The Structures and Microheterogeneity of the Carbohydrate Chains of Human Plasma Ceruloplasmin A STUDY EMPLOYING 500-MHz ‘H-NMR SPECTROSCOPY*

Sialo- and asialoglycopeptides were prepared from recrystallized monodisperse human plasma ceruloplasmin and analyzed by high resolution ‘H-NMR spectroscopy and methylation analysis. This glycoprotein was found to possess only hi- and triantennary N-glycosidic glycans.

NeuAca(2 * G)GalP(l+ 4)GlcNAc/3(1+ Z)Mana(l- 6) As to the microheterogeneity of the carbohydrate units, it should be noted that the biantennary structure may be extended by a fucose residue in an a(1 -+ 6) bond to GlcNAc 1. In the triantennary glycan the 7-8 branch possesses either an a(2 + 3) or an a(2 + 6) linked NeuAc residue. If the NeuAc residue is absent in the 7-8 branch, this branch contains instead a fucose linked in an a(1 + 3) bond to GlcNAc 7.
Ceruloplasmin, the copper-containing protein of human plasma, has been extensively characterized (1,2  In this paper, employing high resolution 'H-NMR spectroscopy which has proved to be particularly advantageous in elucidating the complete primary structures of carbohydrate units of glycoproteins (7, 8 NMR spectroscopy was performed on the sialo-and asialoglycopeptide fractions derived from ceruloplasmin. The NMR spectral data are summarized in Table IV. The interpretation of the NMR spectra was carried out on the basis of our earlier pertinent studies (7,8,16,21). The structural reporter group signals of the Man residues are consistent with the presence of bi-and triantennary structures.
The N-acetyl signal a t S = 2.068 also shows that Fuc is attached to GlcNAc 7 (8,16,22). The occurrence of another Fuc, present to a small extent and in a different type of linkage, is evident from its structural reporter group signals (SH-1 = 4.873 and SCH3 = 1.200) and from the chemical shift of the N-acetyl signal of GlcNAc 2 ( 8 = 2.094) (8). These data establish the presence of Fuc in a ( l -+ 6 ) linkage to GlcNAc 1. The intensity of the signals of the latter type of Fuc suggests that the a(1 + 6) linked Fuc occurs in the biantennary glycopeptide ( d l ) .
Based on the intensity ratios of the various signals in the NMR spectra of the other sialoglycopeptide fractions (a, b, c, and e ) , it can be concluded that they consist primarily of bia ( 2 -+ 6)-sialo biantennary glycopeptides. Fractions b and e also contain small amounts of triantennary glycopeptide with NeuAc in a(2 -+ 6) linkage to Gal 6 and 6', whereas a third NeuAc residue is a ( 2 + 3) linked to Gal 8.
The NMR spectral data of the investigated asialoglycopeptide fractions corroborate the identification of bi-and triantennary glycan structures. For the biantenna, the data are in full agreement with those previously published (8,16,21). The spectrum of the triantenna with Fuc attached to GlcNAc 7 (fraction f ) is given in Fig. 3, while the spectral data of fraction f a r e presented in Table IV. /? Elimination-Exhaustive /?-elimination afforded no evidence of the presence of 0-glycosidically linked heteroglycans. This procedure yielded small quantities of N-glycosidic carbohydrate moieties, possessing bi-and triantennary structures.
Conclusions-Based on the data of methylation analysis and 'H-NMR spectroscopy, the following glycan structures of ceruloplasmin could be established. Compound a was found to possess a biantennary structure without Fuc and compound dl a biantennary structure with Fuc attached to GlcNAc 1 (for structures, see Summary), whereas compounds d2 and dB have a triantennary structure without Fuc but differing in the type of their sialic acid-Gal linkage, and compound & a triantennary structure with Fuc attached to GlcNAc 7 (Fig.  2).
The glycans exhibit several types of microheterogeneity. NeuAc may be attached to Gal 8 in a(2 -+ 3) or a(2 -+ 6) linkage. If NeuAc is absent at this Gal residue, Fuc occurs at GlcNAc 7 in a(1+ 3) linkage. This observation is in support of Hill's exclusion principle regarding NeuAc or Fuc as terminal residues (23). In addition, as a third form of microheterogeneity, Fuc may be present in a(1 -+ 6) linkage to GlcNAc 1. This investigation further shows that it is now feasible to elucidate the complete structure of closely related glycans by high resolution 'H-NMR spectroscopy. by H -NMR I I