JBC, Vol. 252, Issue 7, 2372-2383, Apr, 1977
Glycoprotein biosynthesis in myeloma cells. Characterization on nonglycosylated immunoglobulin light chain secreted in presence of 2-deoxy-D-glucose
P. K. Eagon and E. C. Heath
The biosynthesis and secretion of a glycosylated, K-type immunoglobulin
light chain (K-46) was studied in a mouse myeloma tumor, mineral oil
plasmacytoma-46B. Viable single cell suspensions were prepared from excised
tumors and optimal conditions were established for incorporation of amino
acid and carbohydrate precursors into the protein synthesized and secreted
by the cells. The glucose analog, 2-deoxy-D-glucose, was utilized as an
inhibitor of glycosylation to determine the role of glycosylation in the
biosynthesis, intracellular transport, and export of the protein from the
cell. It was determined that 6 mM 2-deoxyglucose prevents the incorporation
of glucosamine, mannose, and galactose into secreted protein, but permits
the incorporation of leucine at approximately 40% of control values. The
nonglycosylated protein, secreted in the presence of 2-deoxyglucose, was
characterized as a nonglycosylated form of K-46 light chain by the
following criteria: (a) electrophoresis in polyacrylamide gels containing
sodium dodecyl sulfate, (b) reactivity of the nonglycosylated protein with
antisera prepared against native, fully glycosylated, K-46 light chain, (c)
analysis of the protein by gel filtration techniques, (d) behavior of the
protein on lectin-derivatized Sepharose, and (e) analysis of tryptic
peptides derived from the protein. We have concluded that
2-deoxyglucose-inhibited cells synthesize and secrete the normal
polypeptide chain of K-46 devoid of its carbohydrate side chain indicating
that glycosylation is not an essential step in the biosynthesis,
intracellular transport, or export of this protein that is normally
synthesized and secreted in a glycosylated form. Under conditions of
2-deoxyglucose inhibition, the nonglycosylated form of K-46 light chain
constitutes a significantly greater proportion of accumulated intracellular
protein, suggesting that the biosynthesis of the polypeptide chain of K-46
light chain proceeds at a nearly normal rate, but that the absence of the
carbohydrate side chain of the protein retards, but does not prevent, the
intracellular transport of the protein and its export from the tumor cell.
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