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. 2012 Nov 1;23(21):4175–4187. doi: 10.1091/mbc.E12-05-0411

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© 2012 Cline et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 7: — Free glycans in pmm2 morphants. Total free glycans were measured with FACE techniques from the same samples of zebrafish presented in Figures 5 and 6. (A) Free glycans were measured in control embryos, pmm2 morphants, and morphants rescued by coinjection of pmm2 mRNA. Results of three independent experiments, mean ± SEM. Control levels were arbitrarily set as 100%. (B) Representative FACE image showing total free glycans from control embryos, pmm2 morphants, mpi morphants, and pmm2/mpi double morphants. Glucose oligomer standards (G₄–G₇) are shown. As shown previously (Gao et al., 2011), G₃M₉Gn₂ released from the LLO pool by M6P action is glycosidically degraded, resulting in the heterogeneous mixture of free glycans detected. The most abundant glycan detected near the bottom of the gel is likely to be M₂Gn₂, based on migration. (C) Results for four independent experiments as in B, mean ± SEM, with control set as 100%. (D) Schematic showing that dolichol pyrophosphate can be recycled for multiple rounds of LLO synthesis while free glycans are released by the action of M6P. This explains why the molar increase of free glycans in pmm2 morphants vastly exceeds the measured loss of LLO (see Discussion).