Analysis of the microheterogeneity of the IgA1 hinge glycopeptide having multiple O-linked oligosaccharides by capillary electrophoresis.

It was found that the self-aggregation of IgA1 was closely connected with the glycoform of a mucin-type sugar chain on its hinge portion. In this report, normal human serum IgA1 was separated into two subfractions by a jacalin column. The elution condition, 25 mM galactose, used here was similar to that reported for the glycoprotein with a single mucin-type sugar chain per molecule. The IgA1 eluted under this condition was substantially the monomeric form. In contrast, the remaining IgA1 eluted from the column with 0. 8 M galactose was substantially the aggregated form. An analytical method for the microheterogeneity of the IgA1 hinge glycopeptide (HGP33) was developed to determine the difference between these IgA1 fractions by capillary electrophoresis (CE). Native HGP33 from both IgA1 fractions was separated into peaks 1-11, depending on their glycoforms. Because the sialic acid-rich component migrated slowly on CE, the 25 mM fraction was abundant in the sialic acid-rich components (peaks 7-11), but the 0.8 M fraction was abundant in the sialic acid-poor components (peaks 1-4). Comparison of the number of sugar chains per hinge peptide indicated that the 25 mM fraction was relatively well glycosylated. Thus, application of CE analysis to the HGP33 indicated that the monomeric IgA1 was composed of a relatively complete molecule with respect to the glycoform rather than the aggregated IgA1.

Effect of carbohydrate structure on biological activity of artificially N-glycosylated eel calcitonin.

To reveal the function of the carbohydrate portion of glycopeptides and glycoproteins, we chemo-enzymatically synthesized artificially N-glycosylated derivatives of eel calcitonin and studied their three-dimensional structure and biological activity. The CD and NMR spectra in trifluoroethanol-H(2)O solution showed that the glycosylation did not change the three-dimensional structure. All the derivatives retained the strong in vivo hypocalcemic activity of calcitonin. However, the relative activity was dependent on the structure of the attached carbohydrate. The single GlcNAc attachment best enhanced the activity, while larger carbohydrates decreased the activity. This relative activity order of compounds could be partly explained by their calcitonin-receptor binding affinity, though the affinity of the GlcNAc derivative did not exceed that of calcitonin. The enhanced hypocalcemic activity of the GlcNAc derivative was explained by its altered biodistribution. The GlcNAc attachment caused calcitonin to escape from the trap at the liver during the early circulation. Thus, the glycosylation was shown to modulate the biological activity of calcitonin depending on the carbohydrate structure without a change in the peptide backbone conformation.

Human serum immunoglobulin G3 subclass bound preferentially to asialo-, agalactoimmunoglobulin A1/Sepharose.

As we reported before, exoglycosidase treatment of human serum IgA1 changed it to a sticky molecule. In order to examine the presence of the specific binding protein to the sticky IgA1 in human serum, IgA1, asialo-IgA1 (IgA1-S) and asialo-, agalacto-IgA1 (IgA1-SG)/Sepharose column chromatography of normal human serum was carried out. Purified hinge glycopeptide (HGP33) prepared from IgA1 was used for the preparation of HGP/Sepharose. A portion of the serum protein was bound to those columns and eluted with the buffer containing 1.0 M NaCl. About four times the amount of protein was eluted from the IgA1-SG/Sepharose column than that from IgA1/Sepharose. Most of the eluted protein was IgG, and the IgG1 and IgG3 subclasses but neither IgG2 nor IgG4 was dominant. Under the lower salt concentration, a portion of IgG was also bound to the HGP-SG/Sepharose column. The obtained results coincide well with the previous report of the co-present of IgG1 and IgG3 with deposited IgA1 in IgA nephropathy patients (Aucouturier, P., et al. (1989) Clin. Immunol. Immunopathol. 51, 338-347). Thus, the results solved the question of why the IgG3 was co-present with deposited IgA1 in IgA nephropathy patients.