Intracellular features of type II procollagen and chondroitin sulfate proteoglycan synthesis in chondrocytes (original) (raw)
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Journal of Cell Biology, 1989
Immunocytochemical methods were used at the levels of light and electron microscopy to examine the intracellular compartments of chondrocytes involved in extracellular matrix biosynthesis. The results of our studies provide morphological evidence for the compartmentalization of secretory proteins in the ER. Precursors of the large chondroitin sulfate proteoglycan (CSPG), the major proteoglycan species produced by chondrocytes, were present in the Goigi complex. In addition, CSPG precursors were localized in specialized regions of the ER. Link protein, a separate gene product which functions to stabilize extracellular aggregates of CSPG monomers with hyaluronic acid, was segregated similarly. In contrast, type II procollagen, another major secretory molecule produced by . Adjacent sections of a rER-associated, CSPG precursor-containing ER region. Noncounterstained, consecutive sections of a CSPG precursor-containing region continuous with the rER are shown. The same position in the rER lumen (asterisk) and the position of the CSPG precursor-containing region (arrow) are indicated in each micrograph. Note the vesicular buds along the periphery of the immunoreactive compartment. Bar, 0.25 #m.
Secretion of proteoglycans by chondrocytes
Archives of Biochemistry and Biophysics, 1979
Chondrocytes obtained from epiphyseal cartilage of fetal guinea pigs or ear cartilage of young rabbits were cultured in monolayer. The influence of colchicine, cytochalasin B, and p-nitrophenyl-P-D-xylopyranoside on secretion of proteoglycans was investigated. Radioactive sulfate was used as a precursor. As observed previously in other systems, /%Dxylosides initiated the synthesis of free chondroitin sulfate chains, competing with the endogenous proteoglycan core protein acceptor. The molecular weights of the chondroitin sulfate chains synthesized both on the xyloside and on the core-protein acceptor in maximally stimulated cells were similar and significantly lower than in proteoglycans synthesized in the absence of xyloside. The size of the chondroitin sulfate chains synthesized on the xyloside was inversel) related to the concentration of this compound. This finding suggests that the chain length is dependent on the ratio between available acceptor and chain-lengthening enzymes or precursors.
The proteoglycan synthesis repertoire of rabbit chondrocytes maintained in type II collagen gels
Osteoarthritis and Cartilage, 1994
experimental articular cartilage lesions employing cultured rabbit articular chondrocytes requires a i~ d~:tailed knowledge of the phenotypic stability of these cells. A suitable matrix vehicle for use in chondrocyte i-:gtansplantation is a much sought-after component of any transplantation paradigm. We studied the proteoglycan ::~nthesis repertoire of young immature rabbit articular chondrocytes maintained in chick type II collagen gels or ::~bllagen gels supplemented with recombinant human transforming growth factor-fl~ (rhTGFfll). :~::::.:.:,Maintenance of chondrocytes in type II collagen gels increased the percentage 35SO4-1abeled proteoglycans reaching i::~e~tilibrium in the A1D1 or D1 fraction of CsC1 density gradient when compared to chondrocytes maintained in !!:p~lystyrene microwell cultures. Although rhTGFfll supplementation increased the percentage of A1D1/D1 p~oteoglycan by chondrocytes grown on polystyrene, rhTGFfl~ did not augment this percehtage increase in AID1/D1 ~h:en added to collagen II gels. ~ii!i!iRabbit chondrocytes synthesized two core proteins derived from the high-density aggregatable proteoglycans. L~ and ::~i~:have apparent molecular sizes of 480 kDa and 390 kDa, respectively. Both core protein forms were found in the ::~dium fraction, but the predominant core protein form associated with the cell fraction was L I. Maintenance of ;':::~drocytes in collagen II gels increased synthesis of both core proteins. In addition to the large core proteins, three :: :~er core proteins with properties on SDS PAGE characteristic of the small dermatan sulfate proteoglycans, biglycan :;~t decorin, were identified. Synthesis of these core proteins was stimulated by maintenance in collagen gels. :~hermore, they were preferentially retained in the gel matrix. i! !i~i!.:~hondrocytes maintained on glass or in type II collagen gels stained with monoclonal antibodies specific for iii~ndroitin-6-sulfate, chondroitin-4-sulfate and keratan sulfate. However, while chondrocytes grown on glass slides !ii~!~]ed to stain with monoclonal antibody 3B3 in the absence of chondroitinase ABC digestion, chondrocytes grown in ii!!~lagen II gels stained intensely in the absence of enzyme pretreatment. These results were confirmed by Western words: Rabbit, Chondrocytes, Type II collagen gels, Proteoglycan. :ii' .
The Journal of Cell Biology, 1983
We investigated the subcellular sites of glycoprotein oligosaccharide maturation by using lectin conjugates to stain lightly-fixed, saponin-permeabilized myeloma cells. At the electron microscopic level, concanavalin A-peroxidase stains the cisternal space of the nuclear envelope, the rough endoplasmic reticulum, and cisternae along the proximal face of the Golgi stack. Conversely, wheat germ agglutinin-peroxidase stains cisternae along the distal face of the Golgi stack, associated vesicles, and the cell surface. These observations confirm the existence of two qualitatively distinct Golgi subcompartments, show that the lectin conjugates can be employed as relatively proximal or distal Golgi markers under conditions of excellent ultrastructural preservation, suggest that the asymmetric distribution of qualitatively distinct oligosaccharides is a property of underlying cellular components and not simply of the principal secretory product, and suggest that the oligosaccharide structur...
Proceedings of the National Academy of Sciences, 1979
In order to investigate the coordinated synthesis of matrix components by individual chondrocytes, specific antibodies to type I collagen, type II collagen, and chondroitin sulfate proteoglycan core protein were used in simultaneous double immunofluorescence reactions. Extensive accumulation of core protein surrounding chondrocytes and the intracellular accumulation of type II collagen were observed. Extracellular core protein immunofluorescence obscured the intracellular reaction product, but the extracellular immunoreactive material could be removed by digestion with purified testicular hyaluronidase prior to fixation. Subsequent to digestion, core protein and type II collagen were observed in the same chondrocytes within discrete, sometimes identical, cytoplasmic regions, thus demonstrating the simultaneous localization of these two products characteristic of differentiating cartilage.
Structures of N- and O-linked oligosaccharides from chondrosarcoma proteoglycan
Seminars in Arthritis and Rheumatism, 1981
The core protein of cartilage proteoglycan as an intermediate in the biosynthesis of proteoglycan by intact chondrocytes has been tentatively identified. It appears to be largely a monodisperse species of M,-370,000 on SDS-PAGE. The presence of a single predominant core protein intermediate suggests that proteoglycan synthesis proceeds through a rate-limiting step that allows the accumulation of label in this intermediate. The nature of this step is not known, but it may represent the transport time for the movement of newly synthesized core protein from the rough endoplasmic reticulum to the Golgi for the addition of chondroitin-sulfate chains.
Journal of Biological …, 1986
Extraction of stage 22-23 chick embryo limb buds that had been metabolically labeled with [SsS]sulfate yielded heparan sulfate proteoglycan, small chondroitin sulfate proteoglycan, and large chondroitin sulfate proteoglycan (designated PG-M). PG-M constituted over 60% of the total macromolecular [SsS]sulfates. It was larger in hydrodynamic size, richer in protein, and contained fewer chondroitin sulfate chains as compared to the predominant proteoglycan (PG-H, M, 1.5 x lo6) of chick embryo cartilage. The chondroitin sulfate chains were notable for their large size (Mr 2 60,000) and high content of nonsulfated chondroitin units (about 20% of the total hexosamine). Hexosamine-containing chains corresponding in size to Nlinked and O-linked oligosaccharides were also present. The core protein was rich in serine, glutamic acid (glutamine), and glycine which together comprised about 38% of the total amino acids. Following chondroitinase AC I1 (or ABC) digestion, core molecules were obtained which migrated on sodium dodecyl sulfate gel electrophoresis as a doublet of bands with approximately M, = 550,000 (major) and 500,000, respectively. The M, = 550,000 core glycoprotein was structurally different from the core glycoprotein (Mr As cartilage differentiation progresses, type I collagen is gradually replaced by type I1 collagen (2,3), fibronectin disappears (2,4), the rate of [35S]sulfate incorporation into proteoglycans becomes higher in the core mesenchyme region than in the surrounding loose mesenchyme region (5), and a cartilagespecific proteoglycan, PG-H,' becomes detectable in cartilage primordia (6).
Upsala Journal of Medical Sciences, 2009
Versican/PG-M (proteoglycan-mesenchymal) is a large chondroitin sulfate (CS) proteoglycan of the extracellular matrix (ECM) that is constitutively expressed in adult tissues such as dermis and blood vessels. It serves as a structural macromolecule of the ECM, while in embryonic tissue it is transiently expressed at high levels and regulates cell adhesion, migration, proliferation, and differentiation. Knock-in mouse embryonic (Cspg2 D3/D3) fibroblasts whose versican lack the A subdomain of the G1 domain exhibit low proliferation rates and acquire senescence. It was suspected that chondroitin sulfate on versican core protein would be altered when the A subdomain was disrupted, so fibroblasts were made from homozygous Cspg2 D3/D3 mouse embryos to investigate the hypothesis. Analysis of the resulting versican deposition demonstrated that the total versican deposited in the Cspg2 D3/D3 fibroblasts culture was approximately 50% of that of the wild type (WT), while the versican deposited in the ECM of Cspg2 D3/D3 fibroblasts culture was 35% of that of the WT, demonstrating the lower capacity of mutant (Cspg2 D3/D3) versican deposited in the ECM. The analysis of CS expression in the Cspg2 D3/D3 fibroblasts culture compared with wild-type fibroblasts showed that the composition of the non-sulfate chondroitin sulfate isomer on the versican core protein increased in the cell layer but decreased in the culture medium. Interestingly, chondroitin sulfate E isomer was found in the culture medium. The amount of CS in the Cspg2 D3/D3 cell layer of fibroblasts with mutant versican was dramatically decreased, contrasted to the amount in the culture medium, which increased. It was concluded that the disruption of the A subdomain of the versican molecule leads to lowering of the amount of versican deposited in the ECM and the alteration of the composition and content of CS on the versican molecule.