Abstract
We previously reported that the carbohydrate-deficient glycoprotein (CDG) syndrome is an asparagine-N-linked sugar chain transfer deficiency [Yamashita et al. (1993) J. Biol. Chem. 268, 5783-5789]. In order to confirm this hypothesis, we applied electrospray ionization-mass spectrometric analysis to transferrin isoforms purified from patients with the CDG syndrome. Transferrin isoforms containing 4, 2, and 0 sialic acid residues, S4, S2, and S0, were separated by Mono Q anion exchange column chromatography from serum of a patient with the CDG syndrome. The molecular masses of S4•S2, and S0 were determined to be 79, 570±5, 77, 364±6, and 75, 157±6 Da by electrospray ionization mass spectrometry (ESI/MS). The differences between S4 and S2, and between S2 and S0 were both in accordance with the molecular mass of a disialylated biantennary sugar chain {Neu5Acα2→6Ga1β1→•G1cNAc β1→2Man α 1→6 (Neu5Ac α 2→6Gal β 1→4G1cNAc β 1→2Man α 1→3) Man β1→ 4G1cNAcβ1→4G1cNAc} (2, 206 Da), showing that S0 is nonglycosylated, and that S4 and S2 carry 2 and 1 mol of asparagine-N-linked sugar chains, respectively. The nonglycosylated asparagine site of S2 was elucidated to be random by high performance liquid chromatog-raphy-ESI/MS of a tryptic peptide of reduced and pyridylethylated S2. ESI/MS analysis of transferrin purified through one step from serum is applicable for a definite diagnosis of the CDG syndrome.
