Functional analysis of Drosophila β1,4-N-acetlygalactosaminyltransferases

Abstract

Members of the mammalian beta1,4-galactosyltransferase family are among the best studied glycosyltransferases, but the requirements for all members of this family within an animal have not previously been determined. Here, we describe analysis of two Drosophila genes, beta4GalNAcTA (CG8536) and beta4GalNAcTB (CG14517), that are homologous to mammalian beta1,4-galactosyltransferases. Like their mammalian homologs, these glycosyltransferases use N-acetylglucosamine as an acceptor substrate. However, they transfer N-acetylgalactosamine rather than galactose. This activity, together with amino acid sequence similarity, places them among a group of recently identified invertebrate beta1,4-N-acetylgalactosaminyltransferases. To investigate the biological functions of these genes, null mutations were generated by imprecise excision of a transposable element (beta4GalNAcTA) or by gene-targeted homologous recombination (beta4GalNAcTB). Flies mutant for beta4GalNAcTA are viable and fertile but display behavioral phenotypes suggestive of essential roles for GalNAc-beta1,4-GlcNAc containing glycoconjugates in neuronal and/or muscular function. beta4GalNAcTB mutants are viable and display no evident morphological or behavioral phenotypes. Flies doubly mutant for both genes display only the behavioral phenotypes associated with mutation of beta4GalNAcTA. Thus Drosophila homologs of the mammalian beta4GalT family are essential for neuromuscular physiology or development but are not otherwise required for viability, fertility, or external morphology.

Requirement for a core 1 galactosyltransferase in the Drosophila nervous system

“…To confirm the enzymatic activity of C1GalTA, we cloned its C-terminal catalytic domain (amino acids 44-388) into the vector pMT(1B; Haines and Irvine, 2005;Xu et al, 2007), resulting in the plasmid pMT(1B)-C1GalTA: V5:His. pMT(1B) contains the Drosophila metallothionein promoter (pMT) for inducible expression, the BiP signal sequence for secretion, a V5 epitope tag for detection, and a Histag for purification.…”

“…Restriction enzyme sites KpnI and XbaI were included in the PCR primers to facilitate cloning. pMT(1B)-C1GalTA:V5:His was transfected into S2 cells, and concentrated media was used for glycosyltransferase assays, as described previously (Ju et al, 2002b;Haines and Irvine, 2005). A similar strategy was used for CG8708, CG7440, and CG18558.…”

Quantitative Genetic Analysis of Sleep inDrosophila melanogaster

“…Sleep was reduced to wild-type levels when this P element was precisely excised, suggesting that malic enzyme has a role in sleep and waking. In addition to malic enzyme, other metabolic pathways were implicated in sleep: glycolysis (Horton et al 1996) (BG02003, 6-phosphofructo-2-kinase), electron transport (BG01007, CG8776, and BG02419, escargot), and glucosamine metabolism (Haines and Irvine 2005) (BG02565, b4GalNAcTA). One insertion line had effects on both 24-hr sleep and energy stores in this experiment: the insertion in this line, BG02009, is located in the first intron of taiman.…”

Humanization of recombinant glycoproteins expressed in insect cells

“…Interestingly, T. ni (42) and Drosophila (43) possess orthologous genes to human b4GalTs; however, the encoded gene products do not catalyze e‹cient transfer of Gal from UDP-Gal to acceptor substrates. Instead, the insect enzyme catalyzes the transfer of GalNAc from UDP-GalNAc to acceptors more e‹ciently (42,43). Indeed, when a soluble form of a S. frugiperda a-mannosidase I was expressed in Sf9 cells with a T. ni ortholog of human b4GalT, N-glycans modiˆed with a terminal GalNAc, but not Gal, were synthesized (42).…”