Different levels of glycosylation contribute to the heterogeneity of α1(II) collagen chains derived from a transplantable rat chondrosarcoma

Furuto, Donald K.; Miller, Edward J.

doi:10.1016/0003-9861(83)90329-6

Cited by 62 publications

(24 citation statements)

References 20 publications

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“…The ␣3(XI) chain is a posttranslational variant of the ␣1(II) chain (14), whereas the ␣1(XI) and ␣2(XI) chains are distinct gene products (5,22). Type XI collagen plays a critical role in regulating the formation of the collagen fibrils (30,38).…”

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confidence: 99%

A Kruüppel-Associated Box-Zinc Finger Protein, NT2, Represses Cell-Type-Specific Promoter Activity of the α2(XI) Collagen Gene

Tanaka

Tsumaki

Kozak

et al. 2002

Molecular and Cellular Biology

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Type XI collagen is composed of three chains, ␣1(XI), ␣2(XI), and ␣3(XI), and plays a critical role in the formation of cartilage collagen fibrils and in skeletal morphogenesis. It was previously reported that the ؊530-bp promoter segment of the ␣2(XI) collagen gene (Col11a2) was sufficient for cartilage-specific expression and that a 24-bp sequence from this segment was able to switch promoter activity from neural tissues to cartilage in transgenic mice when this sequence was placed in the heterologous neurofilament light gene (NFL) promoter. To identify a protein factor that bound to the 24-bp sequence of the Col11a2 promoter, we screened a mouse limb bud cDNA expression library in the yeast one-hybrid screening system and obtained the cDNA clone NT2. Sequence analysis revealed that NT2 is a zinc finger protein consisting of a Krüppel-associated box (KRAB) and is a homologue of human FPM315, which was previously isolated by random cloning and sequencing. The KRAB domain has been found in a number of zinc finger proteins and implicated as a transcriptional repression domain, although few target genes for KRAB-containing zinc finger proteins has been identified. Here, we demonstrate that NT2 functions as a negative regulator of Col11a2. In situ hybridization analysis of developing mouse cartilage showed that NT2 mRNA is highly expressed by hypertrophic chondrocytes but is minimally expressed by resting and proliferating chondrocytes, in an inverse correlation with the expression patterns of Col11a2. Gel shift assays showed that NT2 bound a specific sequence within the 24-bp site of the Col11a2 promoter. We found that Col11a2 promoter activity was inhibited by transfection of the NT2 expression vector in RSC cells, a chondrosarcoma cell line. The expression vector for mutant NT2 lacking the KRAB domain failed to inhibit Col11a2 promoter activity. These results demonstrate that KRABzinc finger protein NT2 inhibits transcription of its physiological target gene, suggesting a novel regulatory mechanism of cartilage-specific expression of Col11a2.Cartilage is a highly specialized tissue and serves as the template for the growth and development of most bones. Cartilage development is initiated by mesenchymal cell condensation, followed by a series of chondrocyte maturation processes including resting, proliferative, and hypertrophic stages. Cartilage contains an extensive extracellular matrix which includes type II, IX, and XI collagens and provides mechanical strength to resist compression in joints. Type II collagen, a major collagen in cartilage, forms collagen fibrils and provides a structural framework for cartilage matrix. Type XI collagen coassembles stoichiometrically with type II collagen in the fibrils, whereas the type IX collagen is associated with the surface of the fibrils (30,46).The type XI collagen molecule is composed of three subunits: ␣1(XI), ␣2(XI), and ␣3(XI) (32). The ␣3(XI) chain is a posttranslational variant of the ␣1(II) chain (14), whereas the ␣1(XI) and ␣2(XI) chains are distinct gene prod...

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A Kruüppel-Associated Box-Zinc Finger Protein, NT2, Represses Cell-Type-Specific Promoter Activity of the α2(XI) Collagen Gene

Tanaka

Tsumaki

Kozak

et al. 2002

Molecular and Cellular Biology

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“…The ␣3(XI) chain is an overglycosylated form of the ␣1(II) collagen chain (7), whereas the ␣1(XI) and ␣2(XI) chains are distinct gene products (8). The ␣1(XI) and ␣2(XI) chains contain similar large, N-terminal domains (NTDs), 1 comprising a "PARP" or "PARP-like" domain, a variable region, and a minor triple helix ( Fig.…”

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Structural Organization of Distinct Domains within the Non-collagenous N-terminal Region of Collagen Type XI

Gregory¹,

Oxford²,

Chen³

et al. 2000

Journal of Biological Chemistry

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Collagen XI is a heterotrimeric molecule found predominantly in heterotypic cartilage fibrils, where it is involved in the regulation of fibrillogenesis. This function is thought to involve the complex N-terminal domain. The goal of this current study was to examine its structural organization to further elucidate the regulatory mechanism. The amino-propeptide (␣1-Npp) alone or with isoforms of the variable region were recombinantly expressed and purified by affinity and molecular sieve chromatography. Cys-1-Cys-4 and Cys-2-Cys-3 disulfide bonds were detected by liquid chromatographytandem mass spectrometry. This pattern is identical to the homologous ␣2-Npp, indicating that the recombinant proteins were folded correctly. Anomalous elution on molecular sieve chromatography suggested that the variable region was extended, which was confirmed using rotary shadowing; the ␣1-Npp formed a globular "head" and the variable region an extended "tail." Circular dichroism spectra analysis determined that the ␣1-Npp comprised 33% ␤-sheet, whereas the variable region largely comprised non-periodic structure. Taken together, these results imply that the ␣1-Npp cannot be accommodated within the core of the fibril and that the variable region and/or minor helix facilitates its exclusion to the fibril surface. This provides further support for regulation of fibril diameter by steric hindrance or by interactions with other matrix components that affect fibrillogenesis.Collagen XI is a component of fibrils both in cartilage and a wide variety of non-cartilaginous tissues, including brain, muscle, tendon, heart valve, skin, calvaria, and endochondral bone (1, 2). In cartilage it assembles with collagens II and IX to produce an extensive network of thin, heterotypic collagen fibrils (15-25 nm in diameter; Ref. 1) in which the other extracellular matrix components are embedded. The major triple helix of collagen XI is not accessible to antibodies unless the fibrils are disrupted, which led to the model that it is located in the interior of the fibril (1, 3). In non-cartilaginous tissues, collagen XI has been found to co-assemble with chains of the highly homologous type V collagen (4, 5).Collagen XI is a heterotrimeric molecule consisting of ␣1, ␣2, and ␣3 collagen chains (6). The ␣3(XI) chain is an overglycosylated form of the ␣1(II) collagen chain (7), whereas the ␣1(XI) and ␣2(XI) chains are distinct gene products (8). The ␣1(XI) and ␣2(XI) chains contain similar large, N-terminal domains (NTDs), 1 comprising a "PARP" or "PARP-like" domain, a variable region, and a minor triple helix ( Fig. 1a; Refs. 9 and 10). The PARP (proline-arginine rich protein) domain was originally isolated as a distinct protein from bovine cartilage (11) but was later demonstrated to be a fragment of the N terminus of the ␣2(XI) chain (12). Recently, the proteolytic processing site of the ␣1(XI) chain has been identified (13,14), and since nearly all of the domain is removed by this processing event, both the PARP-like and PARP domains are now termed the ...

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“…Type XI collagen, consisting of ␣1(XI), ␣2(XI), and ␣3(XI) chains, co-assembles with type II collagen to form cartilage collagen fibrils, whereas type IX collagen is associated with the surface of these fibrils (1,2). The ␣1(XI) and ␣2(XI) chains are distinct gene products (3,4), whereas the ␣3(XI) chain is a post-translational variant of the ␣1(II) chain (5). It has been postulated that type IX and type XI collagens regulate the collagen network by determining the diameter of cartilage collagen fibrils and their interactions with other matrix components (6 -9).…”

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Identification of an Enhancer Sequence within the First Intron Required for Cartilage-specific Transcription of the α2(XI) Collagen Gene

Liu

Tanaka

et al. 2000

Journal of Biological Chemistry

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Type XI collagen, a heterotrimer composed of ␣1(XI), ␣2(XI) and ␣3(XI), is primarily synthesized by chondrocytes in cartilage and is also present in some other tissues. Type XI collagen plays a critical role in collagen fibril formation and skeletal morphogenesis. We investigated a tissue-specific transcriptional enhancer in the first intron of the ␣2(XI) collagen gene (Col11a2). Transient transfection assays using reporter gene constructs revealed that a 60-base pair (bp) segment within intron 1 increased promoter activity of Col11a2 in rat chondrosarcoma cells but not in either BalB/3T3 cells or undifferentiated ATDC5 cells, suggesting that it contained cell type-specific enhancer activity. In transgenic mice, this 60-bp fragment was also able to target ␤-galactosidase expression to cartilage including the limbs and axial skeleton, with similar localization specificity as the fulllength intron 1 fragment. Competition experiments in gel shift assays using mutated oligonucleotides showed that recombinant Sox9 bound to a 7-bp sequence, CT-CAAAG, within the 60-bp segment. Anti-Sox9 antibodies supershifted the complex of the 60-bp segment with recombinant Sox9 or with rat chondrosarcoma cell extracts, confirming the binding of Sox9 to the enhancer. Moreover, a site-specific mutation within the 7-bp segment resulted in essentially complete loss of the enhancer activity in chondrosarcoma cells and transgenic mice. These results suggest that the 7-bp sequence within intron 1 plays a critical role in the cartilagespecific enhancer activity of Col11a2 through Sox9-mediated transcriptional activation.Cartilage is a highly specialized tissue important for bearing compression loads in joints and also serves as the template for most developing bones. Cartilage contains unique extracellular matrix proteins produced by chondrocytes. The collagen network provides the scaffolding of the cartilage matrix and confers tensile strength important for resisting compression in cartilage. Collagen fibrils in cartilage consist of the major collagen, type II, and minor collagens, type IX and type XI. Type XI collagen, consisting of ␣1(XI), ␣2(XI), and ␣3(XI) chains, co-assembles with type II collagen to form cartilage collagen fibrils, whereas type IX collagen is associated with the surface of these fibrils (1, 2). The ␣1(XI) and ␣2(XI) chains are distinct gene products (3, 4), whereas the ␣3(XI) chain is a post-translational variant of the ␣1(II) chain (5). It has been postulated that type IX and type XI collagens regulate the collagen network by determining the diameter of cartilage collagen fibrils and their interactions with other matrix components (6 -9).Mutations in the type XI collagen genes have been identified and found to cause chondrodysplasia in both humans and mice. For example, mutations in the ␣2(XI) collagen gene (Col11a2) were identified in patients with Stickler syndrome and otospondylo-megaepiphyseal dysplasia (10). A null mutation in the ␣1(XI) gene of cho (chondrodysplasia) mice causes dwarfism with reduced matrix an...

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Different levels of glycosylation contribute to the heterogeneity of α1(II) collagen chains derived from a transplantable rat chondrosarcoma

Cited by 62 publications

References 20 publications

A Kruüppel-Associated Box-Zinc Finger Protein, NT2, Represses Cell-Type-Specific Promoter Activity of the α2(XI) Collagen Gene

A Kruüppel-Associated Box-Zinc Finger Protein, NT2, Represses Cell-Type-Specific Promoter Activity of the α2(XI) Collagen Gene

Structural Organization of Distinct Domains within the Non-collagenous N-terminal Region of Collagen Type XI

Identification of an Enhancer Sequence within the First Intron Required for Cartilage-specific Transcription of the α2(XI) Collagen Gene

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