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。     

Human lysyl oxidase-like 2

Lysyl oxidase like-2 (LOXL2) belongs to the lysyl oxidase (LOX) family, which comprises Cu²⁺- and lysine tyrosylquinone (LTQ)-dependent amine oxidases. LOXL2 is proposed to function similarly to LOX in the extracellular matrix (ECM) by promoting crosslinking of collagen and elastin. LOXL2 has also been proposed to regulate extracellular and intracellular cell signaling pathways. Dysregulation of LOXL2 has been linked to many diseases, including cancer, pro-oncogenic angiogenesis, fibrosis and heart diseases. In this review, we will give an overview of the current understandings and hypotheses regarding the molecular functions of LOXL2.     

Expression of lysyl oxidase isoforms in MC3T3-E1 osteoblastic cells

Covalent intermolecular cross-linking of collagen is initiated by the action of lysyl oxidase (LOX) on the telopeptidyl lysine and hydroxylysine residues. Recently, several LOX isoforms, i.e., LOX-like proteins 1-4 (LOXL1-4), have been identified but their specific tissue distribution and functions are still largely unknown. In this study, mRNA expression of LOX and LOXL1-4 in MC3T3-E1 osteoblastic cells was screened by RT-PCR and quantitatively analyzed by real-time PCR during cell differentiation and matrix mineralization. The results demonstrated that LOX and all LOXLs, except LOXL2, were expressed in this cell line and that the expression pattern during cell differentiation and matrix mineralization was distinct from one another. This indicates that the expression of LOX and its isoforms is highly regulated during osteoblast differentiation, suggesting their distinct roles in collagen matrix stabilization and subsequent mineralization.     

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。     

Purification of enzymatically active human lysyl oxidase and lysyl oxidase-like protein from Escherichia coli inclusion bodies

Lysyl oxidase (LOX) is an extracellular copper dependent enzyme catalyzing lysine-derived cross-links in extracellular matrix proteins. Recent molecular cloning has revealed the existence of a LOX family consisting of LOX and four lysyl oxidase-like proteins (LOXLs; LOXL, LOXL2, LOXL3, and LOXL4). Each member of the LOX family contains a copper-binding domain, residues for lysyl-tyrosyl quinone, and a cytokine receptor-like domain. Very recently, novel functions, such as tumor suppression, cellular senescence, and chemotaxis, have been attributed to this family of amine oxidases, but functional differences among the family members have yet to be determined. For efficient expression and purification, we cloned the cDNAs corresponding to proteolytically processed forms of LOX (LOX-p) and LOXL (LOXL-p1 and LOXL-p2) into a bacterial expression vector pET21a with six continuous histidine codons attached to the 3^′ of the gene. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis in the presence of N-lauroylsarcosinate and Cu²⁺. The purified LOX-p, LOXL-p1, and LOXL-p2 proteins showed specific amine oxidase activity of 0.097, 0.054, and 0.150 U/mg, respectively, which was inhibited by β-aminopropionitrile (BAPN), a specific inhibitor of LOX. Availability of these pure and active forms of LOX and LOXLs will be significantly helpful in functional studies related to substrate specificity and crystal structures of this family of amine oxidases.     

Inhibition of the LOX enzyme family members with old and new ligands. Selectivity analysis revisited

Lysyl oxidase (LOX) enzymes as potential drug targets maintain constant attention in the therapy of fibrosis, cancer and metastasis. In order to measure the inhibitory activity of small molecules on the LOX enzyme family members a fluorometric activity screening method was developed. During assay validation, previously reported non-selective small inhibitor molecules (BAPN, MCP-1, thiram, disulfiram) were investigated on all of the major LOX enzymes. We confirmed that MCP-1, thiram, disulfiram are in fact pan-inhibitors, while BAPN inhibits only LOX-like enzymes (preferably LOX-like-protein-2, LOXL2) in contrast to the previous reports. We measured the LOX inhibitory profile of a small targeted library generated by 2D ligand-based chemoinformatics methods. Ten hits (10.4% hit rate) were identified, and the compounds showed distinct activity profiles. Potential inhibitors were also identified for LOX-like-protein-3 (LOXL3) and LOX-like-protein-4 (LOXL4), that are considered as emerging drug targets in the therapy of melanoma and gastric cancer.     

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.     

Comparative immunocytochemical localization of lysyl oxidase (LOX) and the lysyl oxidase-like (LOXL) proteins: changes in the expression of LOXL during development and growth of mouse tissues

Lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) are extracellular enzymes that deaminate peptidyl lysyl residues involved in the cross-linking of fibrillar collagens and elastin. While LOX is required for the survival of newborn mice, the role of LOXL during development remains unclear. Studies have shown that the same cell types express LOX and LOXL in the same tissues, but no functional differences have been established. We have compared the immunohistochemical localization of LOX and LOXL in various tissues from normal, young adult mice. LOX and LOXL were co-localized in the skin, aorta, heart, lung, liver and cartilage, but were localized to different areas in the kidney, stomach, small intestine, colon, retina, ovary, testis and brain. LOXL expression was further examined in tissues from different developmental stages. In embryonic mice (10.5–14.5 dpc), LOXL immunostaining was abundant in the heart, liver, intestine, and neural tube. LOXL was present in most major organs in late fetal (16.5 dpc) and newborn mice, but generally diminished as animals aged. Immunoreactivity was significantly reduced in the heart, lung, kidney and liver of 2 year-old mice, but remained prevalent in the skin and tongue. LOX and LOXL were also found in the nuclei of cells in a number of tissues. These results indicate that LOXL has a role during mouse development and in the maintenance of adult tissues.     

赖氨酰氧化酶样蛋白4在人类恶性肿瘤发生与发展中的作用

赖氨酰氧化酶样蛋白4(lysyl oxidase like 4, LOXL4)是一种属于赖氨酰氧化酶(lysyl oxidase, LOX)蛋白质家族的分泌型铜依赖性胺氧化酶,参与细胞外基质(extracellular matrix, ECM)的组装和维持。LOXL4蛋白在人类肝癌、胃癌、乳腺癌、宫颈癌、头颈鳞癌、食管癌和结直肠癌中表达上调,而在人类膀胱癌和肺癌中表达下调并抑制肿瘤的生长,表明LOXL4蛋白在不同类型的人类恶性肿瘤中具有促癌或抑癌的双向作用。肿瘤细胞外泌体中的LOXL4蛋白通过催化作用产生过氧化氢,后者直接激活FAK/Src信号通路,并促进细胞基质粘附和细胞迁移。外泌体介导的LOXL4还可以通过激活PI3K/Akt信号通路来促进肿瘤细胞的增殖和免疫逃逸。肿瘤细胞中的 LOXL4可以经外泌体转运至巨噬细胞,进一步通过STAT1和STAT3介导的信号通路激活细胞免疫抑制功能和激活程序性死亡配体 1(programmed death ligand 1, PD-L1)表达,触发巨噬细胞的免疫抑制功能,促进肿瘤细胞的免疫逃逸。此外,LOXL4蛋白还能通过激活p53蛋白和抑制Ras/ERK信号转导通路发挥抑癌功能。本文主要总结了LOXL4蛋白的结构、功能及其在人类恶性肿瘤发生发展的作用机制,进一步探讨LOXL4蛋白在恶性肿瘤研究中的应用前景,为恶性肿瘤的临床诊断、治疗和筛选预后标志物提供理论基础和参考依据。     

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.     

The role of lysyl oxidase-like 2 in the odontogenic differentiation of human dental pulp stem cells

Adult human dental pulp stem cells (hDPSCs) are a unique population of precursor cells those are isolated from postnatal dental pulp and have the ability to differentiate into a variety of cell types utilized for the formation of a reparative dentin-like complex. Using LC-MS/MS proteomics approaches, we identified the proteins secreted from the differentiating hDPSCs in mineralization media. Lysyl oxidase-like 2 (LOXL2) was identified as a protein that was down-regulated in the hDPSCs that differentiate into odontoblast-like cells. The role of LOXL2 has not been studied in dental pulp stem cells. LOXL2 mRNA levels were reduced in differentiating hDPSCs, whereas the levels of other LOX family members including LOX, LOXL1, LOXL3, and LOXL4, are increased. The protein expression and secretion levels of LOXL2 were also decreased during odontogenic differentiation. Recombinant LOXL2 protein treatment to hDPSCs resulted in a dose-dependent decrease in the early differentiation and the mineralization accompanying with the lower levels of odontogenic markers such as DSPP, DMP-1 and ALP. These results suggest that LOXL2 has a negative effect on the differentiation of hDPSCs and blocking LOXL2 can promote the hDPSC differentiation to odontoblasts.     

Expression and purification of enzymatically active forms of the human lysyl oxidase-like protein 4

The lysyl oxidase-like protein 4 (LOXL4) is the latest member of the emerging family of lysyl oxidases, several of which were shown to function as copper-dependent amine oxidases catalyzing lysine-derived cross-links in extracellular matrix proteins. LOXL4 contains four scavenger receptor cysteine-rich domains in addition to the characteristic domains of the LOX family, including the copper-binding domain, the cytokine receptor-like domain, and the residues of the lysyl-tyrosyl quinone cofactor. In an effort to assess its amine oxidase activity, we expressed LOXL4 as recombinant forms attached with hexa-histidine residues at the carboxyl terminus by using an Escherichia coli expression system. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis. The purified LOXL4 proteins showed beta-aminopropionitrile-inhibitable activity of 0.022-0.032 units/mg toward a nonpeptidyl substrate, benzylamine. These results indicate that LOXL4, with the four scavenger receptor cysteine rich domains, may also function as an active amine oxidase. Availability of the pure and active forms of LOXL4 will be significantly helpful in functional studies related to substrate specificity and crystal structure of this amine oxidase, which should provide significant insights into functional differences within the LOX family members.     

The Pro-regions of lysyl oxidase and lysyl oxidase-like 1 are required for deposition onto elastic fibers

These studies were undertaken to determine how lysyl oxidase (LOX) and lysyl oxidase like-1 (LOXL) enzymes are targeted to their substrates in the extracellular matrix. Full-length LOX/LOXL and constructs containing just the pro-regions of each enzyme localized to elastic fibers when expressed in cultured cells. However, the LOXL catalytic domain without the pro-region was secreted into the medium but did not associate with matrix. Ligand blot and mammalian two-hybrid assays confirmed an interaction between tropoelastin and the pro-regions of both LOX and LOXL. Immunofluorescence studies localized both enzymes to elastin at the earliest stages of elastic fiber assembly. Our results showed that the pro-regions of LOX and LOXL play a significant role in directing the deposition of both enzymes onto elastic fibers by mediating interactions with tropoelastin. These findings confirmed that an important element of substrate recognition lies in the pro-domain region of the molecule and that the pro-form of the enzyme is what initially interacts with the matrix substrate. These results have raised the interesting possibility that sequence differences between the pro-domain of LOX and LOXL account for some of the functional differences observed for the two enzymes.     

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.     

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.     

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.