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 Manα-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 Manα-1,2Man linkage formation and that in GPI-anchored proteins the third Man residue is attached by a Manα-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 Manα-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 α1,2-mannosyltransferase rather than incorporation of ManN into an aberrant GPI species.