Sulfated polysaccharides and cell differentiation in the sea urchin embryo

The synthesis of sulfated polysaccharides during the embryonic development of Paracentrotus lividus has been investigated by incorporation of radioactive sulfate, glucose, glucosamine and fucose. The following substances become labelled: fucan sulfate (approximately 60%), heparan sulfate (approximately 20%) and dermatan sulfate (approximately 20%), and possibly a very slight amount of chondroitin sulfate. In animalized and vegetalized embryos, the rate of incorporation is significantly reduced, and furthermore dermatan sulfate is almost absent in animalized embryos. It is concluded that this substance is associated with the differentiation of vegetative cells, possibly the mesenchyme cells.     

Analysis of the proteoglycans synthesized by corneal explants from embryonic chicken. I. Characterization of the culture system with emphasis on stromal proteoglycan biosynthesis

Corneal explants with scleral rims were freshly prepared from day 18 chicken embryos and incubated in vitro for 3 h in the presence of various radioactive precursors. Radiolabeled proteoglycans were isolated from the stromal tissue and culture medium for analysis. Two predominant proteoglycans were identified in corneal stroma. One contains dermatan sulfate and the other contains keratan sulfate; a structural analysis of each is reported in the accompanying paper (Midura, R.J., and Hascall, V.C. (1989) J. Biol. Chem. 264, 1423-1430). A minor keratan sulfate proteoglycan distinct from the major form, a small amount of heparan sulfate proteoglycan, and some sulfated glycoproteins were also detected in stromal extracts. The biosynthesis of the dermatan sulfate proteoglycan was stable in vitro and in ovo, whereas that of the major keratan sulfate proteoglycan was stable only in ovo. Various treatments were tried to maintain a high rate of keratan sulfate synthesis with time in culture. Cooling the corneal explants to 5 degrees C was the only treatment that reduced this decline in keratan sulfate synthesis in vitro to any significant extent. Three major proteoglycans were observed in the culture medium. Two were dermatan sulfate proteoglycan and appeared to be mainly derived from the scleral tissue surrounding the corneal explant. The third proteoglycan contained keratan sulfate. It was smaller in size and lower in charge density compared to the keratan sulfate proteoglycan found in the stroma, but both appeared to have similar core protein sizes. It seems likely that this proteoglycan was synthesized in the stroma and secreted into the medium. A small amount of heparan sulfate proteoglycan and some sulfated glycoproteins were also detected in the medium.     

Calcium-mediated hemagglutination by serum amyloid P component and the inhibition by specific glycosaminoglycans

Human serum amyloid P component (SAP) was found to agglutinate erythrocytes in the presence of calcium ion. The hemagglutination was strongly inhibited by hyaluronic acid as well as by heparan sulfate and dermatan sulfate, but not by chondroitin 4-sulfate and keratan sulfate. A specific binding of SAP to hyaluronic acid, heparan sulfate, and dermatan sulfate was also confirmed by the fact that these glycosaminoglycans blocked the binding of SAP to agarose, a specific ligand of SAP.     

Separation and properties of five glycosaminoglycan sulfatases from rat skin.

Chondroitin sulfates, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, and oligosaccharides derived from these sulfated glycosaminoglycans have been used for the measurement of sulfatase activity of rat skin extracts. Chromatographic fractionation of the extracts followed by specificity studies demonstrated the existence of five different sulfatases, specific for 1) the nonreducing N-acetylglucosamine 6-sulfate end groups of heparin sulfate and keratan sulfate, 2) the nonreducing N-acetylgalactosamine (or galactose) 6-sulfate end groups of chondroitin sulfate (or keratan sulfate), 3) the nonreducing N-acetylgalactosamine 4-sulfate end groups of chondroitin sulfate and dermatan sulfate, 4) certain suitably located glucosamine N-sulfate groups of heparin and heparan sulfate, or 5) certain suitably located iduronate sulfate groups of heparan sulfate and dermatan sulfate. Two arylsulfatases, one of which was identical in its chromatographic behaviors with the third enzyme described above, were also demonstrated in the extracts. These results taken together with those previously obtained from studies on human fibroblast cultures suggest that normal skin fibroblasts contain at least five specific sulfatases and diminished activity of any one may result in a specific storage disease.     

Chromatin proteins from normal,vegetalized, and animalized sea urchin embryos

The chemical composition of the chromatin, the fractional content of histones and nonhistone chromatin proteins (NHP), and the biosynthesis of these proteins in normal, vegetalized, and animalized embryos of the sea urchin Strongylocentrotus droebachiensis at the blastula, mesenchyme blastula, and gastrula stages have been studied. The amount of the NHP in the chromatin from normal and vegetalized embryos increases during early embryonic development while that in animalized embryos remains without change at the mesenchyme blastula stage and then decreases. During development the histone content in all three cases slightly decreases. Polyacrylamide gel electrophoresis reveals that both fractional composition of histones and their biosynthesis in normal, vegetalized, and animalized embryos display no differences. During development, however, some changes occur, so that the relative amount of histones F1 and F2a2 increases, F2b decreases, while F3 and F2a1 remains constant. Histone F1 at the blastula stage consists of two subfractions while at the gastrula stage it consists of three subfractions. The histone F2a1 consists of one and two, respectively. Histone F3 at all stages is made up of three subfractions; histone F2b is made up of two; and the histone F2a2 is electrophoretically homogeneous. Specific radioactivity of the arginine-rich histones F3 and F2a1 tends to increase during development, while that of moderately lysine-rich histones F2b and F2a2 does not change, and that of the lysine-rich histone F1 decreases. The NHP in normal, vegetalized, and animalized embryos at different developmental stages consist of 17 fractions that can be separated by isoelectrofocusing within the 4.5-8.8 pH range. Quantitative changes have been observed in the fractions focused at pH 4.5-6.1 during development and in normal and modified embryos at the gastrula stage.     

The Structure and Development of the Apical Ganglion in the Sea Urchin Pluteus Larvae of Strongylocentrotus droebachiensis and Mespilia globulus

The structure of the apical ganglion is described using transmission electron microscopy and ultrastructural immunohistochemical localization with anti-serotonin, and the development of these nerve cells in animalized and vegetalized embryos, and embryos treated with Ca²⁺-deficient sea water are demonstrated with immunofluorescence microscopy. The axons within the neuropile contain clear and dense-core vesicles, but all the vesicles appear anti-serotonin immunoreactive. The principal type of neuron within the apical ganglions is anti-serotonin immunoreactive. In the animalized embryos, serotonergic neuroblasts and neurons appear in the second quarter of animal-vegetal axis. Serotonergic cells were not detected in vegetalized exogestrulae. Mespilia globulus embryos treated with Ca²⁺-deficient sea water ruptured to form a cellular sheet on the substratum. In ruptured embryos serotonergic neurons formed a ring around the area corresponding to the apical plate of normal embryos. These findings indicated that the serotonergic preoral nerve cells of echinopluteus derive from cells on the periphery of the apical plate.     

A general method for the detection and mapping of submicrogram quantities of glycosaminoglycan oligosaccharides on polyacrylamide gels by sequential staining with azure A and ammoniacal silver.

A sensitive method has been developed for the visualization of nonradiolabeled glycosaminoglycan oligosaccharides resolved by polyacrylamide gel electrophoresis using fixation with azure A followed by staining with ammoniacal silver. This method, which can detect as little as 1-2 ng of a single oligosaccharide species, can be used to stain a few micrograms of a complex oligosaccharide mixture. The combination of gradient polyacrylamide gel electrophoresis and sequential azure A/silver staining can be applied to the analysis of all the complex glycosaminoglycans (i.e., heparin, heparan sulfate, chondroitin/dermatan sulfate, keratan sulfate) and hyaluronate, as well as to comparisons of specificities of the glycosaminoglycan-degrading enzymes. This procedure may be particularly valuable in situations where the availability of glycosaminoglycan is very limited and/or where radiolabeling is impractical or undesirable.     

Albumin Synthesis by Rat Hepatocytes Cultured on Collagen Gels Is Sustained Specifically by Heparin

We investigated the influence of various kinds of glycosaminoglycans (GAGs) in collagen gels on the maintenance of albumin synthesis in primary culture of rat hepatocytes. Among the GAGs examined (heparin, heparan sulfate, keratan sulfate, chondroitin sulfate A, dermatan sulfate, and hyaluronic acid), only heparin-containing collagen gel cultures could significantly sustain albumin synthesis. However, other GAGs, such as heparan sulfate and keratan sulfate, had almost no effect on the maintenance of albumin synthesis. Heparin in collagen gels exhibited a dose-dependent effect on albumin synthesis: heparin at 400 μg/ml-collagen solution maintained albumin synthesis for over 3 weeks. On the other hand, when an equivalent amount of heparin was added directly to the collagen gel culture medium, it prolonged albumin synthesis for only 10 days. The results demonstrate that specific regulation of albumin synthesis by heparin was significantly promoted by coincubating it with collagen, suggesting that some specific interaction between heparin and collagen might be of importance for the maintenance of hepatocyte functions.     

Fractionation and isolation of heparan sulfates using poly-L-lysine-Sepharose

A simple procedure for the isolation of heparan sulfates from pig lung using a poly-L-lysine-Sepharose column is described. Glycosaminoglycans are absorbed on poly-L-lysine-Sepharose at pH 7.5 and eluted with an NaCl linear gradient in the following order: hyaluronic acid (0.32 M NaCl), chondroitin (0.36 M NaCl), keratan sulfate (0.80 M NaCl), chondroitin 4-sulfate (0.86 M NaCl), chondroitin 6-sulfate (0.95 M NaCl), dermatan sulfate (0.91 M NaCl), heparan sulfate (1.2 M NaCl), and heparin (1.35 M NaCl). Based on these observations, isolation of heparan sulfate from pig lung crude heparan sulfate fractions which contain chondroitin sulfates and dermatan sulfate was attempted, using this chromatographic technique.     

Immunochemical characterization and ultrastructural localization of chondroitin sulfates and keratan sulfate in embryonic chick bone marrow

Summary Monoclonal antibodies directed against specific carbohydrate epitopes on chondroitin 4-/dermatan sulfate, chondroitin 6-sulfate, keratan sulfate, and a monoclonal antibody directed against the hyaluronate binding region were used to characterize proteoglycans extracted from embryonic chick bone marrow. About half of the proteoglycans separate into the high density fraction on a CsCl gradient. Glycosaminoglycan-specific antibodies recognize proteoglycans from all fractions; this includes an antibody directed against keratan sulfate. Some proteoglycans, principally in the high buoyant density fraction, contain sites recognized by the antibody specific for the hyaluronate binding region. Within limits of detection, all core proteins belong to the high-molecular-weight category, with weights in excess of 212 kD. Antibodies directed against chondroitin 4-/dermatan sulfate and against keratan sulfate primarily bind to extracellular matrix material located in the extracellular spaces and to matrix elements in the pericellular regions of fibroblastic stromal cells. The antibody that recognizes chondroitin 6-sulfate binds to sites on surfaces of fibroblastic stromal cells and also to extracellular matrix material. Little or no antibody binding is detected on surfaces of granulocytic cells. These studies indicate that chondroitin sulfate and keratan sulfate chains are both present in the proteoglycan extract.     

Detection of age-related changes in the distributions of keratan sulfates and chondroitin sulfates in developing chick limbs: an immunocytochemical study

A panel of four separate monoclonal antibodies, all known to specifically recognize epitopes on keratan sulfate glycosaminoglycans, were employed in an immunocytochemical study of developing chick hind limbs. In addition, two monoclonal antibodies specific for epitopes on chondroitin/dermatan sulfate glycosaminoglycans were employed on equivalent sections to determine the degree of colocalization of keratan and chondroitin/dermatan sulfates. The spatial distributions of keratan sulfate and chondroitin/dermatan sulfate differed to some extent. In younger embryos, high extracellular concentrations of keratan sulfate occurred in joints and articular cartilages, with diminishing amounts being present in epiphyseal and diaphyseal regions. The high concentration of keratan sulfate in joints and articular cartilage corresponded to equally high concentration of chondroitin-6 sulfate. With advancing age, the above mentioned distribution was modified, most notably by increased amounts of keratan sulfate within diaphyseal regions. Finally, the use of four different anti-keratan sulfate monoclonal antibodies made it possible to compare keratan sulfate epitope expression. Differences in keratan sulfate epitopes were noted in some regions of bones, mostly in diaphyseal regions of younger bones and epiphyseal regions of older bones. This pattern of keratan sulfate expression suggests that different types of keratan sulfate may be present and their expression may be developmentally regulated.     

Analysis of glycosaminoglycan-derived oligosaccharides using reversed-phase ion-pairing and ion-exchange chromatography with suppressed conductivity detection

Oligosaccharides prepared from glycosaminoglycans (GAGs) including heparin, heparan sulfate, chondroitin sulfates, dermatan sulfate, and keratan sulfate were analyzed using reverse-phase ion-pairing HPLC and ion-exchange HPLC with suppressed conductivity detection. The results were compared with those obtained by strong anion-exchange HPLC using uv detection. These oligosaccharides were first prepared by enzymatically depolymerizing the GAGs with enzymes including heparin lyase (EC 4.2.2.7), heparan sulfate lyase (EC 4.2.2.8), chondroitin ABC lyase (EC 4.2.2.4), and keratan sulfate hydrolase (EC 3.2.1.103). Analysis was then performed without derivitization under isocratic conditions with a limit of sensitivity in the picomole range. Preliminary studies suggest that this approach may be particularly useful in examining oligosaccharides having no uv chromophore such as those prepared from keratan sulfate.     

Influence of various extracellular matrix components on the behavior of cultured chick embryo dermal cells

Dermal cells isolated from the back of 7-day chick embryos were cultured on homogeneous two-dimensional substrates consisting of one or two extracellular matrix components (type I, III or IV collagen, fibronectin and several glycosaminoglycans: hyaluronate, chondroitin-4, chondroitin-6, dermatan or heparan sulfate). The effect of these substrates on cell behavior was compared with that of culture dish polystyrene. Three parameters of cell behavior were examined: cell proliferation and patterning, spreading (cell surface) and locomotion (velocity and directionality). Data were collected by sequential microphotography and analyzed by computer assisted morphometry. Types I and III collagen, hyaluronate and heparan sulfate had a slowing down effect on cell proliferation and patterning. The inhibitory effect of type I collagen was also detected in mixtures with glycosaminoglycans. The other components had no effect. While the smallest spreading was observed on fibronectin substrate, the largest was recorded on chondroitin-6 sulfate and heparan sulfate. The slowest velocity of locomotion was measured on fibronectin, types I and IV collagen and a mixture of type I collagen and chondroitin-6 sulfate. The fastest speed was recorded on chondroitin-4 sulfate. These effects are discussed in view of our knowledge of the role of the dermis in the development of skin and cutaneous appendages, and in the light of the morphogenetically related microheterogeneous distribution of collagens, fibronectin and various glycosaminoglycans in the developing skin.     

Age-related changes in the content and composition of glycosaminoglycans isolated from the mouse skeletal muscle: normal and dystrophic conditions

Glycosaminoglycans were isolated from the skeletal muscle of either normal or dystrophic mice aged from 3 to 18 weeks. The glycosaminoglycan content of the normal muscle, based on the tissue weight, decreased slightly during the period from 3 to 10 weeks, and remained almost unchanged after 10 weeks. The major glycosaminoglycan in normal muscle was hyaluronate, the relative amount of which increased slightly (from 70% to 80%) with age. Both dermatan sulfate and heparan sulfate were also obtained. The relative amounts of these sulfated glycosaminoglycans tended to decrease with age. On the other hand, the glycosaminoglycan content of the dystrophic muscle was higher than that of normal muscle even at 3 weeks. The proportion of hyaluronate was almost constant (about 65%) throughout the age range examined. The relative amount of dermatan sulfate increased from 20% to 30% with a compensatory decrease in the amount of heparan sulfate. Further, the incorporation of [35S]sulfate into glycosaminoglycans by the dystrophic muscle was reduced to about 60% of the normal. These differences in glycosaminoglycan composition and [35S]sulfate incorporation between the normal and the dystrophic muscles may be related to the progressive muscular dysfunction seen in this disease.     

Does ADP-Ribosylation of Proteins in Nuclei Contribute to Ectoderm Cell Differentiation in Sea Urchin Embryos?

In nuclei of sea urchin embryos, marked increase in ADP-ribosyltransferase activity followed by its decrease occurrs in the pre-hatching and post-hatching periods with peaks of activity at the morula and gastrula stages. Increase in its activity was blocked by cycloheximide in the pre- and post-hatching periods and by actinomycin D only in the post-hatching period. Embryo wall cells (ectoderm cells) isolated from gastrulae exhibited markedly higher activity of this enzyme than archenteron cells and mesenchyme cells. Probably, the increase in the activity of this enzyme in the post-hatching period results from expression of the gene for this enzyme mainly in ectoderm cells. In the post-hatching period, the activity increased more in animalized embryos than in normal ones, and increased little in vegetalized embryos. 3-Aminobenzamide (3-ABA), as well as luminol and nicotinamide, inhibited formation of ectoderm structures more than that of endoderm structures, such as the archenteron, in normal and animalized embryos, but had no appreciable effect on morphogenesis in vegetalized embryos. The reaction catalyzed by ADP-ribosyltransferase probably contributes to ectoderm cell differentiation. Treatment of embryos with 3-ABA in the pre-hatching period had little inhibitory effect on the morphogenesis in the post-hatching period, though it caused death of many embryos.     

A competitive binding assay for measurement of heparan sulfate in tissue digests

Glycosaminoglycans complex with constituents of normal human serum, a finding that was exploited to develop a competitive binding assay for these substances. Heparan sulfate was isolated from renal cortex and radiolabeled with tritiated borohydride. The elution pattern of the radiolabeled material on Sephadex G-25, Bio-Gel P-30, and AG- 1X8 resin was identical to that of unlabeled heparan sulfate. The tritiated heparan sulfate formed radiolabeled precipitates when incubated with serum and zinc acetate. Binding was dose dependent and saturable. Heparin, heparan sulfate, and the chondroitin sulfates, but not hyaluronate or keratan sulfate, competed with the radiolabeled heparan sulfate for binding in a dose-dependent manner. The assay is specific for heparin polysaccharides in chondroitinase ABC-treated samples and is sensitive to microgram quantities.     

Stage Specific Effects on Sea Urchin Embryogenesis of Zn2+, Li+, several Inhibitors of cAMP-Phosphodiesterase and Inhibitors of Protein Synthesis

Treatment of sea urchin embryos for 3 hr starting at the 16-64 cell stage with Li⁺ or 3-isobutyl-1-methylxanthine as well as with other inhibitors of cAMP-phosphodiesterase (PDE) and several inhibitors of protein synthesis, resulted in production of vegetalized embryos with a large exogut. However, the same treatment starting at other stages produced hardly any vegetalized embryos. The specific stage for these substances to cause vegetalization is probably the 16-64 cell stage. Treatment with Zn²⁺ between the times of fertilization and hatching, followed by culture in normal sea water produced animalized embryos with little if any archenteron, but the same treatment followed by culture with ethylenediamine-N, N'-diacetic acid (EDDA), a chelator of Zn²⁺, produced quasi-normal plutei. This chelator did not counteract the animalizing effect of Zn²⁺ when culture with EDDA was started at the post-gastrula stage. Treatment of embryos for a long period (1-3 days) starting at the blastula stage with Li⁺ and the inhibitors of PDE and protein synthesis, as well as with Zn²⁺, produced spherical embryos with little or no archenteron. The stages at which these substances produced abnormal embryos with a poor archenteron are post-hatching stages.     

beta-D xyloside alters dermatan sulfate proteoglycan synthesis and the organization of the developing avian corneal stroma.

Corneal transparency is dependent upon the development of an organized extracellular matrix containing small diameter collagen fibrils with regular spacing, organized as orthogonal lamellae. Proteoglycan-collagen interactions have been implicated in the regulation of collagen fibrillogenesis and matrix assembly. To determine the role of dermatan sulfate proteoglycan in the development and organization of the secondary corneal stroma, its synthesis was disrupted using beta-D xyloside. The secondary corneal stroma contains two different proteoglycans, dermatan sulfate and keratan sulfate proteoglycan. beta-D xyloside interferes with xylose-mediated O-linked proteoglycan synthesis, and thus disrupts dermatan sulfate proteoglycan synthesis. Corneal keratan sulfate proteoglycan, a mannose-mediated N-linked proteoglycan, should not be altered. Biochemical analysis of corneas treated both in vitro and in ovo revealed a reduced synthesis of normally glycosylated dermatan sulfate proteoglycans and an increased synthesis of free xyloside-dermatan sulfate glycosaminoglycans. Keratan sulfate proteoglycan synthesis was unaltered in both cases. Corneal stromas were studied using histochemistry and electron microscopy after in ovo treatment with beta-D xyloside. The observed biochemical alterations in dermatan sulfate proteoglycans translated into disruptions in the organization of beta-D xyloside-treated stromas. There was a reduction in the histochemical staining of proteoglycans, but no alteration in collagen fibril diameter. In addition, focal alterations in collagen fibril packing, and a disruption of lamellar organization were observed in beta-D xyloside-treated corneas. These data suggest that dermatan sulfate proteoglycans are not involved in the regulation of corneal collagen fibril diameter, but are important in the fibril-fibril spacing as well as in lamellar organization, and cohesiveness.     

Highly sulfated glycosaminoglycans inhibit aggrecanase degradation of aggrecan by bovine articular cartilage explant cultures.

The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans was investigated using bovine articular cartilage explant cultures maintained in medium containing 10(-6) M retinoic acid or 40 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha) and varying concentrations (1-1000 microg/ml) of sulfated glycosaminoglycans (heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate) and calcium pentosan polysulfate (10 microg/ml). In addition, the effect of the sulfated glycosaminoglycans and calcium pentosan polysulfate on the degradation of aggrecan by soluble aggrecanase activity present in conditioned medium was investigated. The degradation of 35S-labeled aggrecan and reduction in tissue levels of aggrecan by articular cartilage explant cultures stimulated with retinoic acid or rHuIL-1alpha was inhibited by heparin and heparan sulfate in a dose-dependent manner and by calcium pentosan polysulfate. In contrast, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate did not inhibit the degradation of 35S-labeled aggrecan nor suppress the reduction in tissue levels of aggrecan by explant cultures of articular cartilage. Heparin, heparan sulfate and calcium pentosan polysulfate did not adversely affect chondrocyte metabolism as measured by lactate production, incorporation of [35S]-sulfate or [3H]-serine into macromolecules by articular cartilage explant cultures. Furthermore, heparin, heparan sulfate and calcium pentosan polysulfate inhibited the proteolytic degradation of aggrecan by soluble aggrecanase activity. These results suggest that highly sulfated glycosaminoglycans have the potential to influence aggrecan catabolism in articular cartilage and this effect occurs in part through direct inhibition of aggrecanase activity.     

Purification and characterization of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase from the squid cartilage

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 6 of N-acetylgalactosamine 4-sulfate in chondroitin sulfate and dermatan sulfate, was purified 19,600-fold to apparent homogeneity from the squid cartilage. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed a broad protein band with a molecular mass of 63 kDa. The protein band coeluted with GalNAc4S-6ST activity from Toyopearl HW-55 around the position of 66 kDa, indicating that the active form of GalNAc4S-6ST may be a monomer. The purified enzyme transferred sulfate from PAPS to chondroitin sulfate A, chondroitin sulfate C, and dermatan sulfate. The transfer of sulfate to chondroitin sulfate A and dermatan sulfate occurred mainly at position 6 of the internal N-acetylgalactosamine 4-sulfate residues. Chondroitin sulfate E, keratan sulfate, heparan sulfate, and completely desulfated N-resulfated heparin were not efficient acceptors of the sulfotransferase. When a trisaccharide or a pentasaccharide having sulfate groups at position 4 of N-acetylgalactosamine was used as acceptor, efficient sulfation of position 6 at the nonreducing terminal N-acetylgalactosamine 4-sulfate residue was observed.