Altered proteoglycan synthesis by micromelial limbs induced by 6-aminonicotinamide. Appearance of abnormal forms of cartilage-characteristic proteoglycan (PG-H).

Since administration of 6-aminonicotinamide (10 micrograms) to day-4 chick embryos in ovo was shown to induce micromelial limbs, biosynthesis of cartilage-characteristic proteoglycan-H (PG-H) as an important index of limb chondrogenesis was examined in day-7 normal and micromelial hind limbs by biochemical and immunological methods. (1) Metabolic labelling of the micromelial limbs with [6-3H]glucosamine and either [35S]sulphate or [35S]methionine, followed by analyses of labelled PG-H by glycerol density-gradient centrifugation under dissociative conditions, showed a marked reduction in the PG-H synthesis. (2) PG-H synthesized by the micromelial limbs was much lower than that synthesized by the normal limbs in the biosynthetic ratio of chondroitin sulphate to keratan sulphate and glycoprotein-type oligosaccharide, although no significant difference was observed in the immunological properties of these proteoglycans. (3) The degree of sulphation of chondroitin sulphates of PG-H was lowered in the micromelial limbs as judged by the increase of unsulphated disaccharide (delta Di-OS) released by chrondroitinase ABC digestion, although there were no significant differences between the normal and the micromelial limbs in the average molecular size (Mr = 38,000) of labelled chondroitin sulphates of PG-H. (4) Addition of beta-D-xyloside, an artificial initiator for chondroitin sulphate synthesis, to the micromelial limbs in culture recovered the incorporation of labelled glucosamine into chondroitin sulphate to that comparable with the normal control with beta-D-xyloside, although the incorporation of [35S]sulphate was lower in the micromelia than in the control with beta-D-xyloside. These results suggest that the reduction in the biosynthesis of the PG-H as well as the production of altered forms of PG-H induced by 6-aminonicotinamide during a critical period of limb morphogenesis may be an important factor for the micromelia.     

Abnormal synthesis of cartilage-characteristic proteoglycan in azaserine-induced micromelial limbs.

Administration of azaserine (250 micrograms) to day-4 chick embryos in ovo was shown to induce micromelial limbs. In the present study, biosynthesis of cartilage-characteristic proteoglycan H (PG-H) as an index of limb chondrogenesis was examined in normal and micromelial hind limbs from day-7 chick embryos by biochemical and immunological methods. (1) Metabolic labelling of the micromelial limbs with [6-3H]-glucose and [35S]sulphate, followed by analysis of labelled proteoglycans by glycerol-density-gradient centrifugation under dissociative conditions, showed a marked reduction in PG-H synthesis. (2) PG-H synthesized by micromelial limbs differed from that synthesized by normal limbs in possessing a slower sedimenting velocity and much lower amounts of chondroitin sulphates. (3) The amount of PG-H core protein in micromelial limbs was significantly decreased to about 19% on a per limb basis and about 42% on a per DNA basis of that in normal limbs, as determined by e.l.i.s.a. (4) The transition from PG-M to PG-H during limb formation was retarded in micromelial limbs as judged by an indirect immunofluorescence technique using antibodies against PG-M and PG-H. (5) The deficiency of incorporation of labelled glucose into chondroitin sulphate chains of PG-H in micromelial limbs was partially restored by using [6-3H]-glucosamine as a precursor, suggesting that the synthesis of UDP-N-acetylhexosamine, required for chondroitin sulphate chain synthesis of PG-H in micromelial limbs, was decreased. These results suggest that the reduction in the synthesis of PG-H as well as the production of an abnormal form of PG-H during a critical period of limb morphogenesis may be important factors in explaining the micromelia induced by azaserine.     

Early changes in parathyroid hormone response and proteoglycan synthesis of chick embryonic femur produced by exposure to 6-aminonicotinamide in ovo

The mechanism of induction of micromelia in 6-day-old chick embryo by 6-aminonicotinamide (6-AN) was investigated. Six-day-old chick embryo exposed to 6-AN did not show micromelia when tenfold excess of nicotinamide over 6-AN was co-administered. The ability of nicotinamide to prevent the induction of micromelia was partially offset after 4 hr of exposure to 6-AN and completely disappeared after 6 hr. The length of time necessary for the induction of micromelia was not affected by the concentration of 6-AN. These results indicate that exposure to 6-AN for only a short period of 6 hr is sufficient to commit the limb to micromelia and that cellular components involved in the induction of micromelia alter during this period. During this period, newly synthesized proteoglycan monomers typical of cartilage decreased in average molecular size, and isolated femora did not respond to parathyroid hormone (PTH) but to dibutyryl cyclic AMP to stimulate growth of cartilage in organ culture.     

A large chondroitin sulfate proteoglycan (PG-M) synthesized before chondrogenesis in the limb bud of chick embryo

Extraction of stage 22-23 chick embryo limb buds that had been metabolically labeled with [35S]sulfate yielded heparan sulfate proteoglycan, small chondroitin sulfate proteoglycan, and large chondroitin sulfate proteoglycan (designated PG-M). PG-M constituted over 60% of the total macromolecular [35S]sulfates. It was larger in hydrodynamic size, richer in protein, and contained fewer chondroitin sulfate chains as compared to the predominant proteoglycan (PG-H, Mr congruent to 1.5 X 10(6)) of chick embryo cartilage. The chondroitin sulfate chains were notable for their large size (Mr greater than or equal to 60,000) and high content of nonsulfated chondroitin units (about 20% of the total hexosamine). Hexosamine-containing chains corresponding in size to N-linked and O-linked oligosaccharides were also present. The core protein was rich in serine, glutamic acid (glutamine), and glycine which together comprised about 38% of the total amino acids. Following chondroitinase AC II (or ABC) digestion, core molecules were obtained which migrated on sodium dodecyl sulfate gel electrophoresis as a doublet of bands with approximately Mr = 550,000 (major) and 500,000, respectively. The Mr = 550,000 core glycoprotein was structurally different from the core glycoprotein (Mr congruent to 400,000) of PG-H, as ascertained by tryptic peptide mapping and immunochemical criteria. Immunofluorescent localization of PG-M showed that the intensity of PG-M staining progressively became higher in the core mesenchyme region than in the peripheral loose mesenchyme, closely following the condensation of mesenchymal cells. Since the cell condensation process has been shown to begin with the increase of fibronectin and type I collagen concentration, the similar change in PG-M distribution suggests that PG-M plays an important role in the cell condensation process by means of its interaction with fibronectin and type I collagen.     

Nucleotide-sugar transporter SLC35D1 is critical to chondroitin sulfate synthesis in cartilage and skeletal development in mouse and human

Proteoglycans are a family of extracellular macromolecules comprised of glycosaminoglycan chains of a repeated disaccharide linked to a central core protein. Proteoglycans have critical roles in chondrogenesis and skeletal development. The glycosaminoglycan chains found in cartilage proteoglycans are primarily composed of chondroitin sulfate. The integrity of chondroitin sulfate chains is important to cartilage proteoglycan function; however, chondroitin sulfate metabolism in mammals remains poorly understood. The solute carrier-35 D1 (SLC35D1) gene (SLC35D1) encodes an endoplasmic reticulum nucleotide-sugar transporter (NST) that might transport substrates needed for chondroitin sulfate biosynthesis. Here we created Slc35d1-deficient mice that develop a lethal form of skeletal dysplasia with severe shortening of limbs and facial structures. Epiphyseal cartilage in homozygous mutant mice showed a decreased proliferating zone with round chondrocytes, scarce matrices and reduced proteoglycan aggregates. These mice had short, sparse chondroitin sulfate chains caused by a defect in chondroitin sulfate biosynthesis. We also identified that loss-of-function mutations in human SLC35D1 cause Schneckenbecken dysplasia, a severe skeletal dysplasia. Our findings highlight the crucial role of NSTs in proteoglycan function and cartilage metabolism, thus revealing a new paradigm for skeletal disease and glycobiology.     

Sulfation of p-nitrophenyl-N-acetyl-beta-D-galactosaminide with a microsomal fraction from cultured chondrocytes

Chick embryo chondrocyte microsomes containing intact Golgi vesicles took up 3'-phosphoadenosine-5'-phospho[35S]sulfate ([35S]PAPS) in a time- and temperature-dependent, substrate-saturable manner. When [35S]PAPS and p-nitrophenyl-N-acetyl-beta-D-galactosaminide (pNP-GalNAc) were added to the incubation in the absence of detergent, the microsomes catalyzed the transfer of sulfate from [35S]PAPS to pNP-GalNAc to form pNP-GalNAc-6-35SO4. The apparent Km values for PAPS in the uptake and the pNP-GalNAc sulfation reactions were 2 X 10(-7) and 2 X 10(-6) M, respectively. The sulfation of pNP-GalNAc by the microsomal preparation was inhibited by detergent. The microsomal fraction also catalyzed the transfer of sulfate from [35S]PAPS to oligosaccharides prepared from chondroitin. However, in contrast to the sulfation of pNP-GalNAc, the rate of sulfation of these oligosaccharides was low in the absence of detergent and was markedly stimulated when detergent was added. Sulfation of pNP-GalNAc by the freeze-thawed microsomes was inhibited when the octasaccharide prepared from chondroitin was present in the reaction mixture. As the PAPS that had been internalized in the microsomal vesicles was consumed in the sulfation of pNP-GalNAc, more [35S]PAPS was taken up and the sulfated pNP-GalNAc was released from the vesicles. These observations suggest that pNP-GalNAc may serve as a model membrane-permeable substrate for study of the 6-sulfo-transferase reaction involved in sulfation of chondroitin sulfate in intact Golgi vesicles.     

Acetylcholine receptor clustering associates with proteoglycan biosynthesis in C2 variant and heterkaryon muscle cells

Several lines of evidence have suggested roles for proteoglycans (PGs) in acetylcholine receptor (AChR) clustering on muscle cells. One line of evidence comes from the correlation between a defect in the biosynthesis of glycosaminoglycans (GAGs), the defining carbohydrates of PGs, and the failure of spontaneous AChR clustering in the S27 cell line, a genetic variant of the C2 muscle cell line. Two approaches were used in the present study to investigate whether GAG and AChR clustering defects are causally linked. First, the formation of AChR clusters was examined in two more variant lines, S11 and S26, also isolated from the C2 muscle cell line on the basis of deficiencies in GAG biosynthesis. S11 and S26, like S27, are also defective in AChR clustering. Ion exchange analysis of the GAGs made by the S11, S26, and S27 lines revealed that the defects in GAG biosynthesis differ between the three lines. Second, heterokaryon myotubes formed between pairs of the GAG defective variants were tested for complementation in both AChR clustering and GAG biosynthesis. AChR clusters were conspicuous on individual heterokaryon myotubes, and GAG biosynthesis was restored to near wild type levels in the heterokaryon cultures. Complementation in GAG biosynthesis corroborates the biochemical data that the relevant mutations in the genetic variants are in different genes and establishes that the defects are not dominant. The consistent correlation between GAG defects and the failure of AChR clustering across three independent genetic variants and the complementary association of GAG biosynthesis with AChR clustering in heterokaryon myotubes argues against a chance association of the two phenotypes and for a causal relationship between PGs and AChR clustering. A prominent chondroitin sulfate peak correlated with AChR clustering in the heterokaryon cultures. This is consistent with earlier results suggesting that chondroitin sulfate in general is required for the spontaneous clustering of AChRs in C2 cultures and further suggests that a particular chondroitin sulfate proteoglycan may be essential for the clustering process. © 1996 John Wiley & Sons, Inc.     

Distribution in cesium chloride gradients of proteoglycans of chick embryo brain and characterization of a large aggregating proteoglycan

Proteoglycans were extracted from 14-day chick embryo brains, which had been labelled in vitro with [35S]sulfate or 3H-labelled amino acids. 4.0 M guanidinium chloride (containing proteinase inhibitors) extracted 94% of the 35S-labelled glycoconjugates. Following cesium chloride equilibrium centrifugation, the proteoglycans in each fraction were characterized by chromatography on Sepharose CL-2B. The most dense fraction (D1), which contained no detectable non-proteoglycan proteins, contained a large, aggregating chondroitin sulfate proteoglycan in addition to small chondroitin sulfate and heparan sulfate proteoglycans. The less dense fractions (D2-D6) contained both small chondroitin sulfate and heparan sulfate proteoglycans. Removal of hyaluronate from the D1 sample by digestion with Streptomyces hyaluronidase in the presence of proteinase inhibitors showed that aggregation of the large chondroitin sulfate proteoglycan is hyaluronate-dependent. Aggregation was restored by re-addition of hyaluronate. Reduction and alkylation, which blocked aggregation of a cartilage A1 proteoglycan, did not interfere with aggregation of the large brain proteoglycan.     

Sulfated glycosaminoglycans (GAG) in the developing mouse brain : Quantitative aspects on the metabolism of total and individual sulfated GAG in vivo

Sulfation and desulfation of total glycosaminoglycans (GAG) as well as of chondroitin sulfates (A + C), dermatan sulfate, and heparan sulfate were quantified in the developing cerebrum and cerebellum of mice by labeling with [35S]sulfate combined with chases started 24 hr after [35S]sulfate injection. In both the developing cerebrum and cerebellum, the rate of biosynthesis of total sulfated GAG was highest shortly after birth (2 days), decreased sharply thereafter, and reached a plateau after 14 days. The biosynthetic activities of chondroitin sulfates and heparan sulfate decreased sharply up to 14 days and retained constant levels afterward. By contrast, the rates of biosynthesis of dermatan sulfate increased up to 14 days. The biodegradation rates of total sulfated GAG as well as of chondroitin sulfates, heparan sulfate, and dermatan sulfate were strongly correlated with the corresponding rates of biosynthesis during the first 2 postnatal weeks. Total and individual sulfated GAG showed high degradation rates resulting in half-life times of a few hours up to 1 1/2 days. Thus sulfated GAG are synthesized in excess and the actual net content seems to be co-regulated to a high degree by lysosomal degradation. In both brain parts, a proportional increase of the sulfated GAG content vs the total GAG content from 40% at birth to 90% at 28 days was observed. Since during development heparan sulfate and dermatan sulfate manifested a relative increase in their daily net synthesis besides a decrease of chondroitin sulfates, a developmental increase of the sulfate groups linked to GAG is evidenced. This molecular differentiation resulting in microenvironmental changes may be of high functional significance.     

Characterization of proteoglycans synthesized by rabbit articular chondrocytes in response to transforming growth factor-beta (TGF-beta)

The effect of transforming growth factor-beta (TGF-beta, 1 ng/ml) on proteoglycan synthesis by rabbit articular chondrocytes in culture was studied in the presence of fetal bovine serum. Exposure of confluent cells for 24 h to the factor resulted in a marked increase of 35S-labeled sulfate incorporation in the newly synthesized proteoglycans (PG), as estimated by glycosaminoglycan (GAG) radioactivity (+58%). The onset was observed 6 h after addition of the factor but was significant after 12 h. TGF-beta also enhanced the uptake of [35S]sulfate by chondrocytes, but had no effect on the release of PG by these cells. The effect of TGF-beta on the distribution of PG between the medium and the cell layer was shown to be dependent on the serum concentration in the medium: the relative proportion of cell-layer associated GAG of TGF-beta-treated cells decreased with increasing concentration of fetal bovine serum. The proportion of aggregated PG, the hydrodynamic size of PG monomers and GAG chains were not modified by TGF-beta, but the relative distribution of disaccharides 6- and 4-sulfate in GAG chains was altered by the factor: the proportion of chondroitin 6-sulfate (C6S) was decreased while that of chondroitin 4-sulfate (C4S) was augmented in presence of TGF-beta, leading to a decrease of the ratio C6S/C4S (-11 to -22%, P less than 0.01). The present study indicates that TGF-beta promotes the synthesis of a modified extracellular matrix in cultured articular chondrocytes. This mechanism could be relevant to some aspects of cartilage repair in osteoarticular diseases.     

Embryonic lung morphogenesis in organ culture: experimental evidence for a proteoglycan function in the extracellular matrix

The lung rudiment, isolated from mid-gestation (11 day) mouse embryos, can undergo morphogenesis in organ culture. Observation of living rudiments, in culture, reveals both growth and ongoing bronchiolar branching activity. To detect proteoglycan (PG) biosynthesis, and deposition in the extracellular matrix, rudiments were metabolically labeled with radioactive sulfate, then fixed, embedded, sectioned and processed for autoradiography. The sulfated glycosaminoglycan (GAG) types, composing the carbohydrate component of the proteoglycans, were evaluated by selective GAG degradative approaches that showed chondroitin sulfate PG principally associated with the interstitial matrix, and heparan sulfate PG principally associated with the basement membrane. Experiments using the proteoglycan biosynthesis disrupter, beta-xyloside, suggest that when chondroitin sulfate PG deposition into the ECM is perturbed, branching morphogenesis is compromised.     

Partial characterization of newly synthesized proteoglycans isolated from the glomerular basement membrane

Kidneys were perfused with [35S]sulfate at 4 h in vitro to radiolabel sulfated proteoglycans. Glomeruli were isolated from the labeled kidneys, and purified fractions of glomerular basement membrane (GBM) were prepared therefrom. Proteoglycans were extracted from GBM fractions by use of 4 M guanidine-HCl at 4 degrees C in the presence of protease inhibitors. The efficiency of extraction was approximately 55% based on 35S radioactivity. The extracted proteoglycans were characterized by gel-filtration chromatography (before and after degradative treatments) and by their behavior in dissociative CsCl gradients. A single peak of proteoglycans with an Mr of 130,000 (based on cartilage proteoglycan standards) was obtained on Sepharose CL-4B or CL-6B. Approximately 85% of the total proteoglycans were susceptible to nitrous acid oxidation (which degrades heparan sulfates), and approximately 15% were susceptible to digestion with chondroitinase ABC (degrades chondroitin-4 and -6 sulfates and dermatan sulfate). The released glycosaminoglycan (GAG) chains had an Mr of approximately 26,000. Density gradient centrifugation resulted in the partial separation of the extracted proteoglycans into two types with different densities: a heparan sulfate proteoglycan that was enriched in the heavier fraction (p greater than 1.43 g/ml), and a chondroitin sulfate proteoglycan that was concentrated in the lighter fractions (p less than 1.41). The results indicate that two types of proteoglycans are synthesized and incorporated into the GBM that are similar in size and consist of four to five GAG chains (based on cartilage proteoglycan standards). The chromatographic behavior of the extracted proteoglycans and the derived GAG, together with the fact that the two types of proteoglycans can be partially separated into the density gradient, suggest that the heparan sulfate and chondroitin sulfate(s) are located on different core proteins.     

The relation of protein synthesis to chondroitin sulphate biosynthesis in cultured bovine cartilage.

The effect of cycloheximide on chondroitin sulphate biosynthesis was studied in bovine articular cartilage maintained in culture. Addition of 0.4 mM-cycloheximide to the culture medium was followed, over the next 4h, by a first-order decrease in the rate of incorporation of [35S]sulphate into glycosaminoglycan (half-life, t 1/2 = 32 min), which is consistent with the depletion of a pool of proteoglycan core protein. Addition of 1.0 mM-benzyl beta-D-xyloside increased the rate of incorporation of [35S]sulphate and [3H]acetate into glycosaminoglycan, but this elevated rate was also diminished by cycloheximide. It was concluded that cycloheximide exerted two effects on the tissue; not only did it inhibit the synthesis of the core protein, but it also lowered the tissue's capacity for chondroitin sulphate chain synthesis. Similar results were obtained with chick chondrocytes grown in high-density cultures. Although the exact mechanism of this secondary effect of cycloheximide is not known, it was shown that there was no detectable change in cellular ATP concentration or in the amount of three glycosyltransferases (galactosyltransferase-I, N-acetylgalactosaminyltransferase and glucuronosyltransferase-II) involved in chondroitin sulphate chain synthesis. The sizes of the glycosaminoglycan chains formed in the presence of cycloheximide were larger than those formed in control cultures, whereas those synthesized in the presence of benzyl beta-D-xyloside were consistently smaller, irrespective of the presence of cycloheximide. These results suggest that beta-D-xylosides must be used with caution to study chondroitin sulphate biosynthesis as an event entirely independent of proteoglycan core-protein synthesis, and they also indicate a possible involvement of the core protein in the activation of the enzymes of chondroitin sulphate synthesis.     

Structural characterization of proteoglycans produced by testicular peritubular cells and Sertoli cells

The structural characteristics of proteoglycans produced by seminiferous peritubular cells and by Sertoli cells are defined. Peritubular cells secrete two proteoglycans designated PC I and PC II. PC I is a high molecular mass protein containing chondroitin glycosaminoglycan (GAG) chains (maximum 70 kDa). PC II has a protein core of 45 kDa and also contains chondroitin GAG chains (maximum 70 kDa). Preliminary results imply that PC II may be a degraded or processed form of PC I. A cellular proteoglycan associated with the peritubular cells is described which has properties similar to those of PC I. Sertoli cells secrete two different proteoglycans, designated SC I and SC II. SC I is a large protein containing both chondroitin (maximum 62 kDa) and heparin (maximum 15 kDa) GAG chains. Results obtained suggest that this novel proteoglycan contains both chondroitin and heparin GAG chains bound to the same core protein. SC II has a 50-kDa protein core and contains chondroitin (maximum 25 kDa) GAG chains. A proteoglycan obtained from extracts of Sertoli cells is described which contains heparin (maximum 48 kDa) GAG chains. In addition, Sertoli cells secrete a sulfoprotein, SC III, which is not a proteoglycan. SC III has properties similar to those of a major Sertoli cell-secreted protein previously defined as a dimeric acidic glycoprotein. The stimulation by follicle-stimulating hormone of the incorporation of [35S]SO2(-4) into moieties secreted by Sertoli cells is shown to represent an increased production or sulfation of SC III (i.e. dimeric acidic glycoprotein), and not an increased production or sulfation of proteoglycans. Results are discussed in relation to the possible functions of proteoglycans in the seminiferous tubule.     

The expression of proteoglycans in phorbol ester induced U-937 cells

U-937 monoblastic cells were differentiated into macrophage-like cells in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA). Control cells and differentiated cells were labeled with³⁵S-sulfate and were both found to produce exclusively chondroitin sulfate proteoglycan. No differences in glycosaminoglycan structure or macromolecular properties of the proteoglycans produced in the two different cell systems could be observed. However, the differentiated cells were found to have a lower capacity for chondroitin sulfate proteoglycan synthesis, both under ordinary experimental conditions, and when exposed to stimulators of glycosaminoglycan biosynthesis such as -d-xylosides.Abbreviations SDS sodium dodecyl sulfate - TPA 12-O-tetradecanoylphorbol-13-acetate - PG proteoglycan - GAG glycoaminoglycan - CS chondroitin sulfate - CSPG chondroitin sulfate proteoglycan - NASDAE naphthol AS-D acetate esterase     

Defect in 3′-phosphoadenosine 5′-phosphosulfate synthesis in brachymorphic mice: I. Characterization of the defect

Biosynthesis of the undersulfated proteoglycan found in brachymorphic mouse (bm/ bm) cartilage has been investigated. Similar amounts of cartilage proteoglycan core protein, as measured by radioimmune inhibition assay, and comparable activity levels of four of the glycosyltransferases requisite for synthesis of chondroitin sulfate chains were found in cartilage homogenates from neonatal bm/bm and normal mice, suggesting normal production of glycosylated core protein acceptor for sulfation. When incubated with ³⁵S-labeled 3′-phosphoadenosine 5′-phosphosulfate (PAPS), bm/bm cartilage extracts showed a higher than control level of sulfotransferase activity. In contrast, when synthesis was initiated from ATP and ³⁵SO₄^2?, mutant cartilage extracts showed lower incorporation of ³⁵SO₄^2? into endogenous chondroitin sulfate proteoglycan (19% of control level) and greatly reduced formation of PAPS (10% of control level). Results from coincubations of normal and mutant cartilage extracts exhibited intermediate levels of sulfate incorporation into PAPS and endogenous acceptors, suggesting the absence of an inhibitor for sulfate-activating enzymes or sulfotransferases. Degradation rates of ³⁵S]PAPS and of ³⁵S-labeled adenosine 5′-phosphosulfate (APS) were comparable in bm/bm and normal cartilage extracts. Specific assays for both ATP sulfurylase (sulfate adenylyltransferase; ATP:sulfate adenylyltransferase, EC 2.7.7.4) and APS kinase (adenylylsulfate kinase; ATP:adenylylsulfate 3′-phosphotransferase, EC 2.7.1.25) showed decreases in the former (50% of control) and the latter (10–15% of control) enzyme activities in bm/bm cartilage extracts. Both enzyme activities were reduced to intermediate levels in extracts of cartilage from heterozygous brachymorphic mice (ATP-sulfurylase, 80% of control; APS kinase, 40–70% of control). Furthermore, the moderate reduction in ATP sulfurylase activity in bm/bm cartilage extracts was accompanied by increased lability to freezing and thawing of the residual activity of this enzyme. These results indicate that under-sulfation of chondroitin sulfate proteoglycan in bm/bm cartilage is due to a defect in synthesis of the sulfate donor (PAPS), resulting from diminished activities of both ATP sulfurylase and APS kinase, although the reduced activity of the latter enzyme seems to be primarily responsible for the defect in PAPS synthesis.     

Carriers for enzymatic attachment of glycosaminoglycan chains to peptide

In the previous study, we have found that the endo-beta-xylosidase from Patinopecten had the attachment activities of glycosaminoglycan (GAG) chains to peptide. As artificial carrier substrates for this reaction, synthesis of various GAG chains having the linkage region tetrasaccharide, GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl, between GAG chain and core protein of proteoglycan was investigated. Hyaluronic acid (HA), chondroitin (Ch), chondroitin 4-sulfate (Ch4S), chondroitin 6-sulfate (Ch6S), and desulfated dermatan sulfate (desulfated DS) as donors and the 4-metylumbelliferone (MU)-labeled hexasaccharide having the linkage region tetrasaccharide at its reducing terminals (MU-hexasaccharide) as an acceptor were subjected to a transglycosylation reaction of testicular hyaluronidase. The products were analyzed by high-performance liquid chromatography and enzyme digestion, and the results indicated that HA, Ch, Ch4S, Ch6S, and desulfated DS chains elongated by the addition of disaccharide units to the nonreducing terminal of MU-hexasaccharide. It was possible to custom-synthesize various GAG chains having the linkage region tetrasaccharide as carrier substrates for enzymatic attachment of GAG chains to peptide.     

The effect of chondroitin sulfate molecular weight and degree of sulfation on the activity of a sulfotransferase from chicken embryo epiphyseal cartilages

The transfer of [35S] sulfate from [35S]PAPS, by means of PAPS: chondroitin sulfate sulfotransferase, to various chondroitin sulfates, with different degrees of sulfation and molecular weights is reported. Analyses by digestion with chondroitin AC and specific 4- or 6-sulfatases indicate that the sulfation occurs only in position 6 of the non-sulfated N-acetyl galactosamine moiety. The 50-70% desulfated chondroitin 4/6-sulfates are two times better sulfate acceptors than totally desulfated chondroitin, and the affinity of the sulfotransferase increases markedly from the octa-to the deca-saccharide. These results suggest that sulfation increases sharply only after the growing polysaccharide contains about 10 sugar residues, in the early stages of polymerization, and that the sulfation of chondroitin sulfate may be a process in which the addition of some sulfate groups facilitates further sulfation.     

Synthesis of glycosaminoglycans by islets of Langerhans

Incorporation of (35S)-sulfate into glycosaminoglycans (GAG) of toadfish islets of Langerhans in vitro was examined. (35S)-sulfated GAG were synthesized by a component of the microsomal fraction, and subsequently transferred to the secretion granules, mitochondria and nuclei. The predominant type of GAG synthesized was heparan sulfate, but chondroitin 4- and 6-sulfate and dermatan sulfate were also found.     

Immunohistochemical localization of cyclic AMP during normal and abnormal chick and mouse limb development

This paper describes the immunohistochemical localization of cAMP during limb chondrogenesis in talpid3 chick, brachypod mouse, and normal embryos. Comparisons were made between chick wing buds at Stages 22, 25, and 30, and mouse hind limb buds at Days 11, 12.5 and 14. At Stage 22, the normal mesenchyme in the chick displayed areas of bright fluorescence compared to a lesser intense and more evenly distributed fluorescence in talpid3. Sections of the central region from normal Stage 25 limb buds exhibited an intense fluorescence that was uniformly distributed, whereas, in talpid3 staining was more mosaic with some areas fluorescing brightly and others showing little fluorescence. At Stage 30 the staining pattern was similar between normal and talpid3, with the fluorescence being brighter in the cartilage tissue than in the surrounding soft tissue. Difference in the staining patterns of normal and brachypod limb tissue were not detectable. At Days 11 and 12.5, tissue from both genotypes displayed a very bright, uniform fluorescence. In the 14-day hind limb buds, the staining patterns were comparable to those observed in Stage 30 chick wing buds. However, under in vitro conditions conducive for the expression of the chondrogenic phenotype, differences in the intensity and extensiveness of fluorescent staining were detectable in cultures derived from 12-day normal and brachypod hind limb mesenchyme. Compared to the control, the uneven distribution of immunofluorescence in the talpid3 limb buds and the differences in intensity and extensiveness of fluorescence in the brachypod cultures support the hypothesis that cAMP is involved in limb cartilage differentiation.