Lysyl oxidases

Cloning and characterization of the fourth and the fifth human lysyl oxidase isoenzymes, and the consequences of a targeted inactivation of the first described lysyl oxidase isoenzyme in mice

Joni Mäki

Collagen Research Unit, University of Oulu
Biocenter Oulu, University of Oulu
Department of Medical Biochemistry and Molecular Biology, University of Oulu

Abstract

Lysyl oxidases (EC 1.4.3.13, protein-lysine 6-oxidases) are extracellular copper enzymes that initiate the cross-linking of collagens and elastin by catalyzing oxidative deamination of the ε-amino group in certain lysine and hydroxylysine residues. The cross-links formed are responsible for the tensile strength of collagen fibers and the unique elastic properties of elastin.

Three human lysyl oxidase isoenzymes, lysyl oxidase (LOX), lysyl oxidase-like protein (LOXL), and lysyl oxidase-like 2 protein (LOXL2), have been identified and characterized so far. Two additional human lysyl oxidase isoenzymes, lysyl oxidase-like 3 (LOXL3) and lysyl oxidase-like 4 (LOXL4), proteins were identified, cloned, and partially characterized in this study. Both polypeptides showed a high degree of overall similarity to each other and to the LOXL2 polypeptide, whereas the two polypeptides showed a significant similarity to LOX and LOXL only in the C-terminal region, which contains all amino acid residues thought to be needed for the catalytic activity of the LOX enzyme. The LOXL3 gene is expressed in several tissues, the highest expression levels being in the placenta, heart, ovary, testis, small intestine, and spleen. The LOXL4 gene is likewise expressed in most human tissues studied, the highest levels being seen in the skeletal muscle, testis, and pancreas. Both polypeptides were shown to be secreted extracellular proteins.

The role of the first described LOX isoenzyme was studied by inactivating its gene in mice. Most Lox^-/- embryos died at the end of gestation, and the few live-born pups were cyanotic and died within a few hours, autopsy revealing large aortic aneurysms. Light microscopy demonstrated structural abnormalities in the aortic walls of Lox^-/- embryos, and further analysis by electron microscopy showed highly fragmented elastic fibers, discontinuity in the smooth muscle cell layers, and endothelial cell damage. Doppler ultrasonography of Lox^-/- embryos in utero revealed multiple signs of cardiovascular dysfunction, which contributed to the early death of the Lox^-/- mice. The results indicate that Lox has an essential role in the development and function of the cardiovascular system and that this role cannot be replaced to any significant extent by other lysyl oxidase isoenzymes.

Dedication

Table of Contents

Acknowledgements

Abbreviations

List of orginal articles

1. Introduction

2. Review of the literature

2.1. Collagens and elastin

2.1.1. The collagen family of proteins
2.1.2. Elastin

2.2. Cross-links in collagens and elastin

2.3. The lysyl oxidase isoenzyme LOX

2.3.1. Molecular properties
2.3.2. Catalytic properties
2.3.3. Regulation of LOX
2.3.4. Novel biological roles

2.4. Recently identified lysyl oxidase isoenzymes

2.4.1. Lysyl oxidase-like protein (LOXL)
2.4.2. Lysyl oxidase-like 2 protein (LOXL2)

2.5. Consequences of reduced lysyl oxidase activities

2.5.1. Nutritional copper deficiency
2.5.2. Menkes disease and occipital horn syndrome
2.5.3. Lathyrism

3. Outlines of the present research

4. Materials and methods

4.1. Cloning and characterization of the fourth and the fifth human lysyl oxidase isoenzymes (I, II)

4.1.1. Search for expressed sequence tags and high throughput genomic sequences (I, II)
4.1.2. Isolation of cDNA clones (I, II)
4.1.3. Characterization of the exon-intron organization of the LOXL3 gene (I)
4.1.4. Northern blot analysis (I, II)
4.1.5. Sequence analyses (I-III)
4.1.6. Recombinant expression of the LOXL3 and LOXL4 polypeptides (I, II)
4.1.7. Immunofluorescence staining of the recombinant LOXL3 protein (I)

4.2. Generation and analysis of a mouse strain lacking the Lox gene (III)

4.2.1. Generation of the mutant mice (III)
4.2.2. Light microscopy (III)
4.2.3. Transmission electron microscopy (III)
4.2.4. Ultrasonographic examination of the embryos (III)

5.1. The fourth and the fifth lysyl oxidase isoenzymes (I, II)

5.1.1. Molecular cloning of the cDNAs (I, II)
5.1.2. Molecular characterization of the cDNAs (I, II)
5.1.3. Exon-intron organization of the LOXL3 gene (I)
5.1.4. Expression of the mRNAs in various human tissues (I, II)
5.1.5. Recombinant expression of the LOXL3 and LOXL4 polypeptides in human HT-1080 cells (I, II)

5.2. Inactivation of the Lox gene leads to cardiovascular dysfunction in mice (III)

5.2.1. Generation of mice with an inactivated Lox gene (III)
5.2.2. Histological findings (III)
5.2.3. Ultrasonographic findings (III)

6. Discussion

6.1. Cloning and characterization of the fourth and the fifth lysyl oxidase isoenzymes (I, II)

6.1.1. Primary structures of the LOXL3 and LOXL4 polypeptides (I, II)
6.1.2. The structure of the LOXL3 gene, and expression of LOXL3 and LOXL4 mRNAs in various tissues (I, II)
6.1.3. Recombinant expression of the LOXL3 and LOXL4 polypeptides (I, II)

6.2. Lack of Lox activity leads to a severe cardiovascular dysfunction and perinatal death in mice (III)

7. Future perspectives

List of Tables
1. Transcriptional and posttranscriptional regulation of LOX.

List of Figures
1. Lysyl oxidase oxidatively deaminates a peptidyl lysine to generate a peptidyl α-aminoadipic-δ-semialdehyde (allysine), which spontaneously reacts with corresponding aldehydes to form various di-, tri-, or tetrafunctional cross-links (Kagan 1986).
2. Reactions of lysine and hydroxylysine in the biosynthesis of the cross-links in collagens (A) and elastin (B). After oxidative deamination to form reactive aldehydes, subsequent condensation reactions result in various bi-, tri-, and tetrafunctional cross-links. To simplify, intermediates in the reactions are not shown.
3. Pathway for LOX biosynthesis. The LOX precursor enters into the rough endoplasmic reticulum where its signal peptide is cleaved. The precursor is glycosylated within the endoplastic reticulum at an Asn residue located in the propeptide region. The addition of copper (Cu²⁺) and the formation of lysine tyrosylquinone (LTQ) cofactor (which are discussed in Section 2.3.2.1) may occur in the endoplastic reticulum or during protein trafficking through the Golgi elements. After secretion of the LOX precursor into the extracellular space, the propeptide region is cleaved by procollagen C-proteinase (PCP) between Gly-168 and Asp-169 to obtain an active 32 kDa enzyme. Molecular masses of LOX polypeptide are indicated according to rat LOX enzyme (see Trackman et al. 1990, 1991, 1992).
4. Reaction mechanism of LOX. The RNH substituent represents peptidyl lysine 314 in bovine LOX, the letter B represents a general base, LTQ represents lysine tyrosylquinone cofactor, and Lys^Ald represents the end product of the reaction, a lysine-derived aldehyde (modified from Smith-Mungo & Kagan 1998, and Akagawa & Suyama 2001).
5. The lysyl oxidase family. The predicted signal peptides are represented by black boxes, and four scavenger receptor cysteine-rich regions (SRCR) in LOXL2, LOXL3, and LOXL4, the propeptide region in LOX, and the proline-rich region in LOXL are also indicated. The sites of the putative copper-binding region (Cu), the lysine tyrosylquinone cofactor formation (LTQ), and the cytokine receptor-like domain (CRL) are highly conserved between all lysyl oxidase proteins. The number of amino acids in each protein is indicated on the right.
6. Structure of the aortic wall of the Lox^+/+ and Lox^-/- E18.5 embryos. Upper panels: Sections from the descending aorta at the site of the right crus of the diaphragm, stained with hematoxylin-eosin and illuminated in UV light. The lumen of the aorta is marked with an asterisk, and the single intimal elastic lamella is boxed. Lower panels: Electron micrographs from the site of a single elastic fiber from the boxed sections in upper panels: Amorphous elastin (aEL) in a continuous elastic fiber is seen in the Lox^+/+ aortic wall (left). Despite the high degree of fragmentation of the elastic fiber, the remnants of the amorphous elastin (arrows) are still seen in the Lox^-/- aortic wall (right).

		Next
		Acknowledgements

Homepage of this publication

Library Units | Collections | Databases | Library News | Library Services | Electronic Collection | Links elsewhere | Alphabetical Index

© 2002 Oulu University Library