Abstract
A1 proteoglycan fractions were prepared by isopycnic cesium chloride density gradient centrifugation from early passage cultured fibroblasts derived from the skin of human infants. In five different fibroblast strains, A1 preparations from the medium and the cells, respectively, contained 36-63% and 59-79% of the sulfated proteoglycans applied to the gradients. A subpopulation of A1 proteoglycans was excluded from Sepharose CL-2B columns under associative conditions (0.5 M guanidium chloride (GdmCl]. The excluded proteoglycans comprised 45-55% of the medium and 28-40% of the cell-associated A1 preparations, showed reversible association-dissociation behaviors on Sepharose CL-2B columns, were composed primarily of chondroitin sulfate and dermatan sulfate, and behaved as monomers with a Kav of 0.33 following mild reduction and alkylation. These data indicate that the excluded peaks were supramolecular aggregates composed of proteoglycan subunits. The mechanism of aggregate formation was examined in A1D1 and D1 fractions. Hyaluronic acid induced aggregation of medium A1D1 proteoglycans in one cell strain, but not in two others. Aggregation was not observed with hyaluronic acid in any cell-associated A1D1 fraction. D1 medium proteoglycans chromatographed on Sepharose CL-2B columns under associative conditions showed two types of aggregates; one was disrupted with 4 M GdmCl indicating that a portion of the aggregate formed through noncovalent interactions that may not require interaction with hyaluronic acid. A second type of aggregate persisted in 4 M GdmCl and after mild reduction and alkylation of the core protein but was disrupted by 0.2% sodium dodecyl sulfate, suggesting that aggregation had occurred through proteoglycan interaction with lipids or hydrophobic proteins. These data indicate that human skin fibroblasts synthesize proteoglycans which share some of the properties of chondrocyte proteoglycans, but also have distinctive macromolecular characteristics.
Highlights
From the Department of Pediatrics, Case Western Reserve University at the Ckveiand Metropolitan General Hospital, Cleveland
Synthesized glycosaminoglycans pycnic cesium chloride densitgyradient centrifugation synthesized by fibroblasts may be distributed to an extracelfrom early passage cultured fibroblasts derived from lular pool, associatedwith the cell membraneorretained the skinof human infants
Graphed on Sepharose CL-2B columns under associa- The data in this report indicate that cultured fibroblasts tive conditions showed two types of aggregates; one derived from the dermis of infants synthesize proteoglycans was disrupted wit4h M GdmCl indicating thaat portion of the aggregate formed throughnoncovalent interactions thatmay not require interaction with hyaluronic acid
Summary
Fullthickness biopsies were obtained from the anatomicsites indicated. Strains 14-79, 18-79, and 14-80 were developed from autopsy specimens of three infants who died from a congenital heart malformation. Fibroblasts from bovine nasal septum were used to measure the void volume and blast strainsexamined, the A1 gradient fraction contained 68 glucuronic acid, the bed volume of these columns. The reduced and alkylated sulfated proteoglycans were dialyzed against 0.5 M GdmC1, 0.5 M sodium acetate, pH 7.0, and examined on Sepharose CL-2B columns equilifibroblasts were isolated in the AIDIgradient fraction, while. Proteoglycan Nature of the Fibroblast-sulfated Macronolecules in the A , Fraction-A subpopulation of sulfated macromolecules recovered in theA1 gradient fractionsfrom medium and cell extracts were excluded from associative Sepharose CL-2B columns. Fibroblast dissociative gradients sulfated glycosaminoglycans resistant to digestion with chondroitinase ACII and ABC to estimate theproportion of chondroitin sulfate, dermatan sulfate, and heparan sulfate in the sample
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