Inhibition of lysosomal degradative functions in RPE cells by a retinoid component of lipofuscin.
(9/3510)
PURPOSE: To investigate the effect of the lipofuscin component N-retinylidene-N-retinylethanolamine (A2-E) on degradative functions of lysosomes in human retinal pigment epithelial (RPE) cells and to evaluate its mechanism of action. METHODS: A2-E was coupled to low-density lipoprotein (LDL). Human RPE cell cultures were loaded with the A2-E/LDL complex, and controls were run with medium containing LDL alone. To determine whether A2-E accumulated in lysosomes, cells were fractionated in a Percoll gradient, and protein degradation was determined by metabolic labeling and measurement of the release of low-molecular-weight radioactivity. Lysosomal degradation was distinguished from nonlysosomal degradation by inclusion of NH4Cl in the medium. The metabolism of sulfated glycosaminoglycans was studied by radiosulfate incorporation in pulse-chase experiments. Intralysosomal pH was determined using a fluorescent lysosomotropic pH indicator. RESULTS: A2-E accumulated almost exclusively in the lysosomal compartment. Lysosomal protein degradation was reduced in a dose-dependent fashion in A2-E-treated cells. The selectivity of A2-E on lysosomal function was demonstrated by its lack of effect on degradation of extralysosomal protein. Lysosomal glycosaminoglycan catabolism of RPE cells was also strongly inhibited by A2-E. Lysosomal pH was increased by A2-E. CONCLUSIONS: The findings indicate that accumulation of A2-E in RPE cells interferes with lysosomal functions as exemplified by its inhibitory effect on protein and glycosaminoglycan catabolic pathways. The quaternary amine character of the A2-E apparently causes a perturbation of the acidic intralysosomal milieu, resulting in diminished hydrolase action and consequent accumulation of undegraded material. Such mechanism could be operative in retinal diseases associated with excessive lipofuscin accumulation including age-related macular degeneration. (+info)
A comparative chemical and histochemical study of the chondrodystrophoid and nonchondrodystrophoid canine intervertebral disc.
(10/3510)
The chemical composition of the intervertebral disc of 9-month-old chondrodystrophoid and nonchondrodystrophoid dogs was studied for collagen, noncollagenous protein and glycosaminoglycan. Content of these substances differed significantly between breeds. The differences were most marked in the nucleus pulposus; the noncollagenous protein content of the nonchondrodystrophoid breed was higher than in that of the chondrodystrophoid dogs. The total nitrogen value of the nonchondrodystrophoid nuclei pulposi was less than that of the corresponding chondrodystrophoid discs mainly because of the high collagen content of the latter discs. Histochemically, it was found that the nuclei pulposi of the nonchondrodystrophoid breed contains larger amounts of glycosaminoglycan than in the discs of the chondrodystrophoid breeds. (+info)
Glycosaminoglycans differentially bind HARP and modulate its biological activity.
(11/3510)
Heparin affin regulatory peptide (HARP) is a polypeptide belonging to a family of heparin binding growth/differentiation factors. The high affinity of HARP for heparin suggests that this secreted polypeptide should also bind to heparan sulfate proteoglycans derived from cell surface and extracellular matrix defined as extracellular compartments. Using Western blot analysis, we detected HARP bound to heparan sulfate proteoglycans in the extracellular compartments of MDA-MB 231 and MC 3T3-E1 as well as NIH3T3 cells overexpressing HARP protein. Heparitinase treatment of BEL cells inhibited HARP-induced cell proliferation, and the biological activity of HARP in this system was restored by the addition of heparin. We report that heparan sulfate, dermatan sulfate, and to a lesser extent, chondroitin sulfate A, displaced HARP bound to the extracellular compartment. Binding analyses with a biosensor showed that HARP bound heparin with fast association and dissociation kinetics (kass = 1.6 x 10(6) M-1 s-1; kdiss = 0.02 s-1), yielding a Kd value of 13 nM; the interaction between HARP and dermatan sulfate was characterized by slower association kinetics (kass = 0.68 x 10(6) M-1 s-1) and a lower affinity (Kd = 51 nM). Exogenous heparin, heparan sulfate, and dermatan sulfate potentiated the growth-stimulatory activity of HARP, suggesting that corresponding proteoglycans could be involved in the regulation of the mitogenic activity of HARP. (+info)
Formation of HNK-1 determinants and the glycosaminoglycan tetrasaccharide linkage region by UDP-GlcUA:Galactose beta1, 3-glucuronosyltransferases.
(12/3510)
While expression-cloning enzymes involved in heparan sulfate biosynthesis, we isolated a cDNA that encodes a protein 65% identical to the UDP-GlcUA:glycoprotein beta1, 3-glucuronosyltransferase (GlcUAT-P) involved in forming HNK-1 carbohydrate epitopes (3OSO3GlcUAbeta1,3Gal-) on glycoproteins. The cDNA contains an open reading frame coding for a protein of 335 amino acids with a predicted type II transmembrane protein orientation. Cotransfection of the cDNA with HNK-1 3-O-sulfotransferase produced HNK-1 carbohydrate epitopes in Chinese hamster ovary (CHO) cells and COS-7 cells. In vitro, a soluble recombinant form of the enzyme transferred GlcUA in beta-linkage to Galbeta1,3/4GlcNAcbeta-O-naphthalenemethanol, which resembles the core oligosaccharide on which the HNK-1 epitope is assembled. However, the enzyme greatly preferred Galbeta1, 3Galbeta-O-naphthalenemethanol, a disaccharide component found in the linkage region tetrasaccharide in chondroitin sulfate and heparan sulfate. During the course of this study, a human cDNA clone was described that was thought to encode UDP-GlcUA:Galbeta1,3Gal-R glucuronosyltransferase (GlcUAT-I), involved in the formation of the linkage region of glycosaminoglycans (Kitagawa, H., Tone, Y., Tamura, J., Neumann, K. W., Ogawa, T., Oka, S., Kawasaki, T., and Sugahara, K. (1998) J. Biol. Chem. 273, 6615-6618). The deduced amino acid sequences of the CHO and human cDNAs are 95% identical, suggesting that they are in fact homologues of the same gene. Transfection of a CHO cell mutant defective in GlcUAT-I with the hamster cDNA restored glycosaminoglycan assembly in vivo, confirming its identity. Interestingly, transfection of the mutant with GlcUAT-P also restored glycosaminoglycan synthesis. Thus, both GlcUAT-P and GlcUAT-I have overlapping substrate specificities. However, the expression of the two genes was entirely different, with GlcUAT-I expressed in all tissues tested and GlcUAT-P expressed only in brain. These findings suggest that, in neural tissues, GlcUAT-P may participate in both HNK-1 and glycosaminoglycan production. (+info)
Strain variation in glycosaminoglycan recognition influences cell-type-specific binding by lyme disease spirochetes.
(13/3510)
Lyme disease, a chronic multisystemic disorder that can affect the skin, heart, joints, and nervous system is caused by Borrelia burgdorferi sensu lato. Lyme disease spirochetes were previously shown to bind glycosaminoglycans (GAGs). In the current study, the GAG-binding properties of eight Lyme disease strains were determined. Binding by two high-passage HB19 derivatives to Vero cells could not be inhibited by enzymatic removal of GAGs or by the addition of exogenous GAG. The other six strains, which included a different high-passage HB19 derivative (HB19 clone 1), were shown to recognize both heparan sulfate and dermatan sulfate in cell-binding assays, but the relative efficiency of binding to these two GAGs varied among the strains. Strains N40, CA20-2A, and PBi bound predominantly to heparan sulfate, PBo bound both heparan sulfate and dermatan sulfate roughly equally, and VS461 and HB19 clone 1 recognized primarily dermatan sulfate. Cell binding by strain HB19 clone 1 was inhibited better by exogenous dermatan sulfate than by heparin, whereas heparin was the better inhibitor of binding by strain N40. The GAG-binding preference of a Lyme disease strain was reflected in its cell-type-specific binding. Strains that recognized predominantly heparan sulfate bound efficiently to both C6 glioma cells and EA-Hy926 cells, whereas strains that recognized predominantly dermatan sulfate bound well only to the glial cells. The effect of lyase treatment of these cells on bacterial binding was consistent with the model that cell-type-specific binding was a reflection of the GAG-binding preference. We conclude that the GAG-binding preference varies with the strain of Lyme disease spirochete and that this variation influences cell-type-specific binding in vitro. (+info)
Effect of anti-inflammatory drugs on sulphated glycosaminoglycan synthesis in aged human articular cartilage.
(14/3510)
The anti-inflammatory drugs, sodium salicylate, indomethacin, hydrocortisone, ibuprofen, and flurbiprofen, were examined for their effects on sulphated glycosaminoglycan synthesis in aged human cartilage in vitro. Cartilage was obtained from femoral heads removed during surgery and drug effects were found to vary significantly from one head to another. Statistical analysis of the results showed that sodium salicylate exhibits concentration-dependent inhibition of glycosaminoglycan synthesis over the concentration range used. Indomethacin, hydrocortisone, and ibuprofen, at concentrations comparable to those attained in man, caused a statistically significant depression of sulphated glycosaminoglycan synthesis in cartilage from some femoral heads but not others, reflecting the variable response of human articular cartilage to anti-inflammatory drugs. Sodium salicylate and indomethacin at higher doses produced significant (Pless than 0-005) inhibition of sulphated glycosaminoglycan synthesis in all femoral heads studied. The results for flurbiprofen were less conclusive; this compound appears not to inhibit glycosaminoglycan synthesis over the concentration range used. (+info)
Collagen synthesis and deposition in cartilage during disrupted proteoglycan production.
(15/3510)
A simple system was developed to investigate in vitro the possible relationship between collagen and proteoglycan synthesis in cartilage. When production of complete proteoglycan molecules was effectively inhibited with 4-methylumbelliferyl beta-D-xyloside collagen synthesis and distribution were virtually unaffected. (+info)
Uridine diphosphate xylosyltransferase activity in cartilage from manganese-deficient chicks.
(16/3510)
The glycosaminoglycan content of cartilage is decreased in manganese deficiency in the chick (perosis). The activity of xylosyltransferase, the first enzyme in the biosynthetic pathway of sulphated glycosaminoglycans, was studied in the epiphysial cartilage of 4-week-old chicks which had been maintained since hatching on a manganese-deficient diet. Enzymic activity was measured by the incorporation of [14C]xylose from UDP-[14C]xylose into trichloroacetic acid precipitates. Optimal conditions for the xylosyltransferase assay were established and shown to be the same for both control and manganese-deficient cartilage. Assay of the enzyme by using an exogenous xylose acceptor showed no difference in xylosyltransferase activity between control and manganese-deficient tissue. Further, the extent of xylose incorporation was greater in manganese-deficient than in control cartilage preparations, suggesting an increase in xylose-acceptor sites on the endogenous acceptor protein in the deficient cartilage. 35S turnover in the manganese-deficient cartilage was also increased. The data suggest that the decreased glycosaminoglycan content in manganese-deficient cartilage is due to decreased xylosylation of the acceptor protein plus increased degradation of glycosaminoglycan. (+info)