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A novel large heterodimeric dermatan sulfate proteoglycan with core proteins of 460 and 300 kDa, respectively, had been described as a secretory product of human fetal skin fibroblasts (Breuer et al., J. Biol. Chem. 266, 13224-13232 (1991)). Pulse-chase experiments showed a preferential association of the proteoglycan with the cell membrane. Immunogold labeling indicated its localization in fibrils on the cell surface as well as in fibrillar extensions from the cell body. Immunofluorescence studies yielded a fibrillar and punctate staining pattern which was also seen in cultured human and porcine endothelial cells. Dot-like structures were observed in transformed human keratinocytes. Various immunocytochemical double-labeling experiments indicated a remarkable colocalization of the proteoglycan with fibronectin, laminin, perlecan, and type IV collagen whereas only occasionally a colocalization with chondroitin-6-sulfate was found. No evidence for an enrichment of the proteoglycan in vinculin-containing structures was obtained. These results suggest that the proteoglycan is a widely distributed macromolecule which can associate with basement membrane components. Preliminary findings in rat cornea supported this conclusion.

Human skin fibroblasts were metabolically labelled in the presence of affinity-purified antibodies against the core protein of small dermatan sulphate proteoglycan II. The treatment resulted in a dose- and time-dependent accumulation of this proteoglycan in the culture medium, with a 2-3-fold increase found within an experimental period of 4 h. The presence of antibodies was without influence on the rate of biosynthesis of the proteoglycan. However, proteoglycan-antibody complexes were inefficiently endocytosed. Addition of unlabelled proteoglycan, which served as a competitor for uptake, similarly led to an accumulation of newly formed [35S]sulphate-labelled proteoglycans. Proteoglycan accumulation also occurred as a consequence of its binding to collagen fibrils which were physically separated from the cell layer. Together, these results establish the quantitative importance of the secretion-recapture pathway of small dermatan sulphate proteoglycan II in cultured fibroblasts.

Two male patients, aged 6 and 25, both with normal intelligence and absence of neurological abnormalities, exhibited dysostosis multiplex, dwarfism, odontoid anomalies, cloudy corneas, exessive excretion of keratan sulfate, and abnormal urinary oligosaccharides. Leukocytes and fibroblasts of both patients were deficient in acid beta-galactosidase (beta-gal) and normal in N-acetylgalactosamine-6-sulfate sulfatase, the deficient enzyme in classical Morquio syndrome. The beta-gal deficiency was not due to an endogenous inhibitor, and the parents exhibited intermediate activities. Deficient beta-gal activity was observed toward p-nitrophenyl-beta-galactoside, 4-methylumbelliferyl-beta-galactoside (4 MU-beta-gal), lactose, GM1 ganglioside, keratan sulfate, and asialofetuin (ASF). Under standard assay conditions, the residual activity was similar for all substrates tested. Toward p-nitrophenyl-beta-glactoside, the mutant enzyme behaved as a Km variant.

The cellular uptake of N-acetyl-α-D-glucosaminidase, the deficient enzyme in Sanfilippo B disease, and the intracellular fate and metabolic effect of this enzyme have been investigated in Sanfilippo B and normal fibroblasts. For both-genotypes the uptake is highly efficient (up to 0.025 mU/h/mg cell protein), specific and constant over a period of at least 6 days. It is probable that the enzyme protein is taken up by adsorptive pinocytosis. The enzymatic activity as well as the biological activity towards 'SO4-labeled mucopolysaccharides persist in Sanfilippo B cells with a half-life of 34 h, indicating the intralysosomal localization of the pinocytosed enzyme. The data obtained are discussed with regard to a possible enzyme replacement therapy. For Sanfilippo B disease the doses used in the past are considered to be insufficient to cause measurable effects. peerReviewed

Removal of N-sulfated glucosamine residues during degradation of heparan sulfate is accomplished by the sequential action of three enzymes. Action of sulfamidase resuits in the formation of α-glucosaminide residues. Removal of these groups requires conversion to α-N-acetylglucosaminide by the action of an acetyltransferase in the presence of acetyl- CoA, followed by hydrolysis by α-N-acetylglucosaminidase. In fibroblast homogenates from three patients with Sanfilippo syndrome type C (mucopolysaccharidosis III C), a biochemical variant of the Sanfilippo syndrome, complete deficiency of the acetylCoA:α-glucosaminide N-acetyltransferase activity was detected. Activities of all lysosomal hydrolases known so far to degrade mucopolysaccharides, including those of sulfamidase and α-N-acetyl lucosaminidase, were in the range of controls. Acetyl-CoA:α-glucosaminide N-acetyltransferase activity was normal in fibroblasts of patients with other genetic mucopolysaccharidoses, including Sanfilippo syndrome A and B. peerReviewed

Six siblings were followed, three of them suffering from a Sanfilippo disease, type C, as demonstrated by a deficient glucosamine acetyltransferase activity in cultured skin fibroblasts. Clinical and radiological findings were similar in the three affected children and the phenotypical expression of the disease allowed no distinction between the different types of Sanfilippo disease. Ultrastructural studies of the liver demonstrated characteristic intra-vacuolar inclusions.

Cultured arterial fibroblasts were used for a quantitative study on adsorption, uptake and degradation of [35S]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 membrane-associated (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 half-maximum saturation of the glycosaminoglycan pool associated with the cell membrane. 3) After a lag phase, inorganic 35SO4 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.

The small dermatan sulfate proteoglycan decorin is efficiently internalized by a variety of cells of mesenchymal origin. Decorin-binding receptor proteins of 51 and 26 kDa are involved in this process. Uptake is modulated by highly sulfated heparan sulfate which interacts with the receptor proteins, too. Compared with cultured skin fibroblasts, bovine aortic endothelial cells have a lower capacity for decorin endocytosis whereas their apparent concentration of receptor proteins is even higher. The low internalization rate is attributed to the greater occupancy of receptor proteins by heparan sulfate of the plasma membrane and/or the extracellular matrix. Growth of endothelial cells on Falcon 3090 tissue culture inserts made possible to study decorin uptake from the apical and basolateral membrane, respectively. Decorin uptake was at the limit of detection when the proteoglycan was added to the basolateral compartment. Uptake via the apical membrane was at least as efficient as in monolayer cultures on plastic. The basolateral membrane, however, was enriched in receptor proteins, but also in heparan sulfate proteoglycans. It is, therefore, suggested that endothelial cells are especially involved in the clearance of decorin which is present in blood plasma.