polyunsaturated very long chain fatty acid residues in their ceramide moieties. GSLs of this class are expressed differentially in testicular germ cells. More importantly, the neutral subset of this new GSL class strictly correlates with male fertility. These data implicate polyunsaturated, fucosylated GSLs as essential for spermatogenesis and male mouse fertility.

“…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).…”

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confidence: 99%

Disulfide Bonds of GM2 Synthase Homodimers

Li¹,

Yen²,

Allende³

et al. 2000

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 .