Structure determination by 1H NMR spectroscopy of (sulfated) sialylated N-linked carbohydrate chains released from porcine thyroglobulin by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase-F.
The N-linked carbohydrate chains of porcine thyroglobulin were released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase-F (PNGase-F). The resulting oligosaccharides were fractionated by a combination of fast protein liquid chromatography and high performance liquid chromatography and analyzed by 500-MHz 1H NMR spectroscopy. The major acidic compounds are mono- and disialylated, fucosylated diantennary compounds terminated with alpha(2-6)-linked sialic acid on the Man alpha(1-3) branch. The Man alpha(1-6) branch shows a large heterogeneity. It can be terminated with Man-4', GlcNAc-5', or Gal-6', whereas the Gal-6' residue may be extended with Gal alpha(1-3), NeuAc alpha(2-3), or Sia alpha (2-6). In the major structures 8% of alpha(2-6)-linked sialic acid was found as NeuGc instead of NeuAc. The main compounds have sulfated homologues bearing a sulfate group (6-20%) at C-3 of Gal-6' or at C-6 of GlcNAc-5 as follows. [formula: see text]
de Waard
P
P
Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, The Netherlands.
Koorevaar
A
A
Kamerling
J P
JP
Vliegenthart
J F
JF
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D002240:Carbohydrate Sequence; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D012795:Sialoglycoproteins / Q000737:chemistry*; D013431:Sulfates; D013552:Swine; D013954:Thyroglobulin / Q000737:chemistry*
D009844:Oligosaccharides; D012795:Sialoglycoproteins; D013431:Sulfates; D013954:Thyroglobulin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase