Endocytosis of different members of the small chondroitin/dermatan sulfate proteoglycan family.

The family of small interstitial chondroitin/dermatan sulfate proteoglycans consists of at least three different molecular species: biglycan (proteoglycan I), decorin (proteoglycan II), and proteoglycan-100, which has a glycosylated core protein of about 100 kDa. The core protein of decorin has been shown to be responsible for receptor-mediated endocytosis of this proteoglycan species by a variety of mesenchymal cells. It is now demonstrated that skin fibroblasts and articular chondrocytes endocytose biglycan with an efficiency similar to that of decorin. Uptake of biglycan is also mediated by its core protein and can be inhibited by decorin in a partially competitive manner. In human fibroblasts, endosomal proteins of 51 and 26 kDa, which are known to bind decorin core protein, also interact with biglycan. This interaction can be inhibited by decorin. Bovine articular chondrocytes contained binding proteins of 48 and 25 kDa. Proteoglycan-100 can be distinguished from biglycan and decorin by its low clearance rate, which however, exceeds the rate of fluid phase endocytosis.     

Extracellular accumulation of small dermatan sulphate proteoglycan II by interference with the secretion-recapture pathway.

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.     

A study of the interaction in vitro between type I collagen and a small dermatan sulphate proteoglycan.

The interaction between a small dermatan sulphate proteoglycan isolated from human uterine cervix and collagen type I from human and rat skin was investigated by collagen-fibrillogenesis experiments. Collagen fibrillogenesis was initiated by elevation of temperature and pH after addition of proteoglycan, chondroitinase-digested proteoglycan or isolated side chains, and monitored by turbidimetry. Collagen-associated and unbound proteoglycan was determined by enzyme-linked immunosorbent assay after aggregation was complete. (1) The binding of proteoglycan to collagen could be explained by the presence of two mutually non-interacting binding sites, with Ka1 = 1.3 x 10(8) M-1 and Ka2 = 1.3 x 10(6) M-1. The number of binding sites per tropocollagen molecule was n1 = 0.11 and n2 = 1.1. The 0.1 high-affinity binding site per tropocollagen molecule indicates that the strong interaction between proteoglycan and collagen results from a concerted action of tropocollagen molecules in fibrils. Digestion of the proteoglycan with chondroitinase ABC did not affect these binding characteristics. (2) Proteoglycan did not affect the rate of fibrillogenesis, but increased the steady-state A400 by up to 90%. This increase was directly proportional to the saturation of the high-affinity type of binding sites. Neither isolated core protein nor isolated side chains induced a similar high increase in steady-state A400. (3) Electron micrographs showed that the fibril diameter was affected only to a minor extent, if at all, by the proteoglycan, whereas bundles of laterally aligned fibrils were common in the presence of proteoglycan. (4) Results obtained with human and rat collagen were similar.     

Purification and characterization of a small dermatan sulphate proteoglycan implicated in the dilatation of the rat uterine cervix.

Ferritin was purified from chicken liver by two different methods: gel filtration on controlled-pore glass beads, and immunoaffinity chromatography employing a chicken ferritin-specific monoclonal antibody that did not cross-react with horse spleen ferritin. This antibody recognizes intact ferritin and an oligomeric 240 kDa form of the molecule after protein transfer to nitrocellulose, but not the 22 kDa chicken ferritin subunit. Chicken liver ferritin purified by these methods exhibited reduced migration on non-denaturing polyacrylamide gels compared with horse spleen ferritin. These results were consistent with the difference in calculated isoelectric points of chicken and horse ferritin subunits. By two-dimensional gel electrophoresis, chicken ferritin 22 kDa subunits exhibited isoelectric points from 6.1 to 6.6 whereas horse spleen ferritin subunits exhibited isoelectric points of 5.8-6.3. The 240 kDa form of the chicken ferritin molecule had an isoelectric point of 6.6 whereas the 210 kDa form of the horse ferritin molecule had isoelectric points of 5.1 and 4.9. Intact chicken liver ferritin particles were 13.4 +/- 0.8 nm (controlled-pore glass-purified) and 12.5 +/- 0.9 nm (affinity-purified) in diameter when viewed by electron microscopy. Horse spleen ferritin consisted of slightly smaller particles with an average diameter of 11.0 +/- 0.7 nm. However, ferritin from chicken liver and horse spleen co-migrated with an apparent molecular mass of 470 kDa when analysed by Sepharose 4B gel filtration chromatography. These results indicate that, consistent with results from other published purification methods, the chicken ferritin purified by the methods reported here exhibits both structural similarities to, and differences from, horse spleen ferritin.     

Non-uniform influence of transforming growth factor-beta on the biosynthesis of different forms of small chondroitin sulphate/dermatan sulphate proteoglycan.

The influence of transforming growth factor-beta (TGF-beta) on the expression of different forms of small proteoglycans was investigated in human skin fibroblasts and in a human osteosarcoma cell line. TGF-beta was not found to act as a general stimulator of small proteoglycan biosynthesis. In both cell types, an increased expression of the core protein of proteoglycan I was found. However, there was a profound decrease in the expression of a 106 kDa core protein, and either no alteration or a small decrease in the biosynthesis of the collagen-binding small proteoglycan II core protein. These results show that the production of individual members of the small proteoglycan family is differentially regulated.     

Isolation and characterization of dermatan sulphate proteoglycan from human uterine cervix.

Proteoglycans were extracted from human uterine cervix with 4 M-guanidinium chloride in the presence of proteinase inhibitors. They were purified by density-gradient centrifugation in 4 M-guanidinium chloride/CsCl (starting density 1.32 g/ml) followed by DEAE-cellulose and Sepharose chromatography. Only one polydisperse proteoglycan was found. s020,w was 2.1S and the weight-average molecular weight was 73 000 (sedimentation-equilibrium centrifugation) to 110 500 (light-scattering). The core protein was monodisperse, with an apparent molecular weight of 47 000. The proteoglycan contained about 30% protein and probably two or three glycosaminoglycan side chains per molecule. High contents of aspartate, glutamate and leucine were found. The glycan moiety of the proteoglycan was exclusively dermatan sulphate, with a co-polymeric structure with approximately equal quantities of iduronic acid- and glucuronic acid-containing disaccharides.     

Isolation and characterization of a collagen fibril-associated dermatan sulphate proteoglycan from bovine lung

Dermatan sulphate proteoglycans have been extracted from bovine lung with 2.0 M CaCl2 and isolated using CsCl density gradient centrifugation, DEAE ion-exchange chromatography, gel chromatography and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Ultrastructurally these proteoglycans are specifically associated with collagen fibrils. Dermatan sulphate (Mr 15.10(3)-35.10(3), with a strong prevalence for the higher Mr) is link via an O-glycosidic bond to a protein core, which is rich in Asx, Glx and Leu. Of the total uronic acid, 91% is iduronic acid. A part of the glucuronic acid residues is located near the protein core and a large cluster of disaccharides is devoid of glucuronic acid residues. An inhibition enzyme immunoassay has been developed to quantitate the proteoglycan. A model for the interaction between dermatan sulphate proteoglycans and collagen fibrils is proposed.     

Human recombinant interleukin-1 regulates cellular mRNA levels of dermatan sulphate proteoglycan core protein.

Human skin fibroblasts were exposed to various concentrations (from 0.01 to 5.0 units/ml) of human recombinant interleukin-1 alpha and interleukin-1 beta (IL-1 alpha and IL-1 beta). Both IL-1 alpha and IL-1 beta were found to increase dermatan-sulphate-proteoglycan (DSPG) core-protein mRNA levels. Maximal increase (3.0-fold) was seen at 48 h after addition of 1 unit of IL-1 beta/ml. In spite of the elevated DSPG-core-protein mRNA only a slight increase (from 10 to 18%) could be seen in the production of DSPG to cell-culture medium. No changes in the molecular mass of DSPG could be detected.     

Interaction of small dermatan sulfate proteoglycan from fibroblasts with fibronectin

Immunogold labeling was used to localize the core protein of small dermatan sulfate proteoglycan (DS-PG) on the surface of cultured human fibroblasts. At 4 degrees C, DS-PG core protein was uniformly distributed over the cell surface. At 37 degrees C, gold particles either became rearranged in form of clusters or remained associated with fibrils. Double-label immunocytochemistry indicated the co-distribution of DS-PG core protein and fibronectin in the fibrils. In an enzyme-linked immunosorbent assay, binding of DS-PG from fibroblast secretions and of its core protein to fibronectin occurred at pH 7.4 and at physiological ionic strength. Larger amounts of core protein than of intact proteoglycan could be bound. Fibronectin peptides containing either the heparin-binding domain near the COOH-terminal end or the heparin-binding NH2 terminus were the only fragments interacting with DS-PG and core protein. Competition and replacement experiments with heparin and dermatan sulfate suggested the existence of adjacent binding sites for heparin and DS-PG core protein. It is hypothesized that heparan sulfate proteoglycans and DS-PG may competitively interact with fibronectin.     

Immunostaining of a heterodimeric dermatan sulphate proteoglycan is correlated with smooth muscles and some basement membranes

A heterodimeric 760-kDa dermatan sulphate proteoglycan tentatively named PG-760 was characterized as a product of keratinocytes, endothelial cells, and fibroblasts. The two core proteins of 460 kDa and 300 kDa are linked by disulphide bridges, and both carry one or only very few dermatan sulphate chains. Different antisera against PG-760 were used in the present study to investigate the distribution in selected murine tissues by light and electron microscopy. PG-760 immunostaining was observed in cornea (epithelium including basement membrane, stroma, and Descemet's membrane), skin, mucosa of the small intestine, Engelbreth-Holm-swarm (EHS)-tumour (matrix and cells), and the smooth muscle layers of uterus, small intestine, and blood vessels. No staining was observed in capillaries, striated muscles, and liver parenchyma including the central vein. The expression of PG-760 in EHS-tumour was also demonstrated after extraction with 4M guanidine and partial purification by diethylaminoethyl (DEAE)-chromatography. We conclude that this novel proteoglycan exhibits a unique tissue distribution being a constituent of some but not all basement membranes, of some other extracellular matrices, and additionally, of all investigated smooth muscle layers.     

Relationship between dilatation of the rat uterine cervix and a small dermatan sulfate proteoglycan

Cervical linear circumference (lo), extensibility and rate of creep, and the content and concentration of collagen and proteoglycans were determined on uterine cervices of rats at different reproductive stages. The inner circumference increased from 9 +/- 3 (SD) mm at the nongravid stage to a maximum of 41 +/- 5 mm at term; a significant drop to 23 +/- 2 mm occurred by 4 h postpartum with a further drop to 18 +/- 4 mm by 1 day postpartum. The extensibility and rate of creep reached their maxima 1 day before term and returned to the nongravid value by 1 day postpartum. The small (Mr = 95,000) type II dermatan sulfate proteoglycan, the major cervical proteoglycan, increased from 43 +/- 6 micrograms per cervix at the nongravid stage to 196 +/- 33 micrograms at term. The amount of this proteoglycan decreased significantly by 35% to 126 +/- 5 micrograms within 4 h postpartum and declined further to 79 +/- 16 micrograms by 1 day postpartum. The total cervical collagen content increased less than 2-fold during pregnancy, from 3.5 +/- 0.5 to 6.3 +/- 0.7 mg; a decline to 5.8 mg by 1 day postpartum was not significant. The ratio of small proteoglycan: collagen increased 2.5-fold between the nongravid state and term, then returned to the nongravid value by 1 day postpartum. Significant correlations were found between the lo and the amount of small proteoglycan per cervix (r = 0.86; n = 69) and between lo and the ratio of small proteoglycan:collagen (r = 0.83; n = 50) when data from every reproductive stage were combined. A mechanism is proposed whereby the interaction of the proteoglycan with collagen fibers might alter mechanical properties and contribute to cervical dilatation and its rapid reversal.     

Aortic endothelial cells synthesize a large chondroitin sulphate proteoglycan capable of binding to hyaluronate.

Confluent cultures of mouse aortic endothelial (END-D) were incubated with either [35S]methionine or 35SO4 2-, and the radiolabelled proteoglycans in media and cell layers were analysed for their hyaluronate-binding activity. The proteoglycan subfraction which bound to hyaluronate accounted for about 18% (media) and 10% (cell layers) of the total 35S radioactivity of each proteoglycan fraction. The bound proteoglycan molecules could be dissociated from the aggregates either by digestion with hyaluronate lyase or by treatment with hyaluronate decasaccharides. Digestion of [methionine-35S]proteoglycans with chondroitinase and/or heparitinase, followed by SDS/polyacrylamide-gel electrophoresis, indicated that the medium and cell layer contain at least three chondroitin sulphate proteoglycans, one dermatan sulphate proteoglycan, and two heparan sulphate proteoglycans which differ from one another in the size of core molecules. Among these, only the hydrodynamically large chondroitin sulphate species with an Mr 550,000 core molecule was shown to bind to hyaluronate. A very similar chondroitin sulphate proteoglycan capable of binding to hyaluronate was also found in cultures of calf pulmonary arterial endothelial cells (A.T.C.C. CCL 209). These observations, together with the known effects of hyaluronate on various cellular activities, suggest the existence of possible specialized functions of this proteoglycan subspecies in cellular processes characteristic of vascular development and diseases.     

Colorimetric measurement of dermatan sulphate

The periodate-Schiff reaction has been adapted for the measurement of dermatan sulphate. The method is specific for this glycosaminoglycan, provided that glycogen and glycoproteins are removed. Measurements of dermatan sulphate present in the urine of patients affected by various mucopolysaccharidoses indicate a good agreement between the values obtained with enzymic methods and those obtained with the colorimetric method described.     

Biosynthesis and properties of a further member of the small chondroitin/dermatan sulfate proteoglycan family

Human osteosarcoma cells express a 78-kDa proteoglycan core protein to which an asparagine-bound oligosaccharide, O-glycosidically linked oligosaccharides and probably only a single chondroitin 6-sulfate chain of 29-kDa are bound. Prior to O-glycosylation, the N-glycosylated core protein exhibits a mass of 83 kDa. Upon digestion of the secreted proteoglycan with chondroitin ABC lyase a mature core protein with an apparent molecular mass of 106 kDa is obtained. Smaller amounts of core proteins of 101 and 115 kDa can be detected occasionally. The glycosaminoglycan composition and the relative molecular mass of the glycosaminoglycan chain distinguish this proteoglycan, tentatively named proteoglycan 100 (PG-100), from biglycan (small proteoglycan I) and decorin (small proteoglycan II) which are also expressed by osteosarcoma cells. An antiserum against PG-100 shows partial cross-reactivity with decorin, but in contrast to the latter proteoglycan it does not bind to type I collagen fibrils. PG-100 is not a unique product of osteosarcoma cells. It has also been found in the secretions of human skin fibroblasts.     

Dermatan sulphate proteoglycan from human articular cartilage. Variation in its content with age and its structural comparison with a small chondroitin sulphate proteoglycan from pig laryngeal cartilage.

Low molecular mass proteoglycans (PG) were isolated from human articular cartilage and from pig laryngeal cartilage, which contained protein cores of similar size (Mr 40-44 kDa). However, the PG from human articular cartilage contained dermatan sulphate (DS) chains (50% chondroitinase AC resistant), whereas chains from pig laryngeal PG were longer and contained only chondroitin sulphate (CS). Disaccharide analysis after chondroitinase ABC digestion showed that the human DS-PG contained more 6-sulphated residues (34%) than the pig CS-PG (6%) and both contained fewer 6-sulphated residues than the corresponding high Mr aggregating CS-PGs from these tissues (86% and 20% from human and pig respectively). Cross-reaction of both proteoglycans with antibodies to bovine bone and skin DS-PG-II and human fibroblasts DS-PG suggested that the isolated proteoglycans were the humans DS-PG-II and pigs CS-PG-II homologues of the cloned and sequenced bovine proteoglycan. Polyclonal antibodies raised against the pig CS-PG-II were shown to cross-react with human DS-PG-II. SDS/polyacrylamide-gel analysis and immunoblotting of pig and human cartilage extracts showed that some free core protein was present in the tissues in addition to the intact proteoglycan. The antibodies were used in a competitive radioimmunoassay to determine the content of this low Mr proteoglycan in human cartilage extracts. Analysis of samples from 5-80 year-old humans showed highest content (approximately 4 mg/g wet wt.) in those from 15-25 year-olds and lower content (approximately 1 mg/g wet wt.) in older tissue (greater than 55 years). These changes in content may be related to the deposition and maintenance of the collagen fibre network with which this class of small proteoglycan has been shown to interact.     

Self-association of dermatan sulphate proteoglycans from bovine sclera.

1. Two proteodermatan sulphate fractions (I and II) from bovine sclera were studied by gel chromatography, light-scattering and ultracentrifugation under various conditions. 2. Gel chromatography of proteoglycans in the absence or presence of hyaluronate was performed under associative conditions. No effect on the elution profile was noted. 3. Ultracentrifugation experiments (sedimentation-velocity and sedimentation-equilibrium) with proteoglycan I and II in 6 M-guanidine hydrochloride gave molecular weights (Mw) of 160000-220000 and 70000-100000 respectively. As the protein contents were 45% and 60% respectively, it may be calculated that proteoglycan I contained four to five side chains, whereas proteoglycan II contained one or two. Sedimentation-equilibrium runs performed in 0.15 M-NaCl gave an apparent molecular weight (Mw) of 500000-800000 for proteoglycan I and 90000-110000 for proteoglycan II. 4. In light-scattering experiments both proteoglycans I and II yielded high particle weights in 0.15 M-NaCl (3.1 X 10(6) and 3.4 X 10(6) daltons respectively). In the presence of 6 M-guanidine hydrochloride the molecular weights decreased to 410000 and 130000 respectively. The particle weights in 0.15 M-NaCl were not altered by the addition of hyaluronate or hyaluronate oligosaccharides. 5. The dermatan sulphate side chains of scleral proteoglycans (L-iduronate/D-glucuronate ratio 7:13) gave a particle weight of 100000 daltons in 0.15 M-NaCl. In 1.00 M-KCl/0.02M-EDTA the molecular weight was 24000. Addition of free scleral dermatan sulphate chains to a solution of proteoglycan II promoted further multimerization of the macromolecule.     

Endocytosis of the dermatan sulfate proteoglycan decorin utilizes multiple pathways and is modulated by epidermal growth factor receptor signaling

Human skin fibroblasts efficiently internalize the matrikine decorin by receptor-mediated endocytosis, however, very little is known about its intracellular trafficking routes up to lysosomal degradation. In an in vitro system measuring uptake and degradation of [(35)S]sulfate-labeled decorin, endocytosis was blocked by 46% when clathrin assembly/disassembly was inhibited using chlorpromazine. Pharmacological inhibition of EGF receptor signaling caused 34% reduction of decorin uptake, whereas inhibition of the IGF receptor had no effect. Using confocal immunofluorescence microscopy, we determined that only about 5-10% of internalized decorin colocalized with the EGFR. Thus, uptake depends on EGFR signaling rather than trafficking along the same pathway. Decorin passes through early endosomes towards trafficking to lysosomes, since more than 50% of decorin colocalized with EEA1. Moreover, inhibition of endosomal fusion by wortmannin caused a profound inhibition of decorin endocytosis. Overexpression of the clathrin-binding Hrs protein, which has previously been shown to inibit EGFR degradation blocked the degradation of decorin. Cholesterol depletion by filipin inhibited uptake of decorin by 34%, however, nearly no intracellular colocalization was found between decorin and caveolin-1. The combined use of filipin and chlorpromazine had an additive inhibitory effect on decorin endocytosis. Moreover, chlorpromazine diverted decorin from the chlorpromazine-sensitive pathway to an alternative uptake route. The CD44/hyaluronan pathway was excluded as an endocytic route for decorin. Our observations indicate that decorin is taken up by more than one endocytic pathway. Of note, lipid-raft-dependent EGFR signaling modulates decorin uptake, suggesting the presence of a potential feedback regulation mechanism for desensitization of signaling events mediated by decorin.     

Physical properties of chondroitin sulphate/dermatan sulphate proteoglycans from bovine aorta.

Bovine aortic chondroitin sulphate/dermatan sulphate proteoglycans (PG-25, PG-35 and PG-50) were differentially precipitated with ethanol and analysed by a variety of chemical and physical techniques. The glycosaminoglycan chains of PG-25 and PG-35 contained a mixture of glucuronic acid and iduronic acid, whereas the uronic acid component of PG-50 was primarily glucuronic acid. In addition, various amounts of oligosaccharides containing small amounts of mannose, a galactose/hexosamine ratio of 1:1 and an absence of uronic acid were covalently linked to the core protein of all proteoglycans. The weight-average Mr (Mw) values of the proteoglycans determined by light-scattering in 4 M-guanidinium chloride were 1.3 X 10(6) (PG-25), 0.30 X 10(6) (PG-35) and 0.88 X 10(6) (PG-50). The s0 values of the proteoglycans were distributed between 7 and 8 S, and the reduced viscosities, eta sp./c, of all proteoglycans were dependent on the shear rate and polymer concentration. Electron microscopy of spread molecules revealed that PG-25 contained small structural units that appeared to self-associate into large aggregates, whereas PG-35 and PG-50 appeared mainly as monomers consisting of a core with various numbers of side projections. Hyaluronic acid-proteoglycan complexes occurred only with a small proportion of the molecules present in PG-35, and their formation could be inhibited by oligosaccharides. These results suggest the presence in the aorta of subspecies of chondroitin sulphate and dermatan sulphate proteoglycans, which show large variations in their physicochemical and inter- and intra-molecular association properties.