| 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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To find additional genes for human lysyl oxidase isoenzymes, the available databases were searched using sequences of all known human lysyl oxidase isoenzymes. This search resulted in the identification of three high-throughput genomic sequences (AC005033, AC005041 and AL139241) and one expressed tag sequence (AL751493), which were highly similar to the LOX (Hämäläinen et al. 1991), LOXL (Kenyon et al. 1993), and LOXL2 (Saito et al. 1997, Jourdan-Le Saux et al. 1999) sequences. Based on the information obtained from high-throughput genomic sequences AC005033 and AC005041 two primer pairs were synthesized, and then used to generate PCR products of 240 and 527 bp in lenght from a human placenta cDNA library. These PCR products were used as probes to screen the same library. Two LOXL3 cDNA clones covering nucleotides 825-2574 were obtained and sequenced. Based on the information obtained from these two cDNA clones and two high-throughput genomic clones, several primers were tested to obtain the 5’end of the cDNA. By doing this, a 913 bp PCR product including an overlapping 5’region of the cDNA was obtained. To obtain a 5’RACE product, the primers AP1 and HLO406R were used in the first round of PCR, and AP1 and LO4RNA1R in the second round, yielding a product of 388 bp.
Based on the information obtained from sequences AL139241 and AL751493 the full-length LOXL4 cDNA was obtained using several primers, as described in Material and Methods.
The full-length LOXL3 cDNA obtained was 2574 bp in size, and contained an open reading frame of 2226 bp. The 5’ noncoding region was 74 bp in length and the 3’ noncoding region 238 bp. The cloned cDNA had no polyadenylation signal, which indicated that the actual lenght of the 3’ noncoding region is longer than 238 bp. The open reading frame encoded a polypeptide of 753 amino acids (Figure 1 in I). A putative signal peptide was present in the N-terminus, and was likely to be cleaved between amino acids 25 and 26. The size of the processed polypeptide was, therefore, 728 residues, with predicted molecular mass of 80.3 kDa. The nonprocessed LOXL3 polypeptide shared a 55% overall amino acid sequence identity with the LOXL2 polypeptide, whereas its residues 1-528 shared no significant identity with the LOX and LOXL polypeptides. However, the C-terminal residues from 529 to 729 showed a high degree of identity with the LOX, LOXL, and LOXL2 polypeptides, namely 51%, 53%, and 69%, respectively.
The LOXL4 mRNA is at least 2736 bp in size and contains an open reading frame of 2271 bp. The LOXL4 gene is located on chromosome 10 (based on the sequence AL139241) and encodes a polypeptide of 756 amino acids, including a signal peptide of 24 residues (Figure 1 in II). The LOXL4 polypeptide shows a high overall identity with the LOXL2 and LOXL3 polypeptides, the degrees of identity being 63.9% and 65.5%, respectively. The highest identity is found within the lysyl oxidase (LO) domain, being 85% for LOXL2 and 87.4% for LOXL3 (Figure 1 in II). An extensive similarity of the LOXL4 polypeptide to the LOX and LOXL polypeptides is found only within the LO domain, with identities of 51.1% and 50.2%, respectively. In addition, the human LOXL4 polypeptide shows a 93.7% overall identity with a recently reported murine Loxc polypeptide (Ito et al. 2001), indicating that these two cDNAs encode the same polypeptide in different species.
As mentioned above, LOXL3 and LOXL4 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. This region contains a putative copper-binding sequence (Krebs & Krawets 1993) (WVWHECHGHYH, residues 601-611 in the LOXL3, and WVWHQCHRHYH, residues 605-615 in the LOXL4 seqeuence), a putative collagen-related copper-affinity site (Krebs & Krawetz 1993) (GHK, residues 632-634 in the LOXL3, and residues 636-638 in the LOXL4 sequence), as well as lysine and tyrosine residues (Lys-634 and Tyr-670 in the LOXL3, and Lys-638 and Tyr-674 in the LOXL4 sequence) (note an error in Figure 1 and text in I and II), which are involved in the formation of the lysine tyrosylquinone cofactor (Wang et al. 1996), and a growth factor and cytokine binding sequence (Bazan 1989, 1990, Kim et al. 1995) (C-X9-C-X-W-X26-32-C-X10-15-C, residues 666-727 in the LOXL3, and residues 670-731 in the LOXL4 sequence). All these residues are highly conserved in all lysyl oxidase isoenzymes (Figure 1 in I and II). In addition, the putative sites for proteolytic processing by procollagen C-proteinase, Gly-447 and Asp-448, were found in LOXL3 sequence (Cronshaw et al. 1995, Panchenko et al. 1996), but not in the LOXL4 sequence. The 10 cysteine residues present in LOX are entirely conserved in both polypeptides (Figure 1 in I and II).
The N-terminal regions of the LOXL3 and LOXL4 polypeptides contains four repeats of scavenger receptor cysteine-rich (SRCR) repeats (Resnick et al. 1994) that are also found in the LOXL2 polypeptide (Saito et al. 1997, Jourdan-Le Saux et al. 1999) but not in the LOX and LOXL polypeptides (Figure 1 in I and II). In this N-terminal region, all 35 cysteine residues are totally conserved between the processed LOXL2, LOXL3 and LOXL4 polypeptides. In addition, this region of LOXL3 polypeptide contain three putative O-glycosylation and five putative N-glycosylation sites, and the corresponding region of LOXL4 polypeptide contain three putative O-glycosylation and two putative N-glycosylation sites. A bipartite nuclear localization signal (Dingwall et al. 1991, Boulikas 1994) was found in LOXL3 sequence (KKQQQSKPQGEARVRLKG, in residues 293-311), but was not found in any other lysyl oxidase isoenzymes.
The LOXL3 gene has 14 exons and is located on chromosome 2p13 based on the information obtained from the high-throughput sequences AC005033 and AC005041. As suggested by 5’RACE experiments, this gene may have its first intron within the 5’ untranslated region. To verify the existence of this intron, PCR was performed with pools of human placental and fetal cDNAs using two pairs of primers. These reactions gave products with the expected sizes of 971 and 371 bp, thus indicating the presence of this intron.
The sizes of exons 2-13 vary from 112 to 247 bp, and the sizes of the introns vary from 79 to 12,440 bp, intron 4 being the longest (Table 1 in I). All the exon-intron boundaries show the consensus sequence (C/T)AG-exon-GT(A/G), except those of exons 3 and 7, in which the sequence at the 5’ splice donor site is GTC.
The 3’ untranslated region of the LOXL3 gene has a TTATATAAAAA sequence 213 bp downstream of the 3’ end of the cloned cDNA, a TAAATATAT sequence 837 bp downstream of the 3’ end, and a GCAATAAAGT sequence 904 bp downstream of the 3’ end, all of which may act as polyadenylation signals.
Northern analyses indicated the presence of only a single LOXL3 mRNA of about 3.1 kb, and a single LOXL4 mRNA of about 4.0 kb in size. The highest expression levels of LOXL3 mRNA were seen in the placenta, heart, ovary, testis, small intestine, and spleen. However, distinct hybridization signals were obtained from all the tissues examined (Figure 2 in I). The highest expression levels of LOXL4 mRNA among the tissues studied were seen in the skeletal muscle, testis, and pancreas, whereas no signal was detected in the brain or in blood leukocytes (Figure 2a in II).
Recombinant expression of the LOXL3 and the LOXL4 polypeptides were studied by transfecting human HT-1080 cells with a construct that also contained sequences encoding the V5 epitope and a six-histidine tag at its 3’ end. In addition, recombinant LOXL4 polypeptide was also studied by transfecting CHO cells and mouse embryonic fibroblasts with the same construct. Western blots of crude lysates from the transfected cells by antibodies to the V5 epitope showed the expression of a 97 kDa LOXL3 and LOXL4 polypeptides, whereas no staining was seen in lysates from nontransfected cells (Figure 3 in I, and Figure 2b in II). Analysis of concentrated medium samples showed the presence of two major LOXL3 and LOXL4 polypeptides of slightly larger sizes than the polypeptides present in the cell lysates, as well as a very minor 97 kDa polypeptides (Figure 3 in I, and Figure 2b in II). No intracellular or extracellular processing of the polypeptides were detected in HT-1080 (Figure 3 in I, and Figure 2b in II) or CHO cells, or in mouse embryonic fibroblasts (data not shown).
Immunofluorescence staining of the LOXL3 transfected HT-1080 cells by a monoclonal antibody specific for the V5 epitope gave a strong cellular signal, but no signal could be detected in the nuclei (Figure 4 in I).