Analysis of Heparan Sulfate from the Engelbreth-Holm-Swarm (EHS) Tumor

Abstract

The size of the heparan sulfate chains from the Engelbreth-Holm-Swarm (EHS) tumor heparan sulfate proteoglycan (PG) was measured by several techniques in order to resolve uncertainty about their size and the chains were chemically characterized for comparison with other basement membrane heparan sulfate PGs. Heparan sulfate size was determined by gel filtration (Mr = 5.5 – 6.0 X 104), by equilibrium sedimentation centrif-ugation (Mw = 6.8 × 104), and by end group analysis (Mn = 7.1 × 104). A higher molecular weight (HMW) (Mw = 2.13 × 105) calculated from scattering measurements may reflect chain-chain interactions. Forty percent of newly synthesized chains eluted on gel filtration as a lower molecular weight (LMW) shoulder and in vivo turned over faster than the larger species. A large heparan sulfate PG was present after 4 hours of in vivo 35SO4 labeling in both a low density form and a high density, slightly smaller form with large heparan sulfate chains (Mr ∼ 8.0 × 104). Heparan sulfate PG of intermediate size (Kav = 0.3–0.65, Sepharose CL-4B) and of smaller size (Kav = 0.75, CL-4B) were found predominantly as high density species. These PGs contained chains (Mr = 3.5 × 104 and Mr = 1.2 × 104, respectively) which were partially sensitive to chondroitinase ABC (CABC) and may include a hybrid heparan sulfate/chondroitin sulfate PG. Heparan sulfate chains, possibly intracellular degradation products, were also found. Heparan sulfate chains were normal in N-sulfation (58% of hexosamine residues) and in iduronate content (∼ 30%). N-sulfation started within two disaccharides of the linkage region. The EHS heparan sulfate was unusually low in O-sulfation (10% of the total sulfation) and no 6–0 sulfated, N-acetylated glucosamine residues adjacent to N-sulfated block regions were found.