A novel human lysyl oxidase-like gene (LOXL4) on chromosome 10q24 has an altered scavenger receptor cysteine rich domain.

We have identified a novel 14-exon human lysyl oxidase-like gene, LOXL4, on chromosome 10q24. The cDNA and derived amino acid sequence of LOXL4 demonstrates a conserved C-terminal region including the characteristic copper-binding site, lysyl and tyrosyl residues and a cytokine receptor-like domain. One of the four N-terminal SRCR domains contains a 13 amino acid insertion encoded by a short exon not present within the closely homologous LOXL2 and LOXL3 genes. The 3.5-kb LOXL4 mRNA is present in pancreas and testis and at lower levels in several other tissues. Fibroblasts, smooth muscle and osteosarcoma (HOS) cells express LOXL4. No expression was detected in HCT-116 and DLD-1 colon, MCF-7 breast and DU-145 prostate cancer cell lines.     

The LOXL2 gene encodes a new lysyl oxidase-like protein and is expressed at high levels in reproductive tissues

We have reported in this paper the complete cDNA sequence, gene structure, and tissue-specific expression of LOXL2, a new amine oxidase and a member of an emerging family of human lysyl oxidases. The predicted amino acid sequence, from several overlapping cDNA clones isolated from placenta and spleen cDNA libraries, shared extensive sequence homology with the conserved copper-binding and catalytic domains of both lysyl oxidase (LOX) and the lysyl oxidase-like (LOXL) protein. These conserved domains are encoded by five consecutive exons within the LOX, LOXL, and LOXL2 genes that also maintained exon-intron structure conservation. In contrast, six exons encoding the amino-terminal domains diverged both in sequence and structure. Exon 1 of the LOXL2 gene does not encode a signal sequence that is present in LOX and LOXL, suggesting a different processing and intracellular localization for this new protein. Expression of the LOXL2 gene was detected in almost all tissues with the highest steady state mRNA levels in the reproductive tissues, placenta, uterus and prostate. In situ hybridization identified placental syncytial and cytotrophoblasts responsible for the synthesis of LOXL2 mRNA and demonstrated a spatial and temporal expression pattern unique to the LOXL2 gene.     

Cloning and characterization of a fifth human lysyl oxidase isoenzyme: the third member of the lysyl oxidase-related subfamily with four scavenger receptor cysteine-rich domains.

We report the complete cDNA sequence of the human lysyl oxidase-like 4 (LOXL4) gene, a new member of the lysyl oxidase (LO) gene family. The predicted polypeptide is 756 amino acids long, including a 24-residue signal peptide. The C-terminal region contains a LO domain similar to those of LOX, LOXL, LOXL2 and LOXL3. The N-terminal region has four subregions similar to scavenger receptor cysteine-rich domains that are highly conserved with LOXL2 and LOXL3. The LOXL4 mRNA is approximately 4 kb in size and is expressed in many tissues, the highest levels among the tissues studied being in the skeletal muscle, testis and pancreas. Recombinant LOXL4 expressed in HT-1080 cells was secreted into the culture medium with no evident proteolytic processing.     

Cloning and characterization of a human lysyl oxidase-like 3 gene (hLOXL3).

Using the PCR primers generated from human expressed sequence tag (EST), the cDNA of lysyl oxidase-like gene 3 (LOXL3), a new member of human lysyl oxidases gene family, was cloned from the human fetal brain mRNA. The predicted amino acid sequence of the hLOXL3 gene was highly homologous to mLOR2. Bioinformatics analysis shows that hLOXL3 protein is also a member of the scavenger receptor cysteine-rich family, which contains a 25 amino acids signal peptide. The hLOXL3 gene was mapped to human 2p13 locus by BLAST search and at least 14 exons were found. Expression of the hLOXL3 gene was detected in several human tissues and especially high in spleen and testis.     

类赖氨酰氧化酶2与肿瘤转移和发生

类赖氨酰氧化酶2（lysyl oxidase—like 2,LOXL2）是赖氨酰氧化酶（1ysyl oxidase,LOX）基因家族的成员之一,其表达产物能促进胶原沉积。LOXL2的过表达能促进纤维化,并与肿瘤侵袭、转移及不良预后有关。目前大部分学者认为LOXL2是一种转移促进基因,也有实验支持其是一种肿瘤抑制基因。研究发现LOXL2可以通过激活Snail／Ecadherin通路或Src／FAK通路促进转移。LOXL2有望作为肿瘤生物标志物,用于预后判断,成为一个新的治疗靶点。     

A third neurofibromatosis type 1 (NF1) pseudogene at chromosome 15q11.2

Sequences related to the neurofibromatosis type 1 (NF1) gene have been identified on several human chromosomes. In the centromeric region of chromosomes 14 and 15, two NF1 pseudogenes have been described. Sequence comparison between NF1-related exons amplified from two yeast artificial chromosome clones hybridizing to chromosomal region 15q11.2 and published NF1-related sequences localized at 15q11.2 suggested that a third NF1 pseudogene resides in this chromosomal region. The previous localization of an NF1-related locus to the telomeric part of chromosome 15 could not be confirmed by us. Our findings further support pericentromeric spreading of partial NF1 gene copies at chromosome 15q11.2 during evolution. Received: 27 January 1996 / Accepted: 26 May 1997     

Coexpression of the lysyl oxidase-like gene (LOXL) and the gene encoding type III procollagen in induced liver fibrosis

We have isolated a mouse lysyl oxidase-like (LOXL) cDNA from a mouse embryo cDNA library and used this cDNA to measure changes in steady state levels of LOXL mRNA during the development of carbon tetrachloride-induced liver fibrosis in adult mice. These results revealed the coincident appearance of increased steady state levels of LOXL mRNA and type III procollagen mRNA early in the development of liver fibrosis. In contrast, steady state levels of lysyl oxidase mRNA increased throughout the onset of hepatic fibrosis and appeared in parallel with the increased steady state levels of pro-alphaI (I) collagen mRNA. These findings suggest that the LOXL protein (possibly an isoform of lysyl oxidase) is involved in the development of lysine-derived cross-links in collagenous substrates. Moreover, the substrate specificity of the LOXL protein may be different to that of lysyl oxidase and this difference may be collagen-type specific.     

Novel stop and frameshifting mutations in the autosomal dominant polycystic kidney disease 2 (PKD2) gene

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most frequent inherited disorders. The majority of cases are due to mutation of the PKD1 gene, on 16p13.3, while in most of the remainder the disease maps to the PKD2 locus, at chromosome 4q21-q23. Recently, the PKD2 gene has been positionally cloned and three nonsense mutations within the coding sequence of the gene identified. Here we report a systematic mutation screening of all 15 exons of the PKD2 gene in chromosome 4-linked ADPKD families, using heteroduplex and SSCP analyses. We have identified and characterized seven novel mutations, with a detection rate of approximately 90% in the population studied. All of the mutations result in the premature stop of translation: four nonsense changes and three deletions. The deletions are all frameshifting, of four T nucleotides in one case and one G nucleotide in the other two. All mutations are unique and are distributed throughout the gene without evidence of clustering. Comparison of specific mutations with the clinical profile in ADPKD2 families shows no clear correlation. Received: 5 April 1997 / Accepted: 31 July 1997     

人LOXL2启动子的鉴定与初步分析

LOXL2（lysyl oxidase like 2）是赖氨酰氧化酶（LOX）家族的一个重要成员,不仅可促进细胞外基质中胶原蛋白和弹性蛋白的交联,而且在转录调控、细胞信号转导以及细胞粘附等生物学过程中也有重要作用。多篇研究表明,LOXL2在多种肿瘤中高表达,且与多种肿瘤细胞的增殖迁移等生物学行为密切相关。LOXL2的表达调控机制目前仍不清楚。为了进一步研究LOXL2的转录调控机制,本研究克隆鉴定了LOXL2的启动子。首先通过数据库对LOXL2基因结构及潜在启动子区域进行了分析,进而以人的基因组DNA为模板,通过PCR定向克隆策略,构建了5个长度不同并覆盖LOXL2基因转录起始位点附近约1.7 kb的LOXL2基因启动子荧光素酶报告基因重组体。启动子活性分析结果表明,与对照组相比,5个重组体均具有启动子活性(P<0.05),提示LOXL2基因核心启动子定位于转录起始位点附近约185 bp的区域内。转录因子结合位点分析结果表明,LOXL2基因启动子缺乏典型的TATA盒,但含有GC盒以及Sp1、NFkB等潜在的转录因子结合位点。外源转染Sp1表达质粒能显著增强LOXL2基因启动子的活性（P<0.05）,提示Sp1能直接激活LOXL2的转录。     

Lysyl oxidase-like 2 (LOXL2) controls tumor-associated cell proliferation through the interaction with MARCKSL1

Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase gene family that contributes to the invasiveness and metastasis in tumor progression. However, the role of LOXL2 in cellular signaling is incompletely understood. In this study, we investigated a possible mechanism of LOXL2 function in tumor metastases in vitro, using a human breast carcinoma cell line. Myristoylated alanine-rich C kinase substrate-like 1 (MARCKSL1), a modulator in the regulation of cellular homeostasis, was identified as a LOXL2 interacting protein. We examined the binding domains that are required for the interaction between LOXL2 and MARCKSL1. The scavenger-receptor domain of LOXL2 was shown to interact with the N-terminal domain of MARCKSL1. Luciferase activity was noticeably reduced by the transfection of MARCKSL1 in a dose-dependent manner. In addition, over-expression of LOXL2 activates cell growth by inhibiting MARCKSL1-induced apoptosis. The effect of LOXL2 on cell cycle and apoptosis-related components was also confirmed through the silencing of LOXL2 expression. LOXL2 activates the FAK/Akt/mTOR signaling pathways, and MARCKSL1 suppresses LOXL2-induced oncogenesis. These insights supply evidence that LOXL2 promotes cell proliferation and inhibits apoptotic cell death. Taken together, our results indicate an underlying mechanism for an increase of LOXL2-related activity in breast tumor cells.     

The human lysyl oxidase-like 2 protein functions as an amine oxidase toward collagen and elastin

The lysyl oxidase-like 2 (LOXL2) protein is a human paralogue of lysyl oxidase (LOX) that functions as an amine oxidase for formation of lysine-derived cross-links found in collagen and elastin. In addition to the C-terminal domains characteristic to the LOX family members, LOXL2 contains four scavenger receptor cysteine-rich (SRCR) domains in the N-terminus. In order to assess the amine oxidase activity of LOXL2, we expressed a series of recombinant LOXL2 proteins with deletions in the SRCR domains, using an Escherichia coli expression system. All of the purified recombinant LOXL2 proteins, with or without the SRCR domains in the N-terminus, showed significant amine oxidase activity toward several different types of collagen and elastin in in vitro amine oxidase assays, indicating deletion of the SRCR domains does not interfere with amine oxidase activity of LOXL2. Further, amine oxidase activity of LOXL2 was not susceptible to inhibition by β-aminopropionitrile, an irreversible inhibitor of LOX, suggesting a different enzymatic mechanism between these two paralogues.     

Structural and functional diversity of lysyl oxidase and the LOX-like proteins

Lysyl oxidase (LOX) and four lysyl oxidase-like proteins, LOXL, LOXL2, LOXL3 and LOXL4, each contain a copper binding site, conserved lysyl and tyrosyl residues that may contribute to quinone co-factor formation, and a cytokine receptor-like domain. Each protein differs mainly in their N-terminal sequence, which may confer individual functions. Processing of the LOX proteins by BMP-1 and possibly other mechanisms may result in multiple functional forms. Splicing, reported for LOXL3, may also generate additional variants with unique functions. Each LOX, with its individual, developmentally regulated tissue and cell-specific expression and localization, results in a complex structural and functional variation for the LOX amine oxidases. The presence of only two LOX-like proteins in Drosophila, each with distinct spatial and temporal expression, allows for the assignment of individual function to one of these amine oxidases. Comparative expression analysis of each LOX protein is presented to help determine their functional significance.     

A tissue-specific variant of the human lysyl oxidase-like protein 3 (LOXL3) functions as an amine oxidase with substrate specificity

The human lysyl oxidase-like 3 (LOXL3) encodes a member of the emerging family of lysyl oxidase (LOX) that functions as a copper-dependent amine oxidase. The LOXL3 protein contains four scavenger receptor cysteine-rich domains in the N terminus in addition to the C-terminal characteristic domains of the LOX family, such as a copper binding domain, a cytokine receptor-like domain and residues for the lysyl-tyrosyl quinone cofactor. Using BLASTN searches, we identified a LOXL3 variant LOXL3-sv1 that lacked the sequences corresponding to exons 1, 2, 3, and 5 of LOXL3. LOXL3-sv1 showed an exon-intron structure distinct from LOXL3, additionally containing an 80-bp sequence corresponding to intron 3 of LOXL3 in the 5'-UTR and a 561-bp sequence corresponding to the 3'-flanking genomic region of exon 14 in the 3'-UTR. LOXL3-sv1 was predicted to encode a polypeptide of 392 amino acids that contains the C-terminal domains required for amine oxidase activity but lacks the N-terminal SRCR domains 1, 2, and 3. The recombinant LOXL3-sv1 protein showed a beta-aminopropionitrile-inhibitable amine oxidase activity toward elastin and collagen with substrate specificity. In RT-PCR assays with various human tissues, LOXL3-sv1 and LOXL3 showed distinct expression patterns. Further, luciferase reporter assays revealed a strong promoter element in intron 3 that probably functions as a regulatory region for the expression of LOXL3-sv1. These findings strongly indicate that LOXL3 encodes two variants, LOXL3 and LOXL3-sv1, both of which function as amine oxidases with distinct tissue and substrate specificities from one another.     

Genetic mapping of the mouse ferritin light chain gene and 11 pseudogenes on 11 mouse chromosomes

We typed the progeny of two sets of genetic crosses to determine the map locations for loci containing sequences related to the ferritin light chain (Ft11) gene. Twelve loci were positioned on 11 different chromosomes. One of these genes mapped to a position on Chr 7 predicted to contain the expressed gene on the basis of the previously determined position of the human homolog on 19q13.3-q13.4. Received: 23 July 1997 / Accepted: 20 September 1997     

Post-translational modifications of collagen upon BMP-induced osteoblast differentiation

The pattern of collagen cross-linking is tissue specific primarily determined by the extent of hydroxylation and oxidation of specific lysine residues in the molecule. In this study, murine pre-myoblast cell line, C2C12 cells, were transdifferentiated into osteoblastic cells by bone morphogenetic protein (BMP)-2 treatment, and the gene expression of lysyl hydroxylases (LH1, 2a/b, and 3) and lysyl oxidase (LOX)/lysyl oxidase-like proteins (LOXL1-4), and the extent of hydroxylysine were analyzed. After 24 h of treatment, the expression of most isoforms were upregulated up to 96 h whereas LH2a and LOXL2 decreased with time. In the treated cells, both hydroxyproline and hydroxylysine were detected at day 7 and increased at day 14. The ratio of hydroxylysine to hydroxyproline was significantly increased at day 14. The results indicate that LHs and LOX/LOXLs are differentially responsive to BMP-induced osteoblast differentiation that may eventually lead to the specific collagen cross-linking pattern seen in bone.     

Exosomes from hypoxic endothelial cells have increased collagen crosslinking activity through up‐regulation of lysyl oxidase‐like 2

Exosomes are important mediators of intercellular communication. Additionally, they contain a variety of components capable of interacting with the extracellular matrix (ECM), including integrins, matrix metalloproteinases and members of the immunoglobin superfamily. Despite these observations, research on exosome‐ECM interactions is limited. Here, we investigate whether the exosome‐associated lysyl oxidase family member lysyl oxidase‐like 2 (LOXL2) is involved in ECM remodelling. We found that LOXL2 is present on the exterior of endothelial cell (EC)‐derived exosomes, placing it in direct vicinity of the ECM. It is up‐regulated twofold in EC‐derived exosomes cultured under hypoxic conditions. Intact exosomes from hypoxic EC and LOXL2 overexpressing EC show increased activity in a fluorometric lysyl oxidase enzymatic activity assay as well as in a collagen gel contraction assay. Concordantly, knockdown of LOXL2 in exosome‐producing EC in both normal and hypoxic conditions reduces activity of exosomes in both assays. Our findings show for the first time that ECM crosslinking by EC‐derived exosomes is mediated by LOXL2 under the regulation of hypoxia, and implicate a role for exosomes in hypoxia‐regulated focal ECM remodelling, a key process in both fibrosis and wound healing.