As mentioned above, DG on skeletal muscle cells can be differentially glycosylated. In normal muscle, DG localized at the sarcolemma preferentially binds WGA, whereas DG localized at neuromuscular junctions preferentially binds WFA (6
). Substitution of utrophin for dystrophin to ameliorate pathophysiology in specific models of muscular dystrophy coincides with an increase in the WFA-binding fraction of DG and increased binding of WFA to the sarcolemma (6
). In dystrophin-deficient mdx
muscle, WFA binding to skeletal muscle DG is substantially increased, consistent with the increased substitution of utrophin for dystrophin around the extrasynaptic sarcolemma. This is shown in , A
, demonstrating WFA binding to quadriceps muscle sections from wild type and mdx
mice; note the robust WFA binding completely surrounding the myofibers in mdx
mice, compared with the sparse, punctuate WFA binding to neuromuscular junctions in wild type muscle. A similar increase in WFA binding is seen in muscle sections from mice overexpressing constitutively active Akt (D
), which promotes utrophin usage in skeletal muscles of wild type mice (19
). Thus, increasing utrophin usage in skeletal muscle correlates with increased WFA binding to the sarcolemma. Importantly, WFA binding to muscle from mice lacking the α7 integrin chain is comparable with that observed for binding to wild type muscle (, A
), indicating that α7 integrin is not essential for WFA binding to skeletal muscle.
FIGURE 1. WFA binding to sections of murine muscle. Transverse sections of quadriceps from wild type (wt) (A), mdx (B), α7 integrin null (C), or Akt-overexpressing (Akt-DTg) (D), were stained with biotinylated WFA, and bound lectin was detected with fluorescein-streptavidin (more ...)
We thus designed a high throughput screen to identify compounds that would increase WFA binding to cultured mouse myotubes derived from C2C12 cells. We reasoned that any pharmacologic effect that increased WFA binding could result in a concomitant increase in utrophin usage in muscle cells, a long term goal in developing novel therapies for DMD. The screen was mechanism-blind, in that we only measured the end point of increased WFA binding; for example, increased WFA binding could be due to altered expression of glycosyltransferases or of specific glycoprotein acceptors or to increased transport or decreased turnover of WFA binding glycoproteins at the cell surface. For the screen, C2C12 myoblasts were cultured in 384-well plates in growth medium for 2 days, and then a single concentration of test compounds (10 μm) or vehicle was added to duplicate wells for an additional day. On day 3, differentiation medium containing the test compound was added to the cells, and WFA binding to fused myotubes was measured by chemiluminescence after 4 days. We screened the Prestwick library of ~1200 FDA-approved drugs, and an increase in WFA binding of 2-fold or greater was considered a positive response. As a control, we used C2C12 cells stably overexpressing Galgt2 to demonstrate that we could detect increased WFA binding to myotubes derived from these cells compared with C2C12 transfected with vector alone (data not shown).
lists the six compounds that gave a ≥2-fold increase in WFA binding to C2C12 myotubes in two consecutive screens. Importantly, none of the six compounds have similar known mechanisms of action; nor do they group into common families based on structure. We reassayed the six compounds in confirmatory assays to determine dose-response characteristics over a range of concentrations from 10 to 100 μm. Only one of the six compounds, lobeline, demonstrated a consistent dose-dependent effect on WFA binding to C2C12 myotubes in the confirmatory assay format. As shown in A, lobeline treatment increased WFA binding to C2C12 myotubes 2–4-fold compared with vehicle control. The maximum increase in WFA binding was observed at a concentration of 100 μm; although we observed no effect of lobeline on myotube viability at or below this concentration, we did detect loss of C2C12 myotube viability at higher concentrations of lobeline (data not shown), so a maximal concentration of 100 μm was used for experiments with C2C12 cells. Although we observed increased WFA binding to lobeline-treated myotubes, we did not detect increased binding of two other lectins, peanut agglutinin and phytohemagglutinin (PHA) in the screen (data not shown).
FIGURE 2. Lobeline increases WFA binding to myotubes in vitro.
A, dose-response curve of WFA binding to lobeline-treated (filled bars) versus control (DMSO, 0; open bars) C2C12 myotubes. Lobeline or vehicle was added to myoblasts at the time of differentiation (more ...)
We confirmed the effect of lobeline on primary myoblasts cultured from limb muscles of neonatal wild type and mdx mice. As described for C2C12 cells, lobeline was added to myoblast cultures at the indicated concentrations at the time that differentiation was induced, as in the confirmatory assays. Two days after incubation in the presence or absence of lobeline, WFA binding was measured. As shown in , B and C, lobeline increased the binding of WFA to both wild type and mdx myotubes, with the maximal effect observed at 200 μm; this concentration had no effect on the viability of primary muscle cells (data not shown). Thus, lobeline increased binding of WFA to mdx myotubes lacking dystrophin, indicating that the effect of lobeline was not dystrophin-dependent, as well as to cultured C2C12 cells and wild type myotubes.
In designing the screen, we reasoned that, if changes in cell surface glycosylation were required for increased WFA binding, it would be essential to add drugs at the initiation of myotube differentiation, to ensure that new glycoproteins entering the Golgi encountered appropriate glycosyltransferase enzymes; this was the rationale for adding the drugs with the differentiation medium in the original high throughput screen. However, we found that, when lobeline was added to confluent, differentiated C2C12 myotubes, we still observed a 2–4-fold increase in WFA binding (D
), demonstrating that lobeline could increase WFA binding to differentiated cells in vitro
and suggesting that any activity of lobeline might be effective in mature skeletal muscle. In contrast, we saw no increase in WFA binding to C2C12 myoblasts maintained in growth medium in the presence of lobeline (supplemental Fig. 1
), indicating that either the glycoprotein backbones or the glycan structures recognized by WFA are preferentially expressed on differentiated myotubes rather than on myoblasts.
We next asked if specific glycoproteins on muscle cells demonstrated increased WFA binding after lobeline treatment. As shown in A
), treatment of C2C12 cells with lobeline increased the abundance of several glycoproteins that were precipitated with WFA from cell lysates. After these samples were probed with WFA (A
), the blot was reprobed with mAb IIH6, which recognizes O
-mannose glycans that are critical for laminin binding (right
). WFA-binding was detected in control treated cells, but the abundance of WFA binding increased dramatically in lobeline-treated cells. Similarly, lobeline treatment increased the abundance of WFA-binding glycoproteins in primary myotubes derived from wild type (B
) and mdx
) mouse muscle. In primary mouse myotubes, we again observed a dramatic increase in WFA-precipitation of IIH6-reactive glycoproteins, as well as β-DG, after treatment with lobeline. These data demonstrate that lobeline treatment affects multiple glycoproteins that bear GalNAc moieties recognized by WFA, including DG; an increase in WFA-reactive α-DG was also observed in mice overexpressing Galgt2 (6
FIGURE 3. Identification of WFA binding glycoproteins in control-treated versus lobeline-treated cells.
A, equal amounts of cell protein from lysates of C2C12 myotubes treated with 100 μm lobeline (L) or control (C) for 48 h were precipitated with WFA beads, (more ...)
In patients with DMD, loss of dystrophin results in loss of the entire DGC and thus reduced muscle cell adhesion to extracellular matrix. Specifically, reduction in sarcolemmal α-DG results in decreased adhesion to laminin in the extracellular matrix. We asked if lobeline treatment affected laminin binding to proteins from control or treated wild type or mdx myotubes. To detect laminin binding to cell surface proteins, equal amounts of protein from each sample were separated by SDS-PAGE, laminin was added to blots, and bound laminin was detected with anti-laminin (). Samples were also probed with IIH6 mAb. We observed a dramatic increase in the abundance of IIH6 reactivity and in laminin binding to glycoproteins from lobeline-treated cells.
FIGURE 4. Lobeline treatment increases laminin binding. Lysates of surface biotinylated wild type (wt) (A) or mdx myotubes (B) treated with lobeline (L) or control (C) were separated by SDS-PAGE and blotted, laminin was incubated with the blots, and bound laminin (more ...)
To further examine the effect of lobeline on the abundance of DGC/UGC components, as well as other sarcolemmal proteins involved in muscle cell adhesion and function, we compared the abundance of several proteins from control- or lobeline-treated myotubes derived from wild type and mdx
primary muscle cells. Lysates from equal numbers of control- or lobeline-treated cells were separated by SDS-PAGE, and sarcolemmal proteins were examined by immunoblotting. As shown in A
, lobeline treatment of wild type myoblasts increased the abundance of several proteins, including agrin, dystrophin, utrophin, α- and β-DG, sarcospan, sarcoglycans, and β1D integrin, whereas the abundance of caveolin-3 was not affected. A similar effect was found when lobeline-treated mdx
myotubes were examined (B
). In these cells, the increased abundance of agrin, utrophin, and α- and β-DG was particularly striking. Overall, in control-treated mdx
cells, there was increased abundance on a per cell basis of many proteins, including agrin, utrophin, and β1D integrin, compared with control-treated wild type cells; this most likely reflects the compensatory mechanisms that occur in mdx
mouse muscle. However, even with higher base-line abundance of many proteins in mdx
cells, lobeline further increased the abundance of sarcolemmal proteins. Intriguingly, the increased abundance of agrin, α- and β-DG, and utrophin in cells treated with lobeline was consistent with the increased abundance of these proteins with Galgt2 overexpression in various dystrophic mouse models (6
), suggesting that increasing WFA binding to muscle cells by either pharmacologic or genetic approaches results in increasing abundance of sarcolemmal proteins.
FIGURE 5. Lobeline increases abundance of multiple muscle cell proteins.
A, wild type (wt) primary myoblasts differentiated in the presence of 100 μm lobeline (L) or control (C) were solubilized in radioimmune precipitation assay buffer, and 60 μg (more ...)
We next asked what types of glycans are required for WFA binding to myotubes. Because lobeline increased laminin binding to myotube glycoproteins (), an effect involving O
-mannose glycans on muscle cell glycoproteins, and because O
-mannose glycans in general are known to be critical for muscle cell viability and function, we asked if the lobeline-mediated increase in WFA binding required O
-mannose glycans. Although the precise structure of the complete laminin-binding glycans elaborated on O
-mannose structures is not known, it is intriguing that one branch of the O
-mannose glycan bears the SAα2,3Galβ1,4GlcNAc trisaccharide that could be modified to create the Sda tetrasaccharide, SAα2,3Gal (β1,3GalNAc)β1,4GlcNAc (12
). We reduced expression of POMT1/POMT2, the enzyme complex that adds the initiating mannose residue, in C2C12 myoblasts by siRNA; transfections were performed just prior to the addition of differentiation medium with lobeline or vehicle control, and WFA binding was measured after 48 h. We confirmed that reduction in POMT1/POMT2 mRNA resulted in reduced expression of O
-mannose glycans detected by mAb IIH6, demonstrating loss of O
-mannose glycans (A
). However, reduction in POMT1/POMT2 expression resulted in no loss of WFA binding to lobeline-treated cells; rather, there was a modest but reproducible increase in WFA binding to lobeline-treated myotubes in the absence of O
-mannose glycans (B
). We also reduced expression of Large, which modifies O
-mannose structures to create laminin binding glycans, and also observed reduced IIH6 binding but increased WFA binding to lobeline-treated cells (not shown).
FIGURE 6. O-Mannose glycans are not required for increased WFA binding after lobeline.
A, reduction in POMT1/POMT2 by siRNA (+) versus scrambled control (−) in C2C12 cells treated with lobeline (L) or control (C) resulted in loss of mAb IIH6 binding, indicating (more ...)
As mentioned above, muscle cell glycoproteins, such as α-DG, also bear mucin type O
-glycans and N
-glycans; for example, α-DG has three N
-glycosylation sites, and β-DG has one N
-glycosylation site (15
). Thus, we asked if N
-glycans were important for WFA binding to control- and lobeline-treated cells. To globally reduce complex N
-glycans on C2C12 cells, we treated myoblasts with DMNJ, which inhibits mannosidase I and prevents creation of complex N
-glycan structures from high mannose precursors (16
). Complex N
-glycans could bear GalNAc residues, recognized by WFA, either on Sda tetrasaccharides or as terminal structures on N
-glycan branches. As shown in A
, DMNJ had the predicted effect on C2C12 cells because DMNJ treatment abrogated binding of the lectin PHA that recognizes branched complex N
-glycans. Surprisingly, DMNJ also dramatically reduced WFA binding to control- or lobeline-treated cells. As shown in B
, the loss of complex N
-glycans reduced WFA binding by >90% to both control- and lobeline-treated C2C12 myotubes. Thus, complex N
-glycans bear the glycan structures recognized by WFA on muscle cells, and increased WFA binding after lobeline treatment requires expression of complex N
FIGURE 7. Complex N-glycans are required for WFA binding.
A, C2C12 myoblasts were treated with DMNJ or vehicle control, and binding of biotinylated PHA (solid line) was determined by flow cytometry, using biotinylated BSA as a control (dotted line). PHA binding (more ...)
To analyze the effect of lobeline on the abundance of N
-glycans involved in increased WFA binding, N
-glycans of C2C12 cells grown either in the presence or absence of lobeline were analyzed. In order to quantify the abundance of observed N-glycan structures from the treated and control populations, an IDAWG tandem mass spectrometry-based strategy was applied (22
). After characterizing the released N
-glycans and quantifying the abundance of the observed glycan structures, we determined the relative abundance for N
-glycans released from lobeline-treated samples over the control (supplemental Tables 1 and 2
). Specifically, the majority of the N
-glycans released from lobeline-treated C2C12 cells were observed to be elevated over control. A total of 32 glycan masses were observed, of which 30 were able to be quantified, and 84% of the quantified glycans showed an increase of at least 1.5-fold. Of particular interest, two proposed GalNAc-containing structures were elevated 2.8–5.5-fold (); however, glycans bearing the Sda epitope were not substantially elevated.
FIGURE 8. Abundance of N-glycans from lobeline-treated cells relative to control.
N-Glycans were released from lysates of C2C12 cells grown in the presence or absence of lobeline, in both regular 14N medium and 15N medium. Analysis was performed in duplicate with (more ...) N
-Glycosylation of numerous glycoproteins on C2C12 cells has been described (25
); however, in contrast to the extensively studied O
-mannose glycans on α-DG, which are known to participate in laminin binding, no clear functions for N
-glycans on many muscle cell glycoproteins have been proposed. Because increased WFA binding correlated with increased laminin binding to cell surface glycoproteins from lobeline-treated cells (), we asked if loss of WFA-binding glycans would affect laminin binding by muscle cell glycoproteins. Cells were treated with or without DMNJ, and extracts were analyzed by immunoblotting. Loss of complex N
-glycans did not reduce reactivity with IIH6; in fact, reactivity of DMNJ-treated cells with an antibody recognizing an α-DG core epitope actually increased (A
), suggesting that loss of complex N
-glycans may have increased availability of this specific epitope. Also, there was no significant difference in overall IIH6 reactivity in DMNJ-treated versus
control-treated cells. However, there was a significant decrease in laminin binding to glycoproteins from DMNJ-treated cells compared with controls (B
). These data imply that complex N
-glycans may indirectly participate in promoting laminin binding or, alternatively, that loss of complex N
-glycans in DMNJ-treated cells may reduce laminin binding by altering glycoprotein structure or the accessibility of laminin binding sites.
FIGURE 9. Loss of complex N-glycans reduces laminin binding. Cells treated with or without DMNJ were analyzed by immunoblotting to assess effects on laminin binding. A, loss of complex N-glycans did not affect production or processing of α-DG; α-DG (more ...)