©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A New Gene with Sequence and Structural Similarity to the Gene Encoding Human Lysyl Oxidase (*)

(Received for publication, August 19, 1994; and in revised form, January 19, 1995)

Youngho Kim Charles D. Boyd Katalin Csiszar (§)

From the Department of Surgery, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey 08903

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have isolated a number of recombinant clones from a human skin fibroblast cDNA library that contain extensive sequence homology to several coding domains within the human lysyl oxidase mRNA. Using one of these lysyl oxidase-like cDNAs, we obtained several overlapping genomic DNA recombinants. Restriction mapping and DNA sequence analysis revealed that the complete sequence of the lysyl oxidase-like mRNA was encoded by seven exons distributed throughout 25 kilobases of genomic DNA. Exons 2-6 encoded the region of greatest homology to lysyl oxidase. The size of these five exons, moreover, was exactly the same as the size of the corresponding exons within the lysyl oxidase gene. Northern blot analysis also revealed the concomitant appearance of lysyl oxidase and lysyl oxidase-like mRNA in several human tissues. It appears therefore that the genes encoding lysyl oxidase and a lysyl oxidase-like protein share a common evolutionary origin and may also be functionally related.


INTRODUCTION

Lysyl oxidase is a copper-dependent enzyme responsible for the development of lysine-derived cross-links in the structural extracellular matrix proteins, collagen and elastin(1, 2) . This amine oxidase catalyzes allysine cross-links using lysine residues located within the telopeptide and collagenous domains of many procollagens (3) , the biosynthetic precursors of several collagen types. Desmosine and isodesmosine cross-links, in contrast, are the product of the lysyl oxidase-catalyzed deamination of lysine residues distributed throughout tropoelastin, the soluble precursor of insoluble elastin(1, 2) . While the products of lysyl oxidase catalysis have been well characterized, the mechanism(s) by which this enzyme interacts with both procollagen and tropoelastin substrates is unknown.

Lysyl oxidase has been isolated from a number of different tissues from several phylogenetic species as an enzymatically active extracellular matrix protein of 32 kdaltons(2) . Recently, overlapping cDNA recombinants from chicken(4) , mouse(5) , rat(6) , and human(7, 8) tissues have been described that encode a 48-kDa preprolysyloxidase. This enzymatically inactive precursor protein is synthesized from a conserved, single copy gene which has been mapped in humans, to the long arm of chromosome 5 (7, 8) and, in mouse, to chromosome 18 (9, 10, 11) . Both human and mouse lysyl oxidase mRNAs are encoded by seven exons distributed through 14 kb (^1)of both human and mouse genomic DNA(12, 13, 15) . (^2)

Several years ago, Kagan and co-workers (16, 17) and Kuivaniemi (18) reported the presence of several chromatographic variants of lysyl oxidase in both human and bovine tissue. These enzymatically active variants were clearly not precursors of the mature enzyme nor were they derived from any obvious post-translational modification(s) of lysyl oxidase. Moreover, Kagan et al.(19) reported subtle but distinct differences in amino acid composition between isolated variants of lysyl oxidase, implicating the existence of multiple isoforms of the enzyme. Such isoforms could arise from separate lysyl oxidase genes or through alternate usage of exons within the known gene encoding lysyl oxidase and could provide a basis for understanding the mechanism of interaction of lysyl oxidase with multiple substrates.

We have demonstrated very recently that exons within the lysyl oxidase gene are not subject to alternate usage in a variety of human tissues.^2 Kenyon et al.(20) , however, have reported several overlapping cDNA clones that would encode a protein with extensive amino acid sequence homology to the entire sequence of the secreted form of lysyl oxidase. Although the function of this lysyl oxidase-like mRNA is unknown, the existence of such a mRNA suggests that multiple genes may encode structural and functional variants of lysyl oxidase. In support of this hypothesis, this article reports the complete structure of a multiexon gene which has a striking similarity in exon-intron structure, exon sequence homology, and tissue-specific expression to the human lysyl oxidase gene.


MATERIALS AND METHODS

Enzymes and Reagents

Molecular biology grade chemicals, sequencing reagents, T4 DNA ligase, the large fragment of DNA polymerase I, random primer labeling reagents, nylon filters, and antibiotics were purchased from Amersham Corp. Restriction endonucleases were supplied by Pharmacia LKB Biotechnol. Radiochemicals were purchased from ICN (Irvine, CA) and Du Pont NEN. PCR reagents were purchased from Perkin-Elmer Cetus, and oligonucleotides were synthesized by National Biosciences Inc. (Plymouth, MN). A human skin fibroblast cDNA library and a human lung fibroblast genomic library were obtained from Clontech (Palo Alto, CA) and Stratagene (La Jolla, CA), respectively. Clontech also supplied the Multiple Tissue Northern blots, and Bluescript vectors were obtained from Stratagene. Precut nitrocellulose filter circles were purchased from Millipore Corp. (Bedford, MA).

Isolation of cDNA and Genomic DNA Recombinants

A human gt10 skin fibroblast cDNA library was screened using HLO-2, a previously described lysyl oxidase cDNA clone(7) . Plaque-purified phage that resulted in relatively weak autoradiographic signals, using mild post-hybridization wash conditions (2 times SSC, 0.1% SDS at 42 °C), were amplified, insert DNA was released following digestion with EcoRI, and subcloned into the plasmid vector Bluescript SK. Insert DNA fragments were characterized by double-stranded DNA sequencing. Appropriate cDNA fragments were then used to screen a human lung fibroblast -FIX genomic DNA library. Filters were washed in 2 times SSC, 0.1% SDS at 50 °C. After three rounds of screening, four plaque-purified, autoradiographically positive genomic DNA recombinants were amplified, and insert DNA was released following digestion with either SacI or EcoRI and SacI. Genomic DNA fragments were cloned into the Bluescript SK plasmid and characterized by DNA sequencing and restriction mapping.

Determination of Exon and Intron Sizes

The sizes of each exon and exon-intron junctions were determined within plasmid subclones of genomic DNA fragments by DNA sequencing using oligonucleotide primers derived from previously determined cDNA sequence. Exon-derived oligomers were also used to determine the sizes of introns 3, 4, 5, and 6 by PCR. PCR reactions were performed using 100-ng aliquots of plaque-purified, tertiary screened -phage recombinant DNA in 100 µl of reaction mixtures containing final concentrations of the following reagents: 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2 mM MgCl(2), 1 µM each of oligonucleotide primers, 0.2 mM each of dGTP, dATP, TTP, dCTP, and 2.5 units of Taq DNA polymerase. After a predenaturation incubation at 94 °C for 30 s, the phage DNA was denatured at 94 °C for 30 s. Primers were annealed at 54 °C for 30 s, and amplification was carried out at 72 °C for 80 s. This cycle of incubations was then repeated 34 times. The sequences of oligomers used to determine intron sizes were as follows: intron 3 (YK7): 5`-CAGCAGACTTCCTCCCCAAC-3`, (YK23): 5`-CCTCGGCCACCTTCTTGC-3`; intron 4: (YK24): 5`-AGTTTCTGCCTGGAGGAC-3`, (YK26): 5`-CGTCGGTTATGTCGATCC-3`; intron 5: (YK25): 5`-GACACCTACAATGCGGAC-3`, (YK28): 5`-CTGTGTAGTGAATGTTGC-3`; intron 6: (YK27): 5`-CTGACTTCACCAACAACG-3`, (YK29): 5`-TGCCCCTCCCTGCCTGTG-3`.

All these oligonucleotides are derived from exon sequences flanking the appropriate introns. Oligomers YK7, 24, 25, and 27 are all upstream primers. Oligomers YK23, 26, 28, and 29 are downstream primers. The sizes of intron 1 and intron 2 were determined by restriction mapping of -phage recombinants and partial DNA sequencing of plasmid subclones.

Northern Blot Analysis

2-µg aliquots of size-separated poly(A) RNA obtained from adult human lung, kidney, placenta, liver, brain, pancreas, heart, and skeletal muscle were present in each Multiple Tissue Northern (MTN) filter purchased from Clontech Laboratories. Each MTN blot was prehybridized for 4 h at 42 °C in 10 ml of a solution containing 50% formamide, 5 times SSPE, 10 times Denhardt's solution, 2% SDS, and 100 µg/ml denatured salmon sperm DNA. Hybridization, using P-labeled cDNAs prepared by random primer labeling, was carried out in 6 ml of the same solution used for prehybridization at 42 °C for 20 h. The filters were then washed twice in 2 times SSC, 0.1% SDS at room temperature and twice in 0.1 timesSSC, 0.1% SDS at 42 °C. Washed MTN filters were then exposed to Kodak XAR film for varying periods of time at -70 °C. The DNA fragments used for radiolabeling were as follows: a 1.3-kb lysyl oxidase-like cDNA, a previously characterized 1.2-kb cDNA(8) , encoding the carboxyl-terminal end of human lysyl oxidase, and a 2.0-kb beta-actin cDNA provided with the MTN blots by Clontech. The specific activity of all radiolabeled DNA fragments used in these incubations was 5 times 10^9 disintegrations/min/µg.

Primer Extension

Primer extension was performed according to the method of Sambrook et al.(20) using 60 µg of total RNA isolated from confluent cultures of human skin fibroblasts. A P-labeled antisense primer, YK 12 (5`-GATGGTGACCCCTCTGC-3`), that corresponds to sequences -14 to +3 within the lysyl oxidase-like gene, was annealed to total RNA for 2 h at 42 °C. Following extension with 200 units of Moloney murine leukemia virus reverse transcriptase at 37 °C for 1 h, the reaction mixture was treated with RNase A (20 µg/ml) for 15 min at 37 °C, extracted with phenol, ethanol precipitated, and subject to electrophoresis through 6% denaturing polyacrylamide gel.


RESULTS

Isolation of Human Lysyl Oxidase-like cDNAs

A human fibroblast cDNA library was screened with a previously characterized human lysyl oxidase cDNA recombinant, HLO-2(8) , under low stringency hybridization conditions, in an attempt to isolate not only additional lysyl oxidase cDNA recombinants but also cDNA clones that may have partial homology to lysyl oxidase mRNA. 17 autoradiographically positive recombinants were obtained; PCR screening and slot-blot analysis using human lysyl oxidase cDNA-specific oligomers identified 14 of these isolated clones as human lysyl oxidase cDNAs. 3 recombinants however, containing inserts ranging in size from 1.2 to 2.3 kb, did not yield a PCR product with lysyl oxidase cDNA-specific primers and did not hybridize with lysyl oxidase-specific oligomers during slot-blot analysis. Inserts from these three cDNA recombinants were subcloned and sequenced. The nucleotide sequence and derived amino acid sequence agreed with the lysyl oxidase-like sequence recently identified by Kenyon and co-workers(20) . A number of sequence differences which may reflect allelic variants within the lysyl oxidase-like gene, however, were detected. They were: codon 29: CAC (His), previously reported as CAG (Gln), codon 30: GCC (Ala), previously reported as CCC (Pro), codon 95: GCG (Ala), previously reported as CGG (Arg), codon 356: GTG (Val), previously reported as GTA (Val).

Alignment of the derived amino acid sequences for human(7, 8) , rat(6) , mouse(5) , and chicken (4) lysyl oxidases and the open reading frame of the human lysyl oxidase-like cDNAs revealed a similar carboxyl-terminal sequence that exhibited 76% homology between the human lysyl oxidase and lysyl oxidase-like derived amino acid sequences (Fig. 1). This homologous region encompassed the entire sequence of the mature lysyl oxidase, including a copper-binding and other metal-binding domains(22, 23, 24, 25) . The copper-binding domain and the four histidine residues present within this conserved sequence (WEWHSCHQHYH) that are involved in the copper binding coordination complex are strictly conserved. Further, a growth factor and cytokine receptor domain was also identified in both derived amino acid sequences. The amino acid sequence C-X(9)-C-X-W-X-C-X-C (where X(n) is a defined number of any amino acids), is conserved in exon 5 and 6 of both genes, and it agrees with the sequence C-X(9)-C-X-W-X-C-X-C that is a proposed extracellular ligand-binding domain for a number of receptors for cytokines, prolactin, and growth hormone(26, 27) .


Figure 1: Amino acid sequence alignment of the conserved domains of the human (H), rat (R), mouse (M) and chick (C) lysyl oxidase and the predicted lysyl oxidase-like (LOL) protein. Amino acids are indicated in single-letter code and numbered from the amino-terminal end of previously reported sequences for lysyl oxidase (8) and a lysyl oxidase-like (20) protein. Dashes indicate identical amino acids, and * identifies stop codons. Conserved cysteine residues, copper, and putative metal binding sequences are in shaded boxes. The four copper coordinating histidine residues within the copper-binding domain are in reverse font. T bars above the sequences illustrate exon-intron junctions and exon numbers determined for the human and mouse lysyl oxidase and the lysyl oxidase-like gene. A growth factor and cytokine receptor domain is underlined.



From a comparison of the derived amino acid sequences for the human lysyl oxidase-like protein and lysyl oxidases from several different phylogenetic species, it was evident that many of the substitutions within the carboxyl terminus of the lysyl oxidase-like protein are identical to amino acid variations within the analogous domains in lysyl oxidases from different species.

In contrast to the homology with the sequence of mature human lysyl oxidase, very little homology was evident in the region of the amino-terminal domain encoded by human lysyl oxidase-like mRNA. Similarly, no obvious homology was present between the 3`-untranslated regions of both lysyl oxidase and lysyl oxidase-like mRNAs.

The Structure of the Human Lysyl Oxidase-like Gene

A 1.3-kb lysyl oxidase-like cDNA recombinant was radiolabeled and used to screen a human lung fibroblast genomic DNA library. Four overlapping -phage clones were isolated (LOL-G1, G4, G7, and G11, Fig. 2). Using exon-specific primers, DNA sequencing revealed that LOL-G1 and G4 contained exon 1, exon 2 was identified within LOL-G7, and LOL-G11 contained exons 3-7. The complete coding sequence and 5`- and 3`-untranslated regions of the lysyl oxidase-like mRNA were found within these seven exons, distributed through approximately 25 kb of genomic DNA. The structure of this human lysyl oxidase-like gene and the recently determined exon-intron structure of the human lysyl oxidase gene(28) ^2 are presented in Fig. 2.


Figure 2: Structure of the human lysyl oxidase-like gene. Comparison of the exon-intron structure of the lysyl oxidase-like (LOL) and lysyl oxidase (LO) genes. Non-conserved exons are shown in hatched boxes and conserved exons by shaded boxes. Exons are numbered: Ex 1-7. E, EcoRI; P, PstI; H, HindIII; S, SacI. Relative positions of genomic DNA inserts from the phage recombinants LOL-G1, G4, G7, and G11 are indicated. A 1-kb size marker is also indicated. Exact sizes of exons (Ex) and introns (Int) are given in base pairs in the table. The information summarizing the complete structure of the human lysyl oxidase gene was obtained from two previous reports(28) .^2 The sizes of introns 1 and 2 within the lysyl oxidase-like gene were derived from restriction mapping; introns 3-6 were determined by PCR analysis.



The overall structure of the lysyl oxidase and lysyl oxidase-like genes are very similar. Seven exons encode the 5`- and 3`-untranslated regions and the coding domains in each of the lysyl oxidase and lysyl oxidase-like mRNAs. Exons 2-6 encode the regions of greatest homology between the derived amino acid sequences of lysyl oxidase and the lysyl oxidase-like protein. The sizes of the exons 2-6 are also exactly the same in both genes. In contrast, exon 1 is smaller in the lysyl oxidase gene than in the lysyl oxidase-like gene. Conversely, exon 7 is substantially larger in the lysyl oxidase gene than the corresponding exon in the lysyl oxidase-like gene. Exon 7 in the lysyl oxidase gene encodes a large 3`-untranslated region which has little homology to a smaller 3`-untranslated region in exon 7 of the lysyl oxidase-like gene. Similarly, exon 1 in the lysyl oxidase gene shares little homology with exon 1 in the lysyl oxidase-like gene.

Considerable divergence in size and sequence exists between all six introns in both genes. While intron sequence within each intron-exon junction in the lysyl oxidase-like gene conforms to the consensus sequence NCAG/GTRAGT characteristic of exon-intron junctions in eukaryotic genes(29) , no homology between intron sequences in both genes was apparent beyond these consensus sequences.

The positions of four of the six introns within the human lysyl oxidase-like gene resulted in split codons. The first intron interrupted codon 368 (a glycine codon) between the first and second nucleotides. The second intron interrupted codon 405 (encoding serine) between the second and third nucleotides. Similarly, the third intron interrupted codon 450 (a glutamine codon) between the second and third nucleotide. In contrast, the fourth intron interrupted the coding sequence between codons 502 (a glutamine codon) and 503 (a glycine codon), and intron 5 interrupted the coding sequence between codons 534 (a lysine codon) and 535 (a valine codon). Finally, the sixth intron resulted in a split codon 573 (encoding glutamine), interrupting the codon between the second and third nucleotides. These exon-intron junctions interrupt the lysyl oxidase codons at exactly the same positions.

Transcription Initiation Sites and the 5`-Flanking Region

Transcription initiation sites of the lysyl oxidase-like gene were determined by primer extension analysis using total RNA from human skin fibroblasts. The sizes of these DNA fragments identified two transcription initiation sites at positions -51 and -128. A third, less intense band, identified a transcription initiation site at -308 (Fig. 3). Initiation at this latter site was also confirmed by sequence analysis of one of the isolated cDNA clones. Multiple transcription initiation sites established the minimum size of exon 1 in the human lysyl oxidase-like gene as 1200 base pairs.


Figure 3: Transcription initiation sites determined by primer extension analysis. The mRNA start sites were determined using total RNA from human skin fibroblasts. Lane 1 is the primer extension reaction with primer YK 12. Lanes G, A, T, and C are sequencing reactions of unrelated DNA used for size determination.



The nucleic acid sequence of 1.2 kb of the 5`-flanking region of the lysyl oxidase-like gene did not reveal significant sequence homology (35.7%) to the corresponding region of the human lysyl oxidase gene. The lysyl oxidase-like gene promoter contains no typical TATA or CAAT box sequences. Potential transcription factor binding sites including four Sp1 sites, one TF-II-I motif, single Ap2 and Ap4 sites, and an octamer motif were identified. There is one GC box at -588 that overlaps with one of the Sp1 sites (TGGGCGGGGT). The presence of several recognition sequence elements for Hox proteins, zeste-Ubx (CGAGCG, CGCTCG), and zeste-white (CACTCA) suggests that the lysyl oxidase-like gene expression is under developmental regulation(30) . There are putative binding sites for the activator protein malT. The PU box (GAGGAA) that is a binding site for the ets-oncogene-related transcriptional activator is at -598. The nucleotide sequence TGTTCT at -83 is a recognition element of the glucocorticoid receptor, and it is indicative of steroid hormone regulation of the lysyl oxidase-like gene (31) (Fig. 4).


Figure 4: 5`-Flanking region of the human lysyl oxidase-like gene. The first nucleotide preceding the ATG codon is numbered -1. Potential regulatory consensus sequences and transcription factor binding sites are indicated by shaded boxes. Transcription initiation sites determined by primer extension analysis are indicated by arrowheads.



Expression of the Lysyl Oxidase and Lysyl Oxidase-like Genes in Different Human Tissues

Autoradiograms of Multiple Tissue Northern blots incubated with radiolabeled cDNAs for lysyl oxidase, the lysyl oxidase-like protein, and actin are presented in Fig. 5. As expected, two species of lysyl oxidase mRNA were detected in these poly(A) RNA preparations, migrating as mRNAs of 3.8 and 4.8 kb in size. These lysyl oxidase mRNAs were observed in RNA preparations from all tissues analyzed except poly(A) RNA isolated from brain. The lysyl oxidase-like mRNA migrates as a single mRNA-size species of 2.3 kb. This mRNA was also detected in all poly(A) RNA preparations except RNA isolated from brain. A 2.0-kb beta-actin mRNA was detected in all RNA samples. Previously reported heart and skeletal muscle-specific isoforms of beta-actin mRNA (1.6-1.8 kb in size) were also detected (29, 30) .


Figure 5: Northern blot analysis of RNA from several human tissues. MTN blots containing poly(A) RNA isolated from several human tissues were used as described under ``Materials and Methods.'' Panel A, autoradiogram obtained following incubation of the MTN blot with a radiolabeled lysyl oxidase-like cDNA and exposed for 24 h. Panel B, the detection of lysyl oxidase mRNAs in a separate MTN blot exposed for 16 h. Panel C, the MTN blot used in panel A was stripped, rehybridized with an actin cDNA, and exposed for 10 min. The tissue source of each RNA sample on the MTN blots is indicated at the top of each panel. The position of RNA molecular weight markers (in kilobase pairs) is also indicated on each MTN blot.




DISCUSSION

Evolutionary Origins

We have identified a new gene composed of seven exons in about 25 kb of human genomic DNA. Of the seven exons, five contiguous exons encode a protein that is 76% identical and 84% similar to the mature, enzymatically active and secreted form of human lysyl oxidase. In addition to conservation of derived protein and nucleotide coding sequences, the sizes of exons 2-6 are exactly the same as the corresponding exons (exons 2-6) in the lysyl oxidase gene. Conservation of sequence and exon size clearly indicates that at least exons 2-6 within the lysyl oxidase-like gene and the gene encoding lysyl oxidase arose from a common ancestral gene through a process of gene duplication.

Gene duplication is a common mechanism for encoding similar but genetically distinct protein variants. The genes encoding tissue-specific isoforms of cytochrome c oxidase arose through duplication of an ancestral multiexon gene, and, consequently, an identical exon-intron structure was observed within these genes(34, 35) . Similarly, the globin gene cluster evolved from an ancestral gene by consecutive rounds of gene duplication(36) . Variation of intragenic conservation of exon size within the fibrillar collagen genes is also an example of a complex type of gene duplication(37, 38) .

It is evident that all or part of the lysyl oxidase and lysyl oxidase-like genes shared a common ancestor. While exons 2-6 are highly conserved in both sequence and size in both genes, exons 1 and 7 have little sequence homology and no similarity in size. The ancestral gene for lysyl oxidase and the lysyl oxidase-like protein may have contained only exons 2-6. Duplicated copies of this ancestral sequence could have maintained a conserved sequence and size of these five exons and additional exons were added to an evolving multiexon structure through a process of exon shuffling. Alternatively, it is possible that duplication of an ancestral gene involved all seven exons and six introns. Two of those exons and all the intron sequence may have diverged in sequence and in size. For example the size of the first intron of the lysyl oxidase gene is conserved in mouse, rat, and human, and the sequence contains several transcription factor binding sites in all three species. (^3)The same intron in the lysyl oxidase-like gene differs in size and has no sequence homology to the lysyl oxidase intronic sequences suggesting a different role for this part of the gene.

Coding Domains

A comparison of nucleotide sequences within exons 2-6 of the lysyl oxidase-like gene and the amino acid sequence derived from the coding domains within these exons did not reveal any homology to any other copper binding protein or to any other known amino acid sequence other than lysyl oxidase.

Most of the coding domain for the secreted, active form of lysyl oxidase is contained within exon 2-6. Ten of the 12 cysteine residues present in this catalytic domain in human, rat, mouse, and chicken lysyl oxidases are conserved. Six of these exist as disulfide bonds and are responsible for the functional conformation of the mature lysyl oxidase enzyme. The presence of these cysteine residues in the lysyl oxidase-like protein suggests that the tertiary structure of this protein is similar to lysyl oxidase.

The first exon of the lysyl oxidase gene encodes a 5`-untranslated region, a signal peptide, a propeptide sequence, and the first few amino acids of the functional and secreted lysyl oxidase responsible for transport and secretion. Most of this first exon exhibits the greatest divergence among different species. The amino-terminal end of the derived amino acid sequence encoded within exon 1 of the lysyl oxidase-like gene is homologous to the three domains characteristic of a signal sequence(40) . These include a positively charged amino-terminal domain (MALAR) (codons 1-5), a central hydrophobic region (GSRQLGALVWGACL) (codons 6-19), and a carboxyl-terminal region (CVLVHGQ) (codons 20-26). The 3`-end of exon 1 encodes a peptide sequence with little homology to the propeptide sequence of preprolysyl oxidase. However, the presence of putative endopeptidase cleavage sites between arginine residues at positions 85, 86, 93, 94, and 272, 273 suggests that this peptide may function as a propeptide domain. Proteolytic processing at these sites would result in 49-, 48-, and 31-kDa protein products. The divergent sequence within the first exon of the lysyl oxidase-like gene may therefore encode a signal sequence and propeptide domain within a preprolysyl oxidase-like protein that may regulate intracellular and extracellular transport of a secreted protein, the pathway of which, however, may be different from preprolysyl oxidase.

Exon 7 in both the lysyl oxidase and the lysyl oxidase-like genes contains only five nucleotides of coding sequence, a stop codon, and a 3`-untranslated region which is 3.5 kb in length in the lysyl oxidase gene and contains several differentially used polyadenylation signals that result in multiple-sized lysyl oxidase mRNAs.^2 Only one polyadenylation signal was observed in exon 7 of the lysyl oxidase-like gene, and the position of this consensus sequence within the 3`-untranslated region was consistent with the appearance of a 2.3-kb lysyl oxidase-like mRNA. No other polyadenylation signals were detected downstream of the observed consensus sequence, and no other higher molecular weight lysyl oxidase-like mRNAs were detected by Northern blot analysis. Exon 7 seems therefore to be a far shorter exon in the lysyl oxidase-like gene. The difference in size and sequence suggests both separate or divergent evolutionary origins and different potential effects on mRNA stability and other post-transcriptional control mechanisms mediated by 3`-untranslated domains(41) .

Possible Function(s) of a Lysyl Oxidase-like Protein

Sequence and size conservation of exons 2-6 in this new lysyl oxidase-like gene and the presence within exon 1 of a domain encoding a putative signal sequence and a propeptide domain suggests the synthesis of a preprolysyl oxidase-like protein that is secreted into the extracellular matrix. Taken together with observation of concomitant expression of lysyl oxidase and lysyl oxidase-like mRNA in several different human tissue, the data presented in this article supports the possibility that lysyl oxidase and a lysyl oxidase-like protein may have related functions.

Lysyl oxidase has been isolated as an enzymatically active family of polypeptides of 32 kDa that appear in several tissues and in several phylogenetic species as at least four isoforms(17) . The mechanistic and functional basis for these chromatographically isolated variants of lysyl oxidase is unknown. It is clear, however, that these isoforms of lysyl oxidase individually catalyze cross-link formation using both collagen and elastin substrates(17) . Moreover, these isoforms are not proteolytically processed precursors of one another nor do they seem to be post-translationally modified variants of a secreted precursor protein(16, 17, 18) . Kagan and co-workers (19) have shown, however, that subtle differences in amino acid composition exist between the isolated isoforms, suggesting the possible existence of genetically distinct variants of lysyl oxidase. We have recently shown that such variants could not arise through alternate usage of exons within the human lysyl oxidase gene.^2 Therefore, it is possible that the lysyl oxidase-like gene could encode a functional variant of lysyl oxidase. Concomitant appearance of lysyl oxidase and lysyl oxidase-like mRNA in several human tissues suggests that co-ordinate expression of both lysyl oxidase and the lysyl oxidase-like gene could be important to overall lysyl oxidase activity. It is well known, for example, that preparations of lysyl oxidase readily aggregate in vitro and cross-link activity is routinely assayed in the presence of urea(16) . It is possible therefore that in vivo lysyl oxidase is a complex of subunits synthesized from at least two, but possibly as many as four, different genes.

A possible subunit structure for lysyl oxidase would provide the basis for understanding the mechanism(s) whereby lysyl oxidase can mediate multiple catalytic functions. Variation in subunit composition, for example, may explain how lysyl oxidase interacts with procollagen and tropoelastin substrates. A subunit structure may also be important in understanding the recently reported role of lysyl oxidase in ras-mediated tumor suppression(5, 14, 39, 42) . The presence of a cytokine receptor domain in both lysyl oxidase and lysyl oxidase-like proteins may be particularly important to a multifunctional enzyme or enzyme complex that not only plays a significant role in the maintenance of extracellular matrix structure but also functions, either directly or indirectly to maintain an equilibrium between cell proliferation and differentiation.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL 39869 and HL37438. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: kb, kilobase(s); PCR, polymerase chain reaction; MTN, Multiple Tissue Northern.

(^2)
C. D. Boyd, T. J. Mariani, Y. Kim, and K. Csiszar, manuscript submitted.

(^3)
K. Csiszar, P. C. Trackman, D. Samid, and C. D. Boyd, manuscript in preparation.


ACKNOWLEDGEMENTS

We gratefully acknowledge the assistance of Gary Benson in typing this manuscript.


REFERENCES

  1. Kagan, H. M. (1986) in Biology of Extracellular Matrix (Mecham, R. P., ed) Vol. 1, pp. 321-398, Academic Press, Orlando, FL
  2. Kagan, H. M., and Trackman, P. C. (1991) Am. J. Respir. Cell Mol. Biol. 3, 206-210
  3. Eyre, D. R., Paz, M. A., and Gallop, P. M. (1984) Annu. Rev. Biochem. 53, 717-748 [CrossRef][Medline] [Order article via Infotrieve]
  4. Wu, Y., Rich, C. B., Lincecum, J., Trackman, P. C., Kagan, H. M., and Foster, J. A. (1992) J. Biol. Chem. 267, 24199-24206 [Abstract/Free Full Text]
  5. Kenyon, K., Contente, S., Trackman, P. C., Tang, J., Kagan, H. M., and Friedman, R. M. (1991) Science 253, 802 [Medline] [Order article via Infotrieve]
  6. Trackman, P. C., Prott, A. M., Wolanski, A., Tang, S., Offner, G. D., Troxler, R. F., and Kagan, H. M. (1990) Biochemistry 29, 4863-4870 [Medline] [Order article via Infotrieve]
  7. Hamalainen, E. R., Jones, T. A., Sheer, D., Taskinen, K., Pihlajaniemi, T., and Kivirikko, K. L. (1991) Genomics 11, 508-516 [Medline] [Order article via Infotrieve]
  8. Mariani, T. J., Trackman, P. C., Kagan, H. M., Eddy, R. L., Shows, T. B., Boyd, C. D., and Deak, S. B. (1992) Matrix 12, 242-248 [Medline] [Order article via Infotrieve]
  9. Mock, B. A., Contente, S., Kenyon, K., Friedman, R. M., and Kozak, C. A. (1992) Genomics 14, 822-823 [Medline] [Order article via Infotrieve]
  10. Lossie, A. C., Buckwalter, M. S., and Camper, S. A. (1993) Mamm. Genome 4, 177-178 [Medline] [Order article via Infotrieve]
  11. Chang, Y. S., Svinarich, D. M., Yang, T. P., and Krawetz, S. A. (1993) Cytogenet. Cell Genet. 63, 47-49 [Medline] [Order article via Infotrieve]
  12. Contente, S., Csiszar, K., Kenyon, K., and Friedman, R. M. (1993) Genomics 16, 395-400 [CrossRef][Medline] [Order article via Infotrieve]
  13. Csiszar, K., Mariani, T. J., Deak, S. B., and Boyd, C. D. (1993) Genomics 16, 401-406 [CrossRef][Medline] [Order article via Infotrieve]
  14. Hajnal, A., Klemenz, R., and Schafer, R. (1993) Cancer Res. 53, 4670-4675 [Abstract]
  15. Svinarich, D. M., Twomey, T. A., Macauley, S. P., Krebs, C. J., Yang, T. P., and Krawetz, S. A. (1992) J. Biol. Chem. 267, 14382-14387 [Abstract/Free Full Text]
  16. Sullivan, K. A., and Kagan, H. M. (1982) J. Biol. Chem. 257, 13520-13526 [Abstract/Free Full Text]
  17. Williams, M. A., and Kagan, H. M. (1985) Anal. Biochem. 149, 430-437 [Medline] [Order article via Infotrieve]
  18. Kuivaniemi, H. (1985) Biochem. J. 230, 639-643 [Medline] [Order article via Infotrieve]
  19. Kagan, H. M., Sullivan, K. A., Olsson, T. A., III, and Cronlund, A. L. (1979) Biochem. J. 177, 203-214 [Medline] [Order article via Infotrieve]
  20. Kenyon, K., Modi, W. S., Contente, S., and Friedman, R. M. (1993) J. Biol. Chem. 268, 18435-18437 [Abstract/Free Full Text]
  21. Sambrook, J., Flitsch, E. F., and Maniatis, T. (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  22. Berg, J. M. (1986) Science 232, 485-487 [Medline] [Order article via Infotrieve]
  23. Huber, M., Hintermann, G., and Lerch, K. (1985) Biochemistry 24, 6038-6044 [Medline] [Order article via Infotrieve]
  24. Huber, M., and Lerch, K. (1988) Biochemistry 27, 5610-5615 [Medline] [Order article via Infotrieve]
  25. Krebs, C. J., and Krawetz, S. A. (1993) Biochem. Biophys. Act. 1202, 7-12
  26. Bazan, J. F. (1989) Biochem. Biophys. Res. Commun. 164, 788-795 [Medline] [Order article via Infotrieve]
  27. Bazan, J. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6934-6938 [Abstract]
  28. Hamalainen, E., Kemppainen, R., Pihlajaniemi, T., and Kivirikko, K. I. (1993) Genomics 17, 544-548 [CrossRef][Medline] [Order article via Infotrieve]
  29. Shapiro, M. B., and Senapathy, P. (1987) Nucleic Acids Res. 15, 7155-7174 [Abstract]
  30. Wingender, E. (1990) Crit. Rev. Eukaryotic Gene Expression 1, 11-33 [Medline] [Order article via Infotrieve]
  31. Klemsz, M. J., McKercher, S. R., Celada, A., Van Beveren, C., and Maki, R. (1990) Cell 61, 113-124 [Medline] [Order article via Infotrieve]
  32. Giovanna, P., Jardine, K., and McBurney, M. W. (1991) Mol. Cell. Biol. 11, 4796-4803 [Medline] [Order article via Infotrieve]
  33. Lamballe, F., Klein, R., and Barbacid, M. (1991) Cell 66, 967-979 [Medline] [Order article via Infotrieve]
  34. Saccone, C., Pesole, G., and Kadenbach, B. (1991) Eur. J. Biochem. 1, 151-156
  35. Seelans, R. S., and Grossman, L. I. (1993) Genomics 18, 527-536 [Medline] [Order article via Infotrieve]
  36. Higgs, D. R., Wood, W. G., Jarman, A. P., Vickers, M. A., Wilkie, A. O. M., Lamb, J., Vyas, P., and Bennett, J. P. (1990) Ann. N. Y. Acad. Sci. 612, 15-22 [Medline] [Order article via Infotrieve]
  37. Sandell, L. J., and Boyd, C. D. (1990) in Extracellular Matrix Genes (Sandell, L. J., and Boyd, C. D., eds) pp. 1-56, Academic Press, New York
  38. Dorit, R. L., and Gilbert, W. (1991) Curr. Opin. Dev. 4, 464-469
  39. Krzyzosiak, W. J., Shindo-Ocada, N., Teshima, H., Nakajima, K., and Nishimura, S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4879-4883 [Abstract]
  40. von Heijne, G. (1990) J. Membr. Biol. 115, 195-201
  41. Jacob, S. T., Terns, M. P, Hengst-Zhang, J. A., and Vulapali, R. S. (1990) Crit. Rev. EukaryoticGene Expression 7, 49-59
  42. Contente, S., Kenyon, K., Rimoldi, D., and Friedman, R. M. (1990) Science 249, 796-798 [Medline] [Order article via Infotrieve]

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