Development of Rabbit Monoclonal Antibodies for Detection of Alpha-Dystroglycan in Normal and Dystrophic Tissue

<div><p>Alpha-dystroglycan requires a rare <i>O</i>-mannose glycan modification to form its binding epitope for extracellular matrix proteins such as laminin. This functional glycan is disrupted in a cohort of muscular dystrophies, the secondary dystroglycanopathies, and is abnormal in some metastatic cancers. The most commonly used reagent for detection of alpha-dystroglycan is mouse monoclonal antibody IIH6, but it requires the functional <i>O</i>-mannose structure for recognition. Therefore, the ability to detect alpha-dystroglycan protein in disease states where it lacks the full <i>O</i>-mannose glycan has been limited. To overcome this hurdle, rabbit monoclonal antibodies against the alpha-dystroglycan C-terminus were generated. The new antibodies, named 5–2, 29–5, and 45–3, detect alpha-dystroglycan from mouse, rat and pig skeletal muscle by Western blot and immunofluorescence. In a mouse model of fukutin-deficient dystroglycanopathy, all antibodies detected low molecular weight alpha-dystroglycan in disease samples demonstrating a loss of functional glycosylation. Alternately, in a porcine model of Becker muscular dystrophy, relative abundance of alpha-dystroglycan was decreased, consistent with a reduction in expression of the dystrophin-glycoprotein complex in affected muscle. Therefore, these new rabbit monoclonal antibodies are suitable reagents for alpha-dystroglycan core protein detection and will enhance dystroglycan-related studies.</p></div

Development of Rabbit Monoclonal Antibodies for Detection of Alpha-Dystroglycan in Normal and Dystrophic Tissue

Alpha-dystroglycan requires a rare O-mannose glycan modification to form its binding epitope for extracellular matrix proteins such as laminin. This functional glycan is disrupted in a cohort of muscular dystrophies, the secondary dystroglycanopathies, and is abnormal in some metastatic cancers. The most commonly used reagent for detection of alpha-dystroglycan is mouse monoclonal antibody IIH6, but it requires the functional O-mannose structure for recognition. Therefore, the ability to detect alpha-dystroglycan protein in disease states where it lacks the full O-mannose glycan has been limited. To overcome this hurdle, rabbit monoclonal antibodies against the alpha-dystroglycan C-terminus were generated. The new antibodies, named 5–2, 29–5, and 45–3, detect alpha-dystroglycan from mouse, rat and pig skeletal muscle by Western blot and immunofluorescence. In a mouse model of fukutin-deficient dystroglycanopathy, all antibodies detected low molecular weight alpha-dystroglycan in disease samples demonstrating a loss of functional glycosylation. Alternately, in a porcine model of Becker muscular dystrophy, relative abundance of alpha-dystroglycan was decreased, consistent with a reduction in expression of the dystrophin-glycoprotein complex in affected muscle. Therefore, these new rabbit monoclonal antibodies are suitable reagents for alpha-dystroglycan core protein detection and will enhance dystroglycan-related studies.This article is published as Fortunato MJ, Ball CE, Hollinger K, Patel NB, Modi JN, Rajasekaran V, et al. (2014) Development of Rabbit Monoclonal Antibodies for Detection of Alpha-Dystroglycan in Normal and Dystrophic Tissue. PLoS ONE 9(5): e97567. doi: 10.1371/journal.pone.0097567.</p

Detection of αDG core protein from skeletal muscle of normal and dystroglycanopathy mice.

<p>A) Western blot analysis of αDG core protein detection by rabbit aDGct supernatants. Monoclonal antibodies 5–2, 29–5, and 45–3 were tested on replicate Western blots of solubilized murine skeletal muscle. Lane 1 contains normal murine skeletal muscle (LC) and lane 2 contains skeletal muscle from a mouse with a tamoxifen-induced fukutin-deficient dystroglycanopathy (KO). Detection with antibody IIH6 shows glycosylated αDG for comparison. Molecular weight standards are indicated in kDa. B) Wheat germ agglutinin (WGA) purifications of LC and KO mouse brain and heart lysates were conducted and the elution fraction was analyzed by Western blot. Monoclonal antibody media supernatants 5–2, 29–5, and 45–3 were used to detect αDG core protein on replicate blots; detection with IIH6 was performed by reprobing stripped blots. Molecular weight standards are indicated in kDa.</p

Detection of αDG core protein in rat skeletal muscle, heart, and brain tissue.

<p>Detection of rat αDG by aDGct antibody supernatants was analyzed via Western blot and immunofluorescence. A) Monoclonal antibodies 5–2, 29–5, and 45–3 were tested on replicate Western blots of solubilized rat skeletal muscle (lane 1, SkM), rat heart tissue (lane 2, H), and rat brain tissue (lane 3, B). Detection by IIH6 for glycosylated αDG is shown for comparison. Molecular weight standards are indicated in kDa. B) Immunofluorescent detection of αDG in rat tibialis anterior muscle cryosections using antibodies 5–2, 29–5, and 45–3. 20X objective; 100 µm scale bar.</p

Amino acid sequence alignment of the αDG C-terminus.

<p>The mouse αDG C-terminal antigen used for antibody generation is aligned to rat, pig, human, and rabbit species-specific sequences using the Clustal 2.1 multiple sequence alignment tool <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097567#pone.0097567-Singh1" target="_blank">[31]</a>. The chicken αDG C-terminus is also shown for comparison with a non-mammalian sequence. Amino acid numbering is according to the mouse sequence. Aligned sequences are Uniprot No. Q62165 a.a. 484–651 (mouse), F1M8K0 a.a. 484–651 (rat), I3LD20 a.a. 469–635 (pig), Q14118 a.a. 486–653 (human), Q28685 a.a. 486–653 (rabbit), and A4VAR9 a.a. 487–654 (chicken). Amino acids are colored according to similar properties: red, small; blue, acidic; magenta, basic; green, hydroxyl, sulfhydryl or amine. Consensus across all species aligned is indicated below each amino acid residue by symbols: asterisk, conserved residue; colon, residues with strongly similar properties; period, residues with weakly similar properties; no symbol, no conservation of properties. Each residue that is not identical to the corresponding mouse amino acid is highlighted in gray. Horizontal lines are used to group the aDGct monoclonal antibody detection positive species (mouse, rat, pig) with the aDGct antibody detection negative species (human, rabbit fusion). Asn524 (in bold) is the only residue that is conserved in all detection positive species and not in detection negative species.</p

Detection of αDG core protein in a porcine model of Becker muscular dystrophy.

<p>A) Immunoblotting of healthy (N) and BMD-affected (A) pig skeletal muscle homogenates was conducted with monoclonal antibodies 5–2, 29–5, and 45–3. IIH6 detection of glycosylated αDG (reprobe) is shown. Molecular weight standards are indicated in kDa. B) Quantification of signal intensity of αDG detection by Western blot. αDG bands were analyzed; the signal intensity for each sample was normalized to the average αDG expression of all controls on the same blot. The resulting relative αDG expression is plotted for each individual control and BMD-affected sample (with group mean and SEM). C) Immunoblotting of WGA-enriched protein eluted from 500 µg of skeletal muscle lysate from normal and BMD-affected pigs using monoclonal antibody media supernatants 5–2, 29–5, 45–3, and IIH6 on replicate blots. Four hundred micrograms of skeletal muscle lysate (mSkM) from a normal mouse was run along with the pig WGA elutions for comparison. D) Diaphragm muscle from healthy (normal) and BMD-affected pigs (BMD-affected) were stained with 45–3 and IIH6. 20X objective; 100 µm scale bar. E) Quantification of pig αDG in normal and BMD-affected muscle by immunofluorescence. The total number of αDG positive pixels summed from 4 representative images was normalized to the average of all normal images per antibody for each control and BMD-affected sample. Asterisks indicate statistical significance between normal and affected pairs; * P = 0.01–0.05, ** P = 0.001–0.01, *** P = 0.0001–0.001, **** P = <0.0001.</p

Immunofluorescent detection of αDG core protein on muscle sections of normal and dystroglycanopathy mice in the presence and absence of the aDGct antigen.

<p>Calf muscle cryosections from a normal mouse (LC) and a tamoxifen-induced fukutin-deficient dystroglycanopathy mouse (KO) were stained by αDG core monoclonal antibody media supernatants 5–2, 29–5, and 45–3, as well as IIH6, for detection of αDG protein and functional glycan, respectively. Each antibody was pre-incubated with 18 µg of purified aDGct protein antigen or PBS prior to staining. Asterisks mark matching fibers for comparison. Arrowheads mark neuromuscular junctions. 40X objective; 20 µm scale bar.</p