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Glycophorin expression in murine erythroleukaemia cells
J B Ulmer1, E D Dolci, G E Palade
1Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510.
Abstract:
We have identified mature and putative precursor forms of glycophorins expressed in a virus-transformed murine erythroleukaemia (MEL) cell line and compared them with their normal erythroblast counterparts. The following differences were found: (1) the two major MEL cell glycophorins (apparent Mr values 29-30 and 43(x10(3] have greater mobility on polyacrylamide gels than their normal gp-3 and gp-2 counterparts, due at least in part to differences in their oligosaccharide sidechains; (2) MEL cell gp-3 consists of two discrete proteins; and (3) there are more potential glycophorin precursors in MEL cells than in normal mouse erythroblasts. Four proteins, with apparent Mr values of 21, 23, 26 and 27(x10(3], have tentatively been identified as glycophorin precursors, based on the following findings: (1) they are immunologically related to the glycophorins; and (2) their synthesis was induced by dimethyl sulphoxide coincidentally with that of gp-3 and gp-2. They do not appear to be glycoproteins, as evidenced by their lack of incorporation of [3H]galactose, [3H]glucosamine or [3H]mannose. In contrast, gp-3 and gp-2 incorporated [3H]galactose and [3H]glucosamine but not [3H]mannose. Partial characterization of the glycan moieties of MEL cell glycophorins indicates that they consist mostly of tri- and tetrasaccharides, with no indication of any N-linked chains. Hence, the glycans of MEL cell glycophorins are mostly (if not all) O-linked. Furthermore, treatment with N-glycanase did not change their electrophoretic mobility on polyacrylamide gels. MEL cell glycophorins were also shown to be modified by phosphoryl and fatty acyl groups.
Insights
Murine erythroleukaemia (MEL) cells show distinct glycophorin expression compared to normal erythroblasts, with altered oligosaccharide side chains and more precursor forms. These findings highlight differences in erythroid differentiation pathways.
Area of Science:
- Biochemistry
- Cell Biology
- Hematology
Background:
- Glycophorins are key membrane proteins in erythrocytes, crucial for cell structure and function.
- Virus-transformed murine erythroleukaemia (MEL) cells offer a model to study erythroid differentiation.
- Understanding glycophorin expression in normal versus transformed cells can reveal insights into cellular regulation.
Purpose of the Study:
- To identify and characterize mature and precursor forms of glycophorins in MEL cells.
- To compare glycophorin expression and structure between MEL cells and normal erythroblasts.
- To investigate the glycosylation patterns and post-translational modifications of MEL cell glycophorins.
Main Methods:
- Polyacrylamide gel electrophoresis to assess protein mobility.
- Immunological assays to detect glycophorin-related proteins.
- Radiolabeling with [3H]sugars (galactose, glucosamine, mannose) to study glycosylation.
- Treatment with N-glycanase to analyze N-linked glycans.
- Analysis of phosphoryl and fatty acyl group modifications.
Main Results:
- MEL cell glycophorins exhibit altered oligosaccharide side chains and higher electrophoretic mobility compared to normal counterparts.
- MEL cell gp-3 comprises two distinct proteins, and MEL cells possess more potential glycophorin precursors.
- Four proteins (Mr 21-27x10^3) were identified as potential glycophorin precursors, immunologically related and induced by dimethyl sulfoxide.
- Precursors lack glycosylation, while mature MEL cell glycophorins have predominantly O-linked glycans (tri- and tetrasaccharides) and are modified by phosphoryl and fatty acyl groups.
Conclusions:
- Virus-transformed MEL cells display significant differences in glycophorin expression and structure compared to normal erythroblasts.
- The identified precursors suggest a distinct pathway or altered regulation of glycophorin synthesis in MEL cells.
- The characterization of O-linked glycans and other modifications provides a deeper understanding of MEL cell glycophorin biology.