Expression and Characterization of a Lactosaminoglycan‐Carrying Glycoprotein of Zajdela Hepatoma Cell Surface

Abstract

In poorly differentiated hepatoma cells, a glycoprotein carrying lactosaminoglycans is identified, and the structure of its glycan moiety is proposed. After membrane solubilization, protein fractionation by gel filtration, and electroelution, this glycoprotein (GPIII) was identified by its affinity for Datura stramonium lectin and its content in large glycopeptides. As shown by PAGE, GPIII has an apparent molecular mass of 100 kDa and is highly glycosylated (36%). It appears as an integral membrane glycoprotein. It is absent from normal hepatocytes, in that no heavy glycopeptides could be detected that bound to Datura lectin or to specific antiserum. The glycan moiety of GPIII has been analyzed according to carbohydrate composition, glycosidase treatment, affinity chromatography on immobilized pokeweed, Datura and Griffonia lectins, and by NMR and methylation analyses. The glycan is a N-linked tetraantennary lactosaminoglycan of 6.6 kDa, containing Gal, GlcNAc, Man, and NeuNAc in a 16: 14:3:4 molar ratio, with an average of three repeating unitshranch. Its P-Gal residues are in the penultimate position and are linked in PI-4 at least in four structural elements (three peripheral and one internal). It contains a very branched structure with Galal-3Gal/31-4GlcNAc side chains linked in the C6 position to an inner Gal residue in a main branch. a-Gal and NeuNAc residues [mainly NeuNAca(2-3) linkage] are expressed as the nonreducing terminal groups. A possible structural model is proposed for this heterogeneous lactosaminoglycan, although no definitive structure can be established. That this lactosaminoglycan-carrying glycoprotein GPIII is not expressed in hepatocytes suggests its expression to be linked to the undifferentiated and/or malignant state of this hepatoma.Keywords: membrane glycoprotein; protein carrier; malignant transformation; hepatoma cells ; N-glycan chain.Lactosaminoglycans are high-molecular-mass glycans that consist of Gal(~1-4GlcNAc@1-3) repeating units, attached as linear or branched structures to a (Man),(GlcNAc), core. They are sensitive to endo P-galactosidase, and differ either in their number of repeating units or through the presence of branching points in GalV1-6) or in the nature of their non-reducing terminal group (either NeuAc, a-Gal or P-Gal) [l -41.Initially discovered and characterized in erythrocytes [I -3, 5, 61, lactosaminoglycans are also expressed in significant amounts in the hematopoietic systems [7-91, and at the surface of embryonic [lO-12] and transformed cell lines [13-181, and are described in limited amounts in normal adult tissues [2, 171. Furthermore these structures display dramatic changes in their branching patterns and peripheral substitutions during cell differentiation, oncogenesis and embryonic development. Although a relationship seems to exist between these alterations in lactosaminoglycan patterns and the biological processes [4,12, 191, the function of these glycans remains unknown.Correspondence to R. Bourrillon, U.F.R. BiomCdicale des Saints- Although di...

Novel forms of protein glycosylation

“…Thc core structures tire <~cncrai-ly 131,2-xxlosylatcd cmd al,6-f'tlCosylatcd [31 ]-A glycoprotein carrying polylactosaminnglycans has been identificd in Zajdela hepatoma cells and the structttres of its N-glycans have bccn established. The carbohydrate chain is a tetra-antennary lactosaminoglycan of 6.6 kl)a, containing galactose, GIcNAc, mannose and Net,SAc in a 16:14:3:4 ratio, with an average of three repeating N-acctvllactosamine units per branch [22].…”