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Mitsuo Yamauchi - One of the best experts on this subject based on the ideXlab platform.
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Lysyl Hydroxylase 2-induced collagen cross-link switching promotes metastasis in head and neck squamous cell carcinomas
'Elsevier BV', 2021Co-Authors: Kotaro Sato, Masahiko Terajima, Mitsuo Yamauchi, Kshitij Parag-sharma, Adele M. Musicant, Ryan M. Murphy, Matthew R. Ramsey, Hideharu Hibi, Antonio L. AmelioAbstract:Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide and incidence rates are continuing to rise globally. HNSCC patient prognosis is closely related to the occurrence of tumor metastases, and collagen within the tumor microenvironment (TME) plays a key role in this process. Lysyl Hydroxylase 2 (LH2), encoded by the Procollagen-Lysine,2-Oxoglutarate 5-Dioxygenase 2 (PLOD2) gene, catalyzes hydroxylation of telopeptidyl lysine (Lys) residues of fibrillar collagens which then undergo subsequent modifications to form stable intermolecular cross-links that change the biomechanical properties (i.e. quality) of the TME. While LH2-catalyzed collagen modification has been implicated in driving tumor progression and metastasis in diverse cancers, little is known about its role in HNSCC progression. Thus, using gain- and loss-of-function studies, we examined the effects of LH2 expression levels on collagen cross-linking and cell behavior in vitro and in vivo using a tractable bioluminescent imaging-based orthotopic xenograft model. We found that LH2 overexpression dramatically increases HNSCC cell migratory and invasive abilities in vitro and that LH2-driven changes in collagen cross-linking robustly induces metastasis in vivo. Specifically, the amount of LH2-mediated collagen cross-links increased significantly with PLOD2 overexpression, without affecting the total quantity of collagen cross-links. Conversely, LH2 knockdown significantly blunted HNSCC cells invasive capacity in vitro and metastatic potential in vivo. Thus, regardless of the total “quantity” of collagen crosslinks, it is the “quality” of these cross-links that is the key driver of HNSCC tumor metastatic dissemination. These data implicate LH2 as a key regulator of HNSCC tumor invasion and metastasis by modulating collagen cross-link quality and suggest that therapeutic strategies targeting LH2-mediated collagen cross-linking in the TME may be effective in controlling tumor progression and improving disease outcomes
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FKBP65-dependent peptidyl-prolyl isomerase activity potentiates the Lysyl Hydroxylase 2-driven collagen cross-link switch
Scientific Reports, 2017Co-Authors: Yulong Chen, Masahiko Terajima, Priyam Banerjee, Mitsuo Yamauchi, Jiang Yu, Jonathan M. KurieAbstract:Bruck Syndrome is a connective tissue disease associated with inactivating mutations in Lysyl Hydroxylase 2 (LH2/PLOD2) or FK506 binding protein 65 (FKBP65/FKBP10). However, the functional relationship between LH2 and FKBP65 remains unclear. Here, we postulated that peptidyl prolyl isomerase (PPIase) activity of FKBP65 positively modulates LH2 enzymatic activity and is critical for the formation of hydroxylysine-aldehyde derived intermolecular collagen cross-links (HLCCs). To test this hypothesis, we analyzed collagen cross-links in Fkbp10-null and –wild-type murine embryonic fibroblasts. Although LH2 protein levels did not change, FKBP65 deficiency significantly diminished HLCCs and increased the non-hydroxylated lysine-aldehyde–derived collagen cross-links (LCCs), a pattern consistent with loss of LH2 enzymatic activity. The HLCC-to-LCC ratio was rescued in FKBP65-deficient murine embryonic fibroblasts by reconstitution with wild-type but not mutant FKBP65 that lacks intact PPIase domains. Findings from co-immunoprecipitation, protein-fragment complementation, and co-immunofluorescence assays showed that LH2 and FKBP65 are part of a common protein complex. We conclude that FKBP65 regulates LH2-mediated collagen cross-linking. Because LH2 promotes fibrosis and cancer metastasis, our findings suggest that pharmacologic strategies to target FKBP65 and LH2 may have complementary therapeutic activities.
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a scalable Lysyl Hydroxylase 2 expression system and luciferase based enzymatic activity assay
Archives of Biochemistry and Biophysics, 2017Co-Authors: Ashwini K Devkota, Mitsuo Yamauchi, Yulong Chen, William K Russell, G N Phillips, Kevin N Dalby, Jonathan M. KurieAbstract:Abstract Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by Lysyl Hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated using E coli– or insect-based expression systems is either insoluble or enzymatically unstable, and the LH2 enzymatic activity assays that are currently available measure radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems.
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Lysyl Hydroxylase 3 mediated glucosylation in type i collagen molecular loci and biological significance
Journal of Biological Chemistry, 2012Co-Authors: Marnisa Sricholpech, Masahiko Terajima, Irina Perdivara, Hideaki Nagaoka, Megumi Yokoyama, Kenneth B Tomer, Mitsuo YamauchiAbstract:Recently, by employing the short hairpin RNA technology, we have generated MC3T3-E1 (MC)-derived clones stably suppressing Lysyl Hydroxylase 3 (LH3) (short hairpin (Sh) clones) and demonstrated the LH3 function as glucosyltransferase in type I collagen (Sricholpech, M., Perdivara, I., Nagaoka, H., Yokoyama, M., Tomer, K. B., and Yamauchi, M. (2011) Lysyl Hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture. J. Biol. Chem. 286, 8846–8856). To further elucidate the biological significance of this modification, we characterized and compared type I collagen phenotypes produced by Sh clones and two control groups, MC and those transfected with empty vector. Mass spectrometric analysis identified five glycosylation sites in type I collagen (i.e. α1,2-87, α1,2-174, and α2-219. Of these, the predominant glycosylation site was α1-87, one of the major helical cross-linking sites. In Sh collagen, the abundance of glucosylgalactosylhydroxylysine was significantly decreased at all of the five sites with a concomitant increase in galactosylhydroxylysine at four of these sites. The collagen cross-links were significantly diminished in Sh clones, and, for the major cross-link, dihydroxylysinonorleucine (DHLNL), glucosylgalactosyl-DHLNL was diminished with a concomitant increase in galactosyl-DHLNL. When subjected to in vitro incubation, in Sh clones, the rate of decrease in DHLNL was lower, whereas the rate of increase in its maturational cross-link, pyridinoline, was comparable with controls. Furthermore, in Sh clones, the mean diameters of collagen fibrils were significantly larger, and the onset of mineralized nodule formation was delayed when compared with those of controls. These results indicate that the LH3-mediated glucosylation occurs at the specific molecular loci in the type I collagen molecule and plays critical roles in controlling collagen cross-linking, fibrillogenesis, and mineralization.
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Lysyl Hydroxylase 3 mediated glucosylation in type i collagen molecular loci and biological significance
Journal of Biological Chemistry, 2012Co-Authors: Marnisa Sricholpech, Masahiko Terajima, Irina Perdivara, Hideaki Nagaoka, Megumi Yokoyama, Kenneth B Tomer, Mitsuo YamauchiAbstract:Recently, by employing the short hairpin RNA technology, we have generated MC3T3-E1 (MC)-derived clones stably suppressing Lysyl Hydroxylase 3 (LH3) (short hairpin (Sh) clones) and demonstrated the LH3 function as glucosyltransferase in type I collagen (Sricholpech, M., Perdivara, I., Nagaoka, H., Yokoyama, M., Tomer, K. B., and Yamauchi, M. (2011) Lysyl Hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture. J. Biol. Chem. 286, 8846–8856). To further elucidate the biological significance of this modification, we characterized and compared type I collagen phenotypes produced by Sh clones and two control groups, MC and those transfected with empty vector. Mass spectrometric analysis identified five glycosylation sites in type I collagen (i.e. α1,2-87, α1,2-174, and α2-219. Of these, the predominant glycosylation site was α1-87, one of the major helical cross-linking sites. In Sh collagen, the abundance of glucosylgalactosylhydroxylysine was significantly decreased at all of the five sites with a concomitant increase in galactosylhydroxylysine at four of these sites. The collagen cross-links were significantly diminished in Sh clones, and, for the major cross-link, dihydroxylysinonorleucine (DHLNL), glucosylgalactosyl-DHLNL was diminished with a concomitant increase in galactosyl-DHLNL. When subjected to in vitro incubation, in Sh clones, the rate of decrease in DHLNL was lower, whereas the rate of increase in its maturational cross-link, pyridinoline, was comparable with controls. Furthermore, in Sh clones, the mean diameters of collagen fibrils were significantly larger, and the onset of mineralized nodule formation was delayed when compared with those of controls. These results indicate that the LH3-mediated glucosylation occurs at the specific molecular loci in the type I collagen molecule and plays critical roles in controlling collagen cross-linking, fibrillogenesis, and mineralization.
Raili Myllyla - One of the best experts on this subject based on the ideXlab platform.
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Lysyl Hydroxylase 3 Modifies Lysine Residues to Facilitate Oligomerization of Mannan-Binding Lectin
2014Co-Authors: Maija Risteli, Heli Ruotsalainen, Ulrich Bergmann, Umakhanth Venkatraman Girija, Russell Wallis, Raili MyllylaAbstract:Lysyl Hydroxylase 3 (LH3) is a multifunctional protein with Lysyl Hydroxylase, galactosyltransferase and glucosyltransferase activities. The LH3 has been shown to modify the lysine residues both in collagens and also in some collagenous proteins. In this study we show for the first time that LH3 is essential for catalyzing formation of the glucosylgalactosylhydroxylysines of mannan-binding lectin (MBL), the first component of the lectin pathway of complement activation. Furthermore, loss of the terminal glucose units on the derivatized lysine residues in mouse embryonic fibroblasts lacking the LH3 protein leads to defective disulphide bonding and oligomerization of rat MBL-A, with a decrease in the proportion of the larger functional MBL oligomers. The oligomerization could be completely restored with the full length LH3 or the amino-terminal fragment of LH3 that possesses the glycosyltransferase activities. Our results confirm that LH3 is the only enzyme capable of glucosylating the galactosylhydroxylysine residues in proteins with a collagenous domain. In mice lacking the Lysyl Hydroxylase activity of LH3, but with untouched galactosyltransferase and glucosyltransferase activities, reduced circulating MBL-A levels were observed. Oligomerization was normal, however and residual Lysyl hydroxylation was compensated in part by other Lysyl Hydroxylase isoenzymes. Our data suggest that LH3 is commonly involved in biosynthesis of collagenous proteins and the glucosylation of galactosylhydroxylysines residues by LH3 is crucial for the formation of the functional high-molecular weight MBL oligomers.
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The Activities of Lysyl Hydroxylase 3 (LH3) Regulate the Amount and Oligomerization Status of Adiponectin
2012Co-Authors: Heli Ruotsalainen, Chunguang Wang, Maija Risteli, Ulrich Bergmann, Yu Wang, Ari-pekka Kvist, Helmut Pospiech, Karl-heinz Herzig, Marjo Karppinen, Raili MyllylaAbstract:Lysyl Hydroxylase 3 (LH3) has Lysyl Hydroxylase, galactosyltransferase, and glucosyltransferase activities, which are sequentially required for the formation of glucosylgalactosyl hydroxylysines in collagens. Here we demonstrate for the first time that LH3 also modifies the lysine residues in the collagenous domain of adiponectin, which has important roles in glucose and lipid metabolism and inflammation. Hydroxylation and, especially, glycosylation of the lysine residues of adiponectin have been shown to be essential for the formation of the more active high molecular weight adiponectin oligomers and thus for its function. In cells that totally lack LH3 enzyme, the galactosylhydroxylysine residues of adiponectin were not glucosylated to glucosylgalactosylhydroxylysine residues and the formation of high and middle molecular weight adiponectin oligomers was impaired. Circulating adiponectin levels in mutant mice lacking the Lysyl Hydroxylase activity of LH3 were significantly reduced, which indicates that LH3 is required for complete modification of lysine residues in adiponectin and the loss of some of the glycosylated hydroxylysine residues severely affects the secretion of adiponectin. LH mutant mice with reduced adiponectin level showed a high fat diet-induced increase in glucose, triglyceride, and LDL-cholesterol levels, hallmarks of the metabolic syndrome in humans. Our results reveal the first indication that LH3 is an important regulator of adiponectin biosynthesis, secretion and activity and thus might be a potential candidate for therapeutic applications in diseases associated with obesity and insulin resistance.
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Oligomer distribution of recombinant adiponectin is altered in LH3−/− knockout MEF cells.
2012Co-Authors: Heli Ruotsalainen, Chunguang Wang, Maija Risteli, Ulrich Bergmann, Yu Wang, Ari-pekka Kvist, Helmut Pospiech, Karl-heinz Herzig, Marjo Karppinen, Raili MyllylaAbstract:The oligomeric forms of recombinant adiponectin were separated with gel filtration chromatography and quantified by ELISA. Comparison of typical gel filtration elution profiles of recombinant adiponectin produced in (A) wild type and (B) LH3−/− knockout MEFs indicates that HMW and MMW oligomers are not secreted from knockout cells. (C) Double transfection with adiponectin and LH3 constructs normalized the elution profile in LH3−/− knockout MEFs. (D) Recombinant adiponectin produced in LH mutant MEFs formed similar oligomers as the wild type. The adiponectin oligomers corresponding to the peaks seen with gel filtration chromatography were also detected on immunoblot (E) separated under non-reducing and non-heat-denaturing conditions. Equal volumes of concentrated cell culture media were used in all analysis. Representative elution profiles are shown. Abbreviations: WT = wild type; KO = LH3−/− knockout; LH3 = Lysyl Hydroxylase isoform 3; MUT = LH mutant.
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the third activity for Lysyl Hydroxylase 3 galactosylation of hydroxyLysyl residues in collagens in vitro
Matrix Biology, 2002Co-Authors: Chunguang Wang, Jari Heikkinen, Hanne Luosujarvi, Maija Risteli, Lahja Uitto, Raili MyllylaAbstract:Lysyl Hydroxylase (LH, EC 1.14.11.4), galactosyltransferase (EC 2.4.1.50) and glucosyltransferase (EC 2.4.1.66) are enzymes involved in posttranslational modifications of collagens. They sequentially modify Lysyl residues in specific positions to hydroxyLysyl, galactosylhydroxyLysyl and glucosylgalactosyl hydroxyLysyl residues. These structures are unique to collagens and essential for their functional activity. Lysines and hydroxylysines form collagen cross-links. Hydroxylysine derived cross-links, usually as glycosylated forms, occur especially in weight-bearing and mineralized tissues. The detailed functions of the hydroxyLysyl and hydroxyLysyl linked carbohydrate structures are not known, however. Hydroxylysine linked carbohydrates are found mainly in collagens, but recent reports indicate that these structures are also present and probably have an important function in other proteins. Earlier we have shown that human LH3, but not isoforms LH1, LH2a and LH2b, possesses both LH and glucosyltransferase activity (J. Biol. Chem. 275 (2000) 36158). In this paper we demonstrate that galactosyltransferase activity is also associated with the same gene product, thus indicating that one gene product can catalyze all three consecutive steps in hydroxylysine linked carbohydrate formation. In vitro mutagenesis experiments indicate that Cys and aspartates in 144 positions 187-191 of LH3 are important for the galactosyltransferase activity. Our results suggest that manipulation of the gene for LH3 can be used to selectively alter the glycosylation and hydroxylation reactions, and provides a new tool to clarify the functions of the unique hydroxylysine linked carbohydrates in collagens and other proteins. 2002 Elsevier Science B.V. and International Society of Matrix Biology. All rights reserved.
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primary structure tissue distribution and chromosomal localization of a novel isoform of Lysyl Hydroxylase Lysyl Hydroxylase 3
Journal of Biological Chemistry, 1998Co-Authors: Minna Valtavaara, Claude Szpirer, Josiane Szpirer, Raili MyllylaAbstract:We report characterization of a novel isoform of Lysyl Hydroxylase (Lysyl Hydroxylase 3, LH3). The cDNA clones encode a polypeptide of 738 amino acids, including a signal peptide. The amino acid sequence has a high overall identity with LH1 and LH2, the isoforms characterized earlier. Conserved regions are present in the carboxyl-terminal portion of the isoforms and also in the central part of the molecules. Histidine and asparagine residues, which are conserved in the other isoforms and are known to be required for enzymatic activity, are also conserved in the novel isoform. The gene for LH3 (PLOD3) has been assigned to human chromosome 7q36 and rat chromosome 12. Gene expression of LH3 is highly regulated in adult human tissues. A strong hybridization signal, corresponding to an mRNA 2.75 kilobases in size, is obtained in heart, placenta and pancreas on multiple tissue RNA blots. Expression of the cDNA in vitro results in the synthesis of a protein that hydroxylates Lysyl residues in collagenous sequences in a non-triple helical conformation.
Marnisa Sricholpech - One of the best experts on this subject based on the ideXlab platform.
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Lysyl Hydroxylase 3 mediated glucosylation in type i collagen molecular loci and biological significance
Journal of Biological Chemistry, 2012Co-Authors: Marnisa Sricholpech, Masahiko Terajima, Irina Perdivara, Hideaki Nagaoka, Megumi Yokoyama, Kenneth B Tomer, Mitsuo YamauchiAbstract:Recently, by employing the short hairpin RNA technology, we have generated MC3T3-E1 (MC)-derived clones stably suppressing Lysyl Hydroxylase 3 (LH3) (short hairpin (Sh) clones) and demonstrated the LH3 function as glucosyltransferase in type I collagen (Sricholpech, M., Perdivara, I., Nagaoka, H., Yokoyama, M., Tomer, K. B., and Yamauchi, M. (2011) Lysyl Hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture. J. Biol. Chem. 286, 8846–8856). To further elucidate the biological significance of this modification, we characterized and compared type I collagen phenotypes produced by Sh clones and two control groups, MC and those transfected with empty vector. Mass spectrometric analysis identified five glycosylation sites in type I collagen (i.e. α1,2-87, α1,2-174, and α2-219. Of these, the predominant glycosylation site was α1-87, one of the major helical cross-linking sites. In Sh collagen, the abundance of glucosylgalactosylhydroxylysine was significantly decreased at all of the five sites with a concomitant increase in galactosylhydroxylysine at four of these sites. The collagen cross-links were significantly diminished in Sh clones, and, for the major cross-link, dihydroxylysinonorleucine (DHLNL), glucosylgalactosyl-DHLNL was diminished with a concomitant increase in galactosyl-DHLNL. When subjected to in vitro incubation, in Sh clones, the rate of decrease in DHLNL was lower, whereas the rate of increase in its maturational cross-link, pyridinoline, was comparable with controls. Furthermore, in Sh clones, the mean diameters of collagen fibrils were significantly larger, and the onset of mineralized nodule formation was delayed when compared with those of controls. These results indicate that the LH3-mediated glucosylation occurs at the specific molecular loci in the type I collagen molecule and plays critical roles in controlling collagen cross-linking, fibrillogenesis, and mineralization.
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Lysyl Hydroxylase 3 mediated glucosylation in type i collagen molecular loci and biological significance
Journal of Biological Chemistry, 2012Co-Authors: Marnisa Sricholpech, Masahiko Terajima, Irina Perdivara, Hideaki Nagaoka, Megumi Yokoyama, Kenneth B Tomer, Mitsuo YamauchiAbstract:Recently, by employing the short hairpin RNA technology, we have generated MC3T3-E1 (MC)-derived clones stably suppressing Lysyl Hydroxylase 3 (LH3) (short hairpin (Sh) clones) and demonstrated the LH3 function as glucosyltransferase in type I collagen (Sricholpech, M., Perdivara, I., Nagaoka, H., Yokoyama, M., Tomer, K. B., and Yamauchi, M. (2011) Lysyl Hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture. J. Biol. Chem. 286, 8846–8856). To further elucidate the biological significance of this modification, we characterized and compared type I collagen phenotypes produced by Sh clones and two control groups, MC and those transfected with empty vector. Mass spectrometric analysis identified five glycosylation sites in type I collagen (i.e. α1,2-87, α1,2-174, and α2-219. Of these, the predominant glycosylation site was α1-87, one of the major helical cross-linking sites. In Sh collagen, the abundance of glucosylgalactosylhydroxylysine was significantly decreased at all of the five sites with a concomitant increase in galactosylhydroxylysine at four of these sites. The collagen cross-links were significantly diminished in Sh clones, and, for the major cross-link, dihydroxylysinonorleucine (DHLNL), glucosylgalactosyl-DHLNL was diminished with a concomitant increase in galactosyl-DHLNL. When subjected to in vitro incubation, in Sh clones, the rate of decrease in DHLNL was lower, whereas the rate of increase in its maturational cross-link, pyridinoline, was comparable with controls. Furthermore, in Sh clones, the mean diameters of collagen fibrils were significantly larger, and the onset of mineralized nodule formation was delayed when compared with those of controls. These results indicate that the LH3-mediated glucosylation occurs at the specific molecular loci in the type I collagen molecule and plays critical roles in controlling collagen cross-linking, fibrillogenesis, and mineralization.
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Lysyl Hydroxylase 3 glucosylates galactosylhydroxylysine residues in type i collagen in osteoblast culture
Journal of Biological Chemistry, 2011Co-Authors: Marnisa Sricholpech, Irina Perdivara, Hideaki Nagaoka, Megumi Yokoyama, Kenneth B Tomer, Mitsuo YamauchiAbstract:Lysyl Hydroxylase 3 (LH3), encoded by Plod3, is the multifunctional collagen-modifying enzyme possessing LH, hydroxylysine galactosyltransferase (GT), and galactosylhydroxylysine-glucosyltransferase (GGT) activities. Although an alteration in type I collagen glycosylation has been implicated in several osteogenic disorders, the role of LH3 in bone physiology has never been investigated. To elucidate the function of LH3 in bone type I collagen modifications, we used a short hairpin RNA technology in a mouse osteoblastic cell line, MC3T3-E1; generated single cell-derived clones stably suppressing LH3 (short hairpin (Sh) clones); and characterized the phenotype. Plod3 expression and the LH3 protein levels in the Sh clones were significantly suppressed when compared with the controls, MC3T3-E1, and the clone transfected with an empty vector. In comparison with controls, type I collagen synthesized by Sh clones (Sh collagen) showed a significant decrease in the extent of glucosylgalactosylhydroxylysine with a concomitant increase of galactosylhydroxylysine, whereas the total number of hydroxylysine residues was essentially unchanged. In an in vitro fibrillogenesis assay, Sh collagen showed accelerated fibrillogenesis compared with the controls. In addition, when recombinant LH3-V5/His protein was generated in 293 cells and subjected to GGT/GT activity assay, it showed GGT but not GT activity against denatured type I collagen. The results from this study clearly indicate that the major function of LH3 in osteoblasts is to glucosylate galactosylhydroxylysine residues in type I collagen and that an impairment of this LH3 function significantly affects type I collagen fibrillogenesis.
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Lysyl Hydroxylase 3 glucosylates galactosylhydroxylysine residues in type i collagen in osteoblast culture
Journal of Biological Chemistry, 2011Co-Authors: Marnisa Sricholpech, Irina Perdivara, Hideaki Nagaoka, Megumi Yokoyama, Kenneth B Tomer, Mitsuo YamauchiAbstract:Lysyl Hydroxylase 3 (LH3), encoded by Plod3, is the multifunctional collagen-modifying enzyme possessing LH, hydroxylysine galactosyltransferase (GT), and galactosylhydroxylysine-glucosyltransferase (GGT) activities. Although an alteration in type I collagen glycosylation has been implicated in several osteogenic disorders, the role of LH3 in bone physiology has never been investigated. To elucidate the function of LH3 in bone type I collagen modifications, we used a short hairpin RNA technology in a mouse osteoblastic cell line, MC3T3-E1; generated single cell-derived clones stably suppressing LH3 (short hairpin (Sh) clones); and characterized the phenotype. Plod3 expression and the LH3 protein levels in the Sh clones were significantly suppressed when compared with the controls, MC3T3-E1, and the clone transfected with an empty vector. In comparison with controls, type I collagen synthesized by Sh clones (Sh collagen) showed a significant decrease in the extent of glucosylgalactosylhydroxylysine with a concomitant increase of galactosylhydroxylysine, whereas the total number of hydroxylysine residues was essentially unchanged. In an in vitro fibrillogenesis assay, Sh collagen showed accelerated fibrillogenesis compared with the controls. In addition, when recombinant LH3-V5/His protein was generated in 293 cells and subjected to GGT/GT activity assay, it showed GGT but not GT activity against denatured type I collagen. The results from this study clearly indicate that the major function of LH3 in osteoblasts is to glucosylate galactosylhydroxylysine residues in type I collagen and that an impairment of this LH3 function significantly affects type I collagen fibrillogenesis.
Ruud A Bank - One of the best experts on this subject based on the ideXlab platform.
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disentangling mechanisms involved in collagen pyridinoline cross linking the immunophilin fkbp65 is critical for dimerization of Lysyl Hydroxylase 2
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Rutger A F Gjaltema, Miesje M Van Der Stoel, Miriam Boersema, Ruud A BankAbstract:Collagens are subjected to extensive posttranslational modifications, such as lysine hydroxylation. Bruck syndrome (BS) is a connective tissue disorder characterized at the molecular level by a loss of telopeptide lysine hydroxylation, resulting in reduced collagen pyridinoline cross-linking. BS results from mutations in the genes coding for Lysyl Hydroxylase (LH) 2 or peptidyl-prolyl cis-trans isomerase (PPIase) FKBP65. Given that the immunophilin FKBP65 does not exhibit LH activity, it is likely that LH2 activity is somehow dependent on FKPB65. In this report, we provide insights regarding the interplay between LH2 and FKBP65. We found that FKBP65 forms complexes with LH2 splice variants LH2A and LH2B but not with LH1 and LH3. Ablating the catalytic activity of FKBP65 or LH2 did not affect complex formation. Both depletion of FKBP65 and inhibition of FKBP65 PPIase activity reduced the dimeric (active) form of LH2 but did not affect the binding of monomeric (inactive) LH2 to procollagen Iα1. Furthermore, we show that LH2A and LH2B cannot form heterodimers with each other but are able to form heterodimers with LH1 and LH3. Collectively, our results indicate that FKBP65 is linked to pyridinoline cross-linking by specifically mediating the dimerization of LH2. Moreover, FKBP65 does not interact with LH1 and LH3, explaining why in BS triple-helical hydroxylysines are not affected. Our results provide a mechanistic link between FKBP65 and the loss of pyridinolines and may hold the key to future treatments for diseases related to collagen cross-linking anomalies, such as fibrosis and cancer.
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procollagen Lysyl Hydroxylase 2 expression is regulated by an alternative downstream transforming growth factor β 1 activation mechanism
Journal of Biological Chemistry, 2015Co-Authors: Rutger A F Gjaltema, Saskia De Rond, Marianne G Rots, Ruud A BankAbstract:PLOD2 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2) hydroxylates lysine residues in collagen telopeptides and is essential for collagen pyridinoline cross-link formation. PLOD2 expression and subsequent pyridinoline cross-links are increased in fibrotic pathologies by transforming growth factor beta-1 (TGF beta 1). In this report we examined the molecular processes underlying TGF beta 1-induced PLOD2 expression. We found that binding of the TGF beta 1 pathway related transcription factors SMAD:3 and SP1-mediated TGF beta 1 enhanced PLOD2 expression and could be correlated to an increase of acetylated histone H3 and H4 at the PLOD2 promoter. Interestingly, the classical co-activators of SMAD3 complexes, p300 and CBP, were not responsible for the enhanced H3 and H4 acetylation. Depletion of SMAD3 reduced PLOD2 acetylated H3 and H4, indicating that another as of yet unidentified histone acetyltransferase binds to SMAD 3 at PLOD2. Assessing histone methylation marks at the PLOD2 promoter depicted an increase of the active histone mark H3K79me2, a decrease of the repressive H4K2Ome3 mark, but no role for the generally strong transcription-related modifications: H3K4me3, H3K9me3 and H3K27me3. Collectively, our findings reveal that TGF beta 1 induces a SP1- and SMAD3-dependent recruitment of histone modifying enzymes to the PLOD2 promoter other than the currently known TGF beta 1 downstream co-activators and epigenetic modifications. This also suggests that additional activation strategies are used downstream of the TGF beta 1 pathway, and hence their unraveling could be of great importance to fully understand TGF beta 1 activation of genes.
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TGF-ß induces Lysyl Hydroxylase 2b in human synovial osteoarthritic fibroblasts through ALK5 signaling
Cell and tissue research, 2013Co-Authors: Dennis Florian George Remst, Ruud A Bank, Esmeralda N. Blaney Davidson, Elly L. Vitters, Wim B. Van Den Berg, Peter M. Van Der KraanAbstract:Lysyl Hydroxylase 2b (LH2b) is known to increase pyridinoline cross-links, making collagen less susceptible to enzymatic degradation. Previously, we observed a relationship between LH2b and osteoarthritis-related fibrosis in murine knee joint. For this study, we investigate if transforming growth factor-beta (TGF-s) and connective tissue growth factor (CTGF) regulate procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (gene encoding LH2b) and LH2b expression differently in osteoarthritic human synovial fibroblasts (hSF). Furthermore, we investigate via which TGF-s route (Smad2/3P or Smad1/5/8P) LH2b is regulated, to explore options to inhibit LH2b during fibrosis. To answer these questions, fibroblasts were isolated from knee joints of osteoarthritis patients. The hSF were stimulated with TGF-s with or without a kinase inhibitor of ALK4/5/7 (SB-505124) or ALK1/2/3/6 (dorsomorphin). TGF-s, CTGF, constitutively active (ca)ALK1 and caALK5 were adenovirally overexpressed in hSF. The gene expression levels of PLOD1/2/3, CTGF and COL1A1 were analyzed with Q-PCR. LH2 protein levels were determined with western blot. As expected, TGF-s induced PLOD2/LH2 expression in hSF, whereas CTGF did not. PLOD1 and PLOD3 were not affected by either TGF-s or CTGF. SB-505124 prevented the induction of TGF-s-induced PLOD2, CTGF and COL1A1. Surprisingly, dorsomorphin completely blocked the induction of CTGF and COL1A1, whereas TGF-s-induced PLOD2 was only slightly reduced. Overexpression of caALK5 in osteoarthritic hSF significantly induced PLOD2/LH2 expression, whereas caALK1 had no effect. We showed, in osteoarthritic hSF, that TGF-s induced PLOD2/LH2 via ALK5 Smad2/3P. This elevation of LH2b in osteoarthritic hSF makes LH2b an interesting target to interfere with osteoarthritis-related persistent fibrosis.
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ab0120 elevated Lysyl Hydroxylase 2b expression and pyridinoline cross link formation in collagenase induced oa the cause of oa related fibrosis
Annals of the Rheumatic Diseases, 2013Co-Authors: Dennis Florian George Remst, Ruud A Bank, Elly L. Vitters, E Blaney N Davidson, A B Blom, Reinout Stoop, J M Snabel, W B Van Den Berg, Peter M. Van Der KraanAbstract:Background Fibrosis is a major contributor to joint stiffness in osteoarthritis (OA) and associated with joint pain. In different fibrotic diseases there is an increase in the number of pyridinoline cross-links, resulting in harder to degrade collagen. Lysyl Hydroxylase 2b (LH2b) is the enzyme responsible for pyridinoline cross-link formation. Objectives We examined whether LH2b expression and the number of pyridinoline cross-links per triple helix collagen was elevated in the synovium of mice with collagenase-induced OA. In addition, to elucidate the mechanisms by which LH2b is induced and how to prevent its induction, we investigated whether TGF-β-induced LH2b was Smad2P or Smad3P dependent. Methods We induced OA by intra articular injection of collagenase into the right knee joint of C57Bl/6 mice. Mice were sacrificed at day 7, 21, 28 and 42 after collagenase injection and the mRNA was isolated from the synovium for Q-PCR analysis. Paraffin sections of the murine joints were stained immunohistochemically for LH2 to determine the LH2 expression. The number of pyridinoline cross-links per triple helix in synovium was determined with HPCL. All animal experiments were approved by the local animal ethics committee. Human fibroblasts (hSF) were isolated from synovial tissue of knee joints of OA patients undergoing arthroplasty. The hSF were stimulated with TGF-β alone or in combination with the Smad3P inhibitor SIS3 or the ALK5 kinase inhibitor SB-505124 (SB-5). RNA was isolated and the gene expression for LH2b and collagen type 1 (Col1a1) were analyzed with Q-PCR. Results On al measured time points LH2b mRNA expression was significantly upregulated in the synovium of OA-affected knee joints compared to the healthy joints. Histological sections of murine knee joints with collagenase-induced OA showed a mild increase in the thickness of the synovial membrane at day 7 whereas a large increase was seen on later days. Day 7 showed a strong increase in LH2 staining, at later time points there was still a clear increase but less intense than at day 7. There was a significant increase in the number of pyridinoline cross-links per triple helix after day 7 compared to control knee joints. TGF-β upregulated both LH2b and Col1A1 gene expression in hSF. The Smad3P inhibitor SIS3 strongly down-regulated Col1A1 in both the presence as absence of TGF-β. In contrast to Col1a1, LH2b was still induced by TGF-β in the presence of SIS3. SB-5 blocked both TGF-β-induced LH2b and Col1a1. Conclusions We show a strong upregulation of LH2(b) expression and more than a doubling in pyridinoline cross-links per triple helix in the synovium of murine knee joints with collagenase-induced OA. TGF-β, that is elevated during OA, is most likely the driving force of enhanced LH2b expression. We showed previously that TGF-β-induced LH2b relies on ALK5 (Smad2/3) and not ALK1 signaling. Blocking both Smad2 and Smad3 signaling prevented TGF-β-induced LH2b, whereas LH2b was still induced by TGF-β when only Smad3P was inhibited. This suggests that LH2b is mediated through ALK5/Smad2P. We propose that LH2b is responsible for the persistence of fibrosis during OA. Blocking LH2b, or the Smad2P route, in OA may therefore prevent the formation of fibrosis. Disclosure of Interest None Declared
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osteoarthritis related fibrosis is associated with both elevated pyridinoline cross link formation and Lysyl Hydroxylase 2b expression
Osteoarthritis and Cartilage, 2012Co-Authors: Dennis Florian George Remst, Ruud A Bank, Elly L. Vitters, E Blaney N Davidson, A B Blom, Reinout Stoop, J M Snabel, W B Van Den Berg, Peter M. Van Der KraanAbstract:Objective: Fibrosis is a major contributor to joint stiffness in osteoarthritis (OA). We investigated several factors associated with the persistence of transforming growth factor beta (TGF-β)-induced fibrosis and whether these factors also play a role in OA-related fibrosis. Design: Mice were injected intra-articularly (i.a.) with an adenovirus encoding either TGF-β or connective tissue growth factor (CTGF). In addition, we induced OA by i.a. injection of bacterial collagenase into the right knee joint of C57BL/6 mice. mRNA was isolated from the synovium for Q-PCR analysis of the gene expression of various extracellular matrix (ECM) components, ECM degraders, growth factors and collagen cross-linking-related enzymes. Sections of murine knee joints injected with Ad-TGF-β or Ad-CTGF or from experimental OA were stained for Lysyl Hydroxylase 2 (LH2). The number of pyridinoline cross-links per triple helix collagen in synovium biopsies was determined with high-performance liquid chromatography (HPLC). Results: Expression of collagen alpha-1(I) chain precursor (Col1a1), tissue inhibitor of metalloproteinases 1 (TIMP1) and especially procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2b (Plod2b) were highly upregulated by TGF-β but not by CTGF. Elevated expression of Plod2b mRNA was associated with high Lysyl Hydroxylase 2 (LH2) protein staining after TGF-β overexpression and in experimental OA. Furthermore, in experimental OA the number of hydroxypyridinoline cross-links was significant increased compared to control knee joints. Conclusions: Our data show that elevated LH2b expression is associated with the persistent nature of TGF-β-induced fibrosis. Also in experimental OA, LH2b expression as well as the number of hydroxypyridinoline cross-link were significantly upregulated. We propose that LH2b, and the subsequent increase in pyridinoline cross-links, is responsible for the persistent fibrosis in experimental OA. © 2012 Osteoarthritis Research Society International.
Masahiko Terajima - One of the best experts on this subject based on the ideXlab platform.
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Lysyl Hydroxylase 2-induced collagen cross-link switching promotes metastasis in head and neck squamous cell carcinomas
'Elsevier BV', 2021Co-Authors: Kotaro Sato, Masahiko Terajima, Mitsuo Yamauchi, Kshitij Parag-sharma, Adele M. Musicant, Ryan M. Murphy, Matthew R. Ramsey, Hideharu Hibi, Antonio L. AmelioAbstract:Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide and incidence rates are continuing to rise globally. HNSCC patient prognosis is closely related to the occurrence of tumor metastases, and collagen within the tumor microenvironment (TME) plays a key role in this process. Lysyl Hydroxylase 2 (LH2), encoded by the Procollagen-Lysine,2-Oxoglutarate 5-Dioxygenase 2 (PLOD2) gene, catalyzes hydroxylation of telopeptidyl lysine (Lys) residues of fibrillar collagens which then undergo subsequent modifications to form stable intermolecular cross-links that change the biomechanical properties (i.e. quality) of the TME. While LH2-catalyzed collagen modification has been implicated in driving tumor progression and metastasis in diverse cancers, little is known about its role in HNSCC progression. Thus, using gain- and loss-of-function studies, we examined the effects of LH2 expression levels on collagen cross-linking and cell behavior in vitro and in vivo using a tractable bioluminescent imaging-based orthotopic xenograft model. We found that LH2 overexpression dramatically increases HNSCC cell migratory and invasive abilities in vitro and that LH2-driven changes in collagen cross-linking robustly induces metastasis in vivo. Specifically, the amount of LH2-mediated collagen cross-links increased significantly with PLOD2 overexpression, without affecting the total quantity of collagen cross-links. Conversely, LH2 knockdown significantly blunted HNSCC cells invasive capacity in vitro and metastatic potential in vivo. Thus, regardless of the total “quantity” of collagen crosslinks, it is the “quality” of these cross-links that is the key driver of HNSCC tumor metastatic dissemination. These data implicate LH2 as a key regulator of HNSCC tumor invasion and metastasis by modulating collagen cross-link quality and suggest that therapeutic strategies targeting LH2-mediated collagen cross-linking in the TME may be effective in controlling tumor progression and improving disease outcomes
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Pro-metastatic collagen Lysyl Hydroxylase dimer assemblies stabilized by Fe2+-binding
Nature Publishing Group, 2018Co-Authors: Houfu Guo, Masahiko Terajima, Priyam Banerjee, Xin Liu, Chi-lin Tsai, Xiaochao Tan, Mitchell D. Miller, Jovita Byemerwa, Sarah AlvaradoAbstract:Collagen Lysyl Hydroxylases promote cancer progression. Here the authors present the crystal structure of the Lysyl Hydroxylase domain of L230 from Acanthamoeba polyphaga mimivirus, which is of interest for LH inhibitor development, and show that ectopic expression of L230 in tumors promotes collagen cross-linking and metastasis
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FKBP65-dependent peptidyl-prolyl isomerase activity potentiates the Lysyl Hydroxylase 2-driven collagen cross-link switch
Scientific Reports, 2017Co-Authors: Yulong Chen, Masahiko Terajima, Priyam Banerjee, Mitsuo Yamauchi, Jiang Yu, Jonathan M. KurieAbstract:Bruck Syndrome is a connective tissue disease associated with inactivating mutations in Lysyl Hydroxylase 2 (LH2/PLOD2) or FK506 binding protein 65 (FKBP65/FKBP10). However, the functional relationship between LH2 and FKBP65 remains unclear. Here, we postulated that peptidyl prolyl isomerase (PPIase) activity of FKBP65 positively modulates LH2 enzymatic activity and is critical for the formation of hydroxylysine-aldehyde derived intermolecular collagen cross-links (HLCCs). To test this hypothesis, we analyzed collagen cross-links in Fkbp10-null and –wild-type murine embryonic fibroblasts. Although LH2 protein levels did not change, FKBP65 deficiency significantly diminished HLCCs and increased the non-hydroxylated lysine-aldehyde–derived collagen cross-links (LCCs), a pattern consistent with loss of LH2 enzymatic activity. The HLCC-to-LCC ratio was rescued in FKBP65-deficient murine embryonic fibroblasts by reconstitution with wild-type but not mutant FKBP65 that lacks intact PPIase domains. Findings from co-immunoprecipitation, protein-fragment complementation, and co-immunofluorescence assays showed that LH2 and FKBP65 are part of a common protein complex. We conclude that FKBP65 regulates LH2-mediated collagen cross-linking. Because LH2 promotes fibrosis and cancer metastasis, our findings suggest that pharmacologic strategies to target FKBP65 and LH2 may have complementary therapeutic activities.
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Lysyl Hydroxylase 2 is secreted by tumor cells and can modify collagen in the extracellular space
Journal of Biological Chemistry, 2016Co-Authors: Yulong Chen, Masahiko Terajima, Priyam Banerjee, Houfu Guo, Xin Liu, Amin Momin, Hiroyuki Katayama, Samir M Hanash, Alan R Burns, Gregg B FieldsAbstract:Lysyl Hydroxylase 2 (LH2) catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens, which leads to the formation of stable collagen cross-links. Recently we reported that LH2 enhances the metastatic propensity of lung cancer by increasing the amount of stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs), which generate a stiffer tumor stroma (Chen, Y., et al. (2015) J. Clin. Invest. 125, 125, 1147-1162). It is generally accepted that LH2 modifies procollagen α chains on the endoplasmic reticulum before the formation of triple helical procollagen molecules. Herein, we report that LH2 is also secreted and modifies collagen in the extracellular space. Analyses of lung cancer cell lines demonstrated that LH2 is present in the cell lysates and the conditioned media in a dimeric, active form in both compartments. LH2 co-localized with collagen fibrils in the extracellular space in human lung cancer specimens and in orthotopic lung tumors generated by injection of a LH2-expressing human lung cancer cell line into nude mice. LH2 depletion in MC3T3 osteoblastic cells impaired the formation of HLCCs, resulting in an increase in the unmodified lysine aldehyde-derived collagen cross-link (LCC), and the addition of recombinant LH2 to the media of LH2-deficient MC3T3 cells was sufficient to rescue HLCC formation in the extracellular matrix. The finding that LH2 modifies collagen in the extracellular space challenges the current view that LH2 functions solely on the endoplasmic reticulum and could also have important implications for cancer biology.
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Cyclophilin-B Modulates Collagen Cross-linking by Differentially Affecting Lysine Hydroxylation in the Helical and Telopeptidyl Domains of Tendon Type I Collagen.
The Journal of biological chemistry, 2016Co-Authors: Masahiko Terajima, Yulong Chen, Yuki Taga, Masako Nagasawa, Noriko Sumida, Shunji Hattori, Guo Hou-fu, Sirivimol Srisawasdi, Jonathan M. KurieAbstract:Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet 10, e1004465). However, the extent of decrease appears to be tissue- and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all Lysyl Hydroxylase isoforms (isoforms 1-3) and a putative Lysyl Hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with Lysyl Hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation.
