The loss/reduction of N-glycans from proteins usually results in the protein migrating faster upon SDS-PAGE, due to the reduction in hydrodynamic radius caused by the loss of sugars more than compensating for the increased amount of SDS bound to the polypeptide; moreover the extra SDS increases the charge of the polypeptide-SDS complex, which will contribute to faster migration. However, it is well established that in some circumstances (= the “appropriate” size change of N-glycan), then the decrease in hydrodynamic size caused by the loss of sugar is more than compensated for by the increase in hydrodynamic size due to extra SDS binding. The result is that the less glycosylated forms of the proteins now run slower upon SDS-PAGE; the extra charges due to the increased amount of SDS is not sufficient to overcome the increase in size.

However, as noted in the paper, the blots of the FGFRs demonstrate that in the absence or presence of the SiRNA targeting mannose phosphate isomerase (MPI) there is no change in the migration of the either FGFR1 or FGFR2 (Figs 1D, 2A-E, 4B, 5B: http://www.plosone.org/ar...).

Likewise EGFR and c-MET migrate identically whether the cells are treated or not with the SiRNA, as they apparently run at the same position (Figs 4C and 5B, C:
http://www.plosone.org/ar...).

The absence of change in size is interpreted as a selective inhibition of glycosylation due to MPI knockdown. Since this is true for four different glycoproteins, FGFR1, FGFR2, EGFR and c-MET, it is reasonable to conclude that knockdown of MPI is having a very modest effect on N-glycosylation. In the methods the measurement of MPI activity is described, but I could not find data relating to this in the paper. What is the residual MPI activity?

It is established that knockout of MPI does not necessarily abrogate N-glycosylation, due to salvage of mannose from degradation of N-linked glycans.
http://www.jbc.org/conten...
In the present case serum is present with its N-linked glycoproteins, and prior to knockdown the cells had a full complement of N-glycans. Both will provide a source of mannose through salvage, and, moreover, knockdown of MPI is necessarily incomplete. In the absence of MPI, high levels of mannose through exogenous addition are toxic. So the residual level of MPI may be sufficient for normal N-glycan biosynthesis; the higher levels present in untreated cells reflecting perhaps the need to buffer against high mannose. The increased recycling of N-linked glycoproteins (perhaps evidenced by the altered location of receptors observed in Fig 3
http://www.plosone.org/ar...)
would then provide for normal N-glycosylation, but in the context of an altered metabolic state (perhaps akin to a “starved” state).

The interpretation in the present paper though is that a reduction in the mannose pool has a selective effect on particular N-glycans, and so alters the activity of the FGFR. Such an effect would presumably relate to a small reduction in glycan size, which would be insufficient to change the migration of the protein upon SDS-PAGE. In contrast, removal of N-glycans from Ig loops 2 and 3 of FGFR1 (where the FGF ligand and HS-co-receptor bind) increases the affinity of at FGFR1 for both FGF2 and heparin, indicating that some N-glycans in Ig loops 2 and 3 are inhibitory; modelling suggests that this may indeed be the case, since glycosylation of certain asparagines would likely interfere with binding of ligand or co-receptor, or with receptor dimerisation itself.
http://www.jbc.org/conten...
In agreement with the latter in vitro analysis, mutation of asparagines in Ig loops 2 and 3 in the C. elegans FGFR, Egl-15, increases the activity of the receptor in vivo
(http://www.jbc.org/conten...).

The effect of MPI knockdown in the present paper is a reduction in FGFR signalling. A key question is whether this is a direct effect on N-glycosylation of the receptor or an indirect effect. Since structural and in vivo analyses demonstrate that N-glycans act as a brake on FGFR activity and that there is no demonstrable effect on N-glycosylation of FGFR1, FGFR2, EGFR or c-MET, is a more plausible (for now) interpretation that MPI knockdown leads to an altered (“starved”) metabolic state, which would impact in a major way on specific RTK signalling axes?

It would be good to see the entire blots, rather than just the thin slice where the main band appears, which would be consistent with PLOS guidelines on data sharing.

c-MET phosphorylation is directly affected by the scrambled SiRNA control (Fig. 4C) ?

AKT seems somewhat variable. Relative to actin, the levels of AKT seem higher in Fig. 4B than in Fig. 4C?