Derivatives-Ion-Mass-Spectrometry-Lsims-h

Both the amino and hydrazino alkyl benzoates react with the free reducing termini of acid-treated LOS, increasing the hydrophobicity of the released oligosaccharides and allowing them to be separated by reverse-phase HPLC. In lacto-n-neotetraose , these oligosaccharide derivatives now contain a sensitive uv chromophore for subsequent peak detection and improve the quality of the LSIMS spectra compared to underivatized oligosaccharides. However, the amino alkyl benzoates reacted poorly compared to the analogous hydrazino alkyl benzoates with 3-deoxy-manno-2-keto octulosonic acid (KDO), and oligosaccharides with KDO at the reducing terminus, especially when the oligosaccharide also contained phosphoethanolamine. Derivatization with the hydrazino compounds can be carried out quickly and under mild conditions using a minimal amount of reagent, and is therefore suitable for microscale analyses. The chromatographic and mass spectrometric characteristics of these derivatives make them excellent alternatives to permethylation and peracetylation techniques for the structural analysis of complex bacterial oligosaccharides derived from glycolipids.Synthetic polyprenol-pyrophosphate linked oligosaccharides are efficient substrates for mycobacterial galactan biosynthetic enzymes1227 Saskatchewan Drive, Edmonton, Alberta T6G 2G2, Canada.

Mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce a complex cell wall that is critical for their survival. The largest structural component of the cell wall, the mycolyl-arabinogalactan-peptidoglycan complex, has at its core a galactan domain composed of d-galactofuranose residues. Mycobacterial galactan biosynthesis has been proposed to involve two glycosyltransferases, GlfT1 and GlfT2, which elongate polyprenol-pyrophosphate linked glycosyl acceptor substrates using UDP-galactofuranose as the donor substrate. We here report the first chemical synthesis of GlfT1 and GlfT2 acceptor substrates containing pyrophosphate and polyprenol moieties (compounds 3, 4, 22 and 23). The approach involves chemical synthesis of an oligosaccharide, subsequent phosphorylation at the reducing end and coupling to a polyprenol phosphate. These compounds were shown to be substrates for either GlfT1 (22 and 23) or GlfT2 (3 and 4) and all were substantially more active than the corresponding alkyl glycoside substrates reported previously. Mass spectrometric analysis of the products formed from the reaction of 3, 4, 22 and 23 with the respective cognate enzyme and UDP-galactofuranose provide additional evidence for the galactan biosynthetic model in which GlfT1 adds the first two galactofuranose residues with the remainder being installed via GlfT2.

Overall, these results highlight the importance of the pyrophosphate motif in recognition of acceptor substrates by both enzymes and demonstrate a straightforward route for the preparation of such compounds. The work also provides additional support for the process by which this important glycan is biosynthesized using, for the first time, close structural analogs to the natural substrates.N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells.The potential of insect cell cultures and larvae infected with recombinant baculoviruses to produce authentic recombinant glycoproteins cloned from mammalian sources was investigated. A comparison was made of the N-linked glycans attached to secreted alkaline phosphatase (SEAP) produced in four species of insect larvae and their derived cell lines plus one additional insect cell line and larvae of one additional species. These data survey N-linked oligosaccharides produced in four families and six genera of the order Lepidoptera. Lactose-N-neotetraose expressed by recombinant isolates of Autographa californica and Bombyx mori nucleopolyhedroviruses was purified from cell culture medium, larval hemolymph or larval homogenates by phosphate affinity chromatography.

The N-linked oligosaccharides were released with PNGase-F, labeled with 8-aminonaphthalene-1-3-6-trisulfonic acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by fluorescence imaging. The oligosaccharide structures were confirmed with exoglycosidase digestions. Recombinant SEAP produced in cell lines of Lymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyx mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni , H.virescens , B.mori , and Danaus plexippus contained oligosaccharides that were structurally identical to the oligosaccharides attached to SEAP produced in T.ni cell lines. The oligosaccharide structures were all mannose-terminated.