Glyco-Forum section Letter to the Glyco-Forum Deglycosylation of hen ovotransferrin under mild conditions: effect on the immunoreactivity and biological activity

Transferrins belong to a group of homologous iron-binding glycoproteins present in all vertebrates (for review see: Loehr, 1989; Crichton, 1991). Main forms of this protein include serum transferrin from different species, ovotransferrin from avian egg white and lactotransferrin from mammalian milk (Aisen and Listowsky, 1980). These glycoproteins possess a single polypeptide chain with a molecular weight of about 80 kDa folded up to give two similar lobes, Nand C-terminal, each with a single binding site for Fe (III) (Aisen and Listowsky, 1980). Remarkable differences among transferrins can be seen taking into account the carbohydrate moiety, the so-called glycan, attached to the polypeptide backbone. In fact, whereas both human serum transferrin and lactotransferrin present two N-glycans, exclusively located in the C-lobe in human serum transferrin and symmetrically distributed in lactotransferrin, hen ovotransferrin possesses a single N-glycan at the C-lobe (Metz-Boutigue et al., 1984). Moreover, the glycans of most serum transferrin and lactotransferrin are mainly biantennary and terminating in sialic acid residues (Spik et al., 1975, 1982), but interestingly, the glycan of hen ovotransferrin shows a tetra-antennary structure terminating in N-acetylglucosamine (Dorland et al., 1979). As found for transferrins (De Jong et al., 1989; Léger et al., 1989) and many other glycoproteins (Kobata, 1992), glycans could be involved in recognition mechanisms, and in the case of transferrins also in iron delivering (Bomford and Munro, 1985). Being in most instances interested in the purification and characterization of glycans, deglycosylation is usually carried out in harsh conditions for the protein moiety; but in many other cases, where it is necessary to understand the role of glycans in protein structure/function relationship, deglycosylation should be performed in conditions “mild” enough to keep glycoproteins in their native or active state. Thus, in the present paper, a method we already described (D’Andrea et al., 1993), was adopted to obtain exhaustively deglycosylated hen ovotransferrin in “mild” denaturing conditions. Results were then compared with two other standard deglycosylation approaches both based on harsh conditions, i.e., the chemical method reported by Edge et al. (1981), and the “hard” standard enzymatic approach. As shown in Figure 1A, when samples were stained with Coomassie brilliant blue, a main band could be seen, with an apparent molecular weight compatible with that of the full-lenght hen ovotransferrin (OTf) molecule, purified according to Williams (1968). This apparent molecular mass shifted from ∼ 80 kDa (Figure 1A, lanes 2, 5–9) to ∼ 78 kDa (Figure 1A, lanes 3, 4, 10). Thus, efficient deglycosylation was achieved when OTf was treated either with trifluoromethanesulphonic acid (TFMS), or in “hard” enzymatic conditions, or preincubated with 0.1% (w/v) SDS + 0.5 % (v/v) NP-40 (D3) and subsequently treated with peptide N-glycosidase F (PNGase F). This finding was confirmed by lectin blotting; when a duplicate gel was probed with concanavalin A (Con A), a lectin which recognizes both branched mannose and β-N-acetylglucosamine residues, only a faint staining appeared when the sample was subjected to “hard” enzymatic conditions (Figure 1B, lane 4) or was preincubated with detergent mixture D3 and then subjected to digestion with PNGase F (Figure 1B, lane 10). A still fainter band appeared when the sample was chemically treated (Figure 1B, lane 3). Positive staining for lanes 5–9 (Figure 1B) indicated that N-linked side chain was still bound to OTf. Parallel behavior was observed when samples were analyzed by immunoblot. In these experiments, all the OTf samples were recognized by polyclonal anti-native OTf antibodies, obtained following the procedure reported by Campbell et al. (1974), but the sample treated with TMFS (Figure 1C, lane 3), the sample subjected to “hard” enzymatic conditions (Figure 1C, lane 4), and the sample preincubated with 0.1% (w/v) SDS + 0.5% (v/v) NP-40 (D3) and then subjected to digestion with PNGase F (Figure 1C, lane 10) all gave a faint positive band. As determined by the Con A binding assay (Rhodes and Ching, 1993), the much more efficiently deglycosylated OTf forms, that is, the sample TFMStreated, the one treated in “hard” enzymatic conditions, and the sample treated in “mild” enzymatic conditions (detergent mixture D3) underwent a 98%, 92%, and 87% (w/w) of glycan removal, respectively (Table I). Moreover, binding studies showed that the maximum value of bound 125I-OTf molecules per cell (Bmax) was essentially the same being about 3.15 × 105 for native OTf and 2.95 × 105 for OTf deglycosylated in “mild” conditions, respectively (Table II). Iron delivering, measured as 59Fe atoms delived per cell per min did not appreciably changed for both native and “mildly” deglycosylated OTf (1.28 × 104 and 1.13 × 104, respectively; Table II). The other deglycosylated OTf forms were also tested but no one was able neither to bind nor to deliver iron to chick-embryo red blood cells (CERBCs) (data not shown).