Biosynthesis and glycosylation of the human complement regulatory protein decay-accelerating factor

The biosynthesis and oligosaccharide structure of the human complement regulatory glycoprotein decay-accelerating factor (DAF) were studied in erythrocytes and cell lines. Initial information relative to carbohydrate moieties of DAF was obtained by enzymatic digestions. The 74,000 M(r) erythrocyte DAF was lowered 3000 by endoglycosidase F, whereas endoglycosidase H had no effect, indicating one N-linked complex-type unit. Treatment with endo-α-N-acetylgalactosaminidase to remove O-linked oligosaccharides resulted in a 48,000 M(r) molecule (67% of the M(r) shift being due to sialic acid), which decreased to 45,000 M(r) after sequential endoglycosidase F treatment. To additionally define the oligosaccharide structure and identify precursors in biosynthetic pathways, DAF was studied in the HL-60 cell line differentiated by vitamin D toward monocytes. Pulse-chase experiments with [³⁵S]methionine revealed a precursor species of 43,000 M(r) that underwent an early post-translational modification to a 46,000 M(r) intermediate, and subsequently was chased into a mature species of 80,000 M(r) that aligned with ¹²⁵I surface-labeled DAF from these cells. All three forms of DAF were approximately 3000 lower in M(r) in the presence of tunicamycin. The two lower M(r) DAF species were sensitive to endoglycosidases F and H but not to neuraminidase or endo-α-N-acetylgalactosaminidase. In summary, DAF is synthesized as a 43,000 M(r) precursor species containing one N-linked high-mannose unit. Before entering the central region of the Golgi, it is converted to a 46,000 M(r) species by an as yet unknown post-translational modification. The 46,000 M(r) form is converted to the 74,000 M(r) (erythrocyte) or 80,000 M(r) (leukocyte) membrane form of DAF by the addition of multiple, sialylated O-linked oligosaccharide chains (responsible for the large electrophoretic mobility shift) and conversion of the single N-linked high-mannose unit to a complex-type structure. The cell-specific M(r) variation between red and white blood cells arises during this post-translational modification from the 46,000 M(r) biosynthetic intermediate to the mature DAF species expressed on the cell surface.