Collagen cross-linking. Synthesis of collagen cross-links in vitro with highly purified lysyl oxidase.

Abstract Background and Aims Copper is an essential trace element required for many biological processes. Some studies have demonstrated that copper accumulating was related to liver fibrosis and lung fibrosis, but the underlying mechanism is not very clear. Copper is the essential unit of lysyl oxidase (LOXs), which are the key enzymes of crosslinking of extracellular matrix. Method Sprague-Dawley rats were divided into the sham group, unilateral ureteral obstruction (UUO) operated group and UUO treated with copper chelating agents tetrathiomolybdate (TM). Rat kidney fibroblast cells (NRK-49F) were used in vitro. The concentration of copper, the LOXs activity and the degree of cross-linking of extracellular collagen were detected in vivo and vitro. Results (1) The copper concentration in serum, urine and kidney of rats increased significantly at 7 days after UUO surgery; After treatment of TGF-β1, the intracellular copper concentration was increased significantly in cells; The concentration of copper in patients` serum is on the rise with the progression of chronic kidney disease (CKD). (2) The expression of CTR1 was upregulated in the kidneys of UUO rats; The level of CTR1 was increased significantly by TGF-β1 in vitro; (3) Blockage of Smad2/3 suppresses TGF-β1-induced expression of CTR1; (4) Downregulation of CTR1 significantly inhibited the intracellular copper concentration; (5) The activity of LOXs was increased significantly after TGF-β1 treatment; (6) Downregulation of CTR1 significantly inhibited the activity of LOXs and the cross-linking of extracellular collagen induced by TGF-β1 in vitro; (7) The concentration of copper, the degree of collagen cross-linking and the deposition of collagen were decreased in the kidney tissue of UUO rats after treatment with TM. The concentration of intracellular copper, the activity of LOXs and the degree of collagen cross-linking were attenuated with treatment of TM in vitro. Conclusion We firstly found that the intracellular copper accumulating was closely to renal fibrosis. The underlying mechanism was related with the increasing expression of CTR1 and activity of LOXs. Treatment with TM ameliorated the renal fibrosis. This study presented a novel treatment target for renal fibrosis.

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Modulation of lysyl oxidase activity toward peptidyl lysine by vicinal dicarboxylic amino acid residues. Implications for collagen cross-linking.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)31798-2 ◽

1994 ◽

Vol 269 (35) ◽

pp. 22366-22371 ◽

Cited By ~ 1

Author(s):

N. Nagan ◽

H.M. Kagan

Keyword(s):

Amino Acid ◽

Oxidase Activity ◽

Lysyl Oxidase ◽

Cross Linking ◽

Amino Acid Residues ◽

Dicarboxylic Amino Acid ◽

Collagen Cross Linking

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The Interactome of Cancer-Related Lysyl Oxidase and Lysyl Oxidase-Like Proteins

Cancers ◽

10.3390/cancers13010071 ◽

2020 ◽

Vol 13 (1) ◽

pp. 71

Author(s):

Sylvain D. Vallet ◽

Coline Berthollier ◽

Romain Salza ◽

Laurent Muller ◽

Sylvie Ricard-Blum

Keyword(s):

Protein Folding ◽

Cancer Progression ◽

Cellular Response ◽

Lysyl Oxidase ◽

Chaperone Activity ◽

Cross Linking ◽

Binding Assays ◽

Vessel Development ◽

New Interactions

The members of the lysyl oxidase (LOX) family are amine oxidases, which initiate the covalent cross-linking of the extracellular matrix (ECM), regulate ECM stiffness, and contribute to cancer progression. The aim of this study was to build the first draft of the interactome of the five members of the LOX family in order to determine its molecular functions, the biological and signaling pathways mediating these functions, the biological processes it is involved in, and if and how it is rewired in cancer. In vitro binding assays, based on surface plasmon resonance and bio-layer interferometry, combined with queries of interaction databases and interaction datasets, were used to retrieve interaction data. The interactome was then analyzed using computational tools. We identified 31 new interactions and 14 new partners of LOXL2, including the α5β1 integrin, and built an interactome comprising 320 proteins, 5 glycosaminoglycans, and 399 interactions. This network participates in ECM organization, degradation and cross-linking, cell-ECM interactions mediated by non-integrin and integrin receptors, protein folding and chaperone activity, organ and blood vessel development, cellular response to stress, and signal transduction. We showed that this network is rewired in colorectal carcinoma, leading to a switch from ECM organization to protein folding and chaperone activity.

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Collagen cross-linking. Effect of D-penicillamine on cross-linking in vitro.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)32825-9 ◽

1977 ◽

Vol 252 (1) ◽

pp. 254-259

Author(s):

R C Siegel

Keyword(s):

Cross Linking ◽

Collagen Cross Linking

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Chemistry of collagen cross-links: glucose-mediated covalent cross-linking of type-IV collagen in lens capsules

Biochemical Journal ◽

10.1042/bj2960489 ◽

1993 ◽

Vol 296 (2) ◽

pp. 489-496 ◽

Cited By ~ 73

Author(s):

A J Bailey ◽

T J Sims ◽

N C Avery ◽

C A Miles

Keyword(s):

Type Iv Collagen ◽

Cross Linking ◽

Mechanical And Thermal Properties ◽

Maximum Strain ◽

Scanning Calorimetry ◽

Melting Temperatures ◽

Type Iv ◽

Collagen Molecules ◽

Cross Links

The incubation of lens capsules with glucose in vitro resulted in changes in the mechanical and thermal properties of type-IV collagen consistent with increased cross-linking. Differential scanning calorimetry (d.s.c.) of fresh lens capsules showed two major peaks at melting temperatures Tm 1 and Tm 2 at approx. 54 degrees C and 90 degrees C, which can be attributed to the denaturation of the triple helix and 7S domains respectively. Glycosylation of lens capsules in vitro for 24 weeks caused an increase in Tm 1 from 54 degrees C to 61 degrees C, while non-glycosylated, control incubated capsules increased to a Tm 1 of 57 degrees C. The higher temperature required to denature the type-IV collagen after incubation in vitro suggested increased intermolecular cross-linking. Glycosylated lens capsules were more brittle than fresh samples, breaking at a maximum strain of 36.8 +/- 1.8% compared with 75.6 +/- 6.3% for the fresh samples. The stress at maximum strain (or ‘strength’) was dramatically reduced from 12.0 to 4.7 N.mm.mg-1 after glycosylation in vitro. The increased constraints within the system leading to loss of strength and increased brittleness suggested not only the presence of more cross-links but a difference in the location of these cross-links compared with the natural lysyl-aldehyde-derived cross-links. The chemical nature of the fluorescent glucose-derived cross-link following glycosylation was determined as pentosidine, at a concentration of 1 pentosidine molecule per 600 collagen molecules after 24 weeks incubation. Pentosidine was also determined in the lens capsules obtained from uncontrolled diabetics at a level of about 1 per 100 collagen molecules. The concentration of these pentosidine cross-links is far too small to account for the observed changes in the thermal and mechanical properties following incubation in vitro, clearly indicating that another as yet undefined, but apparently more important cross-linking mechanism mediated by glucose is taking place.

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Native cross-links in collagen fibrils induce resistance to human synovial collagenase

Biochemical Journal ◽

10.1042/bj1810639 ◽

1979 ◽

Vol 181 (3) ◽

pp. 639-645 ◽

Cited By ~ 150

Author(s):

C A Vater ◽

E D Harris ◽

R C Siegel

Keyword(s):

Lysyl Oxidase ◽

Collagen Fibrils ◽

Bone Collagen ◽

Collagen Molecule ◽

Cross Linking ◽

Pathological Conditions ◽

Insoluble Material ◽

Induce Resistance ◽

Cross Links

A model system consisting of highly purified lysyl oxidase and reconstituted lathyritic chick bone collagen fibrils was used to study the effect of collagen cross-linking on collagen degradation by mammalian collagenase. The results indicate that synthesis of approx. 0.1 Schiff-base cross-link per collagen molecule results in a 2–3-fold resistance to human synovial collagenase when compared with un-cross-linked controls or samples incubated in the presence of beta-aminopropionitrile to inhibit cross-linking. These results confirm previous studies utilizing artificially cross-linked collagens, or collagens isolated as insoluble material after cross-linking in vivo, and suggest that increased resistance to collagenase may be one of the earliest effects of cross-linking in vivo. The extent of intermolecular cross-linking among collagen fibrils may provide a mechanism for regulating the rate of collagen catabolism relative to synthesis in normal and pathological conditions.

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Mapping the Contact Sites of the Escherichia coli Division-Initiating Proteins FtsZ and ZapA by BAMG Cross-Linking and Site-Directed Mutagenesis

International Journal of Molecular Sciences ◽

10.3390/ijms19102928 ◽

2018 ◽

Vol 19 (10) ◽

pp. 2928 ◽

Cited By ~ 7

Author(s):

Winfried Roseboom ◽

Madhvi Nazir ◽

Nils Meiresonne ◽

Tamimount Mohammadi ◽

Jolanda Verheul ◽

...

Keyword(s):

Site Directed Mutagenesis ◽

Cross Linking ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Binding Interface ◽

Tetrameric Structure ◽

Z Ring ◽

Cross Links ◽

Chemical Cross Linking

Cell division in bacteria is initiated by the polymerization of FtsZ at midcell in a ring-like structure called the Z-ring. ZapA and other proteins assist Z-ring formation and ZapA binds ZapB, which senses the presence of the nucleoids. The FtsZ–ZapA binding interface was analyzed by chemical cross-linking mass spectrometry (CXMS) under in vitro FtsZ-polymerizing conditions in the presence of GTP. Amino acids residue K42 from ZapA was cross-linked to amino acid residues K51 and K66 from FtsZ, close to the interphase between FtsZ molecules in protofilaments. Five different cross-links confirmed the tetrameric structure of ZapA. A number of FtsZ cross-links suggests that its C-terminal domain of 55 residues, thought to be largely disordered, has a limited freedom to move in space. Site-directed mutagenesis of ZapA reveals an interaction site in the globular head of the protein close to K42. Using the information on the cross-links and the mutants that lost the ability to interact with FtsZ, a model of the FtsZ protofilament–ZapA tetramer complex was obtained by information-driven docking with the HADDOCK2.2 webserver.

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Lysyl oxidases: from enzyme activity to extracellular matrix cross-links

Essays in Biochemistry ◽

10.1042/ebc20180050 ◽

2019 ◽

Vol 63 (3) ◽

pp. 349-364 ◽

Cited By ~ 19

Author(s):

Sylvain D. Vallet ◽

Sylvie Ricard-Blum

Keyword(s):

Extracellular Matrix ◽

Molecular Mechanisms ◽

Catalytic Domain ◽

Lysyl Oxidase ◽

Dimensional Structure ◽

Cross Linking ◽

Copper Binding ◽

Molecular Features ◽

Ecm Proteins ◽

Cross Links

Abstract The lysyl oxidase family comprises five members in mammals, lysyl oxidase (LOX) and four lysyl oxidase like proteins (LOXL1-4). They are copper amine oxidases with a highly conserved catalytic domain, a lysine tyrosylquinone cofactor, and a conserved copper-binding site. They catalyze the first step of the covalent cross-linking of the extracellular matrix (ECM) proteins collagens and elastin, which contribute to ECM stiffness and mechanical properties. The role of LOX and LOXL2 in fibrosis, tumorigenesis, and metastasis, including changes in their expression level and their regulation of cell signaling pathways, have been extensively reviewed, and both enzymes have been identified as therapeutic targets. We review here the molecular features and three-dimensional structure/models of LOX and LOXLs, their role in ECM cross-linking, and the regulation of their cross-linking activity by ECM proteins, proteoglycans, and by inhibitors. We also make an overview of the major ECM cross-links, because they are the ultimate molecular readouts of LOX/LOXL activity in tissues. The recent 3D model of LOX, which recapitulates its known structural and biochemical features, will be useful to decipher the molecular mechanisms of LOX interaction with its various substrates, and to design substrate-specific inhibitors, which are potential antifibrotic and antitumor drugs.

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Overhydroxylation of Lysine of Collagen Increases Uterine Fibroids Proliferation: Roles of Lysyl Hydroxylases, Lysyl Oxidases, and Matrix Metalloproteinases

BioMed Research International ◽

10.1155/2017/5316845 ◽

2017 ◽

Vol 2017 ◽

pp. 1-13 ◽

Cited By ~ 6

Author(s):

Marwa Kamel ◽

Mohamed Wagih ◽

Gokhan S. Kilic ◽

Concepcion R. Diaz-Arrastia ◽

Mohamed A. Baraka ◽

...

Keyword(s):

Matrix Metalloproteinases ◽

Treatment Options ◽

Uterine Fibroids ◽

Cross Linking ◽

Ki 67 ◽

Subsequent Formation ◽

Lysyl Oxidases ◽

Cross Links ◽

Collagen Cross Linking

The role of the extracellular matrix (ECM) in uterine fibroids (UF) has recently been appreciated. Overhydroxylation of lysine residues and the subsequent formation of hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) cross-links underlie the ECM stiffness and profoundly affect tumor progression. The aim of the current study was to investigate the relationship between ECM of UF, collagen and collagen cross-linking enzymes [lysyl hydroxylases (LH) and lysyl oxidases (LOX)], and the development and progression of UF. Our results indicated that hydroxyl lysine (Hyl) and HP cross-links are significantly higher in UF compared to the normal myometrial tissues accompanied by increased expression of LH (LH2b) and LOX. Also, increased resistance to matrix metalloproteinases (MMP) proteolytic degradation activity was observed. Furthermore, the extent of collagen cross-links was positively correlated with the expression of myofibroblast marker (α-SMA), growth-promoting markers (PCNA; pERK1/2;FAKpY397; Ki-67; and Cyclin D1), and the size of UF. In conclusion, our study defines the role of overhydroxylation of collagen and collagen cross-linking enzymes in modulating UF cell proliferation, differentiation, and resistance to MMP. These effects can establish microenvironment conducive for UF progression and thus represent potential target treatment options of UF.

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