Primary structure of twenty three neutral and monosialylated oligosaccharides O‐glycosidically linked to the human secretory immunoglobulin A hinge region determined by a combinationof permethylation analysis and 400‐MHz ¹H‐NMR spectroscopy

Abstract

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...

Secretory IgA N- and O-Glycans Provide a Link between the Innate and Adaptive Immune Systems

“…Sensitive analytical procedures have enabled us to identify minor components of the glycan pool in addition to confirming the major glycan structures found by previous investigators (21)(22)(23)(24)(25)(26)(27)(28). The O-glycans on the H chain and the N-glycans on SC presented a wide range of epitopes for adhesin binding.…”

Enzyme‐aided construction of medium‐sized alditols of complete O‐linked saccharides

“…The 'H NMR chemical shifts (Table 2) (9) and (B) GlcNAc/?l-3Ga&%4GlcNAcB1_6(Ga~l-S)GalNAc-ol (5) tural reporter groups of the constructed tetrasaccharide alditol 4 were similar to those determined by a number of groups for the same structure [23-261. The chemical shifts of larger alditols 5-10 were compared to NMR data of related alditols [24,27,28] and to those of oligo-(N-acetyllactosamino)glycans related to the distal end of the molecules studied [14,29]. The chemical shifts of the GalBl-4GlcNAcBl-6Gal sequence of the hexasaccharide aldito19 were also compared to the spectrum of GaljI14GlcNAc~l-6Gal/7l4GlcNAc (Maaheimo et al, unpublished data).…”

The Glycosylation and Structure of Human Serum IgA1, Fab, and Fc Regions and the Role of N-Glycosylation on Fcα Receptor Interactions

“…In contrast, both serine and threonine residues are O-glycosylated in the hinge region of serum IgD (17) and secretory IgA1 (18), although in the latter case no experimental data was presented. These differences in O-glycan site occupancy as well as the finding that the O-glycans of secretory IgA1 are larger and more heterogeneous than in myeloma IgA1 (18,19) may reflect tissue-specific glycosylation.…”

“…The sialylated and neutral glycans identified by NP-HPLC were consistent with earlier reports of the analysis of the desialylated glycan pool (14,16,26,54,55) and confirmed previous findings from this laboratory (14), which indicated that serum IgA1 does not carry the GalNAc neoantigen. The limited repertoire of O-glycan structures attached to serum IgA1, compared with the plasma cells producing IgA1 for mucosal secretion (18,19), indicates tissue-specific O-glycan processing and may reflect the different environments in which these molecules are required to function.…”