Biochemical and structural studies of tenascin/hexabrachion proteins.
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 x 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 alpha-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 beta-structure and a lack of collagen-like or alpha-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 alpha-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.
Taylor
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HC
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710.
Lightner
V A
VA
Beyer
W F
WF
McCaslin
D
D
Briscoe
G
G
Erickson
H P
HP
D000818:Animals; D015816:Cell Adhesion Molecules, Neuronal / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure*; D002478:Cells, Cultured; D002642:Chick Embryo; D002942:Circular Dichroism; D005109:Extracellular Matrix; D005347:Fibroblasts; D005910:Glioma; D006026:Glycoside Hydrolases; D006801:Humans; D008854:Microscopy, Electron; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011487:Protein Conformation; D019063:Tenascin; D014407:Tumor Cells, Cultured / Q000032:analysis
D015816:Cell Adhesion Molecules, Neuronal; D019063:Tenascin; D006026:Glycoside Hydrolases