Structure determination by 1H NMR spectroscopy of (sulfated) sialylated N-linked carbohydrate chains released from porcine thyroglobulin by peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase-F

Abstract

The N-linked carbohydrate chains of porcine thyroglobulin were released by peptide-N4-(N-acetyl-β-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 α(2-6)-linked sialic acid on the Manα(1-3) branch. The Manα(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 Galα(1-3), NeuAcα(2-3), or Siaα(2-6). In the major structures 8% of α(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.