Carbohydrate microheterogeneity of rat serotransferrin

Abstract

(1987) Biochem. Cell Biol. 65,948 -9541 was used to isolate from three DEAE-cellulose chromatographic fractions of diferric rat serotransferrin (rTf) subpopulations having discernible affinities for concanavalin A (ConA). These entities are designated rTf-1 (not retarded by ConA column), rTf-2 (retarded) and rTf-3 (bound). Each rTf type was found to be endowed with carbohydrate sufficient to account for a single diantennary glycan/protein molecule. Glycan structures were determined on the glycopeptides by employing GLC/MS and 400-MHz 'H-NMR spectroscopy.All glycans possessed a common, trimannosyl-N,N-diacetylchitobiose core with or without one L-fucose a-l,6-linked to the Asn-linked GlcNAc. However, there were differences in the antennae. Thus, in rTf-3, both antennae were of the disialylated diantennary N-acetyllactosamine type which is frequently encountered in other plasma glycoproteins. However, the a-1,3-Man-linked antenna in rTf-1 as well as rTf-2 had the sequence: NeuSAc(ct2-3)Gal(fl1-3)[Neu5Ac(ct2-6)]C1cNAc(fll-2)Man.In addition, the a-l,&Man-linked antenna deviated in rTf-2 from the standard structure by having the sequence: Neu5Ac(a2-3)Gal(fl1-3)GlcNAc(P1-2)Man. The possible relevance of the above structures to the ConA binding of rTf is discussed. A further preparation, obtained from the most anionic DEAE-cellulose fraction (peak V) of rTf contained several tetrasialylated diantennary glycans whose precise structures remain to be established in future studies.We have shown recently that the glycan heterogeneity of rat serotransferrin (rTf) is more extensive than previously thought [l]. Evidence for this was obtained by the resolution of iron-saturated rTf (2Fe-rTf) into several components during anion-exchange chromatography as well as affinity chromatography on concanavalin A (ConA) and lentil lectin. Sequential application of these techniques revealed that at least six variants of rTf exist with respect to glycan. They were not an artifact of protein purification, for their presence could be demonstrated after tracing rTf in fresh plasma with radioiron.We have now purified these rTf components in sufficient quantities to enable us to determine their glycan structures. Our results are reported below. MATERIALS AND METHODS Preparation of i+Tf and designation of its componentsPurification of rTf from freshly pooled plasma was undertaken by the three-step procedure previously described [l]. The last step, which consisted of gradient elution from a column of DEAE-cellulose (Balston), yielded five peaks of rTf, referred to as DEAE peak I, 11, etc. in the order of their appearance. The present work concerns rTf in DEAE peak 111 (60-63% of total rTf), DEAE peak IV (30-32%), and DEAE peak V (3-4%). The protein obtained in peaks I11 and IV was used without further purification. However, rTf in DEAE peak V was contaminated with peak IV material. This was removed in two subsequent steps, namely, (a) by chromatofocusing on a PBE 94 column (Pharmacia) that was equilibrated with 10 mM histidine/HCl pH 6.2 and ...

Rat mammary-gland transferrin: nucleotide sequence, phylogenetic analysis and glycan structure

“…In order to determine whether Tf glycosylation is tissuespecific, we compared our results with those obtained previously for rat serum Tf [31]. The structures of the major molecular glycovariants of rat milk Tf ( Figure 5) are of the biantennary Nacetyl-lactosamine type, containing one or two sialic acid residues and zero or one residue of fucose.…”

“…Tf is a major milk whey protein in some species (rabbit and rat) but in man it is virtually undetectable in milk; in contrast, lactotransferrin is present at a high concentration in human milk but is undetectable in rat milk. Mouse milk, however, contains a 3:1 mixture of Tf and lactotransferrin [31]. The biological significance of the high ratio of Tf to lactotransferrin in mouse milk is not well understood, and neither is the differential expression of Tf and lactotransferrin genes in different species.…”

“…As Tf was resolved as a major band after neuraminidase treatment of the milk Tf fraction (Figure 4, lane 2), differences between lanes 2 and 1 suggested that the four bands differ at least in the number of sialic acid residues. Comparison of these Tf glycovariants with those obtained from rat serum Tf (Figure 4, lane 3), which contain one trisialylated biantennary glycan [31], suggests the 1 2 Figure 4 Native PAGE of rat milk Tf fractions using Phast Gel (gradient 10-15%) and Coomassie Blue staining Lane 1, rat milk Tf fraction eluted from the SP-Sephadex column; lane 2, neuraminidase-treated rat milk Tf eluted from the SP-Sephadex column; lane 3, rat serum Tf glycovariant rTF-1 [31]. Table 4 Molar ratios of the monosaccharide methyl ethers present in the methanolysate of the permethylated oligosaccharide alditols released by hydrazinolysis from rmTf glycovariants Molar ratios were calculated on the basis of one residue of 2,4-Me2-Man per mol of oligosaccharide alditol.…”

N-Glycosylation of the carcinoembryonic antigen related cell adhesion molecule, C-CAM, from rat liver: detection of oversialylated bi- and triantennary structures

“…In rat hepatocytes oversialylation of biantennary N-glycan has been described only on the α1,3-bound NeuAcα2,3Galβ1,3Glc-NAcβ1,2Man-antennae of the biantennary N-glycan (Goulut-Chassaing and Bourrillon, 1995). Spik et al (1991) and Baussant et al (1992) also detected oversialylation in biantennary N-glycans of rat plasma glycoproteins in type I chains attached to the α1,3-linked mannose. This corresponds to our findings, as oligosaccharide F3.1 was found to be maximally trisialylated but not tetrasialylated, although it contains two type I chains.…”

“…Oversialylation of N-glycans has been described for factor IX, X, cold insoluble globulin, and fetuin from bovine blood (Takasaki et al, 1979;Mizuochi et al, 1980Mizuochi et al, , 1983Green et al, 1988) and mouse monoclonal IgG (Krotkiewski et al, 1989(Krotkiewski et al, , 1990. In rat, oversialylation has been shown in plasma α1-acid glycoprotein, serotransferrin, and T-kininogen (Yoshima et al, 1981;Spik et al, 1991;Baussant et al, 1992). In all cases, oversialylation was due exclusively to the presence of α2,6-linked sialic acid bound to GlcNAc in NeuAc-α2,3Galβ1-3GlcNAc-antennae (type I-chains).…”