Mannosamine inhibits the synthesis of putative glycoinositol phospholipid anchor precursors in mammalian cells without incorporating into an accumulated intermediate.

Mannosamine (ManN) has been reported to inhibit the formation of Man alpha-1,2Man linkages and to prevent the anchoring of membrane proteins by glycoinositol phospholipids (GPIs). In this paper, the effect of ManN on the synthesis of putative GPI anchor precursors in mammalian cells was studied. Three different cell lines were analyzed: HeLa cells and Thy-1 negative lymphoma mutants B and F that accumulate two-mannosyl GPI (Man2GPI) and three-mannosyl GPI (Man3GPI) species, respectively. ManN did not alter the incorporation of [3H]Man into the Man2GPI in mutant B cells. However, when either HeLa cells or mutant F cells were labeled with [3H]Man in the presence of ManN, [3H]Man incorporation into Man3GPi species was blocked and the TLC profile of [3H]Man-labeled glycolipids resembled that obtained with mutant B cells. Further characterization of the predominant labeled glycolipid that accumulated in ManN-treated HeLa cells by enzymatic and chromatographic criteria and permethylation analysis confirmed the structure as a Man2GPI indistinguishable from the Man2GPI that accumulates in mutant B cells. Based on previous reports that ManN inhibits Man alpha-1,2Man linkage formation and that in GPI-anchored proteins the third Man residue is attached by a Man alpha-1,2Man linkage, these results indicate that the third Man residue that distinguishes the putative Man2GPI precursor from the Man3GPI precursor also is attached by a Man alpha-1,2Man linkage. Furthermore, the observation that Man2GPI rather than a new GPI species containing ManN accumulated during ManN treatment revealed that the mechanism of ManN inhibition primarily involved inhibition of an alpha 1,2-mannosyltransferase rather than incorporation of ManN into an aberrant GPI species.