Enhanced laminin binding by alpha-dystroglycan after enzymatic deglycosylation

Abstract

Carbohydrate modifications are clearly important to the function of alpha-dystroglycan but their composition and structure remain poorly understood. In the present study, we describe experiments aimed at identifying the alpha-dystroglycan oligosaccharides important for its binding to laminin-1 and carbohydrate-dependent mAbs (monoclonal antibodies) IIH6 and VIA4(1). We digested highly purified skeletal muscle alpha-dystroglycan with an array of linkage-specific endo- and exoglycosidases, which were verified for action on alpha-dystroglycan by loss/gain of reactivity for lectins with defined glyco-epitopes. Notably, digestion with a combination of Arthrobacter ureafaciens sialidase, beta(1-4)galactosidase and beta-N-acetylglucosaminidase substantially degraded SiaAalpha2-3Galbeta1-4GlcNAcbeta1-2Man glycans on highly purified alpha-dystroglycan that nonetheless exhibited enhanced IIH6, VIA4(1) and laminin-1 binding activity. Additional results indicate that alpha-dystroglycan is probably modified with other anionic sugars besides sialic acid and suggest that rare alpha-linked GlcNAc moieties may block its complete deglycosylation with currently available enzymes.

Figure 1. Characterization of purified skeletal muscle α-dystroglycan

Shown in (A) are a Coomassie Blue-stained gel (CB) and an autoradiograph from a gel loaded with iodinated α-dystroglycan (¹²⁵I-αDG). Also shown are nitrocellulose transfers loaded with purified α-dystroglycan and blotted with polyclonal (αDG pAb) or mAb (IIH6) to α-dystroglycan, ConA, or laminin-1. Molecular mass standards (×10⁻³) are indicated on the left. Shown in (B) is the amount of ¹²⁵I-α-dystroglycan specifically bound to microtitre wells coated with laminin-1 as a function of increasing α-dystroglycan concentration. Shown in (C) is the amount of laminin-1 bound to microtitre wells coated with purified α-dystroglycan in the presence of increasing concentrations of heparin or N-acetylneuraminic acid (NANA).

Figure 2. Heterogeneous sialoglycosylation of skeletal muscle α-dystroglycan

Shown are identical nitrocellulose transfers loaded with equal amounts of purified α-dystroglycan incubated for 17 h at 37 °C in 50 mM sodium phosphate (pH 7.0) in the absence (Con), or presence of A. ureafaciens, C. perfringens or V. cholerae sialidase and stained with α-dystroglycan mAb IIH6, horseradish peroxidase-conjugated WGA or LFA.

Figure 3. Enzymatic deglycosylation of skeletal muscle α-dystroglycan

α-Dystroglycan was incubated for 20 h at 37 °C in 50 mM sodium phosphate, pH 7, in the absence (−) or presence (+) of all five enzymes provided in the GLYCOPRO (N-glycosidase F, A. ureafaciens sialidase, and endo-O-glycosidase) and PROLINK Extender [β(1-4)galactosidase and β-N-acetylglucosaminidase] enzymatic deglycosylation kits. In (A), equal amounts of control (−) and GLYCOPRO-treated (+) α-dystroglycan were resolved on SDS-polyacrylamide gels and stained with Coomassie Blue, or transferred on to nitrocellulose and stained with chicken polyclonal antibodies to α-dystroglycan core protein (α-DG pAb). Identical gels/transfers containing equal amounts of control and enzyme-treated α-dystroglycan were analysed for residual carbohydrate (Dig-Hz), Stains-All reactivity, or binding to the lectins LFA or ConA. The asterisk marks the 180000 M_r endo-O-glycosidase band present in the enzyme-treated samples. In (B), identically prepared transfers were analysed for laminin-1 binding (Ln-1 O/L), or for reactivity with mAbs IIH6 and VIA4₁.

Figure 4. Skeletal muscle α-dystroglycan contains latent glyco-epitopes reactive with conA and GsII

Shown in (A) are identical nitrocellulose transfers loaded with equal amounts of purified α-dystroglycan incubated for 18 h at 37 °C in 50 mM sodium phosphate (pH 7) in the absence or presence of the indicated enzymes and stained with α-dystroglycan mAb IIH6, PNA, ECA, GsII lectin or ConA. Shown in (B) are the sialylated core 1 (top) and O-mannosyl (bottom) oligosaccharide structures known to modify α-dystroglycan with predicted effect of each glycosidase on oligosaccharide structure and lectin reactivity.

Figure 5. Periodate oxidation of α-dystroglycan

Shown are identical nitrocellulose transfers loaded with equal amounts of skeletal muscle α-dystroglycan incubated at room temperature in 10 mM sodium metaperiodate for the indicated times and analysed for laminin-1 binding (Ln-1 O/L), or for reactivity with chicken polyclonal antibodies to α-dystroglycan (α-DG pAb), mAb IIH6 or ConA.