Acan | GeneID:58968 | Rattus norvegicus
[ ] NCBI Entrez Gene
|Gene ID||58968||Official Symbol||Acan|
|Also Known As||aggrecan 1; aggrecan, structural proteoglycan of cartilage|
|Summary||an extracellular matrix proteoglycan; cleaved by ADAMTS1 [RGD]|
Orthologs and Paralogs
|GeneID:403828||ACAN||XP_536187.1||Canis lupus familiaris|
[ ] Monoclonal and Polyclonal Antibodies
|1||abcam||ab53731||Aggrecan antibody - Cleaved Asp369 (ab53731); Rabbit polyclonal to Aggrecan - Cleaved Asp369|
|2||abcam||ab36861||Aggrecan antibody (ab36861); Rabbit polyclonal to Aggrecan|
|3||abcam||ab3776||Aggrecan FFGxx antibody [BC-14] (ab3776); Mouse monoclonal [BC-14] to Aggrecan FFGxx|
|4||abcam||ab3777||Aggrecan xxPEN antibody [BC-4] (ab3777); Mouse monoclonal [BC-4] to Aggrecan xxPEN|
|5||abcam||ab16320||Aggrecan antibody (ab16320); Rabbit polyclonal to Aggrecan|
|6||abcam||ab3773||Aggrecan ARGxx antibody [BC-3] (ab3773); Mouse monoclonal [BC-3] to Aggrecan ARGxx|
|7||sigma||C8035||Monoclonal Anti-Chondroitin Sulfate antibody produced in mouse ;|
|GO:0005578||Component||proteinaceous extracellular matrix|
|GO:0005201||Function||extracellular matrix structural constituent|
|GO:0005540||Function||hyaluronic acid binding|
|GO:0007417||Process||central nervous system development|
|GO:0030199||Process||collagen fibril organization|
|GO:0030166||Process||proteoglycan biosynthetic process|
- [ ] Cancel M, et al. (2009) "Effects of in vivo static compressive loading on aggrecan and type II and X collagens in the rat growth plate extracellular matrix." Bone. 44(2):306-315. PMID:18849019
- [ ] Agrawal A, et al. (2007) "Normoxic stabilization of HIF-1alpha drives glycolytic metabolism and regulates aggrecan gene expression in nucleus pulposus cells of the rat intervertebral disk." Am J Physiol Cell Physiol. 293(2):C621-C631. PMID:17442734
- [ ] Carulli D, et al. (2007) "Upregulation of aggrecan, link protein 1, and hyaluronan synthases during formation of perineuronal nets in the rat cerebellum." J Comp Neurol. 501(1):83-94. PMID:17206619
- [ ] Popp S, et al. (2003) "Localization of aggrecan and versican in the developing rat central nervous system." Dev Dyn. 227(1):143-149. PMID:12701107
- [ ] Matthews RT, et al. (2002) "Aggrecan glycoforms contribute to the molecular heterogeneity of perineuronal nets." J Neurosci. 22(17):7536-7547. PMID:12196577
- [ ] Doege K, et al. (2002) "A remote upstream element regulates tissue-specific expression of the rat aggrecan gene." J Biol Chem. 277(16):13989-13997. PMID:11834732
- [ ] Rodriguez-Manzaneque JC, et al. (2002) "ADAMTS1 cleaves aggrecan at multiple sites and is differentially inhibited by metalloproteinase inhibitors." Biochem Biophys Res Commun. 293(1):501-508. PMID:12054629
- [ ] Neame PJ, et al. (1987) "Cartilage proteoglycan aggregates. The link protein and proteoglycan amino-terminal globular domains have similar structures." J Biol Chem. 262(36):17768-17778. PMID:3693371
- [ ] Doege K, et al. (1987) "Complete primary structure of the rat cartilage proteoglycan core protein deduced from cDNA clones." J Biol Chem. 262(36):17757-17767. PMID:3693370
- [ ] Doege K, et al. (1986) "Partial cDNA sequence encoding a globular domain at the C terminus of the rat cartilage proteoglycan." J Biol Chem. 261(18):8108-8111. PMID:2424893
Mechanical loads are essential to normal bone growth, but excessive loads can lead to progressive deformities. In addition, growth plate extracellular matrix remodelling is essential to regulate the normal longitudinal bone growth process and to ensure physiological bone mineralization. In order to investigate the effects of static compression on growth plate extracellular matrix using an in vivo animal model, a loading device was used to precisely apply a compressive stress of 0.2 MPa for two weeks on the seventh caudal vertebra (Cd7) of rats during the pubertal growth spurt. Control, sham and loaded groups were studied. Growth modulation was quantified based on calcein labelling, and three matrix components (type II and X collagens, and aggrecan) were assessed using immunohistochemistry/safranin-O staining. As well, extracellular matrix components and enzymes (MMP-3 and -13, ADAMTS-4 and -5) were studied by qRT-PCR. Loading reduced Cd7 growth by 29% (p<0.05) and 15% (p=0.07) when compared to controls and shams respectively. No significant change could be observed in the mRNA expression of collagens and the proteolytic enzyme MMP-13. However, MMP-3 was significantly increased in the loaded group as compared to the control group (p<0.05). No change was observed in aggrecan and ADAMTS-4 and -5 expression. Low immunostaining for type II and X collagens was observed in 83% of the loaded rats as compared to the control rats. This in vivo study shows that, during pubertal growth spurt, two-week static compression reduced caudal vertebrae growth rates; this mechanical growth modulation occurred with decreased type II and X collagen proteins in the growth plate.
The nucleus pulposus is an aggrecan-rich, avascular tissue that permits the intervertebral disk to resist compressive loads. In the disk, nucleus pulposus cells express hypoxia-inducible factor (HIF)-1alpha, a transcription factor that responds to oxygen tension and regulates glycolysis. The goal of the present study was to examine the importance of HIF-1alpha in rat nucleus pulposus cells and to probe the function of this transcription factor in terms of regulating aggrecan gene expression. We found that HIF-1alpha protein levels and mRNA stability were similar at 20 and 2% O(2); there was a small, but significant increase in HIF-1alpha transactivation domain activity in hypoxia. With respect to HIF-1alpha target genes GAPDH, GLUT-1, and GLUT-3, mRNA and protein levels were independent of the oxygen tension. Other than a modest increase in 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase reporter activity, the oxemic state did not change GAPDH, GLUT-1, and GLUT-3 promoter activities. Treatment of cells with 2-deoxyglucose (2-DG), a glycolytic inhibitor, resulted in a significant suppression in ATP synthesis in normoxia, whereas treatment with mitochondrial inhibitors did not affect ATP production and cell viability. However, measurement of the rate of fatty acid oxidation indicated that these cells contained functioning mitochondria. Finally, we showed that when HIF-1alpha was suppressed, irrespective of the oxemic state, there was a partial loss of aggrecan expression and promoter activity. Moreover, when cells were treated with 2-DG, there was inhibition in aggrecan promoter activity. Results of this study indicate that oxygen-independent stabilization of HIF-1alpha in nucleus pulposus cells is a metabolic adaptation that drives glycolysis and aggrecan expression.
Extracellular matrix molecules accumulate around central nervous system neurons during postnatal development, forming so-called perineuronal nets (PNNs). PNNs play a role in restricting plasticity at the end of critical periods. In the adult rat cerebellum, PNNs are found around large, deep cerebellar nuclei (DCN) neurons and Golgi neurons and are composed of chondroitin sulfate proteoglycans (CSPGs), tenascin-R (TN-R), hyaluronan (HA), and link proteins, such as cartilage link protein 1 (Crtll). Granule cells and Purkinje cells are surrounded by a partially organized matrix. Both glial cells and neurons surrounded by PNNs are the site of synthesis of some CSPGs and of TN-R, but only neurons produce HA synthetic enzymes (HASs), thus HA, and link proteins, which are scaffolding molecules for an organized matrix. To elucidate the mechanisms of formation of PNNs, we analyzed by immunohistochemistry and in situ hybridization which PNN components are upregulated during PNN formation in rat cerebellar postnatal development and what cell types express them. We observed that Wisteria floribunda agglutinin-binding PNNs develop around DCN neurons from postnatal day (P)7 and around Golgi neurons from P14. At the same time as their PNNs start to form, these neurons upregulate aggrecan, Crtll, and HASs mRNAs. However, Crtll is the only PNN component to be expressed exclusively in neurons surrounded by PNNs. The other link protein that shows a perineuronal net pattern in the DCN, Bral2, is upregulated later during development. These data suggest that aggrecan, HA, and, particularly, Crtll might be crucial elements for the initial assembly of PNNs.
The localization of aggrecan and mRNA splice variants of versican in the developing rat central nervous system has been examined by using specific polyclonal antibodies to the nonhomologous glycosaminoglycan attachment regions of these hyaluronan-binding chondroitin sulfate proteoglycans. At embryonic day 16 (E16), aggrecan and versican splice variants containing either or both the alpha-and beta-domains are present in the marginal zone and subplate of the cerebral cortex and in the amygdala, internal capsule, and the optic and lateral olfactory tracts. There is strong staining of versican but not of aggrecan in the hippocampus and dentate gyrus by E19, whereas both aggrecan and alpha-versican are present in the fimbria. At E19, aggrecan is seen throughout the cerebral cortex, whereas the distribution of versican is considerably more limited, being confined essentially to the marginal zone and subplate. At 1 week postnatal, both aggrecan and versican are present in the prospective white matter and in the molecular and granule cell layers of the cerebellum, but neither proteoglycan is seen in the external granule cell layer. alpha- but not beta-versican staining is seen in Purkinje cells, and aggrecan staining of Purkinje cells is also rather minimal. In the spinal cord at E13, aggrecan is present in the dorsal root entry zone, ventral funiculus, mantle layer, and floor plate, as well as in the dorsal root ganglia and ventral roots. However, alpha-versican is confined to the dorsal root entry zone and the ependyma surrounding the spinal canal, and beta-versican is not present in spinal cord parenchyma at this developmental stage, being limited to the surrounding connective tissue. By E19, there are significant amounts of all three proteoglycans in the spinal cord. Aggrecan staining is most intense in the lateral funiculus and the fasciculi gracilis and cuneatus, where alpha-versican staining is also strong. In contrast, beta-versican is seen predominantly in the motor columns. Differences in the localization and temporal expression patterns of these chondroitin sulfate proteoglycans suggest that, like neurocan and phosphacan, they have partially complementary roles during central nervous system development.
The perineuronal net forms the extracellular matrix of many neurons in the CNS, surrounding neuron cell bodies and proximal dendrites in a mesh-like structure with open "holes" at the sites of synaptic contacts. The perineuronal net is first detected late in development, approximately coincident with the transformation of the CNS from an environment conducive to neuronal growth and motility to one that is restrictive, suggesting a role for the perineuronal net in this developmental transition. Perineuronal nets show a great degree of molecular heterogeneity. Using monoclonal antibodies Cat-301, Cat-315, and Cat-316, we have shown previously that although all antibodies recognize chondroitin sulfate proteoglycans of similar sizes, each antibody recognizes perineuronal nets on distinct but overlapping sets of neurons in the adult cat CNS. An understanding of the heterogeneity demonstrated by these antibodies is critical to understanding the organization and function of perineuronal nets. Using aggrecan knock-out mice (cmd), we have now determined that all three antibodies recognize aggrecan. Chemical and enzymatic deglycosylation show that the differences revealed by the three antibodies arise from differential glycosylation of aggrecan. We further demonstrate that aggrecan mRNA is expressed relatively late in development and that neurons themselves are likely the predominant cellular sites of aggrecan expression. This work indicates that neurons can directly regulate the composition of their extracellular matrix by regulated synthesis and differential glycosylation of aggrecan in a cell type-specific manner. These results have important implications for the role of regulated microheterogeneity of glycosylation in the CNS.
The regulation of chondrogenesis and of the genes expressed as markers of chondrocyte differentiation is poorly understood. The hyaluronan-binding proteoglycan aggrecan is an essential and specific component of cartilage, but the aggrecan proximal promoter is expressed in an unregulated fashion in vitro. DNA comprising the rat aggrecan gene (83 kb including the 30-kb first intron) was surveyed for active elements, which would impart selective expression to the aggrecan promoter in transfection assays in vitro. A 4.7-kb DNA fragment (P3) with cell-specific enhancer activity was discovered approximately 12 kb upstream of the transcription start site; this active DNA fragment is position- and orientation-independent, and strongly stimulates aggrecan promoter expression in chondrocytes, while weakly suppressing transcription in fibroblasts. Most of this activity has been localized to P3-7, a 2.3-kb internal fragment of P3. Another enhancer element (A23), which is not tissue-specific, was discovered about 70 kb downstream of the transcription start site. Several lines of transgenic mice were created using combinations of these DNA elements to drive the lacZ reporter gene. Neither a short (900 bp) nor a long (3.7 kb) promoter alone showed detectable expression in 14.5-day embryos, whereas placing the P3 tissue-specific enhancer together with P0 gave strong expression restricted to embryonic cartilage of transgenic mice. The A23 downstream enhancer in conjunction with P0 did not confer expression. This is the first report of a gene control region which confers authentic tissue-specific regulation of aggrecan in vitro or in vivo and should greatly facilitate understanding the coordinate regulation of chondrocytic genes.
ADAMTS1 is a secreted protein that belongs to the recently described ADAMTS (a disintegrin and metalloprotease with thrombospondin repeats) family of proteases. Evaluation of ADAMTS1 catalytic activity on a panel of extracellular matrix proteins showed a restrictive substrate specificity which includes some proteoglycans. Our results demonstrated that human ADAMTS1 cleaves aggrecan at a previously shown site by its mouse homolog, but we have also identified additional cleavage sites that ultimately confirm the classification of this protease as an 'aggrecanase'. Specificity of ADAMTS1 activity was further verified when a point mutation in the zinc-binding domain abolished its catalytic effects, and latency conferred by the prodomain was also demonstrated using a furin cleavage site mutant. Suppression of ADAMTS1 activity was accomplished with a specific monoclonal antibody and some metalloprotease inhibitors, including tissue inhibitor of metalloproteinases 2 and 3. Finally, we developed an activity assay using an artificial peptide substrate based on the interglobular domain cleavage site (E(373)-A) of rat aggrecan.
Cartilage proteoglycan aggregates contain two components (proteoglycan monomer and link protein) which interact with each other and with hyaluronic acid. Data from amino acid sequence analysis are presented that shows that a domain of the proteoglycan, the hyaluronic acid binding region, which interacts with link protein and hyaluronic acid is very similar to link protein in terms of its primary structure. However, the pattern of glycosylation in the hyaluronic acid binding region is different from that found in link protein. After removal of N-linked oligosaccharides, the tryptically prepared hyaluronic acid binding region from rat chondrosarcoma has a mass by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of 43 +/- 2 kDa. The COOH-terminal two-thirds of rat chondrosarcoma link protein, starting at residue 105, has 41.3% identity with a similar region in the hyaluronic acid binding region. We show that, in addition to the hyaluronic acid binding region, proteoglycan contains another region with similarity to the two repeating loop structures in the COOH-terminal two-thirds of link protein. This presumably corresponds to the second globular domain reported in rotary shadowing studies of cartilage proteoglycans. We have deduced the positions of all of the disulfide bonds in the hyaluronic acid binding region and find them to be in the same positions as would be expected from comparison of these sequences with link protein.
We have obtained overlapping cDNA clones for the entire coding sequence of the rat cartilage proteoglycan core protein from the Swarm rat chondrosarcoma. These cDNAs hybridize to two sizes of RNA transcripts of 8.2 and 8.9 kilobase pairs, which contain large 3'-untranslated sequences. The total contiguous cDNA is 6.55 kilobase pairs in size, and codes for a 2124-residue protein, including a 19-residue signal peptide. The sequence forms a series of eight structural domains including two globules, (Mr = 37,000 and 22,000) at the NH2 terminus of the molecule, one a complete and one a partial copy of the cartilage link protein. The major feature of the deduced protein sequence is a 1,104-residue segment containing 117 Ser-Gly sequences, the presumed chondroitin sulfate attachment sites. These are arranged in three domains of 428, 503, and 173 amino acids. The first domain contains 11 complete or partial repeats of a 40-residue unit, and the second domain is composed of six copies of a 100-residue repeating sequence. The first pattern is the more highly conserved, and may have given rise to the second. The carboxyl-terminal domain is a third globule which has homology with animal lectins.
We have isolated and sequenced a cDNA clone of 872 base pairs from the 3' end of the mRNA for the large cartilage specific proteoglycan from rat. Identification was confirmed by a comparison with published protein sequence. Hybridization analysis shows the presence of an 8-9-kilobase mRNA for this proteoglycan in rat and chick sternal chondrocytes and rat chondrosarcoma cells, but not in RNA from rat fibroblasts, vitamin A-treated chick chondrocytes, chick crop, or bone. The carboxyl portion of the proteoglycan is deduced to terminate in a globular domain, which includes a region homologous to a chick hepatic lectin, and is possibly involved in binding to N-acetylglucosamine. The clone extends into a region where serines are clustered, probably the start of the chondroitin sulfate-rich region.