Nephrocan, a Novel Member of the Small Leucine-rich Repeat Protein Family, Is an Inhibitor of Transforming Growth Factor-β Signaling

Mochida, Yoshiyuki; Parisuthiman, Duenpim; Kaku, Masaru; Hanai, Jun; Sukhatme, Vikas P.; Yamauchi, Mitsuo

doi:10.1074/jbc.m604787200

Cited by 27 publications

(42 citation statements)

References 50 publications

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“…Super-motifs, S and T , similar to those in SLRP are also present in asporin-like proteins from human and mouse, mouse fibromodulin-like proteins, biglycan-like proteins from sea lamprey, oligodendrocyte-myelin and glycoprotein (OMGP), the FLRT family from human, mouse and Xenopus, and human ECM2 [8,62]. Furthermore, a preliminary analysis indicates that nephrocan, a novel member of the SLRP family [63], contains an STT motif. These observations suggest strongly that "bacterial" and "typical" LRRs evolved from a common precursor.…”

Section: Discussionmentioning

confidence: 99%

Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors

et al. 2007

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Abstract

Background

Toll-like receptors (TLRs) play a central role in innate immunity. TLRs are membrane glycoproteins and contain leucine rich repeat (LRR) motif in the ectodomain. TLRs recognize and respond to molecules such as lipopolysaccharide, peptidoglycan, flagellin, and RNA from bacteria or viruses. The LRR domains in TLRs have been inferred to be responsible for molecular recognition. All LRRs include the highly conserved segment, LxxLxLxxNxL, in which "L" is Leu, Ile, Val, or Phe and "N" is Asn, Thr, Ser, or Cys and "x" is any amino acid. There are seven classes of LRRs including "typical" ("T") and "bacterial" ("S"). All known domain structures adopt an arc or horseshoe shape. Vertebrate TLRs form six major families. The repeat numbers of LRRs and their "phasing" in TLRs differ with isoforms and species; they are aligned differently in various databases. We identified and aligned LRRs in TLRs by a new method described here.

Results

The new method utilizes known LRR structures to recognize and align new LRR motifs in TLRs and incorporates multiple sequence alignments and secondary structure predictions. TLRs from thirty-four vertebrate were analyzed. The repeat numbers of the LRRs ranges from 16 to 28. The LRRs found in TLRs frequently consists of LxxLxLxxNxLxxLxxxxF/LxxLxx ("T") and sometimes short motifs including LxxLxLxxNxLxxLPx(x)LPxx ("S"). The TLR7 family (TLR7, TLR8, and TLR9) contain 27 LRRs. The LRRs at the N-terminal part have a super-motif of STT with about 80 residues. The super-repeat is represented by STTSTTSTT or _TTSTTSTT. The LRRs in TLRs form one or two horseshoe domains and are mostly flanked by two cysteine clusters including two or four cysteine residue.

Conclusion

Each of the six major TLR families is characterized by their constituent LRR motifs, their repeat numbers, and their patterns of cysteine clusters. The central parts of the TLR1 and TLR7 families and of TLR4 have more irregular or longer LRR motifs. These central parts are inferred to play a key role in the structure and/or function of their TLRs. Furthermore, the super-repeat in the TLR7 family suggests strongly that "bacterial" and "typical" LRRs evolved from a common precursor.

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Section: Discussionmentioning

confidence: 99%

Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors

et al. 2007

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“…Interestingly, 5730521E12Rik is identical to nephrocan ( Nepn ), which was recently identified by Mochida et al . as an inhibitor of Transforming Growth Factor-β signaling [52]. However, whether 5730521E12Rik plays an inhibitory role in vivo during endoderm formation and patterning needs to be further investigated.…”

Section: Discussionmentioning

confidence: 99%

A systematic screen for genes expressed in definitive endoderm by Serial Analysis of Gene Expression (SAGE)

et al. 2007

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Background: The embryonic definitive endoderm (DE) gives rise to organs of the gastrointestinal and respiratory tract including the liver, pancreas and epithelia of the lung and colon. Understanding how DE progenitor cells generate these tissues is critical to understanding the cause of visceral organ disorders and cancers, and will ultimately lead to novel therapies including tissue and organ regeneration. However, investigation into the molecular mechanisms of DE differentiation has been hindered by the lack of early DE-specific markers.

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“…The coding sequence of LOX was subcloned from pcDNA3.1/LOX/V5-His by PCR and the primers were designed as follows : for LOX and LOXdm, forward primer, 5Ј-GCGGATCCATGCGTTTCGCCTGGGCTGTGCTC-3Ј, and reverse primer, 5Ј-GCCTCGAGATACGGTGAAATTGTG-CAGCCTGAGGC-3Ј; for mLOX, the same primers used for the full-length LOX and the additional primers forward primer, 5Ј-CTTCTCCGCTGCGACGACCCCTACAATCCCTAC-3Ј, and reverse primer, 5Ј-GTAGGGATTGTAGGGGTCGTCG-CAGCGGAGAAG-3Ј;, and for LOXPP, forward primer, 5Ј-GCG-GATCCATGCGTTTCGCCTGGGCTGTGCTC-3Ј, and reverse primer, 5Ј-GCCTCGAGGCCCACCATGCGATCTATGTG-GCT-3Ј. The PCR products were digested with BamHI and XhoI, ligated into pcDNA3/HA mammalian expression vector (30), and sequenced at the University of North Carolina, Chapel Hill, DNA sequencing facility.…”

Section: Methodsmentioning

confidence: 99%

Lysyl Oxidase Binds Transforming Growth Factor-; and Regulates Its Signaling via Amine Oxidase Activity

Atsawasuwan

Mochida

Katafuchi

et al. 2008

Journal of Biological Chemistry

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121

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Lysyl oxidase (LOX), an amine oxidase critical for the initiation of collagen and elastin cross-linking, has recently been shown to regulate cellular activities possibly by modulating the functions of growth factors. In this study, we investigated the interaction between LOX and transforming growth factor-␤1 (TGF-␤1), a potent growth factor abundant in bone, the effect of LOX on TGF-␤1 signaling, and its potential mechanism. The specific binding between mature LOX and mature TGF-␤1 was demonstrated by immunoprecipitation and glutathione S-transferase pulldown assay in vitro. Both proteins were colocalized in the extracellular matrix in an osteoblastic cell culture system, and the binding complex was identified in the mineral-associated fraction of bone matrix. Furthermore, LOX suppressed TGF-␤1-induced Smad3 phosphorylation likely through its amine oxidase activity. The data indicate that LOX binds to mature TGF-␤1 and enzymatically regulates its signaling in bone and thus may play an important role in bone maintenance and remodeling. Lysyl oxidase (LOX)2 is a copper-dependent amine oxidase that initiates the process of covalent intra-and intermolecular cross-linking in collagen and elastin (1). The critical role of LOX in tissue stability is well exemplified by "lathyrism," the condition where deleterious effects in connective tissues are caused by lathyrogens such as ␤-aminopropionitrile (BAPN) (2). In lathyritic animals, bone is one of the most severely affected tissues revealing kyphoscoliosis, bone deformities, weakening of tendons and ligament attachments, dislocation of joints, impaired bone fracture healing, and ectopic bone exostoses (3, 4). BAPN is a potent and irreversible inhibitor of LOX catalytic activity and thus prevents cross-linking of immature collagen and elastin into mature, stable, and insoluble fibers. Therefore, it has been thought that the phenotypes seen in lathyritic animals are due primarily to the lack of collagen/elastin cross-linking.Recent reports, however, have revealed novel functions for LOX, including the regulation of gene transcription and cellular functions. Although the mechanisms are still not clear, those functions could be associated with its ability to oxidize substrates, other than collagen and elastin, such as basic fibroblast growth factor (5) as well as histone H1 and H2 (6, 7). Thus, lathyritic phenotypes could be due in part to the loss of LOX control of cellular functions. Indeed, several studies have reported that collagen synthesis/expression significantly increased when osteoblasts or chondrocytes were cultured in the presence of BAPN (8 -11), which is suggestive of such functions.In bone, there are several major growth factors, including transforming growth factor-␤ (TGF-␤), bone morphogenetic proteins (BMPs), insulin-like growth factor and platelet-derived growth factor, tumor necrosis factor-␣, and basic fibroblast growth factor. Among those factors, TGF-␤s and BMPs are a group of growth factors that are basic in nature (theoretical pI Ͼ 8.5) and up-regula...

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Nephrocan, a Novel Member of the Small Leucine-rich Repeat Protein Family, Is an Inhibitor of Transforming Growth Factor-β Signaling

Cited by 27 publications

References 50 publications

Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors

Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors

A systematic screen for genes expressed in definitive endoderm by Serial Analysis of Gene Expression (SAGE)

Lysyl Oxidase Binds Transforming Growth Factor-; and Regulates Its Signaling via Amine Oxidase Activity

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