The polypeptide chain of human lactotransferrin possesses two glycosylation sites to which glycans are linked through an N-(P-asparty1)-N-acetylglucosaminylamine bond and which are structurally heterogenous. After chymotryptic or pronase digestions, glycopeptides with five different glycan structures could be isolated. For three of them, the structure has been determined by the application of methanolysis, methylation analysis, hydrazinolysis/nitrous deamination, enzymatic cleavage and 'H-NMR spectroscopy at 360 MHz: Glycopeptides A and B : NeuAc(cr2-6)Gal(~1-4)GlcNAc(~l-2)Man(crl-3) shows that important differences exist between the number and the structure of the glycans present in these four kinds of transferrins.In the case of human lactotransferrin, in 1966, the presence of two glycans linked by a 4-N-(2-acetamido-2-deoxy-B-~-glucopyranosy1)-L-asparagine linkage was described as well as one 0-glycosidically-linked glycan [17]. The presence of two N-glycosidically-linked glycans was confirmed in 1973 and for one of them the structure was proposed [18].Microheterogeneity of the carbohydrate moieties was later demonstrated and partial results concerning different glycan structures were given in general reviews [19-211. In the present paper we describe the complete primary structure of three types of glycans as determined by methanolysis, methylation analysis, hydrazinolysis/nitrous deamination, mass spectrometry, enzymatic cleavage and 'H-NMR spectroscopy at [111._.___
The role of human lactoferrin (hLf) in alimentary iron absorption across intestinal cells was explored using a human differentiated colon carcinoma cell line, HT-29cl.19A. The apical surface of HT-29cl.19A cell monolayers exhibited 1.5 x 10(6) specific binding sites for hLf per cell, with a dissociation constant of 8.3 x 10(-7) M. The apical-to-basolateral transport of 125I-labeled hLf (125I-hLf) or 125I-59Fe-labeled hLf (125I-[59Fe]hLf) was investigated using filter-grown HT-29cl.19A cell monolayers mounted in Ussing chambers. Transport of total (intact plus degraded) hLf, measured by the 125I flux, was not saturable up to an apical hLf concentration of 12.5 microM, whereas immunoreactive hLf transport measured by enzyme-linked immunoabsorbent assay was saturable at 3.75 microM. Lowering the temperature to 4 degrees C reduced the total and immunoreactive hLf fluxes by 77% and 90%, and colchicine (500 microM) reduced these fluxes by 30% and > 97%, respectively. These results indicate that both types of transport are transcellular. Electrophoretic analysis revealed that 125I-hLf transported to the basal compartment consisted of largely degraded fragments (< 2 and 10-20 kDa) and a small portion of intact hLf. At an apical concentration of 3.75 microM diferric 125I-[59Fe]hLf, the immunoreactive hLf flux (64.6 +/- 14.3 ng.h-1.cm-2) constituted approximately 12% of the total hLf flux (552.0 +/- 61.6 ng.h-1.cm-2) and was similar to the [59Fe]hLf-equivalent flux (77.0 +/- 12.3 ng.h-1.cm-2).(ABSTRACT TRUNCATED AT 250 WORDS)
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.
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