The alkali-stable sialoglycopeptides of secretory immunoglobulins A from human milk have been separated from the alkali-labile glycopeptides by gel filtration and from the asialoglycopeptides by ion-exchange chromatography. The structures of five of them have been determined on the basis of the results obtained by methylation analysis, mass spectrometry and 360 MHz 'H-NMR spectroscopy. For glycopeptide B, the following structure has been found :The other glycopeptides can be considered as extensions of this structure. The following extensions to Gal-6' are proposed: NeuAc(a2 -6) (glycopeptide A), Gal(fl1-3) (glycopeptide D) and Fuc(a1-0) (glycopeptide E). Furthermore, in glycopeptide C a fucose residue in (a1 -3) linkage to GlcNAc-5' could be traced.The secretory immunoglobulins of type A (sIgA) from human milk are glycoproteins [l] which are carbohydrate-rich (12 %, w/w) and present an amazing diversity in the structures of their glycans. In fact, sIgA possess both 0-glycosidically and N-glycosidically linked glycans [2,3]. The first ones are located, as in human serum IgA [4,5], in the hinge region of the a chains and we have described in previous papers the structures of some of them [3,6]. The N-glycosidically linked glycans are present on the a chains as in serum IgA [7] and on the junction [8] and secretory [9] pieces.In order to understand the important roles probably played by the glycan moieties in the conformation and behaviour towards proteolytic enzymes of sIgA and in their interaction with receptors, we have investigated further the primary structure of the sIgA glycans, by studying the alkalistable N-glycosidically linked sIgA glycopeptides. These latter fell into two categories, acidic and neutral, according to the presence or absence of sialic acid residues. In the present paper we describe the primary structure of the sialoglycans taking part of the acidic glycopeptide fraction. The neutral fraction of asialoglycans presents a large structural heterogeneity and will be described in a forthcoming paper.
Sialooligosaccharide and asialooligosaccharide alditols, derived from the human milk secretory immunoglobulin A hinge region, have been purified by HPLC using, successively, an amino-bonded silica column and an octadecyl-bonded silica column. Their primary structures were completely resolved by applying a combination of sugar analysis, methylation mass spectrometry and 400-MHz 'H-NMR spectroscopy. In the present report, twenty three oligosaccharide alditols are described and all possess a type two core consisting of the branched trisaccharide: Gal(fl1-3)[GlcNAc(/?1-6)IGalNAc-01. The elongation of this core arises by chain lengthening only on the GlcNAc(p1-6) branch for the sialylated compounds, leading to a dodecasaccharide, and on both branches for the neutral compounds leading to a nonasaccharide. Sixteen of the described oligosaccharide structures are original. Moreover, some of the fucosylated structures were found to support Lea, Leb and X blood group determinants.Secretory immunoglobulin A is present in various secretions including human milk from which it was first isolated by Montreuil et al.[I]. It differs from plasma IgA in that it is predominantly present as a dimer with a junction piece [2] and a secretory component [3]. Secretory and plasma IgA contain both N-and 0-linked oligosaccharides [4-61, the latter being located in the hinge region only. Secretory IgA is richer in carbohydrates than plasma IgA [6] and an extreme heterogeneity of the sialo and asialo forms of the N-linked glycans as well as of the 0-linked glycans has been previously reported [7-91. In a previous report, we have described the structure of the four smallest 0-glycosidically linked glycans from the secretory IgA hinge region corresponding to about 65% of the 0-linked carbohydrates [9]. These glycans were di-, tri-and tetrasaccharides derived from the substitution of the disaccharide core Gal(p1-3)GalNAc-01 by galactose, Nacetylglucosamine and N-acetylneuraminic acid residues. In addition, we observed the presence of larger glycans and, in this paper, we describe the primary structure of twenty three of them as determined by methylation mass spectrometry analysis and 400-MHz 'H-NMR spectroscopy. MATERIALS AND METHODS MaterialsTrypsin treated with diisopropylfluorophosphate was from Miles (Paris, France). Pepsin, the Fractogel TSK HW40S column and silica gel thin-layer chromatography plates (Kieselgel 60F254) were from Merck (Darmstadt, FRG). Bio-Gel P-2 and Bio-Gel P-30 were purchased from Bio-Rad Laboratories (Richmond, CA, USA). The Micropak AX-10 column was from Varian (Walton-on-Thames, UK); the 5 pm Zorbax NH2 column and the 5 pm Zorbax ODS CI8 column were from Du Pont Instruments (Paris, France) and HPLC solvents from FSA Laboratory Supplies (Loughborough, UK). Isolation of the 0-glycosidically linked glycanThe human milk secretory IgA hinge region was purified from 15 g secretory IgA according to the procedure of Frangione and Wolfenstein-Todel [ 101 after a trypsin/pepsin hydrolysis and fractionation of the hydrolysa...
Pure secretory immunoglobulin A was isolated from human milk by fractionation in gradients of pH and (NH4)2S04 concentration followed by gel filtration. The hinge region containing all the 0-glycosidically linked oligosaccharides was isolated en bloc after trypsin and pepsin hydrolysis and separated by gel filtration. The mixture of 0-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 p-elimination several oligosaccharides were separated by a combination of ion-exchange chromatography and gel-filtration chromatography. The complete structure of four of these oligosaccharides was determined by methanolysis, methylation and mass spectrometry.The structure of the four oligosaccharides which are linked to serine or threonine residues of the hinge region are as follows: P-GaL (13 3These oligosaccharides are more complex and heterogenous than the oligosaccharides linked to NAc-01. serine residues of the hinge region from myeloma serum immunoglobulin A1.Human milk represents an important source of secretory immunoglobulins A first identified by immunoelectrophoretic methods by several authors (see [I]) and isolated by Montreuil et al. [2]. This form differs from plasma IgA in predominantly being present as the dimer with a sedimentation coefficient of 12s [3], in possessing the secretory component [4], and being relatively rich in carbohydrates compared to serum IgA [5].Until now, the most extensive results described concern the structures of the oligosaccharide moieties of human plasma IgA [6,7].Less data are available however, on the structure of secretory IgA. The presence of different monosaccharides was demonstrated by Montreuil et al. obtained by Descamps et al. [8] showed that the glycans of secretory IgA were divided into two groups on the basis of the nature of the sugar-peptide linkage.In addition the 0-glycosidically linked oligosaccharides differ from those of plasma IgA, in molar carbohydrate composition and in being linked via N-acetylgalactosamine to both serine and threonine residues.The differing structures, roles and localisation of these two types of IgA have led us to determine the oligosaccharide structures of secretory IgA with a view to comparing them to those obtained for myeloma serum IgA. Since these oligosaccharide structures could play an important part in interactions with cellular receptors and/or in the conformation of secretory IgA molecules we were interested in the determination of their structures. In the present paper we describe the results of the study of 0-glycosidically linked oligosaccharides which we isolated from pure secretory IgA from human milk. Preliminary results have been given in [9]. The results concerning the N-glycosidically linked oligosaccharides will be published at a later date.
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