Isolation of the heparan sulfate proteoglycans from the extracellular matrix of rat skeletal muscle

Abstract

We have previously shown that asymmetric collagen-tailed acetylcholinesterase (AChE) is anchored to the extracellular matrix (ECM) by heparan sulfate proteoglycans (HSPGs). Here we present our studies on the characterization of such PGs from the ECM of rat skeletal muscles. After radiolabeling with 35SO4 for 24h, PGs were extracted from the muscle ECM with 4.0 M guanidine-HCl containing protease inhibitors. PGs were subsequently isolated using sequential DEAE-Sephacel chromatography, digestion with chondroitinase ABC, and Sepharose CL-4B. Two different hydrodynamic size species of HSPGs were found. One type had a Mr of 4-6 X 10(5) (Kav = 0.25) as estimated by gel chromatography in the presence of 1% SDS and accounted for 75% of the total HSPGs. The other HSPG had a Mr 1.5-2.5 X 10(5) (Kav = 0.41). The glycosaminoglycan (GAG) side chains (Mr 20,000 and 12,000) were found composed only of heparan sulfate as determined by nitrous acid oxidation and heparitinase treatment. The large-sized HSPG, which is concentrated in synaptic regions, contains only GAG chains of Mr 20,000, suggesting that each HSPG contains only one kind of heparan sulfate chain in its structure. Our results definitively establish by biochemical criteria that the basement membrane of mammalian skeletal muscle contains HSPGs, the likely matrix receptor for the immobilization of the asymmetric collagen-tailed AChE at the neuromuscular junction.

Heparan sulfate proteoglycans are increased during skeletal muscle regeneration: requirement of syndecan-3 for successful fiber formation

“…Western blot analysis of proteoglycans Protein extracts were prepared with a protocol slightly modified from that previously described by Brandan and Inestrosa (Brandan and Inestrosa, 1987). Briefly, skeletal muscle was homogenized in 4 M guanidine-HCl, 0.05 M sodium acetate (pH 5.8) and 1 mM PMSF at 4°C and maintained under agitation for 18 hours.…”

Caenorhabditis eleganssyndecan (SDN-1) is required for normal egg laying and associates with the nervous system and the vulva

“…Pooled intact radiolabeled proteoglycans were subjected to alkali extraction in the presence of 1.0 M NaBH 4 , as described previously (Brandan and Inestrosa, 1987) to release intact GAG chains. Core proteins were degraded by proteinase K (Sigma) treatment: 50 µg/ml of enzyme at 65°C for 1 hour, in buffer containing 50 mM Tris-HCl pH 7.4, 0.1 M NaCl and 5 mM CaCl 2 (Veiga et al, 1997).…”

“…Core proteins were degraded by proteinase K (Sigma) treatment: 50 µg/ml of enzyme at 65°C for 1 hour, in buffer containing 50 mM Tris-HCl pH 7.4, 0.1 M NaCl and 5 mM CaCl 2 (Veiga et al, 1997). To determine GAG identities, samples were either treated with nitrous acid to specifically hydrolyze heparan sulfate chains, as described by (Brandan and Inestrosa, 1987), or incubated with 50 mU chondroitinase ABC (Seikagaku, Japan) for 18 hours at 37°C to hydrolyze chondroitin/dermatan sulfate chains (Riquelme et al, 2001). …”

Negative inotropic activity of antilaminin IgG: participation of cholinergic mechanisms

“…It is still a matter of discussion if the collagen subunit of this enzyme interacts with laminin or other components of basement membranes. Vigny et al (1983) as well as Brandan & Inestrosa (1987) demonstrated that the asymmetrical form of acetylcholinesterase binds to heparin sulphate proteoglycan and to a lesser extent to laminin and fibronectin contained in the basal lamina present at the neuromuscular junction. However, Grassi et al (1983) were not able to find any binding of the enzyme to basement membrane components.…”