O‐Glycosylation of leukosialin in K562 cells

Abstract

The O-glycosylation of leukosialin, a major sialoglycoprotein found on leukocytes, has been studied in the human erythroleukemic cell line K562. The appearance of its O-linked chains has been followed in pulse-chase experiments with [35S]methionine by immunoprecipitation with an anti-peptide antiserum as well as with a lectin from Sulvia sclureu seeds (SSA) specific for GalNAc-Ser/Thr and the peanut (Aruchis hypogueu) agglutinin (PNA) which recognizes GalPl -+ 3GalNAc-Ser/Thr structures. An O-glycan-free precursor was converted into the fully O-glycosylated mature form within the 1 O-min labeling period and no intermediates carrying only GalNAc-Ser/ Thr structures could be detected. The ionophore monensin was used in order to slow down intracellular traffic and thus O-glycan synthesis. The drug partly inhibited the transport from rough endoplasmic reticulum (RER) to the Golgi and also the cell-surface expression of leukosialin. It was found to have a marked effect on the synthesis of O-linked carbohydrate structures of leukosialin since the amount of O-glycans containing only GalNAc or NeuNAca2 + 6GalNAc was significantly increased after monensin treatment. Under these conditions the biosynthesis of the N-glycan on leukosialin was completely arrested in an endoglycosidase-H-sensitive step of processing, whereas the O-glycans already contained galactose and sialic acid although at a reduced level. On the other hand, the small amounts of leukosialin expressed on the cell surface of monensin-treated cells carried the same glycans as those remaining blocked inside the cell. In addition, immunocytochemical studies using SSA and PNA on untreated K562 cells suggested the absence of detectable amounts of GalNAc-Ser/Thr-bearing glycoproteins in the RER as well as in the Golgi. In contrast Galfll --f 3GalNAc structures could be detected on intracellular membranes which were tentatively identified as the cis-Golgi. Together these results lead us to the following conclusions: N-glycan transfer occurs in the RER before the initiation of O-glycans which takes place at the entrance of the protein into the Golgi; further elongation of O-glycans with galactose and sialic acid follows very rapidly, probably before the final processing of N-glycans to complex-type structures.Leukosialin is a major sialoglycoprotein present at the surface of human leukocytes 111. This glycoprotein has been shown to carry about 90 O-linked oligosaccharides attached to a polypeptide of about 52 kDa. It was found that human leukosialin also contains one N-linked chain. In addition, the structure of the O-linked oligosaccharides present on leukosialin has been determined in detail in different cell lines and was shown to be characteristic for each cell lineage and for a maturational stage in a given cell lineage [2]. In particular, leukosialin from K562 cells carries short and simple O-glycans and can thus serve as a model system to study O-glycosylation as well as the temporal relationship between 0-and Nglycosylation.

Premelanosome Amyloid-like Fibrils Are Composed of Only Golgi-processed Forms of Pmel17 That Have Been Proteolytically Processed in Endosomes

“…Third, we show that ␣Pmel-I labels primarily ERGIC membranes by immunoelectron microscopy. This is consistent with the notion that conventional mucin-like O-glycosylation is initiated by ␣GalNAc transferases in the Golgi (55,56). Moreover, we show that processed forms of Pmel contain both O-linked oligosaccharides and modified N-linked oligosaccharides.…”

Comparison of the carbohydrate‐binding specificities of seven N‐acetyl‐D‐galactosamine‐recognizing lectins

“…Recently, the recognition of aGalNAc-Ser/ Thr structures by SSA has been used to study the initiation of 0-glycan synthesis in the human erythroleukemic K562 cell line. Immunoprecipitation of a highly 0-glycosylated membrane protein by a SSA -anti-SSA complex showed that the lectin binds only those glycoproteins which possess aGalNAcSer/Thr structures [41].…”

Different Fate of a Single Reporter Protein Containing KDEL or KKXX Targeting Signals Stably Expressed in Mammalian Cells

“…We have observed that newly synthesized CD8-K and CD8-E19, in contrast to CD8-S and unmodified CD8, require several hours to undergo the initial O-glycosylation events. There is compelling evidence that these events occur in the cis/medial Golgi compartments (Roth et al, 1994;Piller et al, 1989;Verde et al, 1995;Vassard et al, 1995). This finding may thus support the view of efficient retrieval occurring upstream of these compartments.…”