Glycosaminoglycan metabolism of cultured cornea cells derived from bovine and human stroma and from bovine epithelium

Bleckmann, H.; Kresse, Hans

doi:10.1016/0014-4835(80)90031-7

Cited by 12 publications

(9 citation statements)

References 30 publications

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“…These results led to the conclusion that the recognition marker for uptake of pig skin proteodermatan sulphate (and by analogy for proteoglycans secreted by human fibroblasts) is part of the protein core and does not reside in the glycosaminoglycan moeity of the molecule. Furthermore, the efficiency of uptake of proteoglycans from different mammalian species appears to be similar, as has been suggested previously (Bleckmann & Kresse, 1980). Recent progress in purification and preparation of protein cores from secreted proteoglycans under conditions preserving the uptake properties allowed us to identify the recognition marker of secreted proteoglycans as part of the protein core.…”

Section: Other Methodssupporting

confidence: 76%

Receptor-mediated endocytosis of proteoglycans by human fibroblasts involves recognition of the protein core

Glössl¹,

Schubert-Prinz²,

Gregory³

et al. 1983

Biochemical Journal

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Endocytosis by cultured human skin fibroblasts of 35SO4(2-)-labelled or [3H]leucine-labelled proteoglycans from fibroblast secretions and of 125I-proteodermatan sulphate from pig skin was quantitatively investigated. The following results were obtained. (1) Core proteins prepared by digestion with chondroitin ABC lyase were at least as efficiently endocytosed as native proteoglycans. Pig skin proteodermatan sulphate was a competitive inhibitor of endocytosis of 35SO4(2-)-labelled proteoglycans. (2) Proteoglycans produced in the presence of tunicamycin and native proteoglycans degraded with endoglycosaminidase H were internalized at a normal rate. Several monosaccharides that can be bound by mammalian lectins were unable to influence the internalization of proteoglycans. Treatment of proteoglycans with neuraminidase, however, resulted in an increased clearance rate. (3) Reductive methylation or acetoacetylation of lysine residues was accompanied by a parallel decrease in the rate of proteoglycan endocytosis. Reversal of acetoacetylation normalized the uptake properties. Endocytosis of native proteoglycans was also reduced in the presence of poly-L-lysine, and this reduction in endocytosis was observed as well with proteoglycans synthesized in the presence of the lysine analogue S-2-aminoethylcysteine. These results suggest that the recognition marker required for receptor-mediated endocytosis of proteodermatan sulphate resides in its protein moiety and involves lysine residues.

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Section: Other Methodssupporting

confidence: 76%

Receptor-mediated endocytosis of proteoglycans by human fibroblasts involves recognition of the protein core

Glössl¹,

Schubert-Prinz²,

Gregory³

et al. 1983

Biochemical Journal

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“…More than two decades ago Conrad and Dorfman (24) showed that secretion of highly sulfated keratan sulfate ceased in chick corneas in organ culture in 48 h and that in primary keratocytes the keratan sulfate secretion disappeared after six hours in culture. Since that report absence of keratan sulfate synthesis by cultured keratocytes has been confirmed repeatedly (23)(24)(25)(26)(27)(28)(29). Mammalian corneas in culture appear to secrete keratan sulfate for somewhat longer periods of time, in one case up to 2 weeks in culture; however, as with chick keratocytes, biosynthesis of keratan sulfate was lost rapidly when the cells were separated from the stromal matrix (37)(38)(39).…”

Section: Discussionmentioning

confidence: 92%

“…In healing corneal wounds, rejecting corneal grafts, and other pathologic conditions of the cornea, sulfated KSPG is reduced or has an altered structure (16 -22). Initial studies of keratocytes maintained in cell culture reported that little or no synthesis of the keratan sulfate glycosaminoglycan characteristic of normal cornea KSPG occurs in vitro (23)(24)(25)(26)(27). More recent studies have shown that some keratocyte cultures synthesize the KSPG core proteins but do not add sulfated keratan sulfate to this protein (28,29).…”

mentioning

confidence: 99%

Synthesis of Corneal Keratan Sulfate Proteoglycans by Bovine Keratocytes in Vitro

Funderburgh

Mann

et al. 1996

Journal of Biological Chemistry

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Keratan sulfate proteoglycans (KSPGs) are the major proteoglycans of the cornea and are secreted by keratocytes in the corneal stroma. Previous studies have been able to show only transient secretion of KSPG in cell culture. In this study, cultures of bovine keratocytes were found to secrete the three previously characterized KSPG proteins into culture medium. Reactivity with monoclonal antibody I22 demonstrated substitution of these proteins with keratan sulfate chains. KSPG constituted 15% of the proteoglycan metabolically labeled with [35S]sulfate in keratocyte culture medium. This labeled KSPG contained keratan sulfate chains of 4700 Da compared to 21,000 Da for bovine corneal keratan sulfate. Labeled keratan sulfate from cultures contained nonsulfated, monosulfated, and disulfated disaccharides that were released by digestion with endo-beta-galactosidase or keratanase II. Nonsulfated disaccharides were relatively more abundant in keratan sulfate from culture than in corneal keratan sulfate. These results show that cultured bovine keratocytes maintain the ability to express all three of the known KSPG proteins, modified with keratan sulfate chains and sulfated on both N-acetylglucosamine and galactose moieties. KSPG made in vitro differs from that found in vivo in the length and sulfation of its keratan sulfate chains. The availability of cell cultures secreting corneal keratan sulfate proteoglycans provides an opportunity to examine biosynthesis and control of this important class of molecules.

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“…The GAGs were isolated from three compartments. The GAGs from the extracellular compartment, which contains exocytosed proteoglycans, were obtained from the culture medium [13]. After removal of the medium, cells were washed with PBS and harvested by trypsinization [0.1 % (w/v), 10 min, 37°C].…”

Section: Isolation Of Gagsmentioning

confidence: 99%

“…After removal of the medium, cells were washed with PBS and harvested by trypsinization [0.1 % (w/v), 10 min, 37°C]. Cells were centrifuged and the pericellular GAGs were obtained from the supernatant [13]. Prior to isolation of intracellular GAGs the fibroblasts were homogenized.…”

Section: Isolation Of Gagsmentioning

confidence: 99%

Tilorone-induced lysosomal storage of glycosaminoglycans in cultured corneal fibroblasts: biochemical and physicochemical investigations

Fischer

1995

Biochemical Journal

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Tilorone (2,7-bis[2-(diethylamino)ethoxy]-fluoren-9-one) and several other bis-basic compounds are known to induce lysosomal glycosaminoglycan (GAG) storage. The responsible pathomechanism has not been elucidated yet. The assumption of an unspecific disturbance of lysosomal proenzyme targeting due to elevation of endosomal pH is opposed by the hypothesis of formation of a complex between tilorone and GAGs within the lysosomes, which renders GAGs indigestible to glycosidases. In cultures of bovine corneal fibroblasts the amounts of intracellular GAGs [dermatan sulphate (DS), heparan sulphate (HS) and chondroitin sulphate (CS)] were quantified. The fibroblasts were exposed to tilorone (5 microM), which was found to be readily taken up by the cells and to be accumulated within acidic compartments to finally achieve millimolar concentrations. Under these conditions the GAG storage is predominantly due to the accumulation of DS; however, the DS secretion into the culture medium was not affected. The HS accumulation was much less pronounced, accounting only for 3% of total GAG storage. Ammonium chloride (10 mM), which is known to diminish lysosomal enzyme activity by interfering with the mannose 6-phosphate receptor-mediated transport, prevents both HS and DS breakdown. By means of NMR spectroscopy it was shown that tilorone itself tends to display a concentration-dependent aggregation which was enhanced in the presence of GAGs. The diethylamino groups of tilorone interact physicochemically with DS, and to a smaller extent with HS, but not with chondroitin 4-sulphate. Thus, the strength of the interaction between tilorone and the different GAGs in vitro correlates with the potency of tilorone to inhibit the breakdown of the individual GAGs in cultured bovine fibroblasts. The results support the hypothesis of a specific interaction between tilorone and particular GAGs, rendering these resistant to enzymic degradation.

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Glycosaminoglycan metabolism of cultured cornea cells derived from bovine and human stroma and from bovine epithelium

Cited by 12 publications

References 30 publications

Receptor-mediated endocytosis of proteoglycans by human fibroblasts involves recognition of the protein core

Receptor-mediated endocytosis of proteoglycans by human fibroblasts involves recognition of the protein core

Synthesis of Corneal Keratan Sulfate Proteoglycans by Bovine Keratocytes in Vitro

Tilorone-induced lysosomal storage of glycosaminoglycans in cultured corneal fibroblasts: biochemical and physicochemical investigations

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