Purification, properties and influence of dietary copper on accumulation and functional activity of lysyl oxidase in rat skin

Romero-Chapman, Nadia; Lee, J.; Tinker, David O.; Uriu‐Hare, Janet Y.; Keen, Carl L.; Rucker, Robert R.

doi:10.1042/bj2750657

Cited by 45 publications

(33 citation statements)

References 41 publications

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“…By SDS-PAGE, recombinant human LO co-migrates with purified porcine LO with an apparent molecular mass of 32 kDa, in agreement with the apparent molecular mass of tissue-derived LO purified from several species [1,27,39]. The predicted molecular mass of the mature form of human LO, based on cDNA data, is 29 034 Da.…”

Section: Discussionsupporting

confidence: 67%

Expression of active, human lysyl oxidase in Escherichia coli

1996

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Lysyl oxidase (LO) is a copper amine oxidase of the e~,tracellular matrix which initiates covalent cross-linking in collagens and elastin. Human LO was expressed in Escherichia coli. At 37°C, large amounts of protein were obtained, but in the form of insoluble aggregates. Lowering the growth temperature, and reducing the amount of inducer, resulted in the production of soluble LO, which was active on a [3H]lysine-laheled elastin substrate. LO was also targeted to the periplasm as a fusion protein with the pelb signal peptide. The periplasmic enzyme was soluble, active and inhibited by [$-aminopropionitrile. Production of the carbonyl co-factor is therefore not a limitation in the expression of active LO in bacteria.Aey words: Lysyl oxidase; Copper amine oxidase; Protein e~pression; Bacteria I IntroductionThe formation of lysine-(or hydroxylysine-) derived covaknt cross-links in collagens and elastin is essential for the s ructural integrity and function of connective tissues [1,2]. q he enzyme that initiates cross-linking, lysyl oxidase (EC 1 4,3.13), is a copper-dependent amine oxidase of the extraczllular matrix that oxidatively de-aminates the ~-amino roups of specific lysine (and hydroxylysine) residues, leading t ~ the spontaneous formation of a number of bi-functional, t i-functional and tetra-functional crosslinks [3]. As the tervfinal enzymatic step in the biosynthetic pathway of collagens ~nd elastin, lysyl oxidase (LO) is a potential target in the 9ntrol of fibrotic disease. LO has also been implicated in 11mor suppression, as the product of the ras recision gene after oncogenic transformation of mouse fibroblasts [4]. In ~:ddition, the enzyme has been shown to be a potent chemo-1 tctic agent for unstimulated human peripheral blood monot ytes [5]. Lysyl oxidase (29 kDa) is secreted in precursor form (50 Da), and conversion to the mature form of the enzyme octurs by proteolytic removal of an N-terminal propeptide [6]. We have recently identified [7] the propeptide cleavage site in t,orcine pro-LO as gly-asp, which corresponds to residues 68-169 in the human LO precursor. This sequence also cor-~,~sponds to the C-terminal processing site of fibrillar collagen l recursors by procollagen C-proteinase, the enzyme that has ~ecently been shown to be the same as bone morphogenetic t,rotein-I (BMP-1) [8,9]. The sequence data suggest that BMPmay be required for processing of both pro-LO and the precursor forms of its collagen substrates The nature of the carbonyl co-factor in lysyl oxidase has been the subject of much controversy [11]. Trihydroxyphenylalanine (topa) quinone has been shown to be the co-factor in a number of copper-dependent amine oxidases from E. co~i, yeast, plants and mammals [11 13], where the presence of the Asn-Tyr-Asp consensus sequence appears to be sufficient for the spontaneous conversion of polypeptide bound tyrosine to topaquinone, in the presence of copper and oxygen [12]. This consensus sequence is not present in LO. Very recently, it has been demonstrated [14] that LO cont...

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Section: Discussionsupporting

confidence: 67%

Expression of active, human lysyl oxidase in Escherichia coli

1996

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“…Taken together these studies show that lysyl oxidase activity correlates well with the presence of the 32-kDa molecular form of lysyl oxidase, and not with the 50-kDa molecular form. It is of interest, however, that an active 45-kDa lysyl oxidase has been demonstrated in rat skin tissues (38).…”

Section: Extracellular Proteolytic Conversion Of Lysyl Oxidase Precurmentioning

confidence: 99%

Pre- and Post-translational Regulation of Lysyl Oxidase by Transforming Growth Factor-β1 in Osteoblastic MC3T3-E1 Cells

Feres-Filho

Choi

Han

et al. 1995

Journal of Biological Chemistry

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The final enzymatic step required for collagen crosslinking is the extracellular oxidative deamination of peptidyl-lysine and -hydroxylysine residues by lysyl oxidase. A cross-linked collagenous extracellular matrix is required for bone formation. The goals of this study were to compare the transforming growth factor (TGF)-␤1 regulation of lysyl oxidase enzyme activity and steady state mRNA levels to changes in COL1A1 mRNA levels in MC3T3-E1 osteoblastic cells. TGF-␤1 increased steady state lysyl oxidase and COL1A1 mRNA levels in a dose-and time-dependent manner. The increase in lysyl oxidase mRNA levels was transient, peaking at 12 h and 8.8 times controls in cells treated with 400 pM TGF-␤1. COL1A1 steady state mRNA levels increased maximally to 3.5-fold of controls. Development of increased lysyl oxidase enzyme activity was delayed and was of slightly lower magnitude than the increase in its mRNA levels. This suggested limiting post-translational processing of lysyl oxidase proenzyme. Pulse-labeling/immunoprecipitation studies demonstrated slow proenzyme secretion and proteolytic processing. Development and application of an independent assay for lysyl oxidase proenzyme proteolytic processing activity verified its proportionately lower stimulation by 400 pM TGF-␤1. Thus, lysyl oxidase regulation by TGF-␤1 in osteoblastic cell cultures occurs at both pre-and post-translational levels. This regulation is consistent with increased production of a collagenous extracellular matrix.Lysyl oxidase is the extracellular enzyme that catalyzes the oxidative deamination of lysine and hydroxylysine residues in collagen and elastin precursors to form peptidyl-␣-aminoadipic-␦-semialdehyde, or peptidyl-␦-hydroxy-␣-aminoadipic-␦-semialdehyde, respectively. Spontaneous condensation reactions of these resultant aldehydes leads to the formation of lysine-derived cross-links found in mature collagens and elastin (1).A cross-linked collagen matrix is required for differentiation of osteoblastic cells and for bone mineralization (2). Type I collagen synthesis and accumulation have been shown to be uncoupled processes in developing MC3T3-E1 osteoblastic cells (3). These cells accumulated collagen in the extracellular matrix when the rate of collagen synthesis was decreasing. This suggested post-translational control of collagen fibril accumulation. Similarly, in primary rat calvaria osteoblast cell cultures, TGF-␤1

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“…'~J However, dyschondroplastic cartilage taken from broilers with homocysteine-induced T D had higher levels of collagen cross-links than did normal growth plate.lo9 Homocysteine can partially inhibit lysyl oxidase and not decrease collagen cross-linking, as found in rat skin studies in which lysyl oxidase was not the rate limiting factor in cross-link formation. 126 Other work showed that 1% homocysteine diet in rats caused a copper deficiency.I8 Because a copper deficiency will induce TD, it was hypothesized that a high homocysteine diet in broiler chicks could lead to a copper deficiency, which in turn causes TD. Although Vet Pathol 31:4, 1994 the broilers fed a high-homocysteine diet did not show signs of a copper deficiency (i.e., normal hematocrit and plasma copper concentration), addition of extra copper (250 mglkg) to the diet slightly but significantly reduced the severity of the lesion.…”

Section: Thiram and Antabusementioning

confidence: 99%

Avian Tibial Dyschondroplasia: A Morphological and Biochemical Review of the Growth Plate Lesion and Its Causes

Orth

Cook

1994

Vet Pathol

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Abstract. Avian tibial dyschondroplasia is a disease found in fast growing strains of chickens, ducks, and turkeys worldwide in which growth plate cartilage accumulates in the metaphyseal region of the tibiotarsus; it is similar to mammalian osteochondrosis. Several biochemical and pathologic studies have shown that the growth plate chondrocytes do not reach their expected size in the hypertrophic zone and necrose prematurely. The chondrocytes also produce decreased amounts of extracellular proteins, such as collagen X and fibroblast growth factor-0, that are necessary for cartilage maturation. This immature cartilage becomes highly crosslinked in the collagen molecules and apparently resistant to resorption and vascularization by the metaphyseal vessels. The dyschondroplastic cartilage remains in the metaphysis for several weeks. Not until the growth rate of the birds slows down is the cartilage able to be resorbed and replaced by trabecular bone. Many conditions have been found to induce tibial dyschondroplasia, including copper deficiency; fusarochromanone, thiram, and antabuse intoxication; excessive dietary levels of cysteine and homocysteine; metabolic acidosis; and bird rearing environment. However, the mechanism(s) by which these various methods induce tibial dyschondroplasia is presently not known. Current research is focusing on understanding the development of the disease and whether or not all these methods work by the same physiological chain of events. Recent biochemical evidence suggests that a copper deficiency might be caused by a different mechanism than genetically and thiraminduced tibial dyschondroplasia.Key words: Chickens; copper deficiency; dyschondroplasia; growth plate; homocysteine; osteochondrosis.Endochondral ossification describes the processes by which bones grow longitudinally and several types of fractures heal. Longitudinal growth occurs in the growth plate that links the epiphyseal and the metaphyseal regions within bone. The main physiological events of endochondral ossification are chondrocyte proliferation, extracellular matrix production, chondrocyte hypertrophy, matrix calcification, vascular invasion, matrix degradation, and primary bone formation.The main components of the growth plate include metaphyseal bone, a fibrous peripheral structure, and a layer of cartilage called the physis. The metaphysis functions include 1) vascular penetration into the bottom of the physis, 2) bone formation, and 3) remodeling of calcified cartilage and bone to form trabecular bone.I4 The fibrous peripheral structure is composed of the ossification groove, which provides chondrocytes for latitudinal expansion of the growth plate, and the perichondrial ring, which provides mechanical support for the tenuous chondro-osseous j u n c t i~n . '~~> l~~ The growth plate cartilage is responsible for the rate of growth and actual length of the bone and can be divided into three principal zones: the reserve, proliferative, and hypertrophic zones. Reserve zoneThe reserve zone lies either adjacent to ...

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Purification, properties and influence of dietary copper on accumulation and functional activity of lysyl oxidase in rat skin

Cited by 45 publications

References 41 publications

Expression of active, human lysyl oxidase in Escherichia coli

Expression of active, human lysyl oxidase in Escherichia coli

Pre- and Post-translational Regulation of Lysyl Oxidase by Transforming Growth Factor-β1 in Osteoblastic MC3T3-E1 Cells

Avian Tibial Dyschondroplasia: A Morphological and Biochemical Review of the Growth Plate Lesion and Its Causes

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