Proteoglycan metabolism associated with mouse metanephric development: morphologic and biochemical effects of beta-D-xyloside

Morphology and de novo incorporation of [35S]sulfate into proteoglycans were studied in fetal mouse kidneys at the onset of organogenesis. Branching morphogenesis and nephron development in organ culture and in vivo were associated with de novo synthesis of chondroitin-SO4 and heparan-SO4 proteoglycans. The role of proteoglycan metabolism in metanephrogenesis was then studied by analysis of the effects of p-nitrophenyl-beta-D-xylopyranoside (beta-D-xyloside) on renal development and proteoglycan metabolism. Incubation of fetal kidneys in beta-D-xyloside at concentrations of 1.0 and 0.5 mM, but not at 0.1 mM, caused inhibition of ureteric branching and markedly diminished synthesis of a large Mr 2.0 X 10(6) Da chondroitin-SO4 proteoglycan. Incorporation of [35S]sulfate was stimulated at all beta-D-xyloside concentrations, reflecting synthesis of xyloside initiated dermatan-35SO4 chains. In contrast to dramatic effects on chondroitin-SO4 synthesis and ureteric branching, beta-D-xyloside had no effect on heparan-SO4 synthesis or on development of the glomerulus and glomerular basement membrane. We thus characterize the proteoglycans synthesized early in the course of renal organogenesis and describe observations which suggest an association between metabolism of chondroitin-SO4 proteoglycan and development of the ureter.     

A New C-Xyloside Induces Modifications of GAG Expression,Structure and Functional Properties

Proteoglycans (PGs) are critically involved in major cellular processes. Most PG activities are due to the large interactive properties of their glycosaminoglycan (GAG) polysaccharide chains, whose expression and fine structural features are tightly controlled by a complex and highly regulated biosynthesis machinery. Xylosides are known to bypass PG-associated GAG biosynthesis and prime the assembly of free polysaccharide chains. These are, therefore, attractive molecules to interfere with GAG expression and function. Recently, we have developed a new xyloside derivative, C-Xyloside, that shares classical GAG-inducing xyloside activities while exhibiting improved metabolic stability. We have previously shown that C-Xyloside had beneficial effects on skin homoeostasis/regeneration using a number of models, but its precise effects on GAG expression and fine structure remained to be addressed. In this study, we have therefore investigated this in details, using a reconstructed dermal tissue as model. Our results first confirmed that C-Xyloside strongly enhanced synthesis of GAG chains, but also induced significant changes in their structure. C-Xyloside primed GAGs were exclusively chondroitin/dermatan sulfate (CS/DS) that featured reduced chain size, increased O-sulfation, and changes in iduronate content and distribution. Surprisingly, C-Xyloside also affected PG-borne GAGs, the main difference being observed in CS/DS 4-O/6-O-sulfation ratio. Such changes were found to affect the biological properties of CS/DS, as revealed by the significant reduction in binding to Hepatocyte Growth Factor observed upon C-Xyloside treatment. Overall, this study provides new insights into the effect of C-Xyloside on GAG structure and activities, which opens up perspectives and applications of such compound in skin repair/regeneration. It also provides a new illustration about the use of xylosides as tools for modifying GAG fine structure/function relationships.     

In Vitro Synthesis of Proteoglycans and Collagen in Primary Cultures of Mantle Cells from the Nacreous Mollusk, Haliotis tuberculata: A New Model for Study of Molluscan Extracellular Matrix

: In Mollusca, the mantle produces an organic matrix that mineralizes in time to make shell. Primary mantle cell cultures from the nacreous gastropod Haliotis tuberculata have been established as useful experimental model to investigate in vitro synthesis of both proteoglycans/glycosaminoglycans (PGs/GAGs) and collagen. First, we tested different enzymatic digestion procedures to find the method that gives the highest percentage of viable and adherent cultured cells. Enzymatic digestion with 0.1% pronase plus 0.1% collagenase was routinely used. Six days after the initiation of culture, about 80% of cells were viable, among which 20% were adherent as quantified by the MTT reduction assay. In addition, the protein synthesis estimated by [³H]leucine incorporation remained constant during this period. For the first time, we demonstrated a de novo synthesis of PGs/GAGs and collagen in primary cultures of mantle cells. After 48 hours of labeling, among the [³H]-d-glucosamine macromolecules synthesized, [³H]PGs/GAGs represented 43%, divided into 45% heparan sulfate, 37% chondroitin/dermatan sulfate, and 6% hyaluronic acid. Early elution on anion-exchange chromatography of these PGs/GAGs indicated that most of them appeared as undersulfated GAG molecules. De novo synthesis of collagen represents 4.52% - 0.84% (SD) with respect to the total protein synthesis. Such a model will facilitate studies on the synthesis of PGs/GAGs and collagen as components of the extracellular matrix and its regulation in Mollusca. Both PGs/GAGs and collagen participate in molecular events that regulate cell adhesion, migration, and proliferation. Further studies with this type of in vitro model should provide knowledge about novel aspects of molluscan cell signaling, in relation to extracellular matrix components.     

Inhibition of branching morphogenesis and alteration of glycosaminoglycan biosynthesis in salivary glands treated with β-d-xyloside

The role of glycosaminoglycans (GAGs) in the branching morphogenesis of embryonic mouse salivary glands was investigated by culturing the glands in the presence of xylose derivatives which stimulate synthesis of the xyloselinked classes of GAGs. Branching morphogenesis is inhibited severely, but reversibly, by 0.5–1.0 mM π-nitrophenyl-β-d-xylopyranoside and the inhibition correlates with a stimulation of incorporation of [³H]glucosamine (1.8-fold) and [³⁵S]sulfate (almost 3-fold) into GAGs. The effect of β-xyloside on accumulation of newly synthesized GAG also occurs in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the production of free GAG chains rather than proteoglycan-associated GAGs is being stimulated. The xyloside effects apparently do not result from general cytotoxicity of the derivatives, since similar concentrations of the α-anomer do not alter salivary branching or GAG synthesis, the rudiments resume morphogenesis when returned to control medium, and the effect on GAG synthesis is stimulatory rather than inhibitory. The study suggests that GAG biosynthesis plays an important role in salivary development, and that xylosides provide useful probes for characterizing the molecular events controlling branching morphogenesis.     

Effect of beta-D-xyloside on the glomerular proteoglycans. I. Biochemical studies

The effect of p-nitrophenyl-beta-D-xylopyranoside on glomerular extracellular matrices (glomerular basement membrane and mesangial matrix) proteoglycans was studied. The proteoglycans of rat kidneys were labeled with [35S]sulfate in the presence or absence of beta- xyloside (2.5 mM) by using an isolated organ perfusion system. The proteoglycans from the glomeruli and perfusion medium were isolated and characterized by Sepharose CL-6B chromatography and by their behavior in CsCl density gradients. With xyloside treatment there was a twofold decrease in 35S-labeled macromolecules in the tissues but a twofold increase in those recovered in the medium as compared with the control. The labeled proteoglycans extracted from control kidneys eluted as a single peak with Kav = 0.25 (Mr = approximately 130,000), and approximately 95% of the radioactivity was associated with heparan sulfate proteoglycan (HS-PG), the remainder with chondroitin (or dermatan) sulfate proteoglycan (CS-PG). In the xyloside-treated kidneys, the proteoglycans extracted from the tissue eluted as two peaks, Kav = 0.25 (Mr = approximately 130,000) and 0.41 (Mr = approximately 46,000), which contained approximately 40 and approximately 60% of the total radioactivity, respectively. The first peak contained mostly the HS-PG (approximately 90%) while the second peak had a mixture of HS-PG (approximately 70%) and CS-PG (approximately 30%). In controls, approximately 90% of the radioactivity, mostly HS-PG, was confined to high density fractions of a CsCl density gradient. In contrast, in xyloside experiments, both HS- PG and CS-PG were distributed in variable proportions throughout the gradient. The incorporated 35S activity in the medium of xyloside- treated kidneys was twice that of the controls and had three to four times the amount of free chondroitin (or dermatan) sulfate glycosaminoglycan chains. The data suggest that beta-xyloside inhibits the addition of de novo synthesized glycosaminoglycan chains onto the core protein of proteoglycans and at the same time stimulates the synthesis of chondroitin or dermatan sulfate chains which are mainly discharged into the perfusion medium.     

The interaction of platelet factor four and glycosaminoglycans

The interaction of platelet factor four (PF-4) with glycosaminoglycans (GAG) was evaluated using fluorescence spectroscopy, a radioligand binding assay, and a functional assay utilizing antithrombin III and factor Xa. In these studies, we have (i) characterized the binding parameters for PF-4 to several forms of heparin and to dextran sulfate; (ii) examined the structural features of these glycosaminoglycans which support PF-4 binding; and (iii) examined the effects of selective digestion of the carboxy terminus of PF-4 on binding. The binding of PF-4 to unfractionated porcine intestinal mucosal heparin ([Mr] = 11,000) was specific and saturable, with a molar stoichiometry of PF-4 to heparin of approximately 4:1 and an apparent estimated Kd of 3 X 10(-8) M. Heparin fractions ([Mr] = 6,000) with either low or high affinity for antithrombin III bound to PF-4 with a similar apparent Kd. PF-4 also bound to dextran sulfate ([Mr] = 22,500) with an estimated apparent Kd of 6 X 10(-8) M and a molar stoichiometry of approximately 16:1. Carboxypeptidase Y (CP-Y) digestion of PF-4 progressively decreased GAG binding. After 30 min of digestion, by which time all of the carboxyterminal serine and glutamate, both of the two leucines, and approximately one-quarter of the four lysines were removed, the IC50 for heparin binding shifted from 10 to 150 nM. These studies demonstrated the effect of GAG polymer size and degree of sulfation on the affinity and stoichiometry of PF-4 binding, and the critical importance of the carboxy-terminal amino acids of PF-4 for binding to natural and synthetic GAGs.     

Biosynthesis of proteoglycans by proliferating and differentiating normal human keratinocytes cultured in serum-free medium

Normal human keratinocytes (NHK) were cultured in serum-free medium, containing low (0.1 mM) or high (2 mM) calcium, to obtain proliferating and differentiating cultures, respectively. Proteoglycan (PG) synthesis of proliferating and differentiating NHK was investigated. Cultures were labeled with 35S-sulfate, and the PGs were extracted from medium and cell layer. The newly synthesized PGs were isolated by ion-exchange chromatography on a column of DEAE-Sephacel. The molecular properties of the PGs and the size and composition of glycosaminoglycans (GAGs) were determined. In general, the PGs are relatively small size (Mr 70,000-120,000). The PGs of proliferating cultures are larger in molecular size than the PGs of differentiating cultures, and this is due to the degradation of the GAG chains. The molecular weight of the GAG chains of proliferating NHK ranged from 4,800 to 22,000, and the range for GAGs from differentiating cultures varied from 2,800 to 9,600. By compositional analysis, these PGs proved to contain heparan sulfate, chondroitin sulfate, and dermatan sulfate as determined by nitrous acid degradation, and chondroitinase ACII and ABC digestion. No significant differences were found in the overall GAG composition of the medium secreted PGs of proliferating and differentiating cultures. In contrast, cell-associated PGs of differentiating cells had higher levels of heparan sulfate than those of proliferating cells.     

Chondroitin sulfate proteoglycan synthesis and reutilization of beta-D-xyloside-initiated chondroitin/dermatan sulfate glycosaminoglycans in fetal kidney branching morphogenesis

Branching morphogenesis and chondroitin sulfate proteoglycan synthesis by explanted fetal mouse kidneys were previously shown to be inhibited by p-nitrophenyl beta-D-xylopyranoside (beta-D-xyloside) while glomerular development and heparan sulfate proteoglycan synthesis were unaffected. The metabolic fate of fetal kidney explant proteoglycans was investigated to determine whether or not recovery of proteoglycan synthesis and morphogenesis occur after exposure to beta-D-xyloside. Chondroitin sulfate proteoglycan synthesis resumed within 4 hr of removal of beta-D-xyloside and was enhanced once beta-D-xyloside-initiated chondroitin/dermatan-35SO4 glycosaminoglycans (GAGs) were released from the tissue. Radioactivity incorporated into beta-D-xyloside-initiated chondroitin/dermatan-35SO4 GAGs during labeling in the presence of beta-D-xyloside was reutilized in the synthesis of chondroitin-35SO4 proteoglycan during a 24-hr chase in nonradioactive medium without beta-D-xyloside. Further, highly purified beta-D-xyloside-initiated chondroitin/dermatan-35SO4 GAGs were taken up by kidneys more avidly than was free [35S]sulfate. These 35S-GAGs were degraded and reutilized in the synthesis of chondroitin-35SO4 proteoglycan. Ureteric bud branching resumed 48 hr after beta-D-xyloside was removed from the incubation medium. These findings support the idea that both chondroitin sulfate proteoglycan synthesis and proteoglycan processing may be involved in branching morphogenesis.     

Glycosaminoglycan-lipoprotein interaction

Glycosaminoglycans (GAGs) bound to various proteoglycans (PGs) present in the cardiovascular system have been proposed to perform a wide range of functions. These include conferring viscoelastic properties; interacting with and modulating growth factors and enzymes; and as receptors and co-receptors in lipoprotein metabolism. Binding of apoB-100 lipoproteins, particularly low density lipoproteins (LDL), to GAGs of extracellular matrix PGs in arteries has been proposed to be an initiating event in development of atherosclerosis. This study was initiated with the aim of getting an overview of the binding patterns of different lipoprotein subclasses with individual GAG categories. We thus evaluated the interaction of lipoproteins with GAGs commonly found in the cardiovascular system using a gel mobility-shift assay developed for this purpose. The same procedure was used to measure lipoproteins binding to metabolically [(35)S]-labeled whole PGs prepared from three cell types, arterial smooth muscle cells, THP-1 macrophages and from HepG2 cells. The effect of GAG composition on PGs on lipoprotein binding was evaluated by enzymatic degradation of the carbohydrate chains. Heparan sulfate was found to bind beta very low density lipoproteins (beta-VLDL) and a chylomicron remnant model (beta-VLDL+apoE), but not LDL. Dermatan sulfate was found to bind LDL, but not beta-VLDL or the chylomicron remnant model. Chondroitin sulfate and heparin were found to bind all lipoproteins tested (LDL, beta-VLDL and beta-VLDL+apoE) although with different affinities. We can conclude that each lipoprotein subclass tested binds a specific assortment of the GAGs tested. The observations made contribute to the understanding of new and complex mechanisms by which carbohydrate and lipid metabolism may be linked.     

Glycosaminoglycan variants in the C2 muscle cell line

Using a replica technique, we have isolated and characterized five genetic variants of the C2 mouse muscle cell line that are defective in incorporation of radiolabeled sulfate into glycosaminoglycans (GAGs). The variants incorporate free sulfate into GAGs at 5-20% of wild-type levels. None of the variants is defective in sulfate transport across the cell membrane, and in no case could the deficit in incorporation of sulfate be reversed by addition of an artificial initiator of GAG biosynthesis, p-nitrophenyl beta-D-xyloside. Analysis of the incorporation of [3H]glucosamine into GAGs by the variants revealed three different patterns: one variant incorporated [3H]glucosamine at the wild-type level; one, S27, at a severely reduced level; and three at intermediate levels. Four of the five variants showed marked deficits in their ability to differentiate and fuse. The remaining variant, S27, formed multinucleated myotubes and expressed acetylcholine receptor with a normal time course. Differentiation of the first four variants could not be restored by addition of exogenous GAGs or extracellular matrix. Because of the important roles that GAGs and proteoglycans are thought to play in the differentiation of muscle, these genetic variants should serve as useful tools in functional analyses of these molecules.     

Patterns of synthesis and secretion of sulfated glycosaminoglycans in primary cortical and cerebellar astrocytes in vitro

We determined the amounts of [35S]-glycosaminoglycans (GAGs) found on the intracellular, pericellular and extracellular compartments of primary cultures of astrocytes derived from newborn rat cortex and cerebellum in vitro. Our results show that the greatest portion of newly synthesized GAGs were found in different cellular compartments, depending on the source of the astrocytes. In the cells derived from the cerebellum, the proportion of [35S]-GAGs secreted to the culture medium preponderates over the amount found in the two other compartments, whereas cells derived from the cortex accumulated higher proportions of [35S]-GAGs in the intracellular compartment than in the two other compartments. Cortical and cerebellar glial cells synthesised and secreted heparan sulfate (HS) and chondroitin 4-sulfate (C-4S). HS was predominantly accumulated on the pericellular surface, while C-4S was mostly secreted to the culture medium. Beside the difference on the distribution of total [35S]-GAGs among the three cellular compartments, no difference was observed on the relative proportions of HS and C-4S within each compartment. By defining the source of GAGs, the present study may help to complement and extend information on biosynthesis of these compounds by mammalian glial cells.     

The effect of benzyl beta-D-xyloside on keratan sulphate synthesis in ox articular cartilage

Cartilage from adult bovine hock joints was incubated with [3H]galactose or [35S]sulphate in the presence of benzyl beta-D-xyloside. Radioisotope incorporation into proteoglycan was inhibited by the xyloside; the magnitude of this inhibition depended on the concentration of xyloside used. With 0.2mM xyloside radioisotope incorporation into keratan sulphate was not altered but inhibition was observed at xyloside concentrations of 1.0mM or higher. The decrease in radioisotope incorporation into keratan sulphate in the presence of 1.0mM benzyl beta-xyloside was directly related to a reduction in the average length of the keratan sulphate chains. This effect of beta-xyloside on keratan sulphate biosynthesis was markedly different from its effect on chondroitin sulphate biosynthesis.     

Large disulfide-stabilized proteoglycan complexes are synthesized by the epidermis of axolotl embryos

Proteoglycans (PGs) synthesized by the epidermis during stages crucial to the subepidermal migration of neural crest cells in the trunk of the axolotl (Ambystoma mexicanum, Urodela, Amphibia) embryo were studied. The glycosaminoglycan chains were biosynthetically labeled with [35S]sulfate in vitro during a period corresponding to the onset of migration. After extraction with guanidine HCl, the radiolabeled PGs were separated according to size by molecular-sieve chromatography on Sepharose CL-2B under dissociative conditions. This resulted in the separation of high-molecular-weight PGs, which eluted in the void volume, and low-molecular-weight PGs, eluting in a broad peak with a mean Kav of 0.7. The large PGs were also found to elute in the void volume when chromatographed on a Sephacryl S-1000 column. The low-molecular-weight PGs contained heparan sulfate and chondroitin sulfate (CS) and were not further characterized. The glycosaminoglycan component of the high-molecular-weight PG was completely degraded by chondroitinase ABC, while a large portion was resistant to chondroitinase AC, indicating the presence of dermatan sulfate (DS). These CS/DS chains were of unusually large size (Mr approximately 150,000) as estimated by chromatography on Sepharose CL-4B, relating the elution position to hyaluronan standards. Moreover, the chains were found to have a lower surface charge density than standard CS, and may therefore be undersulfated. After reduction and alkylation the high-molecular-weight PGs were included on both Sepharose CL-2B and Sephacryl S-1000 columns, eluting at Kav 0.2 and 0.4, respectively. Hence, the high-molecular-weight material appears to consist of large PG complexes, stabilized by intermolecular disulfide bonds. A CS/DSPG of similar size as the reduced monomeric form of the high-molecular-weight PG was found in small amounts in the total extract of 35S-labeled material.     

Serglycin is a major proteoglycan in polarized human endothelial cells and is implicated in the secretion of the chemokine GROalpha/CXCL1

Proteoglycan (PG) expression was studied in primary human umbilical vein endothelial cells (HUVEC). RT-PCR analyses showed that the expression of the PG serglycin core protein was much higher than that of the extracellular matrix PG decorin and the cell surface PG syndecan-1. PG biosynthesis was further studied by biosynthetic [(35)S]sulfate labeling of polarized HUVEC. Interestingly, a major part of (35)S-PGs was secreted to the apical medium. A large portion of these PGs was trypsin-resistant, a typical feature of serglycin. The trypsin-resistant PGs were mainly of the chondroitin/dermatan sulfate type but also contained a minor heparan sulfate component. Secreted serglycin was identified by immunoprecipitation as a PG with a core protein of ～30 kDa. Serglycin was furthermore shown to be present in perinuclear regions and in two distinct types of vesicles throughout the cytoplasm using immunocytochemistry. To search for possible serglycin partner molecules, HUVEC were stained for the chemokine growth-related oncogene α (GROα/CXCL1). Co-localization with serglycin could be demonstrated, although not in all vesicles. Serglycin did not show overt co-localization with tissue-type plasminogen activator-positive vesicles. When PG biosynthesis was abrogated using benzyl-β-D-xyloside, serglycin secretion was decreased, and the number of vesicles with co-localized serglycin and GROα was reduced. The level of GROα in the apical medium was also reduced after xyloside treatment. Together, these findings indicate that serglycin is a major PG in human endothelial cells, mainly secreted to the apical medium and implicated in chemokine secretion.     

Isolation of the heparan sulfate proteoglycans from the extracellular matrix of rat skeletal muscle

We have previously shown that asymmetric collagen-tailed acetylcholinesterase (AChE) is anchored to the extracellular matrix (ECM) by heparan sulfate proteoglycans (HSPGs). Here we present our studies on the characterization of such PGs from the ECM of rat skeletal muscles. After radiolabeling with 35SO4 for 24h, PGs were extracted from the muscle ECM with 4.0 M guanidine-HCl containing protease inhibitors. PGs were subsequently isolated using sequential DEAE-Sephacel chromatography, digestion with chondroitinase ABC, and Sepharose CL-4B. Two different hydrodynamic size species of HSPGs were found. One type had a Mr of 4-6 X 10(5) (Kav = 0.25) as estimated by gel chromatography in the presence of 1% SDS and accounted for 75% of the total HSPGs. The other HSPG had a Mr 1.5-2.5 X 10(5) (Kav = 0.41). The glycosaminoglycan (GAG) side chains (Mr 20,000 and 12,000) were found composed only of heparan sulfate as determined by nitrous acid oxidation and heparitinase treatment. The large-sized HSPG, which is concentrated in synaptic regions, contains only GAG chains of Mr 20,000, suggesting that each HSPG contains only one kind of heparan sulfate chain in its structure. Our results definitively establish by biochemical criteria that the basement membrane of mammalian skeletal muscle contains HSPGs, the likely matrix receptor for the immobilization of the asymmetric collagen-tailed AChE at the neuromuscular junction.     

Regulation of glycosaminoglycan biogenesis is critical for sensitive‐period‐dependent vocal ontogeny

In the brain, the extracellular matrix (ECM) plays a central role during neural development and thus modulates critical‐period regulated behavioral ontogeny. The major components of the ECM are glycosaminoglycans (GAGs) including chondroitin sulfate (CS). However, the specific roles of GAGs in behavioral development are largely unknown. It has been shown that xylosides affect the biological functions of GAGs through modulating GAG biosynthesis. Particularly, xylosides affect GAG biosynthesis through priming of GAG chains (priming activity), competing with endogenous core proteins that carry GAG initiation sites (decoy activity), or both. Using birdsong as our model, we investigated, for the first time, how xyloside‐mediated modulation of GAG biogenesis affects song development. Xylosides infused into motor cortex of juvenile birds alter song development by specifically affecting ontogeny of the stereotyped sequence rather than the acoustic structure of syllables. Further analyses reveal that observed changes can be attributed to the priming activity rather than the decoy activity of xylosides. Collectively, these results suggest that regulation of GAG biogenesis through chemical biology approaches may allow promising therapeutic interventions of critical‐period‐dependent central nervous system plasticity. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1401–1412, 2017     

培养的人主动脉平滑肌细胞合成的蛋白聚糖的生化分析

人胎儿主动脉平滑肌细胞(SMC_8)体外培养的第4(T_4)及第10(T_(10))代细胞在含[~(35)S]-硫酸钠的培养液中培养以标记蛋白聚糖(PG)。培养液及细胞层的4mol/L盐酸胍提取液中的PG_8用DEAE-Sephacel离子交换及凝胶过滤柱层析纯化。两代(T_4及T_(10))培养液中均含有一种分子较大及大小类似的硫酸软骨素-PG(CS-PG,在Sepharose CL-4B柱的排阻部位洗脱),其相对含量在T_4为20.8％,T_(10)为12.9％,另外尚均含有一种分子较小及大小类似的硫酸皮肤素-硫酸软骨素-PG(DS-CS-PG,Kd=0.27-0.33,Sepharose CL-4B),其相对含量在T_4为72.7％,T_(10)为81.5％。两代细胞层中除有一种与培养液中大小类似的CS-PG外(其相对含量在T_4为21.7％,T_(10)为10.2％),尚均含有一种分子较小(小于培养液中者)的DS-CS-PG(Kd=0.53,Sepharose CL-4B),其相对含量在T_4为57.4％,T_(10)为75.0％。另外,两代培养液及细胞层中均含有硫酸乙酰肝素-PG(HS-PG),在T_4及T_(10)培养液中分别为6.5％及5.6％;而在细胞层中则分别为20.9％及14.8％。T_(10)的培养液及细胞层中DS-CS-PG的[~(35)S]参入量均高于T_4者;而CS-PG及HS-PG则相反。T_(10)除有PG合成变化外尚有其他衰老迹象(如脂质堆积等),故SMC_8的老化可能与动脉粥样硬化病变形成有关。     

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.     

Soluble complex formation between low-density lipoprotein and glycosaminoglycans. A 2H and 31P-NMR, and quasi-elastic light scattering study

Soluble complex formation between LDL and heparin (HEP) and chondroitin sulfate (CS) has been studied by 2H- and 31P-NMR and light scattering. The 2H-NMR linewidths of [2H]HEP and [2H]C4S increase substantially upon binding to LDL, with the [2H]HEP linewidths broader at low glycosaminoglycan (GAG)/low density lipoprotein (LDL) ratios. Preliminary analysis of the bound C2H3 group correlation times suggests that the observed linewidths are determined by the complex size, and that both [2H]GAGs have similar motions when bound to LDL. The 31P-NMR data demonstrate that large LDL-HEP complexes (diameter approx. 50 nm) are formed only over a narrow range of HEP concentrations, whereas the size of LDL-CS complexes increases continuously over the range of CS concentrations studied, reaching values of 32-35 nm for both C4S and C6S. At the lower protein concentrations studied by light scattering (less than or equal to 1 mg/ml), the same trends are observed, although the mean diameters are less than those estimated by 31P-NMR. Soluble complex formation was unaffected by the presence of 2 mM Ca2+. Dilution studies demonstrate that complex size varies with protein concentration. The binding of GAGs to LDL was also examined by HEP-CS competition studies. HEP has the higher affinity while no differences in binding could be detected between C4S and C6S.     

Age-related changes in hyaluronan, proteoglycan, collagen, and osteonectin synthesis by human bone cells.

Human bone cells grown in culture, representative of a preosteoblastic stage of maturation, produce an extracellular matrix composed of collagen, several noncollagenous glycoproteins, hyaluronan, and four distinct proteoglycans (PGs). The influence of donor age on the levels of expression of these molecules in vitro has not been well characterized. In this study, human bone cells derived from sources ranging from fetal to 60-year-old donors were grown in culture, radiolabeled for 24 h, and the amount of incorporation of [35S]sulfate into PGs, [3H]glucosamine into hyaluronan, [3H]leucine/proline into osteonectin, and [3H]proline into collagen was determined. Cell proliferation was most rapid in fetal-derived bone cells and decreased with increasing age. Total protein and PG synthesis also decreased with increasing age, falling to 1/3 and 1/4, respectively, of fetal levels after age 30. A large chondroitin sulfate PG (Mr approximately 600,000 Da) was the major fetal PG and its levels were highly correlated with cellular proliferation. [3H]Collagen and [35S]decorin levels increased with the increasing age of the donor, reached a maximum in puberty-derived cells, and decreased to 1/3 maximal levels after age 20. The heparan sulfate PG (Mr approximately 400,000 Da) exhibited steady-state levels regardless of donor age. [3H]Osteonectin and [35S]biglycan levels were high in fetal-derived cells and in cells derived from pubescent donors. The percentage of collagen and four proteoglycans associated with the cell layer pool changed with donor age. All fetal-derived PG core proteins possessed more N- and O-linked oligosaccharides than newborn or adult derived PGs.