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GM3, a precursor for synthesis of a- and b-series gangliosides, is elevated in adipocytes of obese model animals and in sera of obese human patients with type 2 diabetes and/or dyslipidemia. GM3 synthase (GM3S)-KO C57BL/6 mice display enhanced insulin sensitivity and reduced development of high-fat diet-induced insulin resistance. However, the pathophysiological roles of GM3 and related gangliosides in the central control of feeding and metabolism remain unclear. We found that a mouse model (KKAy GM3S KO) generated by KO of the GM3S gene in the yellow obese strain, KKAy, displayed significant amelioration of obese phenotype. Whereas KKAy mice were hyperphagic and developed severe obesity, KKAy GM3S KO mice had significantly lower body weight and food intake, and greater glucose and insulin tolerance. The hypothalamic response to intraperitoneal administration of leptin was greatly reduced in KKAy mice, but was retained in KKAy GM3S KO mice. In studies of a cultured mouse hypothalamic neuronal cell line, enhanced leptin-dependent phosphorylation of ERK was observed in GM3S-deficient cells. Furthermore, KKAy GM3S KO mice did show altered coat color, suggesting that GM3S is also involved in melanocortin signaling. Our findings, taken together, indicate that GM3-related gangliosides play key roles in leptin and melanocortin signaling.

Glycosphingolipids (GSLs) are abundant in plasma membranes of mammalian cells, and their synthesis is strictly regulated in the Golgi apparatus. Disruption of GSL homeostasis is the cause of numerous diseases. Hundreds of molecular species of GSLs exist, and the detailed mechanisms underlying their homeostasis remain unclear. We investigated the physiological significance of isoform production for β1,4-N-acetyl-galactosaminyl transferase 1/B4GALNT1 (B4GN1), an enzyme involved in synthesis of ganglio-series GSLs GM2/GD2/GA2. We discovered a new mRNA variant (termed variant 2) of B4GN1 through EST clone search. A new isoform, M1-B4GN1, which has an NH2-terminal cytoplasmic tail longer than that of previously-known isoform M2-B4GN1, is translated from variant 2. M1-B4GN1 has R-based motif (a retrograde transport signal) in the cytoplasmic tail. M1-B4GN1 is partially localized in the endoplasmic reticulum (ER) depending on the R-based motif, whereas M2-B4GN1 is localized in the Golgi. Stability of M1-B4GN1 is higher than that of M2-B4GN1 because of the R-based motif. M2-B4GN1 forms a homodimer via disulfide bonding. When M1-B4GN1 and M2-B4GN1 were co-expressed in CHO-K1 cells, the two isoforms formed a heterodimer. The M1/M2-B4GN1 heterodimer was more stable than the M2-B4GN1 homodimer, but the heterodimer was not transported from the Golgi to the ER. Our findings indicate that stabilization of M1-B4GN1 homodimer and M1/M2-B4GN1 heterodimer by R-based motif is related to prolongation of Golgi retention, but not to retrograde transport from the Golgi to the ER. Coexistence of several B4GN1 isoforms having distinctive characteristics presumably helps maintain overall enzyme stability and GSL homeostasis.

Since the successful molecular cloning in 1998 of GM3 synthase (GM3S, ST3GAL5), the enzyme responsible for initiating biosynthesis of all complex gangliosides, the efforts of our research group have been focused on clarifying the physiological and pathological implications of gangliosides, particularly GM3. We have identified isoforms of GM3S proteins having distinctive lengths of N-terminal cytoplasmic tails, and found that these cytoplasmic tails define subcellular localization, stability, and in vivo activity of GM3S isoforms. Our studies of the molecular pathogenesis of type 2 diabetes, focused on interaction between insulin receptor and GM3 in membrane microdomains, led to a novel concept: type 2 diabetes and certain other lifestyle-related diseases are membrane microdomain disorders resulting from aberrant expression of gangliosides. This concept has enhanced our understanding of the pathophysiological roles of GM3 and related gangliosides in various diseases involving chronic inflammation, such as insulin resistance, leptin resistance, and T-cell function and immune disorders (e.g., allergic asthma). We also demonstrated an essential role of GM3 in murine and human auditory systems; a common pathological feature of GM3S deficiency is deafness. This is the first direct link reported between gangliosides and auditory functions.

GM3, a major lipid component of the plasma membrane outer leaflet in mammalian cells, is synthesized in the luminal side of Golgi by ST3GAL5 protein (ST3G5), a type II membrane protein. Two strains of St3Gal5 knockout mice have been established for studies of GM3 physiological function: St3Gal5-Ex5-KO (lacking exon 5, which contains the catalytic domain of ST3G5), and St3Gal5-Ex3-KO (lacking exon 3, which contains the initiation codons). Results of the present study demonstrate that GM3 synthesis is still present, at a low level, in liver of St3Gal5-Ex3-KO mice. St3Gal5 has two mRNA transcriptional variants: a-type and b-type. When exon 3 is deleted, ST3G5 is not translated from a-type or b-type, as a result of initiation codon deletion or frame shift. Through NCBI database search and real-time PCR analyses of various mouse tissues, we identified a liver-specific St3Gal5 transcriptional variant (c-type) capable of producing artificial ST3G5 (M*-ST3G5) having GM3 synthase activity in the absence of exon 3. St3Gal5-Ex3-KO mice expressed c-type mRNA without exon 3 (c-type-/-) in liver. The transmembrane and catalytic domains of M*-ST3G5 translated from c-type-/- were identical to those from wild-type, although the cytoplasmic regions differed. Expression of M*-ST3G5 in embryonic fibroblasts derived from St3Gal5-Ex3-KO mice led to GM3 synthesis; M*-ST3G5 thus displayed enzyme activity in vivo. Taken together, our findings indicate that expression of liver-specific c-type variant accounts for the residual GM3 synthase activity observed in liver of St3Gal5-Ex3-KO mice.

In the yeast Saccharomyces cerevisiae, structural diversities of complex sphingolipids [inositol phosphorylceramide (IPC), mannosylinositol phosphorylceramide, and mannosyldiinositol phosphorylceramide] are often observed in the presence or absence of hydroxyl groups on the C-4 position of long-chain base (C4-OH) and the C-2 position of very long-chain fatty acids (C2-OH), but the biological significance of these groups remains unclear. Here, we evaluated cellular membrane fluidity in hydroxyl group-defective yeast mutants by fluorescence recovery after photobleaching. The lateral diffusion of enhanced green fluorescent protein-tagged hexose transporter 1 (Hxt1-EGFP) was influenced by the absence of C4-OH and/or C2-OH. Notably, the fluorescence recovery of Hxt1-EGFP was dramatically decreased in the sur2Δ mutant (absence of C4-OH) under the csg1Δcsh1Δ background, in which mannosylation of IPC is blocked leading to IPC accumulation, while the recovery in the scs7Δ mutant (absence of C2-OH) under the same background was modestly decreased. In addition, the amount of low affinity tryptophan transporter 1 (Tat1)-EGFP was markedly decreased in the sur2Δcsg1Δcsh1Δ mutant and accumulated in intracellular membranes in the scs7Δcsg1Δcsh1Δ mutant without altering its protein expression. These results suggest that C4-OH and C2-OH are most probably critical factors for maintaining membrane fluidity and proper turnover of membrane molecules in yeast containing complex sphingolipids with only one hydrophilic head group.

Leucine is a major amino acid in nutrients and proteins and is also an important precursor of higher alcohols during brewing. In Saccharomyces cerevisiae, leucine uptake is mediated by multiple amino acid permeases, including the high-affinity leucine permease Bap2. Although BAP2 transcription has been extensively analyzed, the mechanisms by which a substrate is recognized and moves through the permease remain unknown. Recently, we determined 15 amino acid residues required for Tat2-mediated tryptophan import. Here we introduced homologous mutations into Bap2 amino acid residues and showed that 7 residues played a role in leucine import. Residues I109/G110/T111 and E305 were located within the putative α-helix break in TMD1 and TMD6, respectively, according to the structurally homologous Escherichia coli arginine/agmatine antiporter AdiC. Upon leucine binding, these α-helix breaks were assumed to mediate a conformational transition in Bap2 from an outward-open to a substrate-binding occluded state. Residues Y336 (TMD7) and Y181 (TMD3) were located near I109 and E305, respectively. Bap2-mediated leucine import was inhibited by some amino acids according to the following order of severity: phenylalanine, leucine>isoleucine>methionine, tyrosine>valine>tryptophan; histidine and asparagine had no effect. Moreover, this order of severity clearly coincided with the logP values (octanol–water partition coefficients) of all amino acids except tryptophan. This result suggests that the substrate partition efficiency to the buried Bap2 binding pocket is the primary determinant of substrate specificity rather than structural amino acid side chain recognition.

The ganglioside GM4 is a sialic acid-containing glycosphingolipid mainly expressed in mammalian brain and erythrocytes. GM4 is synthesized by the sialylation of galactosylceramide (GalCer), while the ganglioside GM3 is synthesized by the sialylation of lactosylceramide (LacCer). Recently, the enzyme GM3 synthase was found to be responsible for the synthesis of GM4 in vitro and in vivo, yet the mechanism behind GM4 expression in cells remains unclear. In this study, we attempted to establish GM4-reconstituted cells to reveal the regulation of GM4 synthesis. Interestingly, GM4 was not detected in RPMI 1846 cells expressing LacCer, GalCer, and GM3. Similarly, GM4 was not detected in CHO-K1 cells, even when such cells expressing LacCer and GM3 were stably transfected with the GalCer synthase (GalCerS) gene. GM4 became detectable only when the GM3/GM4 synthase (GM3/GM4S, ST3GAL5) gene was overexpressed in either RPMI 1846 or CHO-K1/GalCerS cells. A mutant of the B16 melanoma cell line, GM-95, lacks GlcCer and LacCer, due to an absence of GlcCer synthase, but carries endogenous LacCer synthase and GM3/GM4S. GalCer became detectable after transfection of GalCerS into GM95 cells, but the GM95/GalCerS reconstituted cells did not express GM4, indicating that competition between the substrates LacCer and GalCer for GM3/GM4S does not cause the failure of GM4 synthesis. These results suggest that the expression machinery of GM4 under physiological conditions is independent from that of GM3.

The Drosophila Toll pathway plays important roles in innate immune responses against Gram-positive bacteria and fungi. To identify previously uncharacterized components of this pathway, we performed comparative, ex vivo, genome-wide RNA interference screening. In four screens, we overexpressed the Toll adaptor protein dMyd88, the downstream kinase Pelle, or the nuclear factor κB (NF-κB) homolog Dif, or we knocked down Cactus, the Drosophila homolog of mammalian inhibitor of NF-κB. On the basis of these screens, we identified the E3 ubiquitin ligase Sherpa as being necessary for the activation of Toll signaling. A loss-of-function sherpa mutant fly exhibited compromised production of antimicrobial peptides and enhanced susceptibility to infection by Gram-positive bacteria. In cultured cells, Sherpa mediated ubiquitylation of dMyd88 and Sherpa itself, and Sherpa and Drosophila SUMO (small ubiquitin-like modifier) were required for the proper membrane localization of an adaptor complex containing dMyd88. These findings highlight a role for Sherpa in Drosophila host defense and suggest the SUMOylation-mediated regulation of dMyd88 functions in Toll innate immune signaling.

GM3 synthase (SAT-I) is the primary glycosyltransferase responsible for the biosynthesis of ganglio-series gangliosides. In this study, we identify three isoforms of mouse SAT-I proteins, named M1-SAT-I, M2-SAT-I, and M3-SAT-I, which possess distinct lengths in their NH2-terminal cytoplasmic tails. These isoforms are produced by leaky scanning from mRNA variants of mSAT-Ia and mSAT-Ib. M2-SAT-I and M3-SAT-I were found to be localized in the Golgi apparatus, as expected, whereas M1-SAT-I was exclusively found in the endoplasmic reticulum (ER). Specific multiple arginines (R) arranged in an R-based motif, RRXXXXR necessary for ER targeting, were found in the cytoplasmic tail of M1-SAT-I, and in vivo GM3 biosynthesis by M1-SAT-I was very low because of restricted transport to the Golgi apparatus. In addition, M1-SAT-I and M3-SAT-I had a long half-life relative to M2-SAT-I. This is the first report demonstrating the presence of an ER-targeting R-based motif in the cytoplasmic tail of a protein in the mammalian glycosyltransferase family of enzymes. The system, which produces SAT-I isoforms having distinct characteristics, is likely to be of critical importance for the regulation of GM3 biosynthesis under various pathological and physiological conditions.