Glycosylation of human leukocyte locus A molecules is dependent on the cell type.

Peripheral blood monocytes and B cells were isolated from a normal donor, and a portion of the B cells was transformed by the Epstein-Barr virus (EBV). Human leukocyte locus A (HLA) class-I and class-II molecules were immunoprecipitated by specific monoclonal antibodies after cell labeling with [3H]mannose. Glycopeptides of HLA molecules were obtained by pronase digestion and were analysed by lectin-affinity chromatography. Complex structures were hydrazinolysed, and their sialic acid content was analysed by ion-exchange chromatography, whereas the high-mannose structures were separated by HPLC. In normal cells, class-I antigens bear principally fucosylated biantennary structures while HLA-DR class-II antigens bear bi-, tri- and tetra-antennary structures and high-mannose structures. HLA antigens are more sialylated on normal B cells than on normal monocytes. An EBV cell line had a very different pattern of HLA-antigen glycosylation when compared with the original B cells. In the transformed cells, the fractions containing biantennary structures are largely decreased. In contrast, an increase of the tri- and tetra-antennary structure fractions is noticed, particularly in class-II molecules, while both triantennary and high-mannose structures are increased in class-I molecules. Moreover, when compared to normal B cells, the complex structures of class-I antigens in the EBV-transformed B-cell line are undersialylated while they are oversialylated in the case of the class-II molecules.

Characterization of N-linked oligosaccharides of an HLA-DR molecule expressed in different cell lines.

In order to determine the factors that influence the glycosylation of an integral membrane protein, we investigated the N-glycosylation of a molecule of the human major histocompatibility complex (MHC) class II, the HLA-DR antigen. This glycoprotein was studied in a human Epstein-Barr-virus-transformed B cell line and in a mouse fibroblastic cell line co-transfected with DR alpha and DR beta genes. We observed that the HLA-DR-antigen glycosylation pattern depends on the cell line in which processing takes place and is closely related to the glycosylation pattern of the overall cellular glycoproteins. Furthermore, when comparing the glycosylation of the separated alpha- and beta-chains, differences were noticed within the same molecule, showing the importance of the individual peptide backbone for the glycosylation process.

Analysis of HLA-DR antigen glycosylation by serial lectin affinity chromatography.

The structure of the N-linked oligosaccharides of HLA-DR molecules from an HLA-DR homozygous B-lymphoblastoid cell line (CA) was characterized by serial lectin affinity analysis. Glycans lectin affinity profile were obtained for the HLA-DR complex and separated alpha- and beta-chains. Two structurally distinct glycosylation patterns were detected for the alpha-chain species, the alpha 1 with high-mannose- and complex-type oligosaccharides and alpha 2 with a complex-type one. In contrast, the beta-chain species contains only complex-type oligosaccharides. Further oligosaccharide heterogeneity is found for each alpha 1-, alpha 2- and beta-chain described. In contrast to murine and guinea-pig Ia antigens, no significant amount of glycopeptides with biantennary structure was found in HLA-DR.