Biochemical and structural studies of tenascin/hexabrachion proteins

Abstract Tenascin is a large, disulfide‐bonded glycoprotein of the extracellular matrix. The predominant form of tenascin observed by electron microscopy is a six‐armed oligomer, termed a hexabrachion. We have determined the molecular mass of the native human hexabrachion to be 1.9 × 10 6 Da by sedimentation equilibrium analysis and by electrophoresis on non‐reducing agarose gels. On reducing polyacrylamide gel electrophoresis (SDS‐PAGE), human tenascin showed a single prominent band at 320 kDa and minor bands of 220 and 230 kDa. The molecular weight of the native human hexabrachion is thus consistent with a disulfide‐bonded hexamer of the 320 kDa subunits. Upon treatment with neuraminidase, the apparent molecular weights of all human and chicken tenascin subunits on reducing SDS‐PAGE were decreased by about 10 kDa. Prolonged incubation with α‐mannosidase, however, caused no apparent change in the apparent molecular weight of tenascin subunits. Sedimentation in a cesium chloride gradient gave a higher buoyant density for human tenascin than for fibronectin, suggesting that it has a higher degree of glycosylation. The far‐UV circular dichroism spectrum indicates a predominance of β‐structure and a lack of collagen‐like or α‐helical structure. When human hexabrachions were reduced and acetylated, the resulting fragments were single arms which sedimented at 6 S in glycerol gradients and migrated at 320 kDa on non‐reducing gels. Treatment of tenascin with trypsin and α‐chymotrypsin also produced large fragments which were fractionated by gradient sedimentation and analyzed by non‐reducing SDS‐PAGE and electron microscopy. We present a structural model for the assembly of the observed fragments into the elaborate native hexabrachion.