Primary structure of horse serotransferrin glycans. Demonstration that heterogeneity is related to the number of glycans and to the presence of N-acetylneuraminic acid and N-acetyl-4-O-acetylneuraminic acid

Three serotransferrin variants Tf 2a, Tf 4b and Tf 5b were isolated in an homogeneous form from a preparation of homozygous horse serotransferrin Tf 0. On the basis of the results concerning molecular mass determination and the carbohydrate analysis, it is concluded that the serotransferrin variant Tf 2a contains only one glycan while variants Tf 4b and Tf 5b contain two glycans. The structure of all of the glycans has been established by combining methylation analysis, mass spectrometry and 400-MHz 1H-NMR spectroscopy. From the obtained results, it appears that the two glycans of Tf 5b variant are, like in human serotransferrin, of the N-acetyllactosaminic biantennary type, fully sialylated by two residues of N-acetylneuraminic acid (Neu5Ac; glycan type I). In contrast, in addition to this structure, two N-acetyllactosaminic biantennary isomeric structures named type II-A and type II-B sialylated by one Neu5Ac residue and one N-acetyl-4-O-acetylneuraminic acid Neu(4,5)Ac2] residue located either at Gal6 or 6' and one N-acetyllactosaminic biantennary structure (named type III) sialylated by two residues of Neu(4,5)Ac2, were identified in variants Tf 2a and Tf 4b. These results demonstrate that in an homozygous preparation of horse serotransferrin Tf 0, the heterogeneity is dependent, on the one hand, on the nature of the neuraminic acid substituting a N-acetyllactosaminic biantennary structure and, on the other hand, on the number of glycans bound to the polypeptide chain. Moreover, the differences which exist in the molecular mass of 77.5 kDA, 80 kDa and 82 kDa for serotransferrin variants Tf 2a, Tf 4b and Tf 5b, respectively, are not completely explained by the structure and the number of the glycans suggesting that the three variants should also differ in their polypeptide chain.