Search

Decorin, a small multifunctional proteoglycan, is expressed by sprouting endothelial cells (ECs) during inflammation-induced angiogenesis in vivo and by human ECs co-cultured with fibroblasts in a collagen lattice. To investigate how decorin is induced, human EA.hy 926 ECs and/or human umbilical vein ECs were treated with interleukin (IL)-10 and IL-6. Both treatments induced decorin mRNA in human ECs. IL-6 and IL-10 led to a dose-dependent mRNA increase with a maximum at 10 and 50 ng/ml, respectively. The combination of both interleukins together had a stronger effect than one alone. Immunostaining demonstrated that both interleukins caused decorin synthesis in ECs and the formation of capillary-like structures in a collagen lattice. However, immunoprecipitations of interleukin-treated ECs cultured on plastic were negative. Only interleukin-stimulated ECs grown on a collagen type I matrix or growth factor-reduced Matrigel were able to synthesize the proteoglycan. Acid-soluble collagen type I did not support decorin protein synthesis. The addition of antibodies to alpha(1) or alpha(2) integrins or the alpha(2) integrin inhibitor rhodocetin led to an inhibition of synthesis. These data show that IL-10 and IL-6 induce decorin mRNA transcription, but additional signals from the extracellular matrix are necessary for its translation.

During glomerular inflammation mesangial cells are the major source and target of nitric oxide that pro-foundly influences proliferation, adhesion, and death of mesangial cells. The effect of nitric oxide on the mRNA expression pattern of cultured rat mesangial cells was therefore investigated by RNA-arbitrarily-primed polymerase chain reaction. Employing this approach, biglycan expression turned out to be down-regulated time- and dose-dependently either by interleukin-1beta-stimulated endogenous nitric oxide production or by direct application of the exogenous nitric oxide donor, diethylenetriamine nitric oxide. There was a corresponding decline in the rate of biglycan biosynthesis and in the steady state level of this proteoglycan. In vivo, in a model of mesangioproliferative glomerulonephritis up-regulation of inducible nitric-oxide synthase mRNA was associated with reduced expression of biglycan in isolated glomeruli. Biglycan expression could be normalized, both in vitro and in vivo, by using a specific inhibitor of the inducible nitric-oxide synthase, l-N6-(l-iminoethyl)-l-lysine dihydrochloride. Further studies showed that biglycan inhibited cell adhesion on type I collagen and fibronectin because of its binding to these substrates. More importantly, biglycan protected mesangial cells from apoptosis by decreasing caspase-3 activity, and it counteracted the proliferative effects of platelet-derived growth factor-BB. These findings indicate a signaling role of biglycan and describe a novel pathomechanism by which nitric oxide modulates the course of renal glomerular disease through regulation of biglycan expression.

Chondroitin sulfate and dermatan sulfate proteoglycans are distinguished by differences in their proportion of d-glucuronosyl and l-iduronosyl residues, the latter being formed by chondroitin-glucuronate 5-epimerase during or after glycosaminoglycan chain polymerization. To investigate the influence of the core protein on the extent of epimerization, we expressed chimeric proteins in 293 HEK cells constructed from intact or modified Met(1)-Gln(153) of decorin (DCN), which normally has a single dermatan sulfate chain at Ser(34), in combination with intact or modified Leu(241)-Ser(353) of CSF-1, which has a chondroitin sulfate attachment site at Ser(309). Transfected DCN(M1-Q153), like full-length DCN, contained approximately 20% l-iduronate. Conversely, transfected CSF-1(L241-S353), attached C-terminally on the DCN prepropeptide, contained almost exclusively d-glucuronate. Transfected intact chimeric DCN(M1-Q153)-CSF-1(L241-S353), with two glycosaminoglycan chains, also contained almost exclusively d-glucuronate in chains at both sites, as did chimeras in which alanine was substituted for serine at either of the glycosaminoglycan attachment sites. Nevertheless, undersulfated intact chimeric proteoglycan was an effective substrate for epimerization of glucuronate to iduronate residues when incubated with microsomal proteins and 3'-phosphoadenylylphosphosulfate. C-terminal truncation constructs were prepared from the full-length chimera with an alanine substitution at the CSF-1 glycosaminoglycan attachment site. Transfected truncations retaining the alanine-blocked site contained chains with essentially only glucuronate, whereas those further truncated by 49 or more amino acids and missing the modified attachment site contained chains with approximately 15% iduronate. This 49-amino acid region contains a 7-amino acid motif that appears to be conserved in several chondroitin sulfate proteoglycans. The results are consistent with a model in which the core protein, possibly via this motif, is responsible for routing to subcellular compartments with or without sufficient access to chondroitin-glucuronate 5-epimerase for the addition of chains with or without iduronate residues, respectively.

Endothelial cells undergoing angiogenesis express decorin, a small multifunctional proteoglycan. We have shown that decorin is causally involved in the formation of capillary-like structures and a decrease in apoptosis in endothelial cells cultured in a collagen lattice. Here we investigate signal transduction pathways mediating the effects of decorin. Reverse transcription-polymerase chain reaction demonstrated that p21 and p27, two inhibitors of cyclin-dependent kinases, were up-regulated by decorin induction. Decorin also increased protein levels of p21 and caused its translocation into the nucleus. p21 synthesis started 6 h after decorin addition and reached a plateau after 18 h, while cyclin A, which was also induced, peaked at 12 h and declined below basal levels within 24 h. These effects were mediated by the Akt/protein kinase B pathway. Akt phosphorylation at Thr-308 increased 4-fold and at Ser-473 1.4-fold within 10 min after decorin addition. Overexpression of dominant negative Akt inhibited the decorin-mediated induction of p21 and cyclin A, but had no effect on p27. These results show that decorin is a signaling molecule in sprouting endothelial cells where it acts via different pathways, one of them involving Akt/protein kinase B.

Biglycan is a member of the small leucine-rich proteoglycan family. Its core protein comprises two chondroitin/dermatan sulfate attachment sites on serine 42 and serine 47, respectively, which are the fifth and tenth amino acid residues, respectively, after removal of the prepro peptide. Because the regulation of glycosaminoglycan chain assembly is not fully understood and because of the in vivo existence of monoglycanated biglycan, mutant core proteins were stably expressed in human 293 and Chinese hamster ovary cells in which i) either one or both serine residues were converted into alanine or threonine residues, ii) the number of acidic amino acids N-terminal of the respective serine residues was altered, and iii) a hexapeptide was inserted between the mutated site 1 and the unaltered site 2. Labeling experiments with [(35)S]sulfate and [(35)S]methionine indicated that serine 42 was almost fully used as the glycosaminoglycan attachment site regardless of whether site 2 was available or not for chain assembly. In contrast, substitution of site 2 was greatly influenced by the presence or absence of serine 42, although additional mutations demonstrated a direct influence of the amino acid sequence between the two sites. When site 2 was not substituted with a glycosaminoglycan chain, there was also no assembly of the linkage region. These results indicate that xylosyltransferase is the rate-limiting enzyme in glycosaminoglycan chain assembly and implicate a cooperative effect on the xylosyl transfer to site 2 by xylosylation of site 1, which probably becomes manifest before the removal of the propeptide. It is shown additionally that biglycan expressed in 293 cells may still contain the propeptide sequence and may carry heparan sulfate chains as well as sulfated N-linked oligosaccharides.

Decorin is a member of the family of the small leucine-rich proteoglycans. In addition to its function as an extracellular matrix organizer, it has the ability to activate the epidermal growth factor receptor, and it forms complexes with various isoforms of transforming growth factor beta (TGF-beta). Decorin is expressed during skeletal muscle differentiation and is up-regulated in dystrophic muscle. In this study we investigated the role of decorin in TGF-beta-dependent inhibition of myogenesis. To probe the function of decorin during myogenesis, C(2)C(12) myoblasts were stably transfected with a plasmid expressing antisense decorin mRNA. The resulting inhibition of decorin expression led to the expression of myogenin, a master transcription factor for muscle differentiation, under growth conditions and accelerated skeletal muscle differentiation as determined by the expression of creatine kinase. In contrast myogenin expression was inhibited by adenovirally induced decorin expression or by adding exogenous decorin. Reduced synthesis of decorin resulted in a 7-fold decreased sensitivity to TGF-beta-mediated inhibition of myogenin expression. In contrast, adenovirally induced decorin expression in wild type cells resulted in a 5-fold increased sensitivity to TGF-beta-mediated inhibition of myogenin expression. Transfection studies with the TGF-beta-dependent promoter of the plasminogen activator inhibitor-1 coupled with luciferase revealed that the transducing receptors for TGF-beta1 and TGF-beta2 were involved in the different responses of wild type and antisense decorin myoblasts. These results demonstrate that a reduction of decorin expression or of decorin availability results in a decreased responsiveness to TGF-beta. These findings strongly suggest a new role for decorin during skeletal muscle terminal differentiation by activating TGF-beta-dependent signaling pathways.

Cell surface-associated heparan sulfate proteoglycans, predominantly perlecan, are involved in the process of binding and endocytosis of thrombospondin-1 (TSP-1) by vascular endothelial cells. To investigate the structural properties of heparan sulfate (HS) side chains that mediate this interaction, the proteoglycans were isolated from porcine endothelial cells and HS chains obtained thereof by beta-elimination. To characterize the structural composition of the HS chains and to identify the TSP-1-binding sequences, HS was disintegrated by specific chemical and enzymatic treatments. Cell layer-derived HS chains revealed the typical structural heterogeneity with domains of non-contiguously arranged highly sulfated disaccharides separated by extended sequences containing predominantly N-acetylated sequences of low sulfation. Affinity chromatography on immobilized TSP-1 demonstrated that nearly all intact HS chains possessed binding affinity, whereas after heparinase III treatment only a small proportion of oligosaccharides were bound with similar affinity to the column. Size fractioning of the bound and unbound oligosaccharides revealed that only a specific portion of deca- to tetradecasaccharides possessed TSP-1-binding affinity. The binding fraction contained over 40% di- and trisulfated disaccharide units and was enriched in the content of the trisulfated 2-O-sulfated L-iduronic acid-N-sulfated-6-O-sulfated glucosamine disaccharide unit. Comparison with the disaccharide composition of the intact HS chains and competition experiments with modified heparin species indicated the specific importance of N- and 6-O-sulfated glucosamine residues for binding. Further depolymerization of the binding oligosaccharides revealed that the glucosamine residues within the TSP-1-binding sequences are not continuously N-sulfated. The present findings implicate specific structural properties for the HS domain involved in TSP-1 binding and indicate that they are distinct from the binding sequence described for basic fibroblast growth factor, another HS ligand and a potential antagonist of TSP-1.

A seventh member of the human beta4-galactosyltransferase family, beta4Gal-T7, was identified by BLAST analysis of expressed sequence tags. The coding region of beta4Gal-T7 depicts a type II transmembrane protein with sequence similarity to beta4-galactosyltransferases, but the sequence was distinct in known motifs and did not contain the cysteine residues conserved in the other six members of the beta4Gal-T family. The genomic organization of beta4Gal-T7 was different from previous beta4Gal-Ts. Expression of beta4Gal-T7 in insect cells showed that the gene product had beta1,4-galactosyltransferase activity with beta-xylosides, and the linkage formed was Galbeta1-4Xyl. Thus, beta4Gal-T7 represents galactosyltransferase I enzyme (xylosylprotein beta1, 4-galactosyltransferase; EC 2.4.1.133), which attaches the first galactose in the proteoglycan linkage region GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser. Sequence analysis of beta4Gal-T7 from a fibroblast cell line of a patient with a progeroid syndrome and signs of the Ehlers-Danlos syndrome, previously shown to exhibit reduced galactosyltransferase I activity (Quentin, E., Gladen, A., Rodén, L., and Kresse, H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1342-1346), revealed two inherited allelic variants, beta4Gal-T7(186D) and beta4Gal-T7(206P), each with a single missense substitution in the putative catalytic domain of the enzyme. beta4Gal-T7(186D) exhibited a 4-fold elevated K(m) for the donor substrate, whereas essentially no activity was demonstrated with beta4Gal-T7(206P). Molecular cloning of beta4Gal-T7 should facilitate general studies of its pathogenic role in progeroid syndromes and connective tissue disorders with affected proteoglycan biosynthesis.

Through its ability to bind extracellular matrix constituents and growth factors the small leucine-rich chondroitin/dermatan sulfate proteoglycan decorin which is present in many types of connective tissues may play an important biological role in remodeling and maintenance of extracellular matrices during inflammation, fibrosis, and cancer growth. In this study we investigated the known binding of decorin to human collagen XIV. This binding was unaffected when the small collagenous moiety of collagen XIV was removed with collagenase. Therefore, fragments covering the large noncollagenous domain NC3 of collagen XIV were expressed in Escherichia coli, each fused to a 26-kDa fragment of glutathione S-transferase. Using radioiodinated decorin as ligand for the immobilized fusion proteins, a binding site that interacted with the decorin core protein could be assigned to the NH2-terminal fibronectin type III repeat of collagen XIV. In addition, an auxiliary binding site located COOH-terminal to this fibronectin type III repeat interacted with the glycosaminoglycan component of decorin.

The chondroitin/dermatan sulfate proteoglycan decorin is known to interact via its core protein with fibrillar collagens, thereby influencing the kinetics of fibril formation and the final diameter of the fibrils. To define the binding site(s) for type I collagen along the core protein, which is mainly composed of leucine-rich repeat structures, decorin cDNAs were constructed and expressed in human kidney 293 cells. The constructs encoded (i) C-terminally truncated molecules, (ii) core proteins with deletions of selected leucine-rich repeats, or (iii) various point mutations. The deletion of the sixth leucine-rich repeat Met176-Lys201 and the mutation E180K drastically interfered with the binding to reconstituted type I collagen fibrils. In contrast, the deletion of the seventh repeat Leu202-Ser222 led at the most to a marginally impaired binding, although the secretion of this proteoglycan was abnormally low. Decorin with two other point mutations in the sixth leucine-rich repeat, Lys187 --> Gln and Lys200 --> Gln, respectively, bound type I collagen either normally or even better than the normal recombinant proteoglycan. These data suggest that a major collagen-binding site of decorin is located within the sixth leucine-rich repeat and that glutamate-180 within this repeat is of special importance for ionic interactions between the two matrix components.

Interactions between the core protein of the small dermatan sulfate proteoglycan decorin and type I collagen have been considered to influence the kinetics of collagen fibrillogenesis and the diameter of and the distance between the fibrils. A variety of recombinant core protein fragments were expressed in Escherichia coli, extracted from inclusion bodies, and renatured in the presence of bovine serum albumin, which was essential for obtaining functional activity. A recombinant protein lacking the first 14 amino acids of the mature core protein (P15-329) interacted with reconstituted type I collagen fibrils and inhibited collagen fibrillogenesis almost as efficiently as intact decorin purified from fibroblast secretions under non-denaturing conditions. Peptides comprising amino acids 15-183 (P15-183) and 185-329 (P185-329) were able to compete for the binding of wild-type decorin, with P15-183 being more active than P185-329. Several other peptides were much less effective. Binding studies using radioactively labeled peptides P15-183 and P185-329 gave direct evidence for the independent binding of both peptides. Peptides 15-183 and 15-125 had the capability of inhibiting collagen fibrillogenesis, whereas peptide 185-329 was inactive. These data suggest (i) that there are at least two separate binding domains for the interaction between decorin core protein and type I collagen and (ii) that binding is not necessarily correlated with an alteration of collagen fibrillogenesis.

The small proteoglycan decorin is known to interact with type I collagen fibrils, thereby influencing the kinetics of fibril formation and the distance between adjacent collagen fibrils. The structurally related proteoglycan biglycan has been proposed not to bind to fibrillar collagens. However, when osteosarcoma cells were cultured on reconstituted type I collagen fibrils, both decorin and biglycan were retained by the matrix. Immunogold labeling at the electron microscopic level showed that both proteoglycans were distributed along collagen fibrils not only in osteosarcoma cell-populated collagen lattices but also in human skin. Reconstituted type I collagen fibrils were able to bind in vitro native and N-glycan-free biglycan as well as recombinant biglycan core protein. From Scatchard plots dissociation, constants were obtained that were higher for glycanated biglycan (8.7 x 10(-8) mol/liter) than for glycanated decorin (7 x 10(-10) mol/liter and 3 x 10(-9) mol/liter, respectively). A similar number of binding sites for either proteoglycan was calculated. Recombinant biglycan and decorin were characterized by lower dissociation constants compared with the glycanated forms. Glycanated as well as recombinant decorin competed with glycanated biglycan for collagen binding, suggesting that identical or adjacent binding sites on the fibril are used by both proteoglycans. These data suggest that, because of its trivalency, biglycan could have a special organizing function on the assembly of the extracellular matrix.

Fibroblasts from young patients exhibiting clinical features of progeroidal syndromes showed decreased biosynthesis of the small proteoglycan decorin. Cells in culture were metabolically labeled, and proteoglycans secreted into the medium were analyzed electrophoretically after immunoprecipitation with antibodies raised against decorin and biglycan. Fluorograms showed regularly a reduction to 15-30% of the normal amount of mature decorin and its core protein after chondroitin ABC lyase treatment. The size of the glycosaminoglycan chains was increased, but there was no obvious anomaly in the secretion kinetics of the mature proteoglycan. In addition, the patients' fibroblasts synthesized an increased amount of biglycan compared to control cells from healthy donors. Northern blot analysis clearly demonstrated a reduction by 85-94% in decorin mRNA, but biglycan mRNA was concomitantly increased, indicating that these alterations occur at the transcriptional level of protein expression. Transcription of decorin in fibroblasts from one of the patients was stimulated up to 3-fold by treatment with interleukin-1 beta. No response to interleukin-1 beta and transforming growth factor-beta was observed in the cells from another patient. In situ hybridization of cultured cells with an antisense decorin probe showed that decorin levels were reduced throughout the cell population. Surprisingly, subsequent examination of cells from one of the patients, now in mid-teenage, revealed a return to normal levels of decorin expression compared to age-matched controls. These studies suggest that, as in Marfan's syndrome where the primary defect concerns the fibrillin gene, reduced decorin expression contributes to the formation of an abnormal matrix and the pathogenesis of these disorders. They also indicate that this abnormality is likely to represent a secondary phenomenon which leads to a fault in the regulation of decorin gene transcription.

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.

Human skin fibroblasts express, in addition to versican, a second large chondroitin sulfate/dermatan sulfate proteoglycan, which has been investigated with the aid of a specific antiserum in cultures of fetal fibroblasts. Its core protein, obtained after chondroitin ABC lyase treatment, exhibits an apparent molecular mass of about 740 kDa in the absence of a reducing agent whereas reduction produces two core proteins of 460 and 300 kDa, respectively. Both subunits carry one or very few dermatan sulfate chains of about 20 kDa which are of similar chemical composition irrespective of the type of subunits to which they are attached. Tryptic peptide maps of [35S]methionine-labeled core proteins indicated that both subunits are related neither to each other nor to versican, suggesting that the proteoglycan exists predominantly as a heterodimeric molecule. It is insensitive to collagenase and does not interact with hyaluronan. Pulse-chase experiments suggested that the core proteins are different gene products. Dimerization begins soon after core protein synthesis but requires more than 2 h for completion. Glycosaminoglycan synthesis occurs immediately prior to secretion. A small proportion of both subunits may be secreted in form of a monomeric proteoglycan. The heterodimeric proteoglycan is a major proteoglycan species of fetal fibroblasts. The secreted product represents 10-20% of [35S]methionine and about 5-10% of [35S]sulfate incorporated into secreted proteoglycans.

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.

The first step of the degradation of p-nitrophenyl-6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside and of keratan sulfate-derived oligosaccharides bearing N-acetylglucosamine-6-sulfate residues at the nonreducing end was considered to be accomplished by the action of a specific sulfatase (Kresse, H., Paschke, E., von Figura, K., Gilberg, W., and Fuchs W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6822-6826). In purification from human placenta, however, this activity co-chromatographed with isoenzyme A of beta-N-acetylhexosaminidase and had the same electrophoretic mobility as the latter enzyme. The activity was precipitated by a specific antiserum against beta-N-acetylhexosaminidase. A pronounced enzyme deficiency was found in Tay-Sachs and Sandhoff fibroblasts. The purified enzyme released p-nitrophenol from the chromogenic substrate as well as a second product which contained equimolar amounts of hexosamine and sulfate. This product had the same electrophoretic and chromatographic behavior as sulfated N-acetylglucosamine. It could be degraded by periodate to a smaller charged fragment. Incubation of keratan sulfate-derived oligosaccharides with beta-N-acetylhexosaminidase A analogously resulted in the liberation of N-acetylglucosamine-6-sulfate. The enzyme showed the highest affinity towards a trisulfated tetrasaccharide and exhibited a similar Km for the sulfated and the unsulfated p-nitrophenyl derivative.

Endocytosis and subsequent degradation of iduronic acid-rich small dermatan sulfate proteoglycan from fibroblast secretions were studied in human fibroblasts. Upon endocytosis of [3H]leucine- and [35S]sulfate-labeled proteoglycan release of free leucine was 10 to 15 times more rapid than that of inorganic sulfate. Within approximately 3 h a steady state was approached between transport of proteoglycan to the compartment of core protein degradation and release of free leucine. No such steady state could be found with respect to the dermatan sulfate chains. In the presence of benzyloxycarbonyl-Phe-Ala-diazomethylketone or of other SH-protease inhibitors the degradation of the protein moiety of endocytosed proteoglycan was much less inhibited than the degradation of the polysaccharide chain. Benzyloxycarbonyl-Phe-Ala-diazomethylketone did not affect the degradation of dermatan sulfate chains taken up by fluid phase endocytosis and the activities of all known dermatan sulfate-degrading enzymes. Percoll gradient centrifugation indicated that also in the presence of the protease inhibitor the partially degraded proteoglycan accumulated in dense lysosomes. The isolation of intracellular dermatan sulfate peptides and molecular size determinations of endocytosed dermatan sulfate proteoglycan supported the conclusion that a critical proteolytic step is required before the dermatan sulfate chain becomes accessible to hydrolytic enzymes.

Biosynthesis and secretion of proteodermatan sulfate produced by cultured human skin fibroblasts were investigated employing immunological procedures. During an incubation period of 10 min in the presence of [3H]leucine, two core protein forms of Mr = 46,000 and 44,000, respectively, were synthesized. They were converted to mature proteodermatan sulfate with a half-time of approximately 12 min. Fifty per cent of total mature proteodermatan sulfate were found in the culture medium after a 35-min chase. Six to eight per cent remained associated with the cell layer after a chase of 6 h. In the presence of tunicamycin, fibroblasts synthesized a single core protein of Mr = 38,000 that was converted to mature proteodermatan sulfate and secreted with similar kinetics as the N-glycosylated species. Subtle differences in the molecular size of core proteins were noted when cell-associated and secreted proteodermatan sulfate were degraded with chondroitin ABC lyase, but core proteins free of N-linked oligosaccharides were identical. Labeling with [3H]mannose revealed that secreted proteodermatan sulfate contains two or three complex-type or two complex-type and one high-mannose-type N-linked oligosaccharide chains. The N-glycans are bound to a 21-kDa fragment of the core protein. After incubation in the presence of [3H]glucosamine, the [3H]galactosamine/[3H]glucosamine ratio was 3.76 and 3.30 for secreted and cell-associated proteodermatan sulfate, respectively. Evidence for the presence of O-linked oligosaccharides could not be obtained. Small amounts of core protein free of dermatan sulfate chains were secreted when the cultures were treated with p-nitrophenyl-beta-D-xyloside.

In cultured human skin fibroblasts, the core protein of the small proteodermatan sulfate becomes phosphorylated post-translationally but before the glycosaminoglycan chains are synthesized. This phosphorylation can occur when the intracellular transport is inhibited by carbonyl cyanide m-chlorophenylhydrazone or when the attachment of asparagine-linked oligosaccharides is prevented by tunicamycin. Serine and glycosaminoglycan chains were identified as phosphorylation sites of secreted proteodermatan sulfate. Upon alkaline borohydride treatment and degradation by chondroitin ABC lyase, the main phosphorylated product co-chromatographed with an unsulfated 3H-labeled hexasaccharide prepared analogously from [3H]galactose/[35S]sulfate-labeled proteodermatan sulfate.

N-Acetylglucosamine-6-sulfate sulfatase, which liberates sulfate from the N-acetylglucosamine 6-sulfate residue at the nonreducing terminus of a 3H-labeled trisaccharide prepared from heparan sulfate, was purified 136-fold from human urine. The final N-acetylglucosamine-6-sulfate sulfatase preparation was free of all lysosomal sulfatases known to act on sulfated polysaccharides and gave a single band in polyacrylamide gel electrophoresis. The enzyme appears to be a glycoprotein with a molecular weight of around 97,000 and displays considerable charge heterogeneity. Multiple forms with pI values between 5.4 and 8.3 with a maximum at pH 7.7 were detected. The enzyme acts on the 3H-trisaccharide with a pH optimum at 5.5 and is active towards the sulfated monosaccharides N-acetylglucosamine 6-sulfate and glucose 6-sulfate. Although predominantly in exosulfatase, the enzyme catalyzes hydrolysis of sulfate from internal N-acetylglucosamine 6-sulfate moieties at a low rate. The Km for the 3H-trisaccharide, N-acetylglucosamine 6-sulfate, and glucose 6-sulfate were 0.15, 1.5, and 7.7 mM, respectively. The enzyme is inhibited by albumin, Hg2+, PO43-, SO42-, and CN-. Enzyme activity was highest in kidney and cultured fibroblasts but could be demonstrated in all human tissues tested.

Two 3'-phosphoadenylylsulfate:keratan sulfate sulfotransferases were purified 600-fold and 340-fold, respectively, from isolated bovine cornea cells. Sulfotransferase I exhibited an apparent Mr = 220,000, whereas an Mr = 140,000 was calculated for sulfotransferase II. The final preparations were both devoid of chondroitin sulfate sulfotransferase activity. The position of sulfation was determined by proton nuclear magnetic resonance spectroscopy. Sixty per cent of the sulfate ester groups formed by sulfotransferase I were linked to the C-6 atom of galactosyl residues, the other ones to the C-6 atom of N-acetylglucosamine. Sulfotransferase II showed a different specificity: 23% of the newly formed sulfate ester groups were on galactosyl and 77% on N-acetylglucosaminyl residues. Both sulfotransferase preparations acted in a cooperative manner. In the presence of both sulfotransferases, the incorporation of [35S]sulfate into keratan sulfate was up to 75% higher than could be expected from the sum of individual activities. From the specific radioactivities of the oligosaccharides produced by digestion with endo-beta-galactosidase, it was also concluded that both enzyme species reacted best with keratan sulfate segments exhibiting a relatively high degree of sulfation.

Biosynthesis and processing of cathepsin B in cultured human skin fibroblasts were investigated using immunological procedures. Upon metabolic labeling with [35S]methionine for 10 min, a precursor form with Mr 44,500 was identified. During an 80-min chase, about 50% of it was converted to an Mr 46,000 form. Further processing yielded mature forms with Mr 33,000 and 27,000, in a final quantitative ratio of about 3:1. Processing of cathepsin B was inhibited by leupeptin, which led to an accumulation of the Mr 33,000 polypeptide. The Mr 33,000 form appeared to be the most active form and showed a half-time of about 12 h. About 5% of newly synthesized enzyme was secreted as precursor, being detectable extracellularly already after 40 min. NH4Cl enhanced the secretion of the precursor about 20-fold. The precursor and the 33-kDa form contained phosphorylated N-linked oligosaccharides. Cleavage by peptide N-glycosidase F or biosynthesis in the presence of tunicamycin yielded a precursor with Mr 39,000. Evidence of a mannose 6-phosphate-dependent transport of cathepsin B in fibroblasts was obtained on the basis of the following results: (i) cathepsin B precursor from NH4Cl-stimulated secretions was internalized in a mannose 6-phosphate inhibitable manner, and (ii) I-cell fibroblasts secreted more than 95% of newly synthesized cathepsin B precursor. In conclusion, cathepsin B from human skin fibroblasts shows an analogous biosynthetic behavior as other lysosomal enzymes.

The influence of chlorate, an inhibitor of sulfate adenylyltransferase, on biosynthesis and secretion of proteoglycans was investigated in cultured human skin fibroblasts. At up to 10 mM concentrations, chlorate caused a reduction of [35S]sulfate incorporation into small chondroitin sulfate/dermatan sulfate proteoglycan by up to 96%. Incorporation of [3H]leucine and [3H] glucosamine was only slightly affected. No influence was seen on the polymerization degree of the polysaccharide chain as judged by gel filtration, and on the kinetics of secretion of the proteoglycan. Concomitant with reduced sulfation, however, was an increased sensitivity toward chondroitin AC lyase which suggests a diminished epimerization of D-glucuronic acid to L-iduronic acid residues. Agarose gel electrophoresis revealed that all polysaccharide chains of control chondroitin sulfate/dermatan sulfate proteoglycan exhibited a similar sulfation degree. Chlorate treatment led to the formation of polysaccharide chains of widely varying degree of sulfation, but fully sulfated chains were synthesized even in the presence of 3 mM chlorate, and sulfate-free chondroitin was not detected. Studying the effects of chlorate treatment on the synthesis of other proteoglycan types it was found that, in cell-associated galactosaminoglycans, 6-sulfation of N-acetylgalactosamine residues was less affected than was 4-sulfation. In case of heparan sulfate the synthesis of sulfamate groups was less impaired than sulfate ester formation. Nitrous acid degradation at pH 4.1 indicated the presence of unsubstituted amino groups. Chlorate treatment may be considered as a means for the production of proteoglycans with defined structural alterations.