Hydroxylysine Residues Research Articles

Sulfilimine bond formation in collagen IV.

The collagen IV network plays a crucial role in providing structural support and mechanical integrity to the basement membrane and surrounding tissues. A key aspect of this network is the formation of intra- and inter-collagen fibril crosslinks. One particular crosslink, an inter-residue sulfilimine bond, has been found, so far, to be unique to collagen IV. More specifically, these crosslinks are primarily formed between methionine and lysine or hydroxylysine residues and can occur within a single collagen fibril or between different collagen fibrils. Due to its significance as the major crosslink in the collagen IV network, the sulfilimine bond plays critical roles in tissue development and various human diseases. While the proposed reaction mechanism for sulfilimine bond formation is supported by experimental evidence, the precise nature of this bond remained uncertain until computational studies were conducted. The process involves the reaction of hypohalous acids (e.g., HOBr, HOCl), produced by a peroxidasin enzyme in the basement membrane, with the sidechain sulfur of methionine or sidechain nitrogen of lysine/hydroxylysine residues in collagen IV, to form halosulfonium or haloamine intermediates, respectively. The halosulfonium/haloamine then reacts with the sidechain amine/sulfide of the lysine (or hydroxylysine) or methionine respectively, eventually resulting in the formation of the sulfilimine (MetSNLys/Hyl) crosslink. The sulfilimine product formed not only plays a crucial role in physiological processes but also finds applications in various industrial and pharmaceutical contexts. In this review, we provide a comprehensive summary of existing studies, including our own research, aimed at understanding the reaction mechanism, protonation states, characteristic nature, and dynamic behavior of the sulfilimine bond in collagen IV. The goal is to offer readers an overview of this critically important biochemical bond.

Exploring the Interplay between Polyphenols and Lysyl Oxidase Enzymes for Maintaining Extracellular Matrix Homeostasis.

Collagen, the most abundant structural protein found in mammals, plays a vital role as a constituent of the extracellular matrix (ECM) that surrounds cells. Collagen fibrils are strengthened through the formation of covalent cross-links, which involve complex enzymatic and non-enzymatic reactions. Lysyl oxidase (LOX) is responsible for catalyzing the oxidative deamination of lysine and hydroxylysine residues, resulting in the production of aldehydes, allysine, and hydroxyallysine. These intermediates undergo spontaneous condensation reactions, leading to the formation of immature cross-links, which are the initial step in the development of mature covalent cross-links. Additionally, non-enzymatic glycation contributes to the formation of abnormal cross-linking in collagen fibrils. During glycation, specific lysine and arginine residues in collagen are modified by reducing sugars, leading to the creation of Advanced Glycation End-products (AGEs). These AGEs have been associated with changes in the mechanical properties of collagen fibers. Interestingly, various studies have reported that plant polyphenols possess amine oxidase-like activity and can act as potent inhibitors of protein glycation. This review article focuses on compiling the literature describing polyphenols with amine oxidase-like activity and antiglycation properties. Specifically, we explore the molecular mechanisms by which specific flavonoids impact or protect the normal collagen cross-linking process. Furthermore, we discuss how these dual activities can be harnessed to generate properly cross-linked collagen molecules, thereby promoting the stabilization of highly organized collagen fibrils.

Insight into the Spatial Arrangement of the Lysine Tyrosylquinone and Cu2+ in the Active Site of Lysyl Oxidase-like 2.

Lysyl oxidase-2 (LOXL2) is a Cu2+ and lysine tyrosylquinone (LTQ)-dependent amine oxidase that catalyzes the oxidative deamination of peptidyl lysine and hydroxylysine residues to promote crosslinking of extracellular matrix proteins. LTQ is post-translationally derived from Lys653 and Tyr689, but its biogenesis mechanism remains still elusive. A 2.4 Å Zn2+-bound precursor structure lacking LTQ (PDB:5ZE3) has become available, where Lys653 and Tyr689 are 16.6 Å apart, thus a substantial conformational rearrangement is expected to take place for LTQ biogenesis. However, we have recently shown that the overall structures of the precursor (no LTQ) and the mature (LTQ-containing) LOXL2s are very similar and disulfide bonds are conserved. In this study, we aim to gain insights into the spatial arrangement of LTQ and the active site Cu2+ in the mature LOXL2 using a recombinant LOXL2 that is inhibited by 2-hydrazinopyridine (2HP). Comparative UV-vis and resonance Raman spectroscopic studies of the 2HP-inhibited LOXL2 and the corresponding model compounds and an EPR study of the latter support that 2HP-modified LTQ serves as a tridentate ligand to the active site Cu2. We propose that LTQ resides within 2.9 Å of the active site of Cu2+ in the mature LOXL2, and both LTQ and Cu2+ are solvent-exposed.

Cleavage of LOXL1 by BMP1 and ADAMTS14 Proteases Suggests a Role for Proteolytic Processing in the Regulation of LOXL1 Function.

Members of the lysyl oxidase (LOX) family catalyze the oxidative deamination of lysine and hydroxylysine residues in collagen and elastin in the initiation step of the formation of covalent cross-links, an essential process for connective tissue maturation. Proteolysis has emerged as an important level of regulation of LOX enzymes with the cleavage of the LOX isoform by metalloproteinases of the BMP1 (bone morphogenetic protein 1) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) families as a model example. Lysyl oxidase-like 1 (LOXL1), an isoform associated with pelvic organ prolapse and pseudoexfoliation (PEX) glaucoma, has also been reported to be proteolytically processed by these proteases. However, precise molecular information on these proteolytic events is not available. In this study, using genetic cellular models, along with proteomic analyses, we describe that LOXL1 is processed by BMP1 and ADAMTS14 and identify the processing sites in the LOXL1 protein sequence. Our data show that BMP1 cleaves LOXL1 in a unique location within the pro-peptide region, whereas ADAMTS14 processes LOXL1 in at least three different sites located within the pro-peptide and in the first residues of the catalytic domain. Taken together, these results suggest a complex regulation of LOXL1 function by BMP1- and ADAMTS14-mediated proteolysis where LOXL1 enzymes retaining variable fragments of N-terminal region may display different capabilities.

Oligomeric States and Hydrodynamic Properties of Lysyl Oxidase-Like 2.

Lysyl oxidase-like 2 (LOXL2) has emerged as a promising therapeutic target against metastatic/invasive tumors and organ and tissue fibrosis. LOXL2 catalyzes the oxidative deamination of lysine and hydroxylysine residues in extracellular matrix (ECM) proteins to promote crosslinking of these proteins, and thereby plays a major role in ECM remodeling. LOXL2 secretes as 100-kDa full-length protein (fl-LOXL2) and then undergoes proteolytic cleavage of the first two scavenger receptor cysteine-rich (SRCR) domains to yield 60-kDa protein (Δ1-2SRCR-LOXL2). This processing does not affect the amine oxidase activity of LOXL2 in vitro. However, the physiological importance of this cleavage still remains elusive. In this study, we focused on characterization of biophysical properties of fl- and Δ1-2SRCR-LOXL2s (e.g., oligomeric states, molecular weights, and hydrodynamic radii in solution) to gain insight into the structural role of the first two SRCR domains. Our study reveals that fl-LOXL2 exists predominantly as monomer but also dimer to the lesser extent when its concentration is <~1 mM. The hydrodynamic radius (Rh) determined by multi-angle light scattering coupled with size exclusion chromatography (SEC-MALS) indicates that fl-LOXL2 is a moderately asymmetric protein. In contrast, Δ1-2SRCR-LOXL2 exists solely as monomer and its Rh is in good agreement with the predicted value. The Rh values calculated from a 3D modeled structure of fl-LOXL2 and the crystal structure of the precursor Δ1-2SRCR-LOXL2 are within a reasonable margin of error of the values determined by SEC-MALS for fl- and Δ1-2SRCR-LOXL2s in mature forms in this study. Based on superimposition of the 3D model and the crystal structure of Δ1-2SRCR-LOXL2 (PDB:5ZE3), we propose a configuration of fl-LOXL2 that explains the difference observed in Rh between fl- and Δ1-2SRCR-LOXL2s in solution.

Dysregulation of Lysyl Oxidases Expression in Diabetic Nephropathy and Renal Cell Carcinoma.

Lysyl oxidases (LOXs) are amino oxidase enzymes that catalyze the oxidative deamination of lysine and hydroxylysine residues to form allysine, the first step towards the development of the final cross-linking reaction in collagens, a crucial macromolecule that reinforces extracellular matrices. Basement membranes are specialized extracellular matrices that are essential components of the glomerular filtration barrier, which also support tubular epithelial cells. Lysyl oxidases are post-translational enzymes indispensable for tissue architecture, participating actively in the development and function of kidneys. The differential expression and dysregulation of these enzymes promote diabetic nephropathy, one of the major complications observed in end-stage renal diseases. In addition, these enzymes act as transcription factors that trigger the epithelial-mesenchymal transition responsible for the generation of different cancers. In the kidney, the expression studies in physiological conditions identified LOXL1 and LOXL2 as constituent proteins of glomerular basement membranes. Besides, LOX and LOXL2 are upregulated in fibrosis and renal cell carcinoma. The current review summarizes the physiological expression of LOXs enzymes in the nephrons, including glomerulus and tubules. Their roles in renal diseases are particularly highlighted in diabetic nephropathy and renal cell carcinoma, two pathophysiological conditions where these enzymes have been demonstrated to participate. The focus of the present study is to describe and discuss the current understanding in this field. The current potential of LOXs enzymes as a biomarker and pharmacological target to kidney diseases that involves extracellular matrix cross-linking enzymes is also discussed. LOXs isoforms and their capacity as therapeutic targets could be used for diagnostic and prognostic purposes and in treating these renal complications.

Collagen molecular phenotypic switch between non-neoplastic and neoplastic canine mammary tissues

In spite of major advances over the past several decades in diagnosis and treatment, breast cancer remains a global cause of morbidity and premature death for both human and veterinary patients. Due to multiple shared clinicopathological features, dogs provide an excellent model of human breast cancer, thus, a comparative oncology approach may advance our understanding of breast cancer biology and improve patient outcomes. Despite an increasing awareness of the critical role of fibrillar collagens in breast cancer biology, tumor-permissive collagen features are still ill-defined. Here, we characterize the molecular and morphological phenotypes of type I collagen in canine mammary gland tumors. Canine mammary carcinoma samples contained longer collagen fibers as well as a greater population of wider fibers compared to non-neoplastic and adenoma samples. Furthermore, the total number of collagen cross-links enriched in the stable hydroxylysine-aldehyde derived cross-links was significantly increased in neoplastic mammary gland samples compared to non-neoplastic mammary gland tissue. The mass spectrometric analyses of type I collagen revealed that in malignant mammary tumor samples, lysine residues, in particular those in the telopeptides, were markedly over-hydroxylated in comparison to non-neoplastic mammary tissue. The extent of glycosylation of hydroxylysine residues was comparable among the groups. Consistent with these data, expression levels of genes encoding lysyl hydroxylase 2 (LH2) and its molecular chaperone FK506-binding protein 65 were both significantly increased in neoplastic samples. These alterations likely lead to an increase in the LH2-mediated stable collagen cross-links in mammary carcinoma that may promote tumor cell metastasis in these patients.

Collagen hydroxylysine glycosylation: non-conventional substrates for atypical glycosyltransferase enzymes.

Collagen is a major constituent of the extracellular matrix (ECM) that confers fundamental mechanical properties to tissues. To allow proper folding in triple-helices and organization in quaternary super-structures, collagen molecules require essential post-translational modifications (PTMs), including hydroxylation of proline and lysine residues, and subsequent attachment of glycan moieties (galactose and glucose) to specific hydroxylysine residues on procollagen alpha chains. The resulting galactosyl-hydroxylysine (Gal-Hyl) and less abundant glucosyl-galactosyl-hydroxylysine (Glc-Gal-Hyl) are amongst the simplest glycosylation patterns found in nature and are essential for collagen and ECM homeostasis. These collagen PTMs depend on the activity of specialized glycosyltransferase enzymes. Although their biochemical reactions have been widely studied, several key biological questions about the possible functions of these essential PTMs are still missing. In addition, the lack of three-dimensional structures of collagen glycosyltransferase enzymes hinders our understanding of the catalytic mechanisms producing this modification, as well as the impact of genetic mutations causing severe connective tissue pathologies. In this mini-review, we summarize the current knowledge on the biochemical features of the enzymes involved in the production of collagen glycosylations and the current state-of-the-art methods for the identification and characterization of this important PTM.

Inhibition of Lysyl Oxidase with β-aminopropionitrile Improves Venous Adaptation after Arteriovenous Fistula Creation.

The arteriovenous fistula (AVF) is the preferred hemodialysis access for end-stage renal disease (ESRD) patients. Yet, establishment of a functional AVF presents a challenge, even for the most experienced surgeons, since postoperative stenosis frequently occludes the AVF. Stenosis results from the loss of compliance in fibrotic areas of the fistula which turns intimal hyperplasia into an occlusive feature. Fibrotic remodeling depends on deposition and crosslinking of collagen by lysyl oxidase (LOX), an enzyme that catalyzes the deamination of lysine and hydroxylysine residues, facilitating intra/intermolecular covalent bonds. We postulate that pharmacological inhibition of lysyl oxidase (LOX) increases postoperative venous compliance and prevents stenosis in a rat AVF model. LOX gene expression and vascular localization were assayed in rat AVFs and human pre-access veins, respectively. Collagen crosslinking was measured in humans AVFs that matured or failed, and in rat AVFs treated with β-aminopropionitrile (BAPN), an irreversible LOX inhibitor. BAPN was either injected systemically or delivered locally around rat AVFs using nanofiber scaffolds. The major endpoints were AVF blood flow, wall fibrosis, collagen crosslinking, and vascular distensibility. Non-maturation of human AVFs was associated with higher LOX deposition in pre-access veins (N=20, P=0.029), and increased trivalent crosslinks (N=18, P=0.027) in human AVF tissues. Systemic and local inhibition of LOX increased AVF distensibility, while reducing wall fibrosis and collagen crosslinking in rat fistulas. Our results demonstrate that BAPN-mediated inhibition of LOX significantly improves vascular remodeling in experimental fistulas.

Reaction of human peroxidasin 1 compound I and compound II with one-electron donors

Human peroxidasin 1 (hsPxd01) is a homotrimeric multidomain heme peroxidase embedded in the extracellular matrix. It catalyses the two-electron oxidation of bromide by hydrogen peroxide to hypobromous acid which mediates the formation of essential sulfilimine cross-links between methionine and hydroxylysine residues in collagen IV. This confers critical structural reinforcement to the extracellular matrix. This study presents for the first time transient kinetic measurements of the reactivity of hsPxd01 compound I and compound II with the endogenous one-electron donors nitrite, ascorbate, urate, tyrosine and serotonin using the sequential stopped-flow technique. At pH 7.4 and 25 °C compound I of hsPxd01 is reduced to compound II with apparent second-order rate constants ranging from (1.9 ± 0.1) × 104 M−1 s−1 (urate) to (4.8 ± 0.1) × 105 M−1 s−1 (serotonin). Reduction of compound II to the ferric state occurs with apparent second-order rate constants ranging from (4.3 ± 0.2) × 102 M−1 s−1 (tyrosine) to (7.7 ± 0.1) × 103 M−1 s−1 (serotonin). The relatively fast rates of compound I reduction suggest that these reactions may take place in vivo and modulate bromide oxidation due to formation of compound II. Urate is shown to inhibit the bromination activity of hsPxd01, whereas nitrite stimulates the formation of hypobromous acid. The results are discussed with respect to known kinetic data of homologous mammalian peroxidases and to the physiological role of human peroxidasin 1.

Down-regulation of GLT25D1 inhibited collagen secretion and involved in liver fibrogenesis

Collagen β (1-O) galactosyltransferase 1 (GLT25D1) has been reported to transfer galactose to hydroxylysine residues via β (1-O) linkages in collagen. However, the role of Glt25d1 in liver fibrogenesis is still unknow. Recently, we generated a Glt25d1 knockout mouse to elucidate the role of Glt25d1 in vivo. However, we found that complete deletion of the Glt25d1 gene resulted in embryonic lethality at E11.5. Histopathological analysis revealed that dysplasia in Glt25d1−/− labyrinth with defects of the vascular network. Immunohistochemical showed that the decrease in proliferation of Glt25d1−/− liver and the developing central nervous system (CNS). The role of Glt25d1 in liver fibrogenesis was explored by Glt25d1+/− mice. Glt25d1+/− mice and wild-type (WT) mice were injected intraperitoneally with the same dose of CCl4. The higher level of serum alanine aminotransferase was observed in Glt25d1+/− mice. Reverse transcription-quantitative polymerase chainreaction demonstrated that the mRNA expression levels of the inflammatory cytokines such as, Tnf-α, Cxcl-1 and Mcp-1, showed a significantly increase in CCl4-treated Glt25d1+/− mice. Collagen-I, collagen-III and α-SMA transcripts accumulation was markedly increased in the Glt25d1+/− mice. However, Masson’s trichrome staining revealed a trend to decrease in the ECM proteins deposition of Glt25d1+/− liver. Immunohistochemistry and Western blots revealed that the protein expression of Collagen-III was reduced and a trend to a decrease in collagen-I was observed in the Glt25d1+/− liver compared with those of WT mice. Our results demonstrate that Glt25d1 knockout results in embryonic lethality and down-regulation of Glt25d1 may inhibit collagen secretion during liver fibrogenesis.

Impaired Gastric Hormone Regulation of Osteoblasts and Lysyl Oxidase Drives Bone Disease in Diabetes Mellitus.

ABSTRACTDiabetic bone disease is a complication of type I and type II diabetes, both of which are increasing in the United States and elsewhere. Increased hip and foot fracture rates do not correlate well with changes in bone mineral density (BMD), whereas studies support the importance of collagen structure to bone strength. Extracellular lysyl oxidase (LOX) catalyzes the oxidative deamination of hydroxylysine and lysine residues in collagens resulting in aldehydes that subsequently form critically important biosynthetic crosslinks that stabilize functional collagens. Although LOX‐dependent biosynthetic crosslinks in bone collagen are deficient in diabetic bone, the expression and regulation of bone LOXs in diabetes have not been comprehensively studied. Here, we found that LOX is profoundly downregulated in bone in diabetes. Moreover, we have identified a novel metabolic regulatory relationship that is dysregulated in diabetes using mouse models. Data indicate that the incretin (gastric hormone) known as glucose‐dependent insulinotropic polypeptide (GIP) that is anabolic to osteoblasts strongly upregulates LOX, and that this regulation is disrupted in the streptozotocin‐induced model of diabetes in mice. In vivo and in vitro studies support that diabetes results in elevated circulating peripheral dopamine, likely also derived from the gut, and is responsible for blocking GIP signaling and LOX levels in osteoblasts. Moreover, peripheral administration of the dopamine D2 receptor antagonist amisulpride to diabetic mice restored trabecular bone structure to near normal and partially reversed downregulation of LOX. Taken together our data identifies a novel metabolic relationship between the gut‐derived hormone GIP and bone‐derived LOX, and points to the importance of LOX dysregulation in the pathology of diabetic bone disease. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Loss of function of Colgalt1 disrupts collagen post-translational modification and causes musculoskeletal defects.

ABSTRACTIn a screen for organogenesis defects in N-ethyl-N-nitrosourea (ENU)-induced mutant mice, we discovered a line carrying a mutation in Colgalt1 [collagen beta(1-O)galactosyltransferase type 1], which is required for proper galactosylation of hydroxylysine residues in a number of collagens. Colgalt1 mutant embryos have not been previously characterized; here, we show that they exhibit skeletal and muscular defects. Analysis of mutant-derived embryonic fibroblasts reveals that COLGALT1 acts on collagen IV and VI, and, while collagen VI appears stable and its secretion is not affected, collagen IV accumulates inside of cells and within the extracellular matrix, possibly due to instability and increased degradation. We also generated mutant zebrafish that do not express the duplicated orthologs of mammalian Colgalt1. The double-homozygote mutants have muscle defects; they are viable through the larvae stage but do not survive to 10 days post-fertilization. We hypothesize that the Colgalt1 mutant could serve as a model of a human connective tissue disorder and/or congenital muscular dystrophy or myopathy.

Development of a Novel In Situ Activity Assay for Lysyl Oxidase Like‐2 (LOXL2)

Lysyl oxidase like‐2 (LOXL2) is a member of the lysyl oxidase (LOX) super family, which are copper‐dependent amine oxidases that initialize collagen and elastin cross‐linking to form a stable and organized matrix. Augmented LOXL2 expression is highly associated with diverse disease processes with a known element of matrix accumulation including fibrosis, pulmonary hypertension, and cancer metastasis. To date, lack of sensitive and specific assays that can detect LOXL2 catalytic function in situ in the extracellular matrix (ECM) of cells and tissue has limited the ability to fully elucidate the role of LOXL2 in pathobiological mechanisms.Current methods to detect LOXL2 enzymatic activity are inefficient and limited in scope. The most widely used assay is hydrogen peroxide based: oxidation of the LOX active site occurs when substrate is added to samples, and releases hydrogen peroxide. The level of hydrogen peroxide is detected using an HRP‐coupled system, that utilizes the conversion of Amplex Red to the fluorescent end product, resorufin. While the results are semi‐quantitative, this assay has limited dynamic range and cannot distinguish hydrogen peroxide arising from other pathways. Moreover, the assay requires LOXL2 in solution. The harsh conditions required to obtain a solubilized ECM sample, where the pertinent LOXL2 activity occurs, cause LOXL2 unfolding and loss of activity, rendering this assay unfeasible. Thus, there is a need to develop a sensitive, specific, and practicable in situ activity assay that overcomes these limitations.This study provides a proof of concept for a novel in situ activity assay for LOXL2. Here, we developed an assay to fluorescently label the products of LOXL2 catalysis. Specifically, LOXL2 catalyzes an oxidative de‐amination of lysine and hydroxylysine residues (found on collagen and elastin) to highly reactive semi‐aldehydes called allysines. In this assay, we labeled the newly generated allysine residues with biotin hydrazide using 1‐ethyl‐3‐(3‐dimethyl‐aminopropyl) carbodiimide hydrochloride (EDC) crosslinker. This biotinylation allows for a robust method of measuring LOXL2 activity through western blotting, immunoprecipitation and immunofluorescence imaging using biotin‐streptavidin interactions.Support or Funding InformationFunded by MedImmune research funds.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigation of Triple-Helix Collagen Hydroxylation by Solid-State NMR Spectroscopy.

Solid-state nuclear magnetic resonance spectroscopy (ssNMR) is an emerging technique in structural methods of studying collagen proteins, capable of identifying features on an atomic length scale in tissues and protein samples without extensive extraction or purification. Hydroxylation is a key posttranslational modification of collagen that gives rise to distinctive signals in the ssNMR spectrum of collagen proteins. Here we outline the type of information that ssNMR can provide and describe the procedures involved in a ssNMR structural study, with particular focus on using dynamic nuclear polarization to enhance sensitivity for detecting hydroxylysine residues by ssNMR.

Glycation of type I collagen selectively targets the same helical domain lysine sites as lysyl oxidase–mediated cross-linking

Nonenzymatic glycation of collagen has long been associated with the progressive secondary complications of diabetes. How exactly such random glycations result in impaired tissues is still poorly understood. Because of the slow turnover rate of most fibrillar collagens, they are more susceptible to accumulate time-dependent glycations and subsequent advanced glycation end-products. The latter are believed to include cross-links that stiffen host tissues. However, diabetic animal models have also displayed weakened tendons with reduced stiffness. Strikingly, not a single experimentally identified specific molecular site of glycation in a collagen has been reported. Here, using targeted MS, we have identified partial fructosyl-hydroxylysine glycations at each of the helical domain cross-linking sites of type I collagen that are elevated in tissues from a diabetic mouse model. Glycation was not found at any other collagen lysine residues. Type I collagen in mouse tendons is cross-linked intermolecularly by acid-labile aldimine bonds formed by the addition of telopeptide lysine aldehydes to hydroxylysine residues at positions α1(I)Lys87, α1(I)Lys930, α2(I)Lys87, and α2(I)Lys933 of the triple helix. Our data reveal that site-specific glycations of these specific lysines may significantly impair normal lysyl oxidase-controlled cross-linking in diabetic tendons. We propose that such N-linked glycations can hinder the normal cross-linking process, thus altering the content and/or placement of mature cross-links with the potential to modify tissue material properties.

Downregulation of Glt25d1 aggravates carbon tetrachloride‑induced acute hepatic injury through activation of the TGF‑β1/Smad2 signaling pathway.

Collagen β (1-O) galactosyltransferase 1 (GLT25D1) has been reported to transfer galactose to hydroxylysine residues via β (1-O) linkages in collagen. The present study investigated the function of the collagen galactosyltransferase activity of GLT25D1 against carbon tetrachloride (CCl4)-induced acute liver injury in vitro. Glt25d1+/− mice and wild-type (WT) mice were injected intraperitoneally with the same dose of CCl4. The grade of hepatic injury and the extent of hepatocyte necrosis in the acute phase were assessed 48 h following CCl4 injection. Hepatocyte necrosis was evaluated by histological examination and by serum alanine aminotransferase and aspartate aminotransferase levels, which were higher in the Glt25d1+/− mice compared with those in the WT mice. Reverse transcription-quantitative polymerase chain reaction was performed, and the results demonstrated that the mRNA expression levels of inflammatory cytokines, including tumor necrosis factor-α and interleukin-6 were significantly increased in the Glt25d1+/− mice. Furthermore, western blot analyses were performed, and the results demonstrated that the protein levels of cleaved caspase-3 and −9 were also markedly increased in the Glt25d1+/− liver, indicating that hepatocyte apoptosis was induced. Additionally, the expression levels of transforming growth factor (TGF)-β1 and phosphorylated small mothers against decapentaplegic (Smad)2 were markedly upregulated, indicating activation of the TGF-β1/Smad2 signaling pathway during CCl4-induced acute liver injury in Glt25d1+/− mice. CCl4 administration also resulted in severe damage to Glt25d1+/− primary hepatocytes in vitro. Taken together, the downregulation of Glt25d1 deteriorated CCl4-induced liver injury in mice, which may involve triggering inflammatory responses, apoptosis and TGF-β1/Smad2 signaling pathway activation.

Insights into the structure and dynamics of lysyl oxidase propeptide, a flexible protein with numerous partners

Lysyl oxidase (LOX) catalyzes the oxidative deamination of lysine and hydroxylysine residues in collagens and elastin, which is the first step of the cross-linking of these extracellular matrix proteins. It is secreted as a proenzyme activated by bone morphogenetic protein-1, which releases the LOX catalytic domain and its bioactive N-terminal propeptide. We characterized the recombinant human propeptide by circular dichroism, dynamic light scattering, and small-angle X-ray scattering (SAXS), and showed that it is elongated, monomeric, disordered and flexible (Dmax: 11.7 nm, Rg: 3.7 nm). We generated 3D models of the propeptide by coarse-grained molecular dynamics simulations restrained by SAXS data, which were used for docking experiments. Furthermore, we have identified 17 new binding partners of the propeptide by label-free assays. They include four glycosaminoglycans (hyaluronan, chondroitin, dermatan and heparan sulfate), collagen I, cross-linking and proteolytic enzymes (lysyl oxidase-like 2, transglutaminase-2, matrix metalloproteinase-2), a proteoglycan (fibromodulin), one growth factor (Epidermal Growth Factor, EGF), and one membrane protein (tumor endothelial marker-8). This suggests new roles for the propeptide in EGF signaling pathway.

Characterisation of peroxidasin activity in isolated extracellular matrix and direct detection of hypobromous acid formation

Peroxidasin is a heme peroxidase that catalyses the oxidation of bromide by hydrogen peroxide to form an essential sulfilimine cross-link between methionine and hydroxylysine residues in collagen IV. We investigated cross-linking by peroxidasin embedded in extracellular matrix isolated from cultured epithelial cells and its sensitivity to alternative substrates and peroxidase inhibitors. Peroxidasin showed peroxidase activity as measured with hydrogen peroxide and Amplex red. Using a specific mass spectrometry assay that measures NADH bromohydrin, we showed definitively that the enzyme releases hypobromous acid (HOBr). Less than 1 μM of the added hydrogen peroxide was used by peroxidasin. The remainder was consumed by catalase activity that was associated with the matrix. Results from NADH bromohydrin measurements indicates that low micromolar HOBr generated by peroxidasin was sufficient for maximum sulfilimine cross-linking, whereas 100 μM reagent HOBr or taurine bromamine was less efficient. This implies selectivity for the enzymatic process. Physiological concentrations of thiocyanate and urate partially inhibited cross-link formation. 4-Aminobenzoic acid hydrazide, a commonly used myeloperoxidase inhibitor, also inhibited peroxidasin, whereas acetaminophen and a 2-thioxanthine were much less effective. In conclusion, HOBr is produced by peroxidasin in the extracellular matrix. It appears to be directed at the site of collagen IV sulfilimine formation but the released HOBr may also undergo other reactions.

Analysis of collagen and elastin cross-links.

Fibrillar collagens represent the most abundant extracellular matrix proteins in vertebrates providing tissues and organs with form, stability, and connectivity. For such mechanical functions, the formation of covalent intermolecular cross-linking between molecules is essential. This process, the final posttranslational modification during collagen biosynthesis, is initiated by conversion of specific lysine and hydroxylysine residues to the respective aldehydes by the action of lysyl oxidases. This conversion triggers a series of condensation reactions with the juxtaposed lysine-aldehyde, lysine, hydroxylysine, and histidine residues within the same and neighboring molecules resulting in di-, tri-, and tetravalent cross-links. Elastin, another class of extracellular matrix protein, is also stabilized by the lysyl oxidase-mediated mechanism but involving only lysine residues leading to the formation of unique tetravalent cross-links. This chapter presents an overview of fibrillar collagen cross-linking, and the analytical methods for collagen and elastin cross-links we have developed.