Abstract
Biglycan is a small dermatan sulfate proteoglycan present in the extracellular matrix of a variety of connective tissues. Sedimentation velocity and equilibrium studies were carried out to determine the monomer molecular weight of biglycan in denaturing solvents and to define the oligomeric states of biglycan in physiologic solvents in the presence and absence of Zn2+. In 6 M guanidine chloride, biglycan is a monomer with s0(20,w) = 2.9 S and Mz = 93,100 (where Mz is z-average molecular weight). In 0.15 M NaCl, 50 mM Tris, pH 7.5, in the absence of divalent metal ions, and at concentrations above 1 mg/ml, biglycan is predominantly dimer (s0(20,w) = 4.8 S). Under these same conditions in solvent containing 5 mM Zn2+, biglycan exists predominantly as a hexamer, with s0(20,w) = 9.4 S and Mz approximately 600,000. In either case, the oligomers dissociate reversibly. In order to determine whether the glycosaminoglycan chains or the core protein was responsible for self-association, sedimentation velocity and sedimentation equilibrium studies were conducted on the isolated components. Dermatan sulfate chains prepared from biglycan, examined in both denaturing and physiologic solvents, show no significant difference in molecular weight (Mz approximately 22,000), whether or not the solvents contain Zn2+. However, biglycan core protein strongly self-associated in physiologic solvents. Thus, the self-association of biglycan appears to be mediated by the core protein and not by its glycosaminoglycan chains.
Highlights
Biglycanis a small dermatan sulfate proteoglycan pernetsa-t positions 1, 4, 6, 11, and 14 [11].Similar leucine-rich ent in the extracellular matrix of a variety of connec- repeats are presentin decorin, fibromodulin, lumican, leucinetive tissues
In ordetro determine whetherthe studies of the bindingof biglycan to othermacromolecules have glycosaminoglycan chains or the core protein was re- utilized solid phase assays, and when attempts bheaevnemade sponsiblefor self-association, sedimentationvelocity to estimatestoichiometry and affinity of binding, biglycan moand sedimentation equilibriumstudies were conducted lecular weights determinedby SDS-PAGE have been used
Solvents contain Zn2+.,biglycan core protein strongly self-associated in physiologic solvents
Summary
Mately 230 amino acids and containasseries of 10 leucine-rich repeats, each 14 residues in length, characterizedby the consensus sequence LXXLXLXXNXLSXLin which leucineis pres-. To prepare biglycan solutions in physiologic solvents in the presence of Zn" or in the absence of divalent metal ions, biglycan was first dissolved in and dialyzed against 4 M GdnHCl, 5 m~ EDTA, 5 m~ EGTA, 50 mM Tris, pH 7.5.Aliquots of this solution were extensivelydialyzed against Chelex 100-treated 0.15 M NaCl, 3 mM NaN,, 50 m~ Tris,pH 7.5,containing either 5 m~ EDTA, 5 mM EGTA or 5 m~ ZnC1,. To examine the self-associationof biglycan in physiologic solvents in the presence and absence of Zn", biglycan in 4 M GdnHCl was dialyzed against either 0.15 M NaCl, 5 mM EDTA, 50 mM Tris, pH 7.5, or 0.15 M NaCI, 5 mM ZnCl,, 50 m~ Tris,pH 7.5, as described above.Sedimentation equilibrium experiments were carried out at biglycan concentrations of 0.2-3.0 mg/ml and at four rotor speeds between 10,000 and 20,000 rpm. Self-association of Biglycan glycan occursin 6 M GdnHCl comparedwith 4 M GdnHCl
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