|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|
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Isoenzymes are distinct forms with the same catalytic activity, differing in their physical and chemical properties. They can be present in different tissues, cell types, subcellular compartments, or developmental stages of the same organism, or in different organisms. In past decade, four lysyl oxidase isoenzymes have been characterized in addition to LOX; lysyl oxidase-like protein (LOXL) (Kenyon et al. 1993, Kim et al. 1995), lysyl oxidase-like 2 protein (LOXL2) (Saito et al. 1997, Jourdan-Le Saux et al. 1999), lysyl oxidase-like 3 protein (LOXL3) (paper I of the present study and Jang et al. 1999, Huang et al. 2001, Jourdan-Le Saux et al. 2001), and lysyl oxidase-like 4 protein (LOXL4) (paper II of the present study and Ito et al. 2001, Asuncion et al. 2001). Polypeptides of all these isoenzymes are highly conserved within their C-terminal ends, which include the copper-binding sites, cytokine receptor-like domains, and lysine tyrosylquinone cofactor sites (see Csiszar 2001 for review).
The emerging members of the lysyl oxidase gene family raise many questions concerning the role of each member with respect to altered lysyl oxidase activities in nutritional copper-deficiency, Menkes and occipital horn diseases, lathyrism, tumor suppression, and fibrotic tissues. The substrate or tissue specificity may vary among these five family members, although no strong evidence for this has been presented so far. The isoenzymes LOXL3 and LOXL4 identified in the present study will be discussed in detail in Chapters 4, 5, and 6.
Kenyon et al. (1993) characterized a novel human cDNA with a predicted protein similar to LOX. This protein was named lysyl oxidase-like protein (LOXL). The LOXL cDNA corresponds to a single polyadenylated mRNA species of 2.5 kb, which shows a concomitant expression with LOX mRNA in several human tissues (Kim et al. 1995). The LOXL polypeptide consists of 574 amino acids, with a calculated molecular mass of 63 kDa. It shows 76% identity with LOX at its C-terminal region (Kenyon et al. 1993, Kim et al. 1995), which includes the copper-binding site and the four histidine residues within the conserved sequence (WEWHSCHQHYH) that are involved in the copper binding coordination complex. Furthermore, a growth factor and cytokine receptor-like sequence are present in this region in both the LOX and LOXL polypeptides. This highly homologous region encompasses the mature 32 kDa form of LOX (Kim et al. 1995). The LOXL gene contains 7 exons and has been mapped to chromosome 15q24 in humans (Szabo et al. 1997) and chromosome 9 in mice (Tchernev et al 1997, Wydner et al. 1997).
The LOXL protein has been identified as a secreted protein that is expressed in the extracellular matrix in active fibrotic diseases and in the early stromal reaction of breast cancer (Decitre et al. 1998). Coincident appearance of increased steady-state levels of LOXL and COL3A1 mRNAs was detected in the early development of liver fibrosis, suggesting that the LOXL protein is involved in the development of lysine-derived cross-links in collagenous substrates. In contrast, steady-state levels of LOX mRNA were increased throughout the onset of hepatic fibrosis and appeared in parallel with increased stedy-state levels of COL1A1 mRNA (Kim et al. 1999). A specific antibody against LOXL has been used to identify proteins immunochemically distinct from LOX in various cells and in bovine aorta. These proteins had molecular weights of approximately 68, 52, 42, and 30 kDa (Decitre et al. 1998). Recently, Borel et al. (2001) isolated and characterized a 56 kDa LOXL protein from bovine aorta. This LOXL precursor was cleaved by a furin-like activity and was largely inactive. However, further processing by BMP-1 in vitro led to an enzyme that was active on elastin and collagen substrates (Borel et al. 2001).
Lysyl oxidase-like 2 protein (LOXL2) was orginally cloned, characterized, and named WS9-14 by Saito et al. (1997) for its possible association with Werner syndrome, which is characterized by premature aging (Murano et al. 1991). However, it was subsequently reported that WS9-14 mRNA corresponds to LOXL2 mRNA, with the exception that the WS9-14 transcript encodes one additional scavenger receptor cysteine-rich domain (SRCR) in its 5’end region (Jourdan-Le Saux et al. 1999, Csiszar 2001).
The LOXL2 mRNA encodes an 87 kDa polypeptide, which contains four SRCR domains that are found in several secreted and cell surface proteins (Saito et al. 1997). The LOXL2 polypeptide shares a 48% identity with the LOX polypeptide in its amino acid sequence from residue 546 to 751 in its C-terminal region. This region contains all conserved amino acid sequences needed for the proper function of the mature 32 kDa form of the LOX enzyme (Saito et al. 1997, Jourdan-Le Saux et al. 1999). The LOXL2 gene has been mapped to chromosome 8p21 in humans (Jourdan Le Saux et al. 1998) and 14 in mice (Jourdan-Le Saux et al. 2000), and it consists of at least 11 exons (Jourdan-Le Saux et al. 1999).
LOXL2 is abundantly expressed in senescent fibroblasts and several adherent tumour cell lines, but is down-regulated in several nonadherent tumour cells. This suggests that it may be involved in cell adhesion and that a loss of this protein may be associated with the loss of tumour cell adhesion. LOXL2 may, therefore, play a role in metastasis (Saito et al. 1997). LOXL2 shows similar spatial expression with LOX and LOXL in human placenta and fetal tissues in early pregnancy. However, this pattern diverges during gestation (Hein et al. 2000). In full-term human placenta, LOX is expressed predominantly in the amniotic epithelium, with little expression in the placenta, while LOXL shows the highest expression in the placenta and lowest expression in the amnion. LOXL2 expression differs in that it is detected predominantly in chorionic cytotrophoblasts of the membranes with only low expression levels in the amnion and placenta (Hein et al. 2000, see also Casey & MacDonald 1997, Jourdan-Le Saux 1999).