Kinetics of intracellular processing of chondroitin sulfate proteoglycan core protein and other matrix components.

Campbell, Steven C.; Schwartz, Nancy B.

doi:10.1083/jcb.106.6.2191

Cited by 35 publications

(25 citation statements)

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“…Previous studies have shown that these two glycosyltransferase activities are localized in different compartments (15,19). Note that galactosylation in N4-expressed protein is evident after chondroitinase treatment, in agreement with the observations that xylosylation is a rate-limiting step in GAG biosynthesis and that, after translocation of the core protein to the Golgi, successive reactions proceed quickly without allowing observation of partial intermediate forms (10). The present results imply that, until the precursor is translocated from the xylosylation-competent compartment to the next one, the core protein cannot be galactosylated, and that translocation requires information inherent in the second lectin exon.…”

Section: Resultssupporting

confidence: 69%

“…Cell Culture and Transfection-Chondrocytes were prepared from sterna of 15-day-old chick embryos and grown in F12 media with 10% fetal calf serum supplemented with gentamicin at a density of 5 ϫ 10 6 cells per dish as described previously (10). COS-1 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum at 37°C and 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

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Role of the C-terminal G3 Domain in Sorting and Secretion of Aggrecan Core Protein and Ubiquitin-mediated Degradation of Accumulated Mutant Precursors

Domowicz¹,

Pirok²,

Novak³

et al. 2000

Journal of Biological Chemistry

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Aggrecan is a complex multidomain macromolecule that undergoes extensive processing and post-translational modification. A thorough understanding of the events and signals that promote translocation of aggrecan through the secretory pathway is lacking. To investigate which features of the C-terminal G3 region are necessary for successful translocation of the core protein, a number of deletion constructs based on the chick aggrecan cDNA sequence were prepared and transiently expressed in COS-1 cells and the natural host, embryonic chick chondrocytes; stable cell lines were established as well. The present results clearly establish a precise requirement for that portion of the G3 C-lectin domain encoded by exon 15 for: (i) translocation from the endoplasmic reticulum (ER) to the Golgi, (ii) secretion from the cell, (iii) galactosylation of chondroitin sulfate (CS) chains, (iv) generation of Ca ؉2 -dependent galactose binding ability. Furthermore, in the absence of this subdomain there is excess accumulation in the ER of expression products leading to a stress-related response involving the chaperones Grp78 and protein disulfide isomerase, followed by degradation via a ubiquitin-proteosome pathway. All of these events in the model system faithfully mimic the naturally occurring nanomelic mutant, which also elicits a ubiquitin-mediated degradation response due to the accumulation of the truncated core protein precursor. This study represents the first report of the mode of degradation of overexpressed or misfolded proteoglycans and suggests that, although proteoglycans follow different glycosylation pathways from other glycoproteins, they are monitored by an ER surveillance system similar to that which detects other misfolded proteins.Aggrecan, the major proteoglycan of the cartilage extracellular matrix, contains two globular domains in the N-terminal portion, G1 and G2, and one C-terminal globular domain, G3. Between the G2 and G3 globular domains, glycosaminoglycan chains (GAGs) 1 are covalently attached to an extended core protein. Functionally, it is well established that the G1 domain is responsible for interaction with hyaluronan in the extracellular matrix. The C-terminal G3 domain has been implicated in synthesis and maturation of aggrecan, because its absence is associated with the avian chondrodystrophy, nanomelia. The nanomelic chick bears a mutation in the aggrecan gene that introduces an early stop codon into the translated sequence, resulting in synthesis of a truncated core protein, which is neither glycosylated nor secreted by chondrocytes (1). The mutant precursor is modified by addition of N-linked oligosaccharides and the chondroitin sulfate chain initiating xylose, but does not acquire mature CS chains, consistent with the conclusion that it progresses no further than the endoplasmic reticulum (ER) in the secretory pathway (2). These studies suggested a previously unrecognized role for the C-terminal globular domain: that it might contain recognition or retention signals or that proper folding o...

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Section: Resultssupporting

confidence: 69%

Section: Methodsmentioning

confidence: 99%

Role of the C-terminal G3 Domain in Sorting and Secretion of Aggrecan Core Protein and Ubiquitin-mediated Degradation of Accumulated Mutant Precursors

Domowicz¹,

Pirok²,

Novak³

et al. 2000

Journal of Biological Chemistry

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“…An overlapping localisation or an interaction of xylosyltransferase with galactosyltransferase I is possible since a similar antibody preparation to that used in the electron microscopy study described above could immunoprecipitate both xylosyltransferase and galactosyltransferase activities [96]; galactosyltransferase I bound to semipurified and immobilised xylosyltransferase [97] and a subcellular fractionation experiment suggested that both enzymes are present in rough and smooth membranes [98]. It has been proposed that the membrane-bound galactosyltransferase I may help 'dock' a soluble xylosyltransferase/core-protein complex to the Golgi membranes, so allowing further processing and translocation of the core protein to occur [99] (presumably such a soluble form resulting from proteolytic loss of the transmembrane domain). However, since data on xylosyltransferase localisation was performed at different times and in different ways, it is still difficult to come up with a unifying conclusion as to where it is located or with which proteins it may interact.…”

Section: Localisation Of the Xylosyl Transfer Reactionmentioning

confidence: 98%

The never-ending story of peptide O -xylosyltransferase

Wilson

2004

Cellular and Molecular Life Sciences (CMLS)

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In a journey lasting 40 years from the first reports on its activity in the 1960s to its purification and the cloning of relevant complementary DNAs, peptide O-xylosyltransferase has finally arrived at the same point as many other enzymes. This enzyme, whose systematic name is UDP-alpha-D-xylose:proteoglycan core protein beta-D-xylosyltransferase (EC 2.4.2.26), catalyses the first step in the biosynthesis of chondroitin, dermatan and heparan sulphates in the endoplasmic reticulum and/or the cis-Golgi cisternae. Analyses of their primary structure show that peptide O-xylosyltransferases are members of glycosyltransferase family 14 and so are homologous to beta1,6- N-acetylglucosaminyltransferases involved in O-glycan and poly- N-acetyllactosamine branching. Furthermore, vertebrates appear to have two rather similar genes encoding xylosyltransferase I and xylosyltransferase II, but enzymatic activity was only detected for a recombinant form of the first isoform. On the other hand, Caenorhabditis and Drosophila have each only one peptide O-xylosyltransferase gene. In the worm sqv-6 mutant, a mutation of the xylosyltransferase gene is associated with defective vulval morphogenesis, indicative of the importance of the glycosaminoglycan chains of proteoglycans in animal development. There remain, however, open questions, for instance, on the enzyme's intracellular localisation and structure-function relationships.

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“…4c). A first-order fit of these data revealed that 3 the core protein had a short intracellular half-life of about -CP 12 min (Campbell & Schwartz, 1988) In a second set of experiments, cultures were preincubated with ascorbate for 3 h, at which time the secretion rates of types II and IX collagen had increased to their maximum (Fig. 3) above.…”

Section: Pg-h Core Protein Translocation and Secretionmentioning

confidence: 99%

“…For example, different secretory proteins are transported out of the RER at markedly different rates but move through the remaining portion of the secretory pathway at similar rates, suggesting that protein translocation from the RER is a selective, rate-limiting step in protein secretion (Mitchell & Hardingham, 1981;Fitting & Kabat, 1982;Lodish et al, 1983;Fries et al, 1984;Fellini et al, 1984;Campbell & Schwartz, 1988). In addition, abnormal secretory proteins resulting from mutations or pharmacological treatments exit the RER very slowly, if at all, indicating that the RER also has the ability to discriminate between normal and abnormal secretory proteins (Jimenez et al, 1974;Jimenez & Yankowski, 1978;Kreis & Lodish, 1986;Gothing et al, 1986;Sifers et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

Independent secretion of proteoglycans and collagens in chick chondrocyte cultures during acute ascorbic acid treatment

Pacifici

1990

Biochemical Journal

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The mechanisms regulating the secretion of proteoglycans and collagens in chondrocytes, in particular those operating at the level of the rough endoplasmic reticulum (RER), are largely unknown. To examine these mechanisms, I studied the effects of acute ascorbate treatment on the secretion of two collagen types (types II and IX) and two proteoglycan types (PG-H and PG-Lb, the major keratan sulphate/chondroitin sulphate proteoglycan and the minor chondroitin sulphate proteoglycan respectively in cartilage) in scorbutic cultures of chick vertebral chondrocytes. I found that the scorbutic chondrocytes synthesized underhydroxylated precursors of types II and IX collagen that were secreted very slowly and accumulated in the RER. When the cultures were treated acutely with ascorbate, both macromolecules underwent hydroxylation within 1-1.5 h of treatment, and began to be secreted at normal high rates starting at about 2 h. Proteoglycan synthesis and secretion, however, remained largely unaffected by ascorbate treatment. Both the half-time of newly synthesized PG-H core protein in the RER and its conversion into completed proteoglycan were unchanged during treatment. Similarly, the overall rates of synthesis and secretion of both PG-H and PG-Lb remained at control levels during treatment. The data indicate that secretion of types II and IX collagen is regulated independently of secretion of PG-H and PG-Lb. This may be mediated by the ability of the RER of the chondrocyte to discriminate between procollagens and proteoglycan core proteins.

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Kinetics of intracellular processing of chondroitin sulfate proteoglycan core protein and other matrix components.

Cited by 35 publications

References 44 publications

Role of the C-terminal G3 Domain in Sorting and Secretion of Aggrecan Core Protein and Ubiquitin-mediated Degradation of Accumulated Mutant Precursors

Role of the C-terminal G3 Domain in Sorting and Secretion of Aggrecan Core Protein and Ubiquitin-mediated Degradation of Accumulated Mutant Precursors

The never-ending story of peptide O -xylosyltransferase

Independent secretion of proteoglycans and collagens in chick chondrocyte cultures during acute ascorbic acid treatment

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