Metabolism of Sulfated Glycosaminoglycans in Cultivated Bovine Arterial Cells. II. Quantitative Studies on the Uptake of³⁵SO₄-Labeled Proteoglycans

Abstract

Cultured arterial fibroblasts were used for a quantitative study on adsorption, uptake and degradation of [ 35 S]proteoglycans derived from secretions of cultured arterial or skin fibroblasts. The following results were obtained: 1) Proteoglycans added to the culture medium are integrated into the pool of cell membraneassociated (trypsin-removable) glycosaminoglycans by a saturable process, which depends on time and temperature.2) Up to 17% of the added proteoglycans are taken up by the cells within 24 h. The uptake exhibits saturation kinetics, characteristic for adsorptive pinocytosis. Proteoglycan concentrations required for half-maximum uptake are higher than for haii'-maximum saturation of the glycosaminoglycan pool associated with the cell membrane.3) After a lag phase, inorganic 3S S0 4 appears in the culture medium as a degradation product of the internalized proteoglycans. Pinocytosed proteoglycans are catabolized more rapidly than proteoglycans which remain inside the cell after their biosynthesis. 4) Pinocytosis exhibits specificity, the individual proteoglycans being internalized at different rates. The highest rate of uptake was measured for a dermatan-sulfate-rich proteoglycan. No competition of uptake between a dermatan-sulfate-rich and a heparan-sulfate-rich proteoglycan was observed. 5) Optimum pinocytosis requires an intact protein moiety and, presumably, undegraded carbohydrate chains of the proteoglycans. Abbreviations: CS, Chondroitin sulfate; DS, dermatan sulfate; HS, heparan sulfate. Nomenclature: Glycosaminoglycans (CS, DS, HS) are defined as [ 35 S]sulfate-containing polysaccharides characterized by the NaCl molarity by which they are eluted from Dowex 1X2 as compared with authentic glycosaminoglycan preparations and by their susceptibility to Chondroitin lyases. A covalent linkage of glycosaminoglycans to a protein or polypeptide moiety is not excluded. The term proteoglycan is used for glycosaminoglycan-containing macromolecules which are excluded from Sepharose 4B gel or are eluted from it with a volume distinctly smaller than that of glycosaminoglycans.

Endocytosis and degradation of chondroitin sulphate by liver endothelial cells

“…The uptake of fluorescein-labelled proteoglycan ( Fig. 5) (Kresse et al, 1975;Prinz et al, 1978;. These reports suggest that proteoglycans and glycosaminoglycans are recognized by two different receptor species.…”

Structure and metabolism of sulphated glycosaminoglycans in cultures of human fibroblasts. Structural characteristics of co-polymeric galactosaminoglycans in sequential extracts of fibroblasts during pulse-chase experiments

“…4 indicate that there is a selective secretion and/or degradation of IdUA-rich glycans, since the retained material was relatively GlcUA-rich. Moreover, there is no evidence that IdUA-rich proteoglycans are pinocytosed and degraded to a greater extent than GlcUA-rich ones within the same population; the receptors for pinocytosis of proteoglycans appear to be specific for the protein portion rather than the glycan side chains (Kresse et al, 1975b;Prinz et al, 1977).…”

Increased glycosaminoglycan accumulation as a genetic characteristic in cell cultures of one variety of dominant dystrophic epidermolysis bullosa.

“…35SO=-labeled compounds to be used f'or the endocytosis studies were pre- Endocytosis of labeled material was determined by incubating confluent normal and DDEB cells at 37°C for 7 h at pH 6.8 in 2.0 ml of medium containing the 35SO--labeled compounds as described by Kresse et al. (12). Briefly, after incubating each of the cell types with either control-derived or DDEB-derived labeled material, the medium was removed and the cell layer was quickly washed five times with Hanks' balanced salt solution.…”

“…Briefly, after incubating each of the cell types with either control-derived or DDEB-derived labeled material, the medium was removed and the cell layer was quickly washed five times with Hanks' balanced salt solution. The cells were then harvested by trypsinization and centrifugation and washed one additional time after which they were solubilized for determination of radioactivity and protein (12). Ethanol-soluble degradation products were determined in the culture medium by adding three volumes of ethanol and allowing the medium to stand at 25°C for at least 3 h. After centrifugation, the radioactivity of the supernate was determined (11,12).…”

“…The cells were then harvested by trypsinization and centrifugation and washed one additional time after which they were solubilized for determination of radioactivity and protein (12). Ethanol-soluble degradation products were determined in the culture medium by adding three volumes of ethanol and allowing the medium to stand at 25°C for at least 3 h. After centrifugation, the radioactivity of the supernate was determined (11,12). Endocytosis was defined as the sum of the intracellular radioactivity plus the extracellular labeled ethanol-soluble material minus the ethanolsoluble radioactvity of a medium blank (i.e., the same amount of 35SOm-labeled material added to 2 ml of conditioned medium from the same cell lines and incubated at 37°C for 7 h) (11).…”