Structural and functional stability of the mature transferrin receptor from human placenta.

The transferrin receptor (TfR) is a N- and O-glycosylated transmembrane protein mediating the cellular iron uptake by binding and internalization of diferric transferrin. In this study, rate constants and dissociation constants of 125I-ferri-transferrin binding to the human TfR were examined dependent on receptor glycan composition, pH, bivalent cations, and temperature. To do so, purified human placental TfR was noncovalently immobilized to polystyrene surfaces and subjected to alterations in various parameters. We found that transferrin binding was clearly dependent on a receptor pretreatment with buffers of various pH in that most of the TfR molecules irreversibly lost transferrin binding activity below pH 6.5. However, the dissociation constant of the remaining active binding sites was not affected. Similarly, we were able to define the thermal stability of the receptor as a function of transferrin binding ability. Binding of transferrin was completely lost provided that the receptor was pretreated at temperatures of at least 65 degrees C. Treatment with EDTA also caused an irreversible loss of transferrin binding activity, indicating that the functionally active conformation of the mature TfR depends on bivalent cations. In order to examine the role of the receptor glycans, we enzymatically removed the sialic acid residues, the hybrid and oligomannosidic N-glycans, or all types of N-glycans. In contrast to the parameters described above, all desialylated and N-deglycosylated TfR variants had exactly the same transferrin binding properties as the native TfR. To assess changes in the secondary structure of the receptor, circular dichroic spectra were recorded from TfR at pH 5.0, from heat pretreated receptor and from deglycosylated TfR. Since the receptor did not exhibit detectable changes in the CD spectrum of the deglycosylated receptor, it can be concluded that the N-linked carbohydrates of the mature, fully processed TfR are not essential for transferrin binding and conformational stability.

Structure of the HNK-1 carbohydrate epitope on bovine peripheral myelin glycoprotein P0.

The HNK-1 carbohydrate epitope, expressed by many neural recognition molecules, is involved in cell interactions that control cell type-specific neurite outgrowth and regeneration. It is also the target for autoimmune IgM antibodies in demyelinating neuropathies of the peripheral nervous system in humans. Despite its acknowledged importance in cell interactions, the HNK-1 carbohydrate structure, when expressed on glycoproteins, is still unknown. Here, we describe the structure of one of the predominant HNK-1-bearing glycans of bovine P0. The epitope consists of the sulfated trisaccharide SO4-3GlcAbeta1-3Galbeta1-4GlcNAc, attached to the alpha1-6 arm of a diantennary core with a bisecting N-acetylglucosamine. It is the first example of a terminal 3-sulfated glucuronic acid on an asparagine-linked carbohydrate. Because the similarity between the glycoprotein-derived structure and the glycosphingolipids carrying HNK-1 is restricted to the terminal sulfated trisaccharide, we conclude that this element is sufficient for HNK-1 immunoreactivity. Knowledge of the HNK-1 structure on proteins is an important prerequisite for the elucidation of its functional role in development and disease.

Direct calibration ELISA: a rapid method for the simplified determination of association constants of unlabeled biological molecules.

We present a novel method for the rapid determination of association constants. The method is based on the direct calibration of an enzyme-linked immunosorbent assay (dcELISA) and does not require any external calibration. It combines kinetic and equilibrium binding experiments and can be performed on a single microtiter plate. The absorbance data are evaluated by several linearized plots without the need for sophisticated computations. The dcELISA has been used to analyze the binding of a monoclonal antibody, OKT9, to its cognate antigen, the human transferrin receptor, and yielded an association constant of Ka = 2.2 x 10(9) l/mol and a complex formation rate constant of kc = 2.7 x 10(-4) s-1. A 26% larger association constant was obtained with a radioimmunoassay (RIA)-based Scatchard analysis using 125I-labeled OKT9. By quantifying the binding of the same iodinated antibody with the dcELISA we were able to verify that the iodination modifies the binding properties of the antibody. The dcELISA thus appears to be superior to all methods requiring covalent modifications. In principle, the direct calibration method can also be combined with all other solid phase assays. It should thus expand their scope in quantifying the binding properties of biologically important molecules.

Selective reentry of recycling cell surface glycoproteins to the biosynthetic pathway in human hepatocarcinoma HepG2 cells.

Return of cell surface glycoproteins to compartments of the secretory pathway has been examined in HepG2 cells comparing return to the trans-Golgi network (TGN), the trans/medial- and cis-Golgi. Transport to these sites was studied by example of the transferrin receptor (TfR) and the serine peptidase dipeptidylpeptidase IV (DPPIV) after labeling these proteins with the N-hydroxysulfosuccinimide ester of biotin on the cell surface. This experimental design allowed to distinguish between glycoproteins that return to these biosynthetic compartments from the cell surface and newly synthesized glycoproteins that pass these compartments during biosynthesis en route to the surface. Reentry to the TGN was measured in that surface glycoproteins were desialylated with neuraminidase and were monitored for resialylation during recycling. Return to the trans-Golgi was traced measuring the transfer of [3H]fucose residues to recycling surface proteins by fucosyltransferases. To study return to the cis-Golgi, surface proteins were metabolically labeled in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM). As a result surface proteins retained N-glycans of the oligomannosidic type. Return to the site of mannosidase I in the medial/cis-Golgi was measured monitoring conversion of these glycans to those of the complex type after washout of dMM. Our data demonstrate that DPPIV does return from the cell surface not only to the TGN, but also to the trans-Golgi thus linking the endocytic to the secretory pathway. In contrast, no reentry to sites of mannosidase I could be detected indicating that the early secretory pathway is not or is only at insignificant rates accessible to recycling DPPIV. In contrast to DPPIV, TfR was very efficiently sorted from endosomes to the cell surface and did not return to the TGN or to other biosynthetic compartments in detectable amounts, indicating that individual surface proteins are subject to different sorting mechanisms or sorting efficiencies during recycling.

The L2/HNK-1 carbohydrate is carried by the myelin associated glycoprotein and sulphated glucuronyl glycolipids in muscle but not cutaneous nerves of adult mice.

We have previously shown that myelinating Schwann cells associated with motor, but not sensory, axons in peripheral nerves of adult mice express the L2/HNK-1 carbohydrate epitope. This carbohydrate structure carried by glycolipids and neural cell adhesion molecules has been suggested to specifically foster regrowth of motor as opposed to sensory axons after infliction of a lesion. To determine which molecular components may be the carriers of the L2 carbohydrate in motor axon-associated myelinating Schwann cells, we have isolated the purely sensory, cutaneous branch and the mixed sensory and motor muscle branch of the femoral nerve of adult mice, isolated the myelin fraction thereof and analysed the molecules expressing the L2 carbohydrate by several immunochemical methods. L2 immunoreactivity in myelin of the muscle branch was four to five times higher than that of the cutaneous branch. The 110 kDa L2-immunoreactive glycoprotein in myelin of the muscle branch, which is not L2-immunoreactive in the cutaneous branch, was identified as the myelin-associated glycoprotein by a combination of immunoprecipitation and Western blot analysis. Myelin extraction with organic solvents additionally revealed the two L2-carrying glycolipids, which amounted to approximately 40 ng glycolipid/mg dry weight in myelin of the muscle branch, whereas no significant amounts of the L2 glycolipids were found in myelin of the cutaneous branch. These observations suggest an astonishing degree of differential regulation of carbohydratesynthesizing activities in myelinating Schwann cells.

Enzymatic modeling of the oligosaccharide chains of glycoproteins immobilized onto polystyrene surfaces.

A method for the modification of the oligosaccharide moiety of even small amounts of purified glycoproteins by enzymatic glycosylation and deglycosylation is described. The method includes noncovalent immobilization of the glycoproteins onto the polystyrene surface of the wells of microtiter plates used as reaction tubes, deglycosylation or glycosylation by incubation either with exoglycosidases or endoglycosidases or with glycosyltransferases, and the characterization of the modified glycan structures by probing them with lectins. Placental transferrin receptor employed as a model glycoprotein was modified in amounts of as little as 100 ng removing sialic acid residues, hybrid-type glycans or all types of N-glycans with neuraminidase, endo-beta-N-acetylglucosaminidase H or peptide-N4-(acetyl-beta-glucosaminyl) asparagine amidase. Asialotransferrin receptor was alpha-2,6-sialylated with alpha-2,6-sialyltransferase from rat liver, but could not be alpha-2,3-sialylated with alpha-2,3-sialyltransferase from porcine liver. Changes in the structure and in the relative amount of the oligosaccharides could be monitored semiquantitatively with high sensitivity by the binding of digoxigenin-labeled lectins and anti-digoxigenin Fab fragments. The method is easy to use, does not require immobilization of the enzymes employed, offers simple separation of the enzymes and the product, and leaves the protein intact for further studies.

Structure of the N-linked oligosaccharides of the human transferrin receptor.

Human transferrin receptor was isolated from placenta and from the hepatocarcinoma cell line Hep G2. Asparagine-linked oligosaccharides were released by treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Oligosaccharide alditols were fractionated by anion-exchange high-performance liquid chromatography and by high-pH anion-exchange chromatography. Glycans from placental transferrin receptor were further characterized, after desialylation, by methylation analysis and, in part, by liquid secondary-ion mass spectrometry. Sialylation of placental transferrin receptor was examined by lectin affinity blotting with Sambucus nigra agglutinin and Maackia amurensis agglutinin. In order to trace possible inter-individual differences in N-glycosylation of the receptor, two preparations of placental transferrin receptor purified from two donors were compared. The results demonstrate that human transferrin receptor from placenta predominantly carries diantennary and triantennary N-acetyllactosaminic glycans as well as hybrid-type species, the galactose residues of which being almost completely substituted with (alpha 2-3)-linked sialic acid residues. Distinct differences were noted in the glycosylation pattern of the receptor from different individuals. Transferrin receptor from donor A carried predominantly diantennary and triantennary complex-type glycans, in part fucosylated at the innermost N-acetylglucosamine residue, in addition to small amounts of bisected and of incomplete diantennary species. Placental transferrin receptor from donor B predominantly carried triantennary N-acetyllactosaminic glycans without fucose and hybrid-type oligosaccharides with four or five mannose residues. Distinct from placental transferrin receptor, the receptor from Hep G2 cells contained larger amounts of oligomannosidic glycans with six to nine mannose residues and tetrasialylated complex-type oligosaccharides apart from mono-, di- and trisialylated species.