M. Kimura et al., CHARACTERIZATION OF A DEAMINATED NEURAMINIC ACID-CONTAINING GLYCOPROTEIN FROM THE SKIN MUCUS OF THE LOACH MISGURNUS-ANGUILLICAUDATUS, The Journal of biological chemistry, 269(51), 1994, pp. 32138-32143
Using NMR spectroscopy and mass spectrometry, the major sialic acid of
the skin mucus of the leach, Misgurnus anguillicaudatus was found to
be 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN). We have subse
quently devised a method to isolate a KDN-containing glycoprotein prep
aration from leach skin mucus. The method involves the sonication of t
he skin mucus with 0.05 M Tris-HCl, pH 8.0, to solubilize the glycopro
tein, followed by DE52-cellulose chromatography of the extract, Nuclea
se P-1 treatment, and Sepharose CL-4B gel filtration. The purified gly
coprotein preparation was found to contain 38.5% KDN, 0.4% NeuAc, 24.6
% GalNAc, 3.3% Gal, and 28.2% amino acids (w/w). The amino acid compos
ition of this glycoprotein preparation revealed that it is unusually r
ich in Thr, and 6 amino acids, Thr, Ser, Glu (or Gin), Pro, Gly and Al
a, account for 83% of the total amino acids. This glycoprotein is extr
emely poor in Cys, Met, Tyr, Phe, Arg, and Trp. Treatment of this glyc
oprotein with alkali resulted in the destruction of 83% of Thr suggest
ing that most of the sugar chains in this glycoprotein are linked thro
ugh Thr. Alkaline borohydride treatment of 100 mg of the glycoprotein
preparation, followed by Sephadex G-25 (superfine) gel filtration, yie
lded two major oligosaccharide alditols, I (15.4 mg) and II (15.6 mg).
Using Liquid secondary ion mass spectrometry and methylation analysis
, I was identified to be KDN alpha 2-->6GalNAc-ol and II, KDN alpha 2-
->6(KDN alpha 2-->3)-GalNAc-ol. KDN alpha 2-->GalNAc is structurally s
imilar to NeuAc alpha 2-->6GalNAc found in ovine submaxillary glycopro
tein while II represents a novel structure which contains two sialic a
cids linked to a GalNAc through both alpha 2-->3 and alpha 2-->6 linka
ges. This structure has never been found in mucin type glycoproteins i
ncluding mammalian epithelial mucin glycoproteins. This is the first r
eport of the presence of a mucin type glycoprotein which contains ISDN
instead of NeuAc or NeuGc in fish skin mucus.