Life is sweet! A novel role for N-glycans in Drosophila lifespan

Abstract

N-glycans are post-translational modifications in which the sugar chain is covalently linked to protein by a GlcNAcβ1-N-asparagine linkage. Drosophila melanogaster and other invertebrates, but not vertebrates, synthesize large amounts of "paucimannose" N-glycans that contain only three or four mannose residues. The enzyme UDP-GlcNAc:α3-D-mannoside β1,2-N-acetylglucosaminyltransferase I (GnTI, encoded by the Mgat1 gene) controls the synthesis of paucimannose N-glycans. Either deletion or neuron-specific knockdown of Mgat1 in wild type flies results in pronounced defects in locomotion, structural defects in the adult central nervous system and a severely reduced lifespan. We have recently shown that neuronal expression of a wild-type Mgat1 transgene in Mgat1-null flies rescues the structural defects in the brain (fused β-lobes) and the shortened lifespan and, surprisingly, results in a dramatic 135% increase in mean lifespan relative to genetically identical controls that do not express the transgene. In this review, we discuss various approaches that can be used to determine the roles of paucimannose N-glycans in Drosophila longevity and in the adult CNS.

Figure 1

AN-glycan structures. The figure shows the major N-glycan structures in D. melanogaster. The abbreviated names assigned to these structures are used in the synthetic scheme shown in (B). M = Man; Gn = N-acetylglucosamine. Oligomannose N-glycans have from five to nine Man residues; only M9Gn2 and M5Gn2 are shown. M4Gn2 and M3Gn2 are paucimannose N-glycans. The remaining five structures have a GlcNacβ1-2Manα1-3 moiety and are therefore dependent on prior GnTI (Mgat1) action. Four of these GnTI-dependent structures are hybrid N-glycans (italics) that carry a GlcNacβ1-2 moiety on the Manα1-3 arm but no GlcNac residues on the other arm(s). The fifth GnTI-dependent structure (Gn2M3Gn2) is a complex N-glycan in which both the Manα1-3 and Manα1-6 arms carry the GlcNacβ1-2 moiety. The R group is defined in the figure. Fucosylated N-glycan synthesis in wild-type D. melanogaster. Reactions shown by continuous arrows have been established by in vitro assays whereas discontinuous arrows are based on other evidence.,, The figure shows the conversion of oligomannose N-glycans (M9-5Gn2) to hybrid (GnM5-3Gn2, Gn2M4Gn2, fucosylated GnM3Gn2), paucimannose (fucosylated and non-fucosylated M3-4Gn2) and complex (Gn2M3Gn2) N-glycans. The major structure in wild-type flies is M3Gn2F⁶ (box with thick continuous lines)., Other structures present in relatively large amounts (32–68% of M3Gn2F⁶) are boxed with thin continuous lines. The remaining structures (boxes with discontinuous lines) are present in amounts less than 10% of M3Gn2F⁶. Unboxed structures have not been detected by mass spectrometry but are included in the figure on the basis of other evidence. GnTI adds GlcNac in β1-2 linkage to the Manα1-3 arm of M5Gn2 to form the hybrid N-glycan GnM5Gn2., Two Man residues are removed from GnM5Gn2 by α3,6-mannosidase II (MaseII) to form the truncated hybrid N-glycans GnM4Gn2 and GnM3Gn2. A specific β-N-acetylglucosaminidase (FDL in the fly) not found in vertebrates, removes the GlcNac added by GnTI to form paucimannose N-glycans. GnTII (encoded by Mgat2) acts on GnM3Gn2 to initiate the synthesis of complex N-glycans (Gn2M3Gn2); this is a minor pathway in plants, insects and C. elegans because the specific β-N-acetylglucosaminidase competes more effectively for substrate than GnTII. The substrates, products and reactions of core α1,6-fucosyltransferase (6FucT) and core α1,3-fucosyltransferase (3FucT) are shown. In Drosophila, both 6FucT and 3FucT are dependent on prior GnTI action. 6FucT cannot act on structures with a core α1,3-linked Fuc and must therefore act before 3FucT to make the small amounts of M3Gn2F3F6 and GnM3Gn2F³F⁶ present in wild-type Drosophila., Mgat1-null flies make very small amounts of M3-4Gn2F^X and relatively larger amounts of M3-4Gn2 indicating that a GnTI-independent α-mannosidase (MaseIII) acts on M5Gn2 upstream of GnTI., Such a mannosidase has been reported in Spodoptera frugiperda but not in Drosophila. Analyses of N-glycan structures in Mgat1-null and fdl-null flies show that the only N-glycan absent or greatly decreased in both null flies is M3Gn2F (M3Gn2F³ + M3Gn2F⁶).

Figure 1

AN-glycan structures. The figure shows the major N-glycan structures in D. melanogaster. The abbreviated names assigned to these structures are used in the synthetic scheme shown in (B). M = Man; Gn = N-acetylglucosamine. Oligomannose N-glycans have from five to nine Man residues; only M9Gn2 and M5Gn2 are shown. M4Gn2 and M3Gn2 are paucimannose N-glycans. The remaining five structures have a GlcNacβ1-2Manα1-3 moiety and are therefore dependent on prior GnTI (Mgat1) action. Four of these GnTI-dependent structures are hybrid N-glycans (italics) that carry a GlcNacβ1-2 moiety on the Manα1-3 arm but no GlcNac residues on the other arm(s). The fifth GnTI-dependent structure (Gn2M3Gn2) is a complex N-glycan in which both the Manα1-3 and Manα1-6 arms carry the GlcNacβ1-2 moiety. The R group is defined in the figure. Fucosylated N-glycan synthesis in wild-type D. melanogaster. Reactions shown by continuous arrows have been established by in vitro assays whereas discontinuous arrows are based on other evidence.,, The figure shows the conversion of oligomannose N-glycans (M9-5Gn2) to hybrid (GnM5-3Gn2, Gn2M4Gn2, fucosylated GnM3Gn2), paucimannose (fucosylated and non-fucosylated M3-4Gn2) and complex (Gn2M3Gn2) N-glycans. The major structure in wild-type flies is M3Gn2F⁶ (box with thick continuous lines)., Other structures present in relatively large amounts (32–68% of M3Gn2F⁶) are boxed with thin continuous lines. The remaining structures (boxes with discontinuous lines) are present in amounts less than 10% of M3Gn2F⁶. Unboxed structures have not been detected by mass spectrometry but are included in the figure on the basis of other evidence. GnTI adds GlcNac in β1-2 linkage to the Manα1-3 arm of M5Gn2 to form the hybrid N-glycan GnM5Gn2., Two Man residues are removed from GnM5Gn2 by α3,6-mannosidase II (MaseII) to form the truncated hybrid N-glycans GnM4Gn2 and GnM3Gn2. A specific β-N-acetylglucosaminidase (FDL in the fly) not found in vertebrates, removes the GlcNac added by GnTI to form paucimannose N-glycans. GnTII (encoded by Mgat2) acts on GnM3Gn2 to initiate the synthesis of complex N-glycans (Gn2M3Gn2); this is a minor pathway in plants, insects and C. elegans because the specific β-N-acetylglucosaminidase competes more effectively for substrate than GnTII. The substrates, products and reactions of core α1,6-fucosyltransferase (6FucT) and core α1,3-fucosyltransferase (3FucT) are shown. In Drosophila, both 6FucT and 3FucT are dependent on prior GnTI action. 6FucT cannot act on structures with a core α1,3-linked Fuc and must therefore act before 3FucT to make the small amounts of M3Gn2F3F6 and GnM3Gn2F³F⁶ present in wild-type Drosophila., Mgat1-null flies make very small amounts of M3-4Gn2F^X and relatively larger amounts of M3-4Gn2 indicating that a GnTI-independent α-mannosidase (MaseIII) acts on M5Gn2 upstream of GnTI., Such a mannosidase has been reported in Spodoptera frugiperda but not in Drosophila. Analyses of N-glycan structures in Mgat1-null and fdl-null flies show that the only N-glycan absent or greatly decreased in both null flies is M3Gn2F (M3Gn2F³ + M3Gn2F⁶).