Expression and characterization of a lactosaminoglycan-carrying glycoprotein of Zajdela hepatoma cell surface--structural analysis of the carbohydrate moiety.

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 units/branch. Its beta-Gal residues are in the penultimate position and are linked in beta1-4 at least in four structural elements (three peripheral and one internal). It contains a very branched structure with Gal alpha1-3Gal beta1-4GlcNAc side chains linked in the C6 position to an inner Gal residue in a main branch. Alpha-Gal and NeuNAc residues [mainly NeuNAc alpha(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.

Structural differences between complex-type Asn-linked glycan chains of glycoproteins in rat hepatocytes and Zajdela hepatoma cells.

Using serial lectin-affinity chromatography, glycosidase digestion, and NMR and methylation analysis, the structures of complex N-linked glycan chains (M(r) range 2000-3500) of rat hepatocytes and poorly differentiated chemically transformed Zajdela ascites hepatoma cells were determined and compared. The results revealed considerable differences between the two cell types: (i) hepatoma cells only expressed tri- and/or tetra-antennary complex N-linked glycan chains, whereas hepatocytes displayed large amounts of bi-antennary N-linked structures and smaller amounts of tri-/tetra-antennary structures; (ii) 20% of the glycan chains in hepatoma cells contained a bisecting GlcNAc residue which was beta (1,4)-linked to the beta-mannosyl residue of the core and was not detected in the hepatocytes; (iii) hepatoma cells expressed a high proportion of the fucosylated or not GlNAc beta (1,6) Man alpha 1-->branch, whereas hepatocytes only contained a little of this branch; (iv) hepatoma cells, but not hepatocytes, exhibited a repeating (Gal beta(1,4) GlcNAc beta (1,3)) sequence characteristic of poly-N-acetyllactosaminoglycans. These glycans were capped by both alpha-galactosyl and sialyl residues; (v) The alpha (2,3)/alpha (2,6)-linkage ratio of sialic acid was significantly higher in hepatoma cells (4/1 vs. 2/1 in hepatocytes); (vi) Only hepatocytes expressed an unusual structure in which a sialyl residue was alpha (2,6)-linked to a GlcNAc residue located within a NeuAc alpha (2,3) Gal beta (1,3) GlcNAc branch which was beta (1,4)-linked to Man alpha 1,3-->. The differences between these complex N-linked glycan chains in hepatocytes and hepatoma cells seem to be both quantitative and qualitative, since some glycan structures were only present in one cell type.

Identification of peanut agglutinin receptors related to the state of tumoral liver cell differentiation.

The relationship between cell differentiation/tumorisation and plasma membrane glycoproteins was approached using peanut agglutinin (PNA) a lectin specific for the Gal-beta(1,3)GalNAc sequence and a homologous cell system consisted of normal rat hepatocytes (HyC) and a poorly differentiated hepatoma (ZHC). This work is focused on the molecular nature of PNA receptors. PNA bound strongly to ZHC, but bound very weakly, if at all to hepatocytes. After sialidase treatment this binding was slightly enhanced in ZHC and HyC. The total number of binding sites on ZHC was 9.6 x 10(6)/cell and 1.2 x 10(7)/cell before and after sialidase treatment respectively. In contrast, this number could not be calculated on HyC, even after sialidase treatment. The PNA receptors were isolated and identified from ZHC using affinity chromatography on immobilized PNA and lectin overlay. Two bands were revealed after SDS-PAGE of PNA receptors: a major one with a relative molecular mass of 160 kDa and a minor one of 110 kDa. The latter disappeared after sialidase treatment of ZHC suggesting the possibility that these two bands could be less and more sialylated forms of the PNA receptors, respectively. In contrast no PNA receptors could be detected on HyC. These PNA receptors could be considered O-linked glycoproteins containing the Gal-beta(1,3)GalNAc disaccharide because: i) PNA carbohydrate specificity toward this disaccharide found in this glycoprotein type; ii) their carbohydrate composition with Gal and GalNAc but not man residues; iii) their sensitivity to alkaline treatment; and iv) strong inhibition of PNA binding to ZHC with the Gal-beta(1,3)GalNAc structure. The absence of PNA receptors on HyC appeared to be related to the absence of this glycoprotein containing the disaccharide but not to the change or failure of glycosylation of the polypeptide chain of PNA receptors. The relationship between the presence of PNA receptors and differentiation/tumorisation phenomena as well as the mechanism that induced the expression of these receptors are discussed.