Biochemical SocietyTransactions ( 1 991 ) 19 377s Physical studies 6 n the protein core of skin dermatan sulphate proteoglycan I1 (decorin). PAUL G. SCOTT Department of Oral Biology, University of Alberta, Edmonton, Alberta T6G 2N8, CANADA. The protein core of the ubiquitous small proteoglycan - DSPGII (dermatan sulphate proteoglycan 11), which can be isolated from skin and other fibrous connective tissues, carries a single dermatan sulphate chain attached to serine at residue 4 [l] and 3 Nlinked oligosaccharides . In vitro DSPGII retards precipitation of collagen fibrils and localizes on their surfaces  in an analogous manner to that seen in vivo . It has been suggested to function in regulation of fibril diameters  or in preventing calcification of soft connective tissues . The protein core is a typical globular protein whose circular dichroism spectrum suggests a high proportion of P-sheet and pturn . However its interaction with collagen in vitro is unaffected by thermal denaturation but is sensitive to reduction of the disulphides, of which there are 3 . Here I describe further studies on the physical properties of the protein core and its interaction with collagen in vitro. DSPGII was purified from bovine skin and the (g1yco)protein core prepared by digestion with chondroitinase ABC . Circular dichroism spectra of samples dissolved in 0.05 M NaH,PO, adjusted to pH 7.0 with NaOH were recorded in a JASCO J-20A spectropolarimeter. Fluorescence spectra and fluorescence yields ( ..282nrn, A ,348nm) were measured in a Perkin-Elmer LS-5 spectrofluorimeter in the same buffer. Fragments of the core protein were prepared by digestion with CNBr [ 8 ] and isolated bx gel-filtration chromatography on Sephadex and SephacrylTn columns. The in vitro fibrillogenesis assay was as described . Heating intact DSPGII or its protein core (Fig. 1A) above 37OC causes a rapid fall in the ellipticity at 205nm and a mid-point for the transition can be measured at 43.5'C. The reduced and alkylated core shows no significant change in ellipticity between 5 and 50'C and is apparently all random coil. The fluorescence spectrum of intact DSPGII (not shown) can be attributed almost completely to its single tryptophan residue. The fluorescence yield of free tryptophan decreases monotonically between 23 and 65OC, whereas that of DSPGII (Fig. 1B) or the protein core (not shown) shows an upward trend first detectable at about 35OC, with a mid-point at 43'C (average of 4 determinations), followed by a monotonic decrease after 48OC. In the presence of 2mercaptoethanol a similar denaturation curve is seen but with a mid-point at 27.5OC. The exact coincidence of denaturation temperatures measured by two independent methods, namely circular dichroism and the intrinsic fluorescence of the single tryptophan residue, is strong evidence for the existence of a single folding domain for this globular protein. The disulphide bridges clearly contribute to the stability of the native structure and indeed may
20 40 TEMPERATURE (OCI
Fig. 1. Effect of temperature on c.d. sDectra [A) and fluorescence vields ( B ) . (M) intact protein core. ( w )reduced and alkylated protein core. DSPGII in the ) and absence (-) of 1% presence ((v/v) 2-mercaptoethanol. (. .. ...) tryptophan - 2.5pM. Fluorescence is in arbitrary units.
preserve sufficient of the tertiary structure for interaction of collagen [ 3 ] . However the present studies were unable to detect any inhibition of fibrillogenesis by a CNBr digest of the protein core of DSPGII or the isolated peptides carrying the two near-Nterminal disulphides or the near-C-terminal disulphide (not shown). Interaction of this proteoglycan with collagen may therefore be a function of the whole protein core rather than a specific "collagen-binding" domain. I thank C. Dodd, E. Edwards and S. Lehocky for technical assistance and the Canadian Medical Research Council for financial support.
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Chopra, R.K., Pearson, C.H., Pringle, G.A., Fackre, D.S. & Scott, P.G. (1985) Biochem. J. 2 3 1 , 277-279. Scott, P.G. & Dodd, C.M. (1990) Connective Tissue Res. 2 4 , 225-236. Scott, P.G., Winterbottom, N., Dodd, C.M., Edwards, E. & Pearson, C.H. (1986) Biochem. Biophys. Res. Comm. 138, 1348-1354. Scott, J . E . (1988) Biochem. J. 2 5 2 , 313323. Scott, J.E., Orford, C.R. & Hughes, E.W. (1981) Biochem. J. 1 9 5 , 573-581. Scott, J.E. & Haigh, M. (1985) Biosci. Rep. 5 , 71-81. Pearson, C.H., Winterbottom, N., Fackre, D.S., Scott, P.G. & Carpenter, M.R. (1983) J. Biol. Chem. 2 5 8 , 1510-1514. Scott, P.G. , Nakano, T. , Dodd, C.M., Pringle, G.A. & Kuc, I.M. (1989) Matrix 9 , 284-292.