The N-linked oligosaccharides of aminopeptidase N from Manduca sexta: site localization and identification of novel N-glycan structures.
Mass spectrometric studies on the N-linked glycans of aminopeptidase 1 from Manduca sexta have revealed unusual structures not previously observed on any insect glycoprotein. Structure elucidation of these oligosaccharides was carried out by high-energy collision-induced dissociation (CID) using a matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) tandem mass spectrometer. These key experiments revealed that three out of the four N-linked glycosylation sites in this protein (Asn295, Asn623 and Asn752) are occupied with highly fucosylated N-glycans that possess unusual difucosylated cores. Cross-ring fragment ions and 'internal' fragment ions observed in the CID spectra, showed that these fucoses are found at the 3-position of proximal GlcNAc and at the 3-position of distal GlcNAc in the chitobiose unit. The latter substitution has only been previously observed in nematodes. In addition, these core structures can be decorated with novel fucosylated antennae composed of Fucalpha(1-3)GlcNAc. Key fragment ions revealed that these antennae are predominantly found on the upper 6-arm of the core mannose. The paucimannosidic N-glycan (Man(3)GlcNAc(2)), commonly found on other insect glycoproteins, is the predominant oligosaccharide found at the remaining N-glycosylation site (Asn609).
Stephens
Elaine
E
Department of Chemistry, University of Cambridge, UK. es287@cam.ac.uk <es287@cam.ac.uk>
Sugars
Jane
J
Maslen
Sarah L
SL
Williams
Dudley H
DH
Packman
Len C
LC
Ellar
David J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D018826:CD13 Antigens / Q000737:chemistry*; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D005643:Fucose / Q000737:chemistry; D006020:Glycopeptides / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry; D006031:Glycosylation; D007477:Ions; D007834:Lasers; D018402:Manduca; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry*; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D011134:Polysaccharides; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
D006020:Glycopeptides; D007477:Ions; D009005:Monosaccharides; D009844:Oligosaccharides; D010455:Peptides; D011134:Polysaccharides; D005643:Fucose; D001216:Asparagine; D006026:Glycoside Hydrolases; D018826:CD13 Antigens; D014357:Trypsin