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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.

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.

Publisher Summary This chapter discusses the functions of L2/HNK-1 (L2) carbohydrate in the nervous system. The L2 carbohydrate is carried by several neural recognition molecules. Its receptors are present on cell surface and extracellular matrix recognition molecules. Thus, the L2 carbohydrate is an important ligand in cell interactions in the nervous system and in the immune system, possibly triggering interactions between the cells of the neural and lymphoid lineages. The finely tuned regulation of L2 expression by the subpopulations of myelinating Schwann cells in the adult peripheral nervous system of mice raises the possibility that it may favor target preferential reinnervation by motor axons. The fact that recognition molecules can be both receptors and donors of functionally important carbohydrates points to an intricate network of multiple combinations in cell-to-cell and cell-to-matrix interactions in the nervous system, particularly because carbohydrate structures are subject to finely tuned-developmentally and cell type-specifically regulated synthetic mechanisms. The L2 carbohydrate is an eminent example of the ingenuity of nature to use carbohydrate structures in functional contexts.

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.