Proteoglycan synthesis by primary chick skeletal muscle during in vitro myogenesis

The proteoglycans synthesized by primary chick skeletal muscle during in vitro myogenesis were compared with those of muscle-specific fibroblasts. Cultures of skeletal muscle cells and muscle fibroblasts were separately labeled using [35S] sulfate as a precursor. The proteoglycans of the cell layer and medium were separately extracted and isolated by ion-exchange chromatography on DEAE-Sephacel followed by gel filtration chromatography on Sepharose CL-2B. Two cell layer-associated proteoglycans synthesized both by skeletal muscle cells and muscle fibroblasts were identified. The first, a high molecular weight proteoglycan, eluted from Sepharose CL-2B with a Kav of 0.07 and contained exclusively chondroitin sulfate chains with an average molecular weight greater than 50,000. The second, a relatively smaller proteoglycan, eluted from Sepharose CL-2B with a Kav of 0.61 and contained primarily heparan sulfate chains with an average molecular weight of 16,000. Two labeled proteoglycans were also found in the medium of both skeletal muscle and muscle fibroblasts. A high molecular weight proteoglycan was found with virtually identical properties to that of the high molecular weight chondroitin sulfate proteoglycan of the cell layer. A second, smaller proteoglycan had a similar monomer size (Kav of 0.63) to the cell layer heparan sulfate proteoglycan, but differed from it in that this molecule contained primarily chondroitin sulfate chains with an average molecular weight of 32,000. Studies on the distribution of these proteoglycans in muscle cells during in vitro myogenesis demonstrated that a parallel increase in the relative amounts of the smaller proteoglycans occurred in both the cell layer and medium compared to the large chondroitin sulfate proteoglycan in each compartment. In contrast, muscle-derived fibroblasts displayed a constant ratio of the small proteoglycans of the cell layer and medium fractions, compared to the larger chondroitin sulfate proteoglycan of the respective fraction as a function of cell density. Our results support the concept that proteoglycan synthesis is under developmental regulation during skeletal myogenesis.     

Structure and interactions of proteoglycans in the extracellular matrix produced by cultured human fibroblasts.

Subconfluent cultures of human embryonic skin fibroblasts were labelled with [35S]sulphate for 3 days, after which cell-free extracellular matrix was isolated. A chondroitin sulphate proteoglycan (CSPG) and a heparan sulphate proteoglycan (HSPG) were purified from the matrix. Chromatography on Sepharose CL-2B gave peak Kav. values of 0.35 and 0.38 respectively for the CSPG and the HSPG. The polysaccharide chains released from the two PGs were of similar size (Kav. 0.50 on Sepharose CL-4B). Approx. 50% of the CSPG showed affinity for hyaluronic acid (HA). However, it differed immunologically from the HA-aggregating CSPG of human articular cartilage, and had a larger core protein (apparent molecular mass 290 kDa) than had the cartilage PG. Neither metabolically [35S]sulphate-labelled PGs, isolated from the medium of fibroblast cultures, nor chemically 3H-labelled polysaccharides (HA, CS, HS and heparin) were incorporated into the extracellular matrix when added to unlabelled cell cultures. These results indicate that the matrix PGs are not derived from the PGs present in the medium and that an interation between polysaccharide chains and matrix components is not sufficient for incorporation of PGs into the matrix. Incubation of cell-free 35S-labelled matrix with unlabelled polysaccharides did not lead to the release of any 35S-labelled material, supporting this conclusion. Furthermore, so-called 'link proteins' were not present in the fibroblast cultures, indicating that the CSPGs were anchored in the matrix in a manner different from the link-stabilized association of CSPG with HA in chondrocyte matrix. The identification of a proteinase, secreted by fibroblasts in culture, that after activation with heparin has the ability to release 35S-labelled PGs from the matrix may also indicate that the core proteins are important for the association of the PGs to the matrix.     

Effect of low-density lipoproteins on the synthesis and secretion of proteoglycans by human endothelial cells in culture.

We studied the effect of low-density lipoproteins (LDL) on the synthesis and secretion of proteoglycans by cultured human umbilical-vein endothelial cells. Confluent cultures were incubated with [35S]sulphate or [3H]glucosamine in lipoprotein-deficient serum in the presence and in the absence (control) of LDL (100-400 micrograms/ml), and metabolically labelled proteoglycans in culture medium and cell layer were analysed. LDL increased accumulation of labelled proteoglycans in medium and cell fractions up to a concentration of 200 micrograms/ml. At this concentration of LDL the accumulations of proteoglycans in medium and cell layer were 65% and 32% respectively above control for 35S-labelled proteoglycans, and 55% and 28% respectively above control for 3H-labelled proteoglycans. At concentrations above this LDL was found to depress the accumulation of proteoglycans in medium and cell layer. Gel filtration on Sepharose CL-4B showed that in both control and LDL-treated cultures the cell layer contained a large (Kav. = 0) and a small (Kav. = 0.35) heparan sulphate proteoglycan, whereas the culture medium contained a large heparan sulphate proteoglycan (Kav. = 0) and a smaller isomeric chondroitin sulphate proteoglycan (control, Kav. = 0.35; LDL-treated, Kav. = 0.17). The relative increase in hydrodynamic size of the isomeric chondroitin sulphate proteoglycan (Mr 150,000 compared with 90,000) in the medium of cultures exposed to LDL was partly attributable to the larger size of the glycosaminoglycan side chains (Mr 39,000 compared with 21,000). The isomeric chondroitin sulphate proteoglycan in LDL-treated culture was relatively enriched in chondroitin 6-sulphate compared with that in control cultures (39% compared with 29%). Pulse-chase studies showed that LDL treatment did not alter the turnover rate of proteoglycans as compared with controls, implying that the elevation in proteoglycan accumulation in LDL-treated cultures was due to enhanced synthesis. These results demonstrate that LDL can modulate proteoglycan synthesis by cultured vascular endothelial cells, resulting in the secretion of a larger isomeric chondroitin sulphate proteoglycan enriched in chondroitin 6-sulphate.     

Arterial chondroitin sulfate proteoglycan: localization with a monoclonal antibody

We generated a monoclonal antibody (Mab) against a large chondroitin sulfate proteoglycan (CSPG) isolated from bovine aorta. This Mab (941) immunoprecipitates a CSPG synthesized by cultured monkey arterial smooth muscle cells. The immunoprecipitated CSPG is totally susceptible to chondroitinase ABC digestion and possesses a core glycoprotein of Mr approximately 400-500 KD. By use of immunofluorescence light microscopy and immunogold electron microscopy, the PG recognized by this Mab was shown to be deposited in the extracellular matrix of monkey arterial smooth muscle cell cultures in clusters which were not part of other fibrous matrix components and not associated with the cell's plasma membrane. With similar immunolocalization techniques, the CSPG antigen was found enriched in the intima and present in the medial portions of normal blood vessels, as well as in the interstitial matrix of thickened intimal lesions of atherosclerotic vessels. Immunoelectron microscopy revealed that this CSPG was confined principally to the space within the extracellular matrix not occupied by other matrix components, such as collagen and elastic fibers. These results indicate that this particular proteoglycan has a specific but restricted distribution in the extracellular matrix of arterial tissue.     

Biosynthesis of heparan sulfate proteoglycans of developing chick breast skeletal muscle in vitro

Previous studies have reported an increase in heparan sulfate glycosaminoglycan (HSGAG) during skeletal muscle differentiation in culture. We have investigated this phenomenon further in relation to the heparan sulfate proteoglycans (HSPG) produced by myogenic cultures. Pulse-chase analysis indicated an approx. 3-fold increase in heparan sulfate synthesis in myotube cultures over that in proliferating or aligning myoblast cultures. Muscle fibroblast culture heparan sulfate synthesis was higher than that of myoblasts but was lower than myotubes. The turnover rates appeared to be the same for all stages of development, with a t1/2 of approx. 5 h. Enrichment for heparan sulfate by Sepharose CL-4B and DEAE-Sephacel chromatography indicated an increase in the hydrodynamic size of the proteoglycan produced by myotubes over that from myoblasts, with a shift in Kav from 0.14-0.19 to 0.07. Fibroblasts synthesized the smallest proteoglycan, with a Kav of 0.22. All of the proteoglycans contained similar sized glycosaminoglycan chains with an estimated molecular weight of 30,000-40,000. Localization of the heparan sulfate proteoglycan in myotube cultures by trypsin sensitivity indicated much of the intact proteoglycan to be closely associated with the cell surface, while internalized material appeared in a degraded form.     

Aggregated proteoglycan synthesis in organ cultures of human nucleus pulposus.

Proteoglycans isolated under associative conditions in the presence of protease inhibitors from human nucleus pulposus contained 17% aggregate and 83% non-aggregating monomer (Kav = 0.5 on Sepharose CL-2B). Isolated aggregate after reduction and alkylation was resolved into two components (Kav = 0.15 and 0.43) on Sepharose CL-2B. Labeled proteoglycans isolated from parallel samples pulsed with [35S]sulfate and chased for up to 18 h were present largely as aggregated material (up to 78%). Reduction and alkylation of the labeled samples gave a labeled proteoglycan monomer with Kav = 0.15. Both the labeled and unlabeled chondroitin sulfate chains had the same distribution on Sepharose CL-6B and equivalent molecular weights (Mr = 2.0 x 10(3)). After chondroitinase ABC digestion, the unlabeled keratan sulfate-protein core was polydisperse with a Kav = 0.38 on Sepharose CL-4B while the labeled keratan sulfate-protein core had a Kav = 0.05. This indicates that the newly synthesized proteoglycan had a large core protein and suggests that the proteoglycans present in nucleus pulposus are originally synthesized as large molecular weight, aggregating proteoglycans.     

Synthesis by Schwann cells of basal lamina and membrane-associated heparan sulfate proteoglycans

Primary cultures that contain only Schwann cells and sensory nerve cells synthesize basal lamina. The assembly of this basal lamina appears to be essential for normal Schwann cell development. In this study, we demonstrate that Schwann cells synthesize two major heparan sulfate-containing proteoglycans. Both proteoglycans band in dissociative CsCl gradients at densities less than 1.4 g/ml, and therefore, presumably, have relatively low carbohydrate-to-protein ratios. The larger of these proteoglycans elutes from Sepharose CL-4B in 4 M guanidine hydrochloride (GuHCl) at a Kav of 0.21 and contains heparan sulfate and chondroitin sulfate chains of Mr 21,000 in a ratio of approximately 3:1. This proteoglycan is extracted from cultures by 4 M GuHCl but not Triton X-100 and accumulates only when Schwann cells are actively synthesizing basal lamina. The smaller proteoglycan elutes from Sepharose CL-4B at a Kav of 0.44 and contains heparan sulfate and chondroitin sulfate chains of Mr 18,000 in a ratio of approximately 4:1. This proteoglycan is extracted by 4 M GuHCl or by Triton X-100. The accumulation of this proteoglycan is independent of basal lamina production.     

Heparan sulfate-degrading enzymes induce modulation of smooth muscle phenotype.

Macrophages cocultured with rabbit aortic smooth muscle cells at a ratio of 1:3 degraded all the 35S-labeled heparan sulfate proteoglycan from the smooth muscle surface into free sulfate (Kav of 0.84 on Sepharose 6B). Concomitantly, the same macrophages induced a decrease in the volume fraction of myofilaments (Vvmyo) of the smooth muscle cells and a decrease in alpha-actin mRNA as a percentage of total actin mRNA. Both macrophage lysosomal lysate at neutral pH and heparinase degraded cell-free 35S-labeled matrix deposited by smooth muscle cells into fragments which eluted at a Kav of 0.63 and which were identified as heparan sulfate chains by their complete degradation in the presence of low pH nitrous acid. At acid pH the macrophage lysosomal lysate completely degraded the heparan sulfate to free sulfate (Kav 0.84). Both macrophage lysosomal lysate and commercial heparinase at neutral pH induced smooth muscle phenotypic change while other enzymes such as trypsin and chondroitin ABC lyase had no effect. It was therefore suggested that the active factor present in the macrophages is a lysosomal heparan sulfate-degrading endoglycosidase (heparinase). Only a small amount of heparan sulfate-degrading activity was released into the incubation medium by living macrophages, and there was no heparinase activity on their isolated plasma membranes, although proteolytic enzymes were evident in both instances. In pulse-chase studies, high Vvmyo smooth muscle cells were seen to constantly internalize and degrade 35S-labeled heparan sulfate proteoglycan from their own pericellular compartment, suggesting that this may be the mechanism by which smooth muscle phenotype is maintained under normal circumstances and that removal of heparan sulfate from the surface of smooth muscle cells and its degradation by macrophages temporarily interrupts this process, inducing smooth muscle phenotypic change.     

Human glomerular epithelial cell proteoglycans

Proteoglycans synthesized by cultures of human glomerular epithelial cells have been isolated and characterized. Three types of heparan sulfate were detected. Heparan sulfate proteoglycan I (HSPG-I; Kav 6B 0.04) was found in the cell layer and medium and accounted for 12% of the total proteoglycans synthesized. HSPG-II (Kav 6B 0.25) accounted for 18% of the proteoglycans and was located in the medium and cell layer. A third population (9% of the proteoglycan population), heparan sulfate glycosaminoglycan (HS-GAG; Kav 6B 0.4-0.8), had properties consistent with single glycosaminoglycan chains or their fragments and was found only in the cell layer. HSPG-I and HSPG-II from the cell layer had hydrophobic properties; they were released from the cell layer by mild trypsin treatment. HS-GAG lacked these properties, consisted of low-molecular-mass heparan sulfate oligosaccharides, and were intracellular. HSPG-I and -II released to the medium lacked hydrophobic properties. The cells also produced three distinct types of chondroitin sulfates. The major species, chondroitin sulfate proteoglycan I (CSPG-I) eluted in the excluded volume of a Sepharose CL-6B column, accounted for 30% of the proteoglycans detected, and was found in both the cell layer and medium. Cell layer CSPG-I bound to octyl-Sepharose. It was released from the cell layer by mild trypsin treatment. CSPG-II (Kav 6B 0.1-0.23) accounted for 10% of the total 35S-labeled macromolecules and was found predominantly in the culture medium. A small amount of CS-GAG (Kav 6B 0.25-0.6) is present in the cell extract and like HS-GAG is intracellular. Pulse-chase experiments indicated that HSPG-I and -II and CSPG-I and -II are lost from the cell layer either by direct release into the medium or by internalization where they are metabolized to single glycosaminoglycan chains and subsequently to inorganic sulfate.     

Immunocytochemical localization of native chondroitin-sulfate in tissues and cultured cells using specific monoclonal antibody

Chondroitin-sulfate containing proteoglycan (CSPG) of the extracellular matrix (ECM) was visualized in chick tissues and cell cultures with a monoclonal antibody, CS-56. Cultured cells of various origins contained dense punctate layers of CSPG on both the substrate and the cell surface, as determined by immunofluorescent and immunogold staining. Under culture conditions the CSPG-containing matrix was usually excluded from stable cell-to-substrate focal contacts. The substrate-attached CSPG exhibited remarkable chemical stability but could be successfully removed by pronase or chondroitinases ABC and AC. Incubation of living cells with CS-56 antibodies resulted in the clustering of surface CSPG into patches, indicating that the surface-bound CSPG is free to move laterally along the plasma membrane. The unique properties of the CSPG-containing ECM revealed by CS-56 antibodies and their relationships to specific types of cell contacts are discussed.     

Nerve Growth Factor-Induced Changes in the Structure of Sulfated Proteoglycans in PC 12 Pheochromocytoma Cells

Structural changes in proteoglycans (PGs) were examined during the neuritogenesis of PC12 cells induced by nerve growth factor (NGF). (1) A heparan sulfate (HS) PG and a chondroitin sulfate (CS) PG were synthesized by PC12 cells, irrespective of the presence of NGF or the duration of culture. PGs released from PC12 cells into the culture medium were mostly CSPGs. (2) In the absence of NGF, the apparent molecular mass of HSPG prepared from PC12 cells after 3 days of culture was in the range of 90-190 kDa for the intact form (Kav = 0.38 on Sepharose CL-6B), 12 kDa for HS, and 61 kDa for the core protein. In the presence of NGF, these values were 90-190 kDa, 10 kDa, and 51 kDa and 61 kDa, respectively. The intact forms of cell-associated CSPG had apparent molecular mass ranges of 120-150 kDa and 120-190 kDa (Kav = 0.38 and 0.34), with CSs of 15 kDa and 20 kDa in the presence and absence of NGF, respectively. The apparent molecular mass of the core protein of cell-associated CSPG was 92 kDa, irrespective of the presence of NGF. The molecular sizes of cell-associated PGs and their glycosaminoglycans remained unchanged during culture. (3) CSPGs released by PC12 cells into the culture medium were separated into two peaks (I and II) by column chromatography on DEAE-cellulose. The peak II fraction prepared from the medium with NGF after 3 days of culture consisted of CSPG with Kav = 0.22 on Sephacryl S-300 [40-84 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteoglycans in primate arteries. III. Characterization of the proteoglycans synthesized by arterial smooth muscle cells in culture

Near confluent monolayers of arterial smooth muscle cells derived from Macaca nemestrina were labeled with Na2[35S]O4 and the newly synthesized proteoglycans present in the culture medium and cell layer were extracted with either 4 M guanidine HCl (dissociative solvent) or 0.5 M guanidine HCl (associative solvent) in the presence of protease inhibitors. The proteoglycans in both compartments were further purified by cesium chloride density gradient ultracentrifugation. Two size classes of proteoglycans were observed in the medium as determined by chromatography on Sepharose CL-2B. The large population (Kav = 0.31) contained predominantly chondroitin sulfate chains with Mr = approximately 40,000. The smaller population (Kav = 0.61) contained dermatan sulfate chains of similar Mr (approximately 40,000). When tested for their ability to aggregate, only proteoglycans in the large-sized population were able to aggregate. A chondroitin sulfate containing proteoglycan with identical properties was isolated from the cell layer. In addition, the cell layer contained a dermatan sulfate component which eluted later on Sepharose CL-2B (Kav = 0.78) than the dermatan sulfate proteoglycan present in the medium. Electron microscopy of the purified proteoglycans revealed a bottlebrush structure containing a central core averaging 140 nm in length with an average of 8 to 10 side projections. The length of the side projections varied but averaged between 70 and 75 nm. Similar bottlebrush structures were observed in the intercellular matrix of the smooth muscle cell cultures after staining with Safranin 0. This culture system provides a model to investigate parameters involved in the regulation of synthesis and degradation of arterial proteoglycans.     

Proteoglycans synthesized by chondrocytes of human nonarthritic and osteoarthritic cartilage

Chondrocyte cultures were developed from the cell outgrowths of explanted human nonarthritic and osteoarthritic human cartilage. Two significant differences in sulfated proteoglycan synthesis were demonstrated between the chondrocytes obtained in this manner. With 35SO4 to measure newly synthesized proteoglycan, we found that chondrocytes derived from osteoarthritic cartilage secreted significantly less (P less than 0.05) high density proteoglycan into the culture medium than did chondrocytes from nonarthritic cartilage after 20 hr of radiolabeling. This reduced amount of high density proteoglycan was sustained when chondrocytes were maintained in unlabeled culture medium ("chase" medium). In addition, the osteoarthritic chondrocytes secreted an increased amount of low density proteoglycan when compared with their nonarthritic counterparts. The elution profile of secreted high density proteoglycan isolated from the osteoarthritic chondrocyte culture medium was assessed by gel filtration on Sepharose CL-2B and revealed the presence of two proteoglycan subpopulations (Kav, 0.25, 0.58), whereas only one proteoglycan series (Kav, 0.37) was seen in the high density fraction of nonarthritic chondrocyte culture medium. Similar gel filtration profiles were also obtained when chondrocytes were maintained in chase medium. The results of this study demonstrated that stable differences in proteoglycan synthesis, but not in intracellular processing, exist between nonarthritic and osteoarthritic chondrocytes. The findings are noteworthy in that these differences were not previously apparent when organ-cultured cartilage was used to assess putative alterations in proteoglycans between the two groups.     

The role of myonuclei in muscle regeneration: An in vitro study

It is well established that during muscle regeneration, the satellite cells which are in a state of mitotic arrest, can initiate cell division to produce myoblasts which subsequently fuse to form myotubes. However, whether myonuclei, contained within damaged myotubes, or “freed” as a result of the trauma, play any role in muscle regeneration remains unresolved. In myogenic cultures, it is possible to obtain renewed myogenesis when initial cultures are sub-cultured. The aim of this study, was to obtain evidence of the participation by myonuclei of primary cultures in myogenesis which occurs subsequently in secondary cultures. In culture, myonuclei can be labelled with H³-thymidine and their ultimate fate, either as “free” myonuclei or myonuclei associated with disrupted myotubes can be followed unequivocally. Three types of experiments are performed: (i) Primary myogenic cultures containing only myotubes are subcultured. (ii) Primary myogenic cultures containing myotubes with labelled myonuclei are disrupted and subcultured. (iii) Primary myogenic cultures containing myotubes with unlabelled myonuclei are mixed with labelled mononucleated myogenic cells and sub-cultured. In all instances no evidence of myogenesis from myonuclei is obtained. It is concluded that myonuclei, which were rendered postmitotic during myogenesis, remain so when muscle is disrupted and cannot re-enter the mitotic cycle.     

Expression of the Chondroitin Sulfate Proteoglycans of Amyloid Precursor (Appican) and Amyloid Precursor-Like Protein 2

Abstract: The Alzheimer amyloid precursor (APP) protein is a member of a family of glycoproteins that includes the amyloid precursor-like proteins (APLPs). Previously, we showed that in C6 glioma cell cultures, secreted APP nexin II occurs as the core protein of a chondroitin sulfate proteoglycan (CSPG). Here, we report that among seven untransfected cell lines, expression of secreted APP CSPG was restricted to two cell lines of neural origin, namely, C6 glioma and Neuro-2a neuroblastoma (N2a) cells. Addition of dibutyryl cyclic AMP in N2a cultures, a treatment that induces the neuronal phenotype in these cells, resulted in a significant reduction in the amount of the secreted APP CSPG, although secretion of APP was only marginally affected. Growth in the presence of serum increased the size of the secreted APP CSPG, suggesting that the number and/or length of the chondroitin sulfate (CS) chains attached to the core APP varies with growth conditions. Extensive mapping with epitope-specific anti-bodies suggested that a CS chain is attached within or proximal to the Aβ sequence of APP. In contrast to the restricted expression of the APP CSPG, expression of secreted APLP2 CSPGs was observed in all cell lines examined. After chondroitinase treatment, two core proteins of ∼100 and 110 kDa were obtained that reacted with an APLP2-specific antiserum, suggesting that non-transfected cell lines contain at least two endogenous APLP2 CSPGs, probably derived by alternative splicing of the APLP2 KPI domain. The fraction of the APLP2 proteins in the CSPG form was dependent on the particular cell line examined. The proteoglycan nature of APP and APLP2 suggests that addition of the CS glycosaminoglycan chains is important for the implementation of the biological function of these proteins. However, the differential expression of these two proteoglycans suggests that their physiological roles and their possible involvement in Alzheimer's disease may differ.     

Cellular distribution of the Ia-associated chondroitin sulfate proteoglycan

The Ia-associated chondroitin sulfate proteoglycan (CSPG) found in anti-Ia and anti-invariant chain immunoprecipitates was originally detected in [35S] sulfate-labeled extracts derived from unseparated populations of splenocytes. To determine whether the CSPG was produced only by a subpopulation of spleen cells, we examined various cell populations for their ability to produce the CSPG. We found that B lymphocytes were the predominant source of CSPG in the spleen. The synthesis of the Ia-associated CSPG in spleen cell cultures was not diminished by the depletion of T cells or adherent cells. Moreover, the CSPG was readily detected in lysates derived from the Lyb-5- B cell subsets of xid mice, splenocytes from athymic (nude) mice, and in vitro B cell hybridomas. Peritoneal exudate macrophages from indomethacin-treated mice were also found to be capable of producing the CSPG. In all of the studies performed to date, no dissociation of the synthesis of the CSPG from the synthesis of Ia was observed in any cell type. We therefore tentatively conclude that all cells that synthesize conventional Ia molecules also synthesize the CSPG. Finally, we have been able to use anion exchange chromatography to prepare proteoglycan-enriched fractions to isolate the CSPG. This purification step has allowed us to convincingly demonstrate that the CSPG can be labeled with amino acids, and is a necessary step for detecting amino acid-labeled CSPG. This purification step method was used in the accompanying report to begin a quantitative examination of the Ia/CSPG complex, to monitor the kinetics of CSPG synthesis and association with Ia, and to determine its subcellular localization.     

Xyloside inhibits synthesis of the class II-associated chondroitin sulfate proteoglycan and antigen presentation events

An alternative form of the human invariant chain exists as a chondroitin sulfate proteoglycan (CSPG) with invariant chain as the core protein. The selective inhibitor of proteoglycan synthesis, p-nitrophenyl beta-D-xyloside was used to study the role of this CSPG in class II biology. At xyloside concentrations of 2.5 and 5.0 mM, CSPG synthesis was completely inhibited with marginal inhibition of protein synthesis. The inhibitory effect on CSPG synthesis was completely reversible. The number of class II molecules on the cell surface was not affected by xyloside, but biosynthesis and appearance of newly synthesized class II molecules at the cell surface were both decreased by xyloside. Recognition of influenza virus-infected cells by class II-restricted, virus-specific cytotoxic T lymphocytes was not diminished by the presence of xyloside in the effector phase of the cytotoxicity assay. However, sensitization of target cells was markedly inhibited when target cells were exposed to virus in the presence of xyloside. These results are consistent with the hypothesis that the CSPG form of invariant chain has a role in antigen processing.     

Partial characterization of proteoglycans synthesized by human glomerular epithelial cells in culture

Confluent adult and fetal human glomerular epithelial cells were incubated for 24 h in the presence of [3H]-amino acids and [35S]sulfate. Two heparan-35SO4 proteoglycans were released into the culture medium. These 35S-labeled proteoglycans eluted as a single peak from anion exchange chromatographic columns, but were separable by gel filtration on Sepharose CL-6B columns. The larger heparan-35SO4 proteoglycan eluted with the column void volume and at a Kav of 0.26 from Sepharose CL-4B columns. The most abundant medium heparan-35SO4 proteoglycan was a high buoyant density proteoglycan similar in hydrodynamic size (Sepharose CL-6B Kav 0.23) to those previously described in glomerular basement membranes and isolated glomeruli. Heparan-35SO4 chains from both proteoglycans were 36 kDa. A smaller proportion of Sepharose CL-6B excluded dermatan-35SO4 proteoglycan was also synthesized by these cells. The predominant protein cores of both medium heparan-35SO4 proteoglycans were approximately 230 and 180 kDa. A hybrid chondroitin/dermatan-heparan-35SO4 proteoglycan with an 80-kDa protein core copurified with the smaller medium heparan-35SO4 proteoglycan. This 35S-labeled proteoglycan appeared as a diffuse, chondroitinase ABC sensitive 155-kDa fluorographic band in sodium dodecyl sulfate-polyacrylamide gels after the Sepharose CL-6B Kav 0.23 35S-labeled proteoglycan fraction was digested with heparitinase. The heparitinase generated heparan sulfate proteoglycan protein cores and the 155-kDa hybrid proteoglycan fragment had molecular weights similar to those previously identified in rat glomerular basement membrane and glomeruli using antibodies against a basement membrane tumor proteoglycan precursor (Klein et al. J. Cell Biol. 106, 963-970, 1988). Thus, human glomerular epithelial cells in culture are capable of synthesizing, processing, and releasing heparan sulfate proteoglycans which are similar to those synthesized in vivo and found in the glomerular basement membrane. These proteoglycans may belong to a family of related basement membrane proteoglycans.     

The effects of epidermal growth factor on neural crest cells in tissue culture

Epidermal growth factor (EGF) stimulates the release of hyaluronic acid (HA) and chondroitin sulfate proteoglycan (CSPG) from quail trunk neural crest cultures in a dose-dependent fashion. It also promotes the expression of cell-associated heparan sulfate proteoglycan (HSPG) as detected by immunofluorescence and immunoprecipitation of the 3H-labeled proteoglycan. Furthermore, EGF stimulates [3H]thymidine incorporation into total cell DNA. These results raise the possibility that EGF or an analogous growth factor is involved in regulation of neural crest cell morphogenesis.