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...

Human serotransferrin (Tf) presents a microheterogeneity based on the existence of biantennary and triantennary glycans of the N-acetyl-lactosaminic type. By affinity chromatography on a concanavalin A-Sepharose column in well-defined conditions, human serotransferrin isolated from healthy donors was resolved into three carbohydrate molecular variants: Tf-I (less than 1%), Tf-II (17 +/- 2%) and Tf-III (82 +/- 3%) containing two triantennary glycans, one triantennary and one biantennary glycans and two biantennary glycans respectively. In addition, two 'isomers' of the triantennary glycans containing the third antenna beta-1,4-linked to the alpha-1,3-mannose residue or beta-1,6-linked to the alpha-1,6-mannose residue were characterized by methylation analysis in the ratio 1:1 in both Tf-I and Tf-II variants. On concanavalin A crossed immuno-affinity electrophoresis, the patterns exhibited by each of the three purified variants or by a mixture of these variants were compared with the patterns given by transferrin present in sera from nonpregnant and pregnant women. The results suggest that the relative proportions of transferrin carbohydrate variants was unchanged when the concentration of transferrin was increased in serum from normal donors, whereas in the serum of pregnant women, especially in the last 3 months of pregnancy, when the serum concentration of transferrin reached 4.5-5 g/l, the relative proportions of the carbohydrate variants Tf-I and Tf-II increased from 1 to 6 +/- 1% and from 17 +/- 2 to 26 +/- 3% respectively while that of Tf-III decreased from 82 +/- 3 to 67 +/- 3%. The binding of the three transferrin carbohydrate variants to the receptor of the syncytiotrophoblast plasma membranes was determined by using Scatchard-plot analysis. The number of binding sites remained constant with an increase in the number of triantennary glycans whereas a decrease up to 6-fold in the affinity constant was observed. Detection of the transferrin-receptor complex by immunoblotting in the presence of non-dissociating detergents revealed the existence of only one type of receptor or of a receptor possessing similar properties involved in the binding of each of the three serotransferrin carbohydrate variants.

Hemoglobins are ancient O(2)-binding proteins, ubiquitously found in eukaryotes. They have been categorized as symbiotic, nonsymbiotic and truncated hemoglobins. We have investigated the cellular localization of nonsymbiotic hemoglobin proteins during somatic embryogenesis in Cichorium hybrid leaves (Cichorium intybus L. var. sativum x C. endivia var. latifolia) using immunolocalization technique. These proteins were detected during the two steps of culture: induction and expression. In leaves, hemoglobins colocalised with plastids, which were dispersed in the parietal cytoplasm as well as in the two guard cells of a stomata, but not in epidermis cells. Upon induction of embryogenesis, in the dark, this pattern disappeared. During the induction phase, where competent cells reinitiate the cell cycle and prepare for mitosis, hemoglobins appeared initially near chloroplasts, and then in the vicinity of vascular vessels especially in the phloem and in cells surrounding the xylem vessels. When leaf fragments were transferred to another medium for the expression phase, hemoglobins were observed in the majority of the leaf blade cells and in small young embryos but not in the older ones. Hemoglobins were also detected in other leaves cells or tissues all along the process. The role of these nonsymbiotic hemoglobins during somatic embryogenesis is discussed.