Secondary structure of pig skin proteodermatan sulfate: a perspective from Raman spectroscopic studies in aqueous solution

Raman spectroscopic studies of pig skin proteodermatan sulfate in H2O are indicative of a well defined secondary structure consisting of alpha-helical, beta-turn, and possibly "random" structures. The above conclusion is surprisingly close to the secondary structure of the "core" protein of pig skin proteodermatan sulfate proposed in the previous paper (V. Renugopalakrishnan et al., Biopolymers 28, 1923-1933, 1989) from FT-IR and CD spectroscopic studies in aqueous solution.     

Post-translational phosphorylation of proteodermatan sulfate

In cultured human skin fibroblasts, the core protein of the small proteodermatan sulfate becomes phosphorylated post-translationally but before the glycosaminoglycan chains are synthesized. This phosphorylation can occur when the intracellular transport is inhibited by carbonyl cyanide m-chlorophenylhydrazone or when the attachment of asparagine-linked oligosaccharides is prevented by tunicamycin. Serine and glycosaminoglycan chains were identified as phosphorylation sites of secreted proteodermatan sulfate. Upon alkaline borohydride treatment and degradation by chondroitin ABC lyase, the main phosphorylated product co-chromatographed with an unsulfated 3H-labeled hexasaccharide prepared analogously from [3H]galactose/[35S]sulfate-labeled proteodermatan sulfate.     

Secondary structure of a core protein from pig skin proteodermatan sulfate: CD and Fourier transform IR spectroscopic studies in solution

The secondary structure of a 38 kDa core protein from pig skin proteodermatan sulfate (PDS), was investigated in solution using CD and Fourier transform (FT) ir spectroscopy. Both techniques generally have provided complementary data on the secondary structures of proteins. CD spectral analysis has shown that the core protein contains 60% beta-turn and alpha-helical structures, the rest being "unordered" structure. FT ir data do not permit calculation of quantitative contributions of substructures, at the present time, to the overall secondary structure of the core protein. CD spectrum of the intact PDS is similar to the core protein CD spectrum.     

Influence of monensin on biosynthesis, processing and secretion of proteodermatan sulfate by skin fibroblasts

The influence of monensin on biosynthesis, processing and secretion of proteodermatan sulfate from human skin fibroblasts was studied with the aid of a specific immunological procedure. Double-labeling experiments with [3H]leucine and [35S]sulfate indicated that monensin caused a dose-dependent parallel decrease of sulfate incorporation into total and of secretion of 3H-labeled proteodermatan sulfate. Compared with the untreated control, a greater proportion of incorporated [35S]sulfate than of incorporated [3H]leucine became secreted. Other monensin effects were a moderate intracellular accumulation of glycosaminoglycan-free core protein, a reduced chain length and a greatly reduced epimerization of D-glucuronic to L-iduronic acid residues. In contrast to the formation of N-acetylgalactosamine 4-sulfate residues 6-sulfation was not affected. Conversion of high-mannose-type oligosaccharides to complex-type N-glycans which normally occurred concomitantly with glycosaminoglycan biosynthesis was inhibited. Withdrawal of monensin made possible an additional sulfation of intracellularly accumulated proteodermatan sulfate. The newly formed sulfate esters did not cluster at the non-reducing ends of the glycosaminoglycan chains. Cells preexposed to monensin and labeled with [3H]glucosamine either in the absence or continuous presence of the drug incorporated similar amounts of 3H radioactivity into proteodermatan sulfate. The results suggest that epimerization of D-glucuronic acid residues and 4-sulfation occur predominantly in the trans cisternae of the Golgi apparatus whereas chain polymerisation and 6-sulfation take place predominantly in the cis Golgi complex.     

Biosynthesis of proteodermatan sulfate in cultured human fibroblasts

Biosynthesis and secretion of proteodermatan sulfate produced by cultured human skin fibroblasts were investigated employing immunological procedures. During an incubation period of 10 min in the presence of [3H]leucine, two core protein forms of Mr = 46,000 and 44,000, respectively, were synthesized. They were converted to mature proteodermatan sulfate with a half-time of approximately 12 min. Fifty per cent of total mature proteodermatan sulfate were found in the culture medium after a 35-min chase. Six to eight per cent remained associated with the cell layer after a chase of 6 h. In the presence of tunicamycin, fibroblasts synthesized a single core protein of Mr = 38,000 that was converted to mature proteodermatan sulfate and secreted with similar kinetics as the N-glycosylated species. Subtle differences in the molecular size of core proteins were noted when cell-associated and secreted proteodermatan sulfate were degraded with chondroitin ABC lyase, but core proteins free of N-linked oligosaccharides were identical. Labeling with [3H]mannose revealed that secreted proteodermatan sulfate contains two or three complex-type or two complex-type and one high-mannose-type N-linked oligosaccharide chains. The N-glycans are bound to a 21-kDa fragment of the core protein. After incubation in the presence of [3H]glucosamine, the [3H]galactosamine/[3H]glucosamine ratio was 3.76 and 3.30 for secreted and cell-associated proteodermatan sulfate, respectively. Evidence for the presence of O-linked oligosaccharides could not be obtained. Small amounts of core protein free of dermatan sulfate chains were secreted when the cultures were treated with p-nitrophenyl-beta-D-xyloside.     

Isolation and characterization of cartilage proteoglycans immunoreactive with an antibody to skin proteodermatan sulfate core protein

Low density proteoglycans showing cross-reaction with an antibody to skin proteodermatan sulfate (PDS) core protein were isolated from the bovine articular cartilage, by CsC1 density gradient centrifugation followed by repeated DEAE-cellulose chromatography. The size and amino acid composition of the core proteins of the immunoreactive proteoglycans, eluted at 0.25M and 0.5M NaC1 on DEAE-cellulose column, were quite similar to that of PDS. The glycosaminoglycan components of both proteoglycans were shown to be composed of a hybrid structure of chondroitin sulfate and dermatan sulfate, based on chondroitinase treatments followed by two-dimensional electrophoresis.     

Light scattering studies of the binding of bovine serum albumin to a cationic polyelectrolyte

Poly(dimethyldiallylammonium chloride) (PDMDAAC) exhibits a strong electrostatic interaction with bovine serum albumin (BSA) at pH 8.0 in 0.16M NaCl. Electrophoretic, dynamic, and static light scattering suggest that the mode of binding of BSA to PDMDAAC depends upon the weight concentration ratio (r) of BSA to PDMDAAC. When r is smaller than ca. 10, the system exhibits characteristics of cooperative binding, in that the BSA molecules are inhomogeneously distributed among the polymer chains, and free PDMDAAC molecules coexist with complex. When r reaches ca. 10, the amount of free PDMDAAC is too small to be observed. Further increase in r leads to a secondary binding process along with an increase in the amount of free protein. Hydrophobic interactions among the bound BSA are proposed as the driving force for the cooperative binding. © 1996 John Wiley & Sons, Inc.     

Organization of glycosaminoglycan sulfation in the biosynthesis of proteochondroitin sulfate and proteodermatan sulfate

Although the intermediates for sulfation of proteochondroitin and proteodermatan have been known for several decades, organizational aspects of this formation have not been clearly defined. Work in several laboratories, including our own, have indicated a pattern which strongly suggests that sulfation ordinarily takes place together with glycosaminoglycan polymerization in the same Golgi sites, and with close relationship to aspects of polymer elongation, polymer modification and polymer termination. the organization of sulfation together with polymerization may be a major factor controlling the location, type, and degree of sulfation, which in turn may direct specific functions of these proteoglycans.     

Light scattering studies of tetramethyl ammonium gellan

Tetramethyl ammonium (TMA) gellan does not gel. Light scattering studies suggest that in solutions of TMA gellan, in tetramethyl ammonium chloride (TMACI), the gellan molecules assemble end to end to produce elongated fibrous structures. Such fibrils are envisaged as resulting from double-helix formation between the ends of neighbouring gellan molecules. Fibrils with molecular weights ranging from (1.06 ± 0.06) × 10⁵ to (4.5 ± 0.1) × 10⁶ have been observed. The molecular weights obtained depended upon the pore size of the filters used to clarify the solutions. The formation of strong gels, in the presence of gel promoting cations, is attributed to a localized ordered lateral association, or crystallization of regions of these fibrils. It is suggested that such a model for gelation may be of general applicability to a number of polysaccharide systems.     

Light scattering studies of supercoiled and nicked DNA

Static and dynamic light scattering measurements were made of solutions of pGem1a plasmids (3730 base pairs) in the relaxed circular (nicked) and supercoiled forms. The static structure factor and the spectrum of decay modes in the autocorrelation function were examined in order to determine the salient differences between the behaviors of nicked DNA and supercoiled DNA. The concentrations studied are within the dilute regime, which is to say that the structure and dynamics of an isolated DNA molecule were probed. Static light scattering measurements yielded estimates for the molecular weight M, second virial coefficient A₂, and radius of gyration R_G. For the nicked DNA, M = (2.8 ± 0.4) × 10⁶g/mol, A₂ = (0.9 ± 0.2) × 10⁻³ mol cm³/g², and R_G = 90 ± 3 nm were obtained. For the supercoiled DNA, M = (2.5 ± 0.4) × 10⁶ g/mol, A₂ = (1.2 ± 0.2) × 10⁻³ mol cm³/g², and R_G = 82 ± 2.5 nm were obtained. The static structure factors for the nicked and supercoiled DNA were found to superpose when they were scaled by the radius of gyration. The intrinsic stiffness of DNA was evident in the static light scattering data. Homodyne intensity autocorrelation functions were collected for both DNAs at several angles, or scattering vectors. At the smallest scattering vectors the probe size was comparable to the longest intramolecular distance, while at the largest scattering vectors the probe size was smaller than the persistence length of the DNA. Values of the self-diffusion coefficients D were obtained from the low-angle data. For the nicked DNA, D = (2.9 ± 0.3) × 10⁻⁸ cm²/s, and for the supercoiled DNA, D = (4.11 ± 0.21) × 10⁻⁸ cm²/s. The contribution to the correlation function from the internal dynamics of the DNA was seen to result in a strictly bimodal decay function. The rates of the faster mode Γ_int, reached plateau values at low angles. For the nicked DNA, Γ_int = 2500 ± 500 s⁻¹, and for the supercoiled DNA, Γ_int = 5000 ± 500 s⁻¹. These rates correspond to the slowest internal relaxation modes of the DNAs. The dependence of the relaxation rates on scattering vector was monitored with the aid of cumulants analysis and compared with theoretical predictions for the semiflexible ring molecule. The internal mode rates and the dependence of the cumulants moments reflected the difference between the nicked DNA and the supercoiled DNA dynamical behavior. The supercoiled DNA behavior seen here indicates that conformational dynamics might play a larger role in DNA behavior than is suggested by the notion of a branched interwound structure. © 1996 John Wiley & Sons, Inc.     

Isolation and some structural analyses of a proteodermatan sulphate from calf skin.

A proteodermatan sulphate was isolated from 0.15 M-NaCl and 0.45 M-NaCl extracts of newborn-calf skin. The proteoglycan was separated from collagen and hyaluronic acid by precipitation with cetylpyridinium chloride and CsCl-density-gradient centrifugation. Further purification was performed by ion-exchange, affinity and molecular-sieve chromatography. The proteoglycan bound to concanavalin A-Sepharose in 1 M-NaCl. It gave a positive reaction with periodic acid/Schiff reagent and contained 8.3% of uronic acid. The dermatan sulphate, the only glycosaminoglycan component, was composed of 74% iduronosylhexosamine units and 26% glucuronosylhexosamine units. The Mr was assessed to be 15000-20000 by gel chromatography. The core protein was found to be a sialoglycoprotein that had O-glycosidic oligosaccharides with N-acetylgalactosamine at the reducing termini. The molar ratio of oligosaccharide chains to dermatan sulphate was approx. 3:1. From these results the proposed structure of proteodermatan sulphate is: one dermatan sulphate chain (average Mr 17500), three O-glycosidic oligosaccharide chains and probably N-glycosidic oligosaccharide chain(s) bound to one core-protein molecule (Mr 55000).