Heterogeneity of the Glycans O‐Glycosidically Linked to the Hinge Region of Secretory Immunoglobulins from Human Milk

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Abstract
Pure secreto IgA was isolated from human milk by fractionation in gradients of pH and (NH4)2SO4 concentration followed by gel filtration. The hinge region containing all the O-glycosidically linked oligosaccharides was isolated en bloc after trypsin and pepsin hydrolysis and separated by gel filtration. The mixture of O-glycosidically linked oligosaccharides contained N-acetylneuraminic acid (NeuAc), fucose (Fuc), galactose (Gal), N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) in the molar ratio of 0.2:0.5:2.5:2:1, respectively. After .beta.-elimination, several oligosaccharides were separated by a combination of ion-exchange chromatography and gel-filtration chromatography. The complete structure of 4 of these oligosaccharides was determined by methanolysis, methylation and mass spectrometry. The structure of the 4 oligosaccharides which are linked to serine or threonine residues of the hinge region are as follows: .beta.-Gal-(1 .fwdarw. 3)-GalNAc-ol; .alpha.-HeuAc-(2 .fwdarw. 3)-.beta.-Gal-(1 .fwdarw. 3)-GalNAc-ol; .beta.-Gal-(1 .fwdarw. 3)-[.beta.-GlcNAc-(1 .fwdarw. 6)]-GalNAc-ol; .beta.-Gal-(1 .fwdarw. 3)-[.beta.-Gal-(1 .fwdarw. 4)-.beta.-GlcNAc-(1 .fwdarw. 6)]-Gal-NAc-ol. These oligosaccharides are more complex and heterogenous than the oligosaccharides linked to serine residues of the hinge region from myeloma serum IgA1.