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Mouse aggrecan, a large cartilage proteoglycan: protein sequence, gene structure and promoter sequence.

ABSTRACT: Seven genomic clones for mouse aggrecan core protein have been isolated including 3 kb of 5'- and 7 kb of 3'-flanking sequences. All exon sequences and their intron boundary sequences in these clones were identified and mapped by DNA sequencing. The gene spans at least 61 kb and contains 18 exons. Exon 1 encodes 5'-untranslated sequence and exon 2 contains a translation start codon, methionine. The coding sequence is 6545 bp for a 2132-amino-acid protein with calculated M(r) = 259,131 including an 18-amino-acid signal peptide. There is a strong correlation between structural domains and exons. Notably, the chondroitin sulphate domain consisting of 1161 amino acids is encoded by a single exon of 3.6 kb. Although link protein has similar structural domains and subdomains, the sequence identity and the organization of exons encoding the subdomains B and B' of G1 and G2 domains revealed a strong similarity of mouse aggrecan to both human versican and rat neurocan. Primer extension analysis identified four transcription start sites which are close together. The promoter sequence showed high G/C content (65%) and contained several consensus binding motifs for transcription factors including Sp-1 and the glucocorticoid receptor. There are stretches of sequences similar to the promoter region of both the type-II collagen and link protein genes. These sequences may be important for cartilage gene expression.

SUBMITTER: Watanabe H

PROVIDER: S-EPMC1136944 | biostudies-other | 1995 Jun

REPOSITORIES: biostudies-other

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Project description:BackgroundRheumatoid arthritis (RA) is an autoimmune disease of the synovial joints. The autoimmune character of RA is underscored by prominent production of autoantibodies such as those against IgG (rheumatoid factor), and a broad array of joint tissue-specific and other endogenous citrullinated proteins. Anti-citrullinated protein antibodies (ACPA) can be detected in the sera and synovial fluids of RA patients and ACPA seropositivity is one of the diagnostic criteria of RA. Studies have demonstrated that RA T cells respond to citrullinated peptides (epitopes) of proteoglycan (PG) aggrecan, which is one of the most abundant macromolecules of articular cartilage. However, it is not known if the PG molecule is citrullinated in vivo in human cartilage, and if so, whether citrulline-containing neoepitopes of PG (CitPG) can contribute to autoimmunity in RA.MethodsCitPG was detected in human cartilage extracts using ACPA+ RA sera in dot blot and Western blot. Citrullination status of in vitro citrullinated recombinant G1 domain of human PG (rhG1) was confirmed by antibody-based and chemical methods, and potential sites of citrullination in rhG1 were explored by molecular modeling. CitPG-specific serum autoantibodies were quantified by enzyme-linked immunosorbent assays, and CitPG was localized in osteoarthritic (OA) and RA cartilage using immunohistochemistry.FindingsSera from ACPA+ RA patients reacted with PG purified from normal human cartilage specimens. PG fragments (mainly those containing the G1 domain) from OA or RA cartilage extracts were recognized by ACPA+ sera but not by serum from ACPA- individuals. ACPA+ sera also reacted with in vitro citrullinated rhG1 and G3 domain-containing fragment(s) of PG. Molecular modeling suggested multiple sites of potential citrullination within the G1 domain. The immunohistochemical localization of CitPG was different in OA and RA cartilage.ConclusionsCitPG is a new member of citrullinated proteins identified in human joints. CitPG could be found in both normal and diseased cartilage specimens. Antibodies against CitPG may trigger or augment arthritis by forming immune complexes with this autoantigen in the joints of ACPA+ RA patients.

Project description:Although ADAMTS-4 and ADAMTS-5 are the principal aggrecanases in mice and humans, mice lacking both these enzymes (ADAMTS-4/-5Dcat) are healthy, viable, with no overt skeletal phenotype, suggesting the presence of an alternative aggrecanase in these mice. We previously identified a novel aggrecanase activity that was regulated by retinoic acid, but not IL-1a. The upregulated activity cleaved aggrecan at E↓A bonds. This study aimed to identify the alternative aggrecanase. Femoral head cartilage from TS-4/5Dcat mice was stimulated with IL-1a or retinoic acid and total RNA was analysed by microarray. Candidate, upregulated genes identified by Quantile Normalisation included ADAMTS, MMPm cathepsin and calpain families as putative aggrecanases. Three criteria, including quantitative RT-PCR analyses, sensitivity to retinoic acid treatment and sensitivity to TIMP-3 inhibition, were then used as readouts to identify candidate extracellular proteinases whose mRNA expression levels were increased by retinoic acid, but not by IL-1a treatment. Thereafter, gene silencing was used to identify the most likely aggrecanase in chondrocytes from TS-4/5Dcat mice. Aggrecanase activity was monitored by Western blotting and immunohistochemistry with neoepitope antibodies. The microarray analyses identified ADAMTS-9, MMP-11 and calpain-5 as candidate aggrecanases that were upregulated by retinoic acid. While qPCR confirmed this finding, calpain 5 was excluded from the candidate list because it was not inhibited by TIMP-3. MMP-11 was also excluded because it was upregulated by IL-1a. ADAMTS-9 therefore emerged as a candidate for the novel aggrecanase. Gene silencing confirmed ADAMTS-9 as the putative aggrecanase. Immunohistochemistry revealed that ADAMTS-9 in mouse knee joints was expressed in the proliferative zone of both wildtype and TS-4/5Dcat growth plates, and also in the calcified cartilage of the wildtype hypertrophic zone. In conclusion, ADAMTS-9 is an aggrecanase that cleaves at FREEE1467↓1468GLGS in the chondroitin sulphate-rich region of aggrecan.

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Mouse aggrecan, a large cartilage proteoglycan: protein sequence, gene structure and promoter sequence.

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