From the Department of Medicine,
§ Division of Dermatology, ¶ Barnes-Jewish Hospital,
and Departments of §§ Pediatrics and
¶¶ Cell Biology and Physiology, Washington University School
of Medicine, St. Louis, Missouri 63110, the ** University of
Texas Health Science Center, Tyler, Texas 75708, the
University of Bristol, Collagen
Research Group, Langford B540 5DS, United Kingdom, and the
Pulmonary and Critical Care Medicine, Brigham and
Women's Hospital, Harvard Medical School,
Boston, Massachusetts 02115
Received for publication, October 3, 2002, and in revised form, December 6, 2002
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ABSTRACT |
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Lysyl oxidase (LOX) is an enzyme responsible for
the cross-linking of collagen and elastin both in vitro and
in vivo. The unique functions of the individual members of
this multigene family have been difficult to ascertain because of
highly conserved catalytic domains and overlapping tissue expression
patterns. To address this problem of functional and structural
redundancy and to determine the role of LOX in the development of
tissue integrity, Lox gene expression was deleted by
targeted mutagenesis in mice. Lox-targeted mice
(LOX Lysyl oxidases are a group of enzymes whose members continue to be
discovered and their functions defined. Classically, lysyl oxidase
(LOX,1 EC 1.4.3.13) is a
copper-containing monoamine oxidase extracellular enzyme that
catalyzes the conversion of the epsilon amino group of specific lysine
residues in collagen and elastin to reactive aldehyde groups (1). This
oxidative deamination of lysine produces reactive "allysine"
residues (lysine aldehyde residues), which participate in the formation
of covalent cross-links. In vitro, LOX can convert various
non-peptidyl amine substrates to their corresponding aldehydes. Two
lines of evidence support the role of lysyl oxidase in cross-linking of
collagen and elastin. First, lysyl oxidase(s), presumably LOX, has been
extracted from bovine aortas using 6 M urea and used as an
reagent to demonstrate elastin and collagen cross-linking by tritium
release using immature elastin and collagen substrates (2, 3). Second,
administration of Four additional lysyl oxidases have been recently described. All five
family members are located on different human chromosomes (11-21) and
mouse chromosomes (22-28) (Table I).
Messenger RNA expression of all human lysyl oxidase family members
appears to be most abundant in adult tissues by Northern blotting
(Table I). Under normal conditions, human LOX mRNA expression is
low in most tissues with 442 molecules/picogram embryonic skin
fibroblast total RNA or about 8% of the mRNA molecules that encode
for /
) died soon after parturition, exhibiting
cardiovascular instability with ruptured arterial aneurysms and
diaphragmatic rupture. Microscopic analysis of the aorta
demonstrated fragmented elastic fiber architecture in homozygous
mutant null mice. LOX activity, as assessed by desmosine (elastin
cross-link) analysis, was reduced by ~60% in the aorta and lungs of
homozygous mutant animals compared with wild type mice. Immature
collagen cross-links were decreased but to a lesser degree than elastin
cross-links in LOX
/
mice. Thus, lysyl oxidase appears
critical during embryogenesis for structural stability of the aorta and
diaphragm and connective tissue development.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminopropionitrile (BAPN), a naturally occurring
inhibitor of lysyl oxidases, results in lathyrism, presumably due to
impaired collagen and elastin cross-linking (4-7). Inhibition by BAPN is dependent upon the carbonyl cofactor in the lysyl oxidases, which in
LOX is lysyl-tyrosine quinone (8-10).
-actin (29). LOXL1 mRNA expression appears greater than LOX
mRNA expression in many adult mouse tissues, while expression of
LOXL2, LOXL3, and LOXL4 mRNA appears to be low overall.
Lysyl Oxidase family member comparison
The cDNA of lysyl oxidase encode proteins with ~60-85% similarity within the catalytic region to LOX (Table I). Human LOX has the shortest open reading frame of 417 amino acids, while human LOXL2 has the longest, encoding 774 amino acids. All five members of the lysyl oxidase family contain the catalytic lysyl oxidase domain ("LO domain") comprising ~205 amino acids in the carboxyl-terminal half of the protein. The LO domain consists of 4 conserved histidine residues that coordinate copper binding and contains conserved lysine and tyrosine residues that form the cofactor lysyl-tyrosine quinone. The three newest members, LOXL2, LOXL3, and LOXL4, also have four scavenger receptor cysteine-rich domains that mediate ligand binding in many other proteins. Table I summarizes LOX steady state mRNA tissue expression.
Thus, while all LOX family members appear to have the structural requirements to be active enzymes, recombinant proteins have not been generated to assess activity. Lysyl oxidase (presumably LOX) has been purified from bovine aorta. Of note, protein expression was also low with mg amounts purified from kilograms tissue (30). Bovine aortic lysyl oxidase has been shown to cross-link both collagen and elastin substrates and other amine substrates in vitro.
With a multiplicity of lysyl oxidase-like enzymes, the contribution of
any individual gene product to substrate cross-linking and tissue
integrity is unknown. To further complicate the situation, mRNA
expression patterns of different lysyl oxidases overlap in many
tissues. To better understand the contribution of LOX to extracellular
matrix formation and tissue development, LOX-deficient mice were
generated by targeted mutagenesis. In this report, we demonstrate that
mice homozygous for the targeted Lox allele
(LOX/
) die at parturition (or within the first hours of
life). LOX
/
mice are of equal weight and size to their
littermate controls, but die following birth trauma, cardiovascular
events, and/or diaphragmatic rupture. These results suggest that LOX is
required for extracellular matrix development in specific tissues.
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EXPERIMENTAL PROCEDURES |
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Lox Gene Targeting--
BAC clones were obtained by hybridizing
a 129/SvJ filter library (Genome Systems, St. Louis, MO) using a
cDNA probe from the 3'-untranslated region of the Lox
gene. Positive clones were further screened by an exon 1 oligo probe,
and a 5-kb HindIII fragment containing exons 1-4 was cloned
into litmus 29 (New England BioLabs, Beverly, MA). This 5-kb
HindIII fragment was reduced in size by digestion with
BssHII. This size reduction permitted mutagenesis at the ATG
codon where the ATG codon and surrounding bases were converted to a
NotI restriction sites by site-directed mutagenesis (QuikChange mutagenesis, Stratagene, La Jolla, CA). The
BssHII fragment was then replaced, and again, reduced in
size by digestion with NdeI and XhoI
(polylinker). The 480-bp outside probe generated by PCR is located
between the NdeI site and the 3' HindIII site. The 5' HindIII site was now unique and an additional 2.3-kb
5' HindIII fragment (isolated from the same
Lox containing BAC clone) was added to the construct. The
PGK-Neo (phosphoglycerate kinase-neomycin phosphotransferase) cassette
was cloned into an EcoRV site of a modified pBS246 vector
(Invitrogen). In the modified pBS246 vector, the 5' LOXP region
was modified to a LOXP' site, and the 3' LOXP site was left unmodified
(the LOXP' and LOXP sites were incorporated for future gene replacement
experiments). A NotI fragment of the modified pBS246 vector
including the PGK-Neo cassette was cloned into the NotI site
of the mutagenized ATG initiation codon of the Lox gene
construct. This construct deletes the ATG initiation codon and replaces
it with the PGK-Neo cassette in the opposite transcriptional
orientation. Correctly targeted RW4 embryonic stem cells (129/SvJ),
carrying this mutation (frequency of five percent), were injected into
C57BL/6 blastocysts and chimeric mice were produced. This mutation was
transmitted into the germline and mice homozygous for this mutation
(LOX/
) were generated in a mixed 129/SvJ × C57BL/6 background.
Southern Blot Analysis--
DNA from mice tails was prepared by
standard techniques (31, 32). The DNA was digested with
HindIII, size-fractionated on an agarose gel, and
alkali-transferred to a positively charged nylon membrane. The
Lox outside probe was labeled by random primer extension
using [-32P]dCTP, denatured, hybridized to the
DNA-containing membrane, washed, and autoradiograms were perform as
described by Hornstra and Yang (31).
Northern Blot Analysis-- Poly(A)+ RNA from whole mice embryos was isolated using the Poly(A)+ pure RNA isolation kit (Ambion, Austin, TX). Thirty micrograms of mRNA was size-fractionated on formaldehyde-MOPS-agarose gel, SSC-transferred to a nylon membrane, and UV cross-linked. The blot was sequentially hybridized to cDNA probes of the Lox gene, Loxl gene, and Gapdh gene as described above in Southern blotting.
Microscopy: Dissecting, Light, and Transmission Electron Microscopy-- Gross dissection, microscopy, and photography of mutant animals were performed at ×5-10 magnifications. Perinatal mice were sacrificed; the thorax and abdomen were gently dissected and were perfused through the left ventricle with phosphate-buffered saline, followed by Histochoice fixative. The mice were fixed in Histochoice for 2-3 days and processed into slides. Hematoxylin and eosin and Hart's elastic stains were performed on both transverse and sagittal sections. For electron microscopy, perinatal animals were prepared as above except that 2.5% glutaraldehyde in phosphate-buffered saline was used for fixation. The thoracic aorta was removed and incubated overnight in glutaraldehyde at 4 °C. Fixed aortas were processed at the Washington University electron microscopy core facility. Aortas were stained with tannic acid in addition to standard processing, thin-sectioning, and staining.
Tissue Desmosine Assay-- Tissue samples from perinatal mice were collected from the thoracic aorta and total lung. Tissue samples containing from 0.05 to 1 mg of protein were placed in secure-lock centrifuge tubes containing 500 µl of 6 N HCl at 100 °C for 24 h. The hydrolysates were evaporated to dryness in a Savant vacuum concentrator and re-dissolved in 200 µl of water. Desmosine was quantified by radioimmunoassay in 10-40 µl of sample as described previously (33). Hydroxyproline was determined in 20-80 µl of sample by amino acid analysis on a Beckman 6300 analyzer. Protein was determined in 1-10 µl of the hydrolysate using a ninhydrin-based assay (34).
Analysis of Tissue Collagen Cross-links--
The small tissue
samples obtained at birth were suspended in phosphate-buffered saline
and reduced with sodium borohydride as described previously (35), prior
to hydrolysis in 6 N HCl at 110 °C for 16 h. The
hydrolysate was eluted from a CF1 cellulose column (Whatman) to
separate the cross-linking amino acids from the standard amino acids.
The cross-linking amino acids were then quantified using an amino acid
analyzer (Alpha Plus, Amersham Biosciences, Loughborough, UK)
using a modified gradient configured for maximum separation of the
cross-links (36). The location of the cross-links had previously been
confirmed with authentic compounds. Quantification was based on the
ninhydrin color and known leucine equivalents.
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RESULTS |
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Lox Gene Targeting Results in Perinatal Lethality--
A
gene-targeting vector was designed to eliminate Lox
expression by converting the translation initiation codon (ATG) into a
translationally inactive NotI restriction site and inserting the phosphoglycerate kinase promoter driving the neomycin antibiotic resistance gene (PGK-Neo) (Fig.
1A). This construct was
electroporated into embryonic stem cells (129/SvJ), and clones
undergoing homologous recombination were injected into blastocysts.
Resulting chimeric mice were bred to C57BL/6J mice yielding germ line
transmission of the targeted allele. Breeding of heterozygous null LOX
mice resulted in 0 of 400 animals that were homozygous for the targeted allele at 3 weeks of age. Timed matings were set up to determine the
fate of LOX/
mice. LOX
/
mice were
observed by genotype late in gestation and at parturition with proper
Mendelian inheritance (Fig. 1B). Grossly,
LOX
/
mice were of similar size to wild type and
heterozygous littermates at late gestation and parturition, except the
LOX
/
mice died shortly after delivery. Examination of
total embryo Poly(A)+ RNA by Northern analysis demonstrated
essentially absent levels of the LOX mRNA (Fig. 1C) in
LOX
/
mice. LOXL1 mRNA expression was
detectable in total embryos, without compensatory changes in the
absence of LOX.
|
Gross Aortic and Diaphragmatic Abnormalities in Lox Null Mutant
Mice--
Wild type and LOX/
mice were of similar
size, limb length, musculature, and overall gross morphology. Major
differences were found in the thorax and abdomen in
lox
/
versus wild type mice (Fig.
2, A and B). The
diaphragm was often ruptured in LOX
/
mice, allowing
herniation of abdominal contents into the thorax. In Fig.
2B, the liver and stomach, no longer constrained by the diaphragm, displaced the heart from the mid-chest and compressed the
lungs. In contrast, the diaphragm in wild type mice distinctly separated the thorax and abdominal contents. Following removal of the
sternum and anterior distal ribs (Fig. 2, C and
D), the wild type mice had an intact diaphragm, while in
LOX
/
mice we observed the muscular rim of the diaphragm
connected to the body wall, but only fragments of the central tendon
were visible. This suggests a critical weakness in the tensile strength of the collagen-rich central tendon of the diaphragm. Diaphragmatic rupture was found to be either unilateral (left or right) or bilateral in different mice. Diaphragmatic rupture occasionally occurred in
LOX
/
mice in the last days of gestation but most
frequently occurred at birth when the animals started breathing.
|
Most lox/
mice had hemothorax and/or hemoperitoneum.
The descending aorta of LOX
/
mice was always markedly
tortuous (Fig. 2D). This contrasted with the uniformly
straight path of the aorta in heterozygous and wild type mice (Fig.
2C). The aortic curvature in the LOX
/
mice
represents a lengthening of the aorta in vivo in the absence of LOX. While LOX is critical for aorta and diaphragmatic development, other organs, including the limbs and skeletal system of
LOX
/
mice, appeared grossly normal.
Microscopic Arterial Abnormalities in LOX/
Mice--
Microscopic analyses of transverse sections from neonatal
mice stained with trichrome demonstrated similar morphology
between the wild type and LOX
/
mice at low power (×40)
in the skin, musculature, vertebrae, and lungs (Fig.
3, upper panels). Higher power
view (Fig. 3, lower panels, ×200) of the lungs following
Hart's elastic stain revealed less prominent staining of elastic
fibers around blood vessels (labeled V) and conducting
airways (labeled C) in the LOX
/
mice as
compared with wild type mice.
|
Light microscopy (×400) of the descending thoracic aorta demonstrated
significant abnormalities at term in the absence of LOX (Fig.
4). Hematoxylin and eosin staining shows
areas of disrupted smooth muscle cell contact in the
LOX/
mice (clear spaces, arrows), not
observed in the wild type (arrow). trichrome staining
reveals discontinuity of collagen (arrow) in the aortic
lamellae as well as impaired continuity of smooth muscle cells in the
LOX null aorta, while the wild type aorta had concentric blue lamellae
and cohesive cell contact (arrow). Hart's elastic stain
demonstrates a well developed internal elastic lamina and concentric
lamellae in wild type (arrow). LOX
/
mice,
however, had a fragmented and discontinuous internal elastic lamina
with similarly fragmented and irregular lamellae (arrow). Aorta wall thickness was more variable and often significantly thinner
in LOX
/
than in wild type mice. Histologically, elastin
fiber abnormalities in LOX
/
mice appear as early as
E14.5 and progress in parallel with the development of elastic fibers
(data not shown). Aortic rupture is a late event in development
occurring most frequently in the perinatal period.
|
Defects in the aortas of full-term LOX/
mice were even
more pronounced when examined by transmission electron microscopy
stained with tannic acid to accentuate the extracellular matrix (Fig. 5). In wild type mice, the internal
elastic lamina just below the luminal endothelial cell layer is well
developed with a nearly continuous thick band of elastin (upper
arrows). Proceeding away from the lumen, nearly linear and
continuous concentric lamellae are visualized between cell layers
(lower arrows). In contrast, the internal elastic lamina is
poorly developed, fragmented, and discontinuous in LOX
/
mice (upper arrows). The concentric lamellae are similarly
fragmented and discontinuous with increased space between smooth muscle
cells in the LOX
/
mice (lower arrows). Only
irregular collections of matrix were seen between the
LOX
/
cells, and some of the cells appeared less
polarized when compared with wild type. Collagen fibers were of similar
diameters in all genotypes (data not shown). These histological
abnormalities in the LOX
/
mice likely predispose to the
aortic rupture with hemothorax and hemoperitoneum that was
observed.
|
Biochemical Analysis of Elastin and Collagen Cross-links and
Hydroxyproline--
LOX activity was assessed by quantifying elastin
cross-linking using a desmosine radioimmunoassay (33) in tissue samples from litters of full-term mice. Compared with wild type mice, LOX+/ mice had 86 and 103% of desmosine in aorta and
lung tissues respectively. LOX
/
mice had only 39% of
the desmosine cross-links in aortic hydrolysates and 45% of the
desmosine in lung hydrolysates when compared with wild type (Table
II). Thus, desmosine analysis agrees with
the ultrastructural studies in documenting decreased elastin
content.
|
Hydroxyproline was measured by amino acid analysis from litters of
full-term mice to quantify collagen content in aortic and lung
hydrolysates. LOX/
mice aortas had 74%, and lungs had
68% hydroxyproline content as compared with wild type mice.
Heterozygous LOX (LOX+/
) aortas and lungs had 97 and 93%
of hydroxyproline content as wild type mice (Table
III). Immature collagen cross-links were analyzed in both the whole body and lungs of mice. As shown in Table
IV, there are statistically
significant decreases in DHLNL (dihydroxylysinonorleucine), and
HLNL (hydroxylysinonorleucine) of 43 and 39%, respectively, in
LOX
/
, as compared with wild type total body collagen
cross-links. Collagen cross-link data are normalized to moles of
collagen as determined by hydroxyproline content. In
LOX+/
mice, DHLNL is 100% of wild type, while HLNL is
only 64% of wild type content. This could represent a greater role for
LOX in HLNL cross-links, with a decrement observed with quantitative
reduction in LOX (50%) in heterozygous mice. Collagen cross-link data
from the lungs was also determined. The analysis of E20.5 lungs was at
the limit of assay detection and showed only a trend toward decreased
HLNL in heterozygote and homozygous null mice (14 and 32%,
respectively). Lung DHLNL was not different between the genotypes. These data demonstrate a quantitative reduction of elastin-derived cross-links, collagen content, and immature collagen cross-links in
LOX
/
mice, all of which support and help explain the
phenotypic abnormalities observed. Yet, incomplete loss of collagen and
elastin cross-linking likely explains the fact that not all organs were
grossly abnormal.
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DISCUSSION |
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LOX gene targeting results in decreases in both collagen and elastin cross-links in vivo, confirming the catalytic capacity of LOX demonstrated in vitro. Despite the presence of five lysyl oxidase enzymes, LOX deficiency is lethal, suggesting that other lysyl oxidases are unable to effectively compensate during development for the physiological stresses associated with parturition and life ex utero. LOXL1 mRNA does not increase to compensate for LOX deficiency in the total embryo. The inability of the other lysyl oxidases to compensate may relate to either distinct substrate specificities of individual enzymes or temporally and spatially unique expression patterns.
The consequences of decreased lysyl oxidase activity appear most
significant in the development of the aorta and diaphragm, two organs
where tensile strength requirements increase significantly at birth
(37). LOX/
mice survive in utero, probably
due to feto-maternal circulation, low systemic blood pressure, and low
intrathoracic pressure. At parturition, the fetus is subjected to many
physiologic stresses, including physical trauma from passage through
the birth canal, increased systemic blood pressure (37), and conversion
of liquid filled to air filled lungs with the onset of breathing
requiring diaphragmatic contraction. It is precisely at this time
shortly after birth when the majority of LOX
/
mice die,
likely due to the inability to handle increased stress placed on the
vascular bed and diaphragm.
Although elastin cross-links were significantly decreased in the
aorta and lungs of LOX/
mice, they were not completely
eliminated. This suggests that other enzyme(s) participate in
cross-linking elastin to produce the ~40% of desmosine that remain
in these organs. Whether LOX catalyzes a specific subset of lysine
residues within tropoelastin or whether it contributes 60% of total
activity on random cross-links is not known. LOX
/
mice
also have 40% reduced immature collagen cross-links (DHLNL and HLNL)
in the whole mouse. Although the exact relationship between collagen
cross-links and tensile strength is unknown, 40% overall reduction in
cross-linking could certainly impair mechanical strength, particularly
if it were greater in individual tissues. The combined reduction in
elastin and collagen cross-linking resulted in loss of structural
integrity of the aorta. Impaired collagen cross-linking most likely
explains the diaphragmatic abnormalities, since this is the major
matrix component of this tissue. Due to the small size of these organs
at birth, detection of collagen cross-links in these individual tissues
was below the sensitivity of the assay.
Morphologically, the elastin defects were more apparent which is likely
because 36 of 40 lysine residues in tropoelastin are cross-linked in
mature elastin, while there are only one to two collagen cross-links
per triple helical collagen unit (38). In addition, the inability to
cross-link tropoelastin renders this precursor molecule sensitive to
trypsin-like proteases (38). LOX/
mice clearly show a
decreased quantity of elastin by electron microscopy, and the decreased
desmosine cross-links appear to correlate with decreased elastin
deposition and presumably increased tropoelastin turnover. In contrast,
morphological changes in collagen are less apparent, since collagen
cross-links are not required for collagen fibril assembly.
LOX
/
mice had a 30% reduction in hydroxyproline
content, which may be influenced by decreased hydroxyproline from both
collagen and elastin. Collagen cross-links are reduced by 40% in
LOX
/
mice, which certainly appears to have influenced
the tensile strength of some connective tissues.
Insight into the physiologic relevance of impaired elastin and collagen
in LOX/
mice can be elucidated from other loss of
function mice. Elastin null mutant mice die by P4.5 likely due to an
obliterative aortic occlusive disease (39). Mice heterozygous or
haploinsufficient for the elastin gene have 2.5 times more aortic
lamellar units compared with control mice (40, 41). This increase in
lamellar units is also observed in humans with supravalvular aortic
stenosis characterized by haploinsuffiency of the elastin gene. Thus,
elastin appears to regulate smooth muscle proliferation and the
quantity of aortic lamellar units (40), but primary deficiency of
elastin does not correlate with arterial rupture. Also, we do not
observe smooth muscle proliferation and increased lamellar units in
LOX
/
mice.
Ehlers-Danlos syndrome Type IV is characterized by a primary collagen defect (42). Type IV or the vascular type of Ehlers-Danlos syndrome in many patients is a consequence of mutations in the COL3A1 gene. The disease phenotype includes arterial rupture, thin skin, bruising, uterine rupture, and small joint hyperextensibility. Most Col3a1 gene-targeted mice die perinatally due to blood vessel rupture, and those that make into adulthood have one-fifth of the normal life span also dying from aortic rupture (43). Aortic aneurysm formation occurs between the media and the adventitia (43), where collagen is relatively more prominent than elastin at this interface. Therefore, the lethal phenotypic changes in LOX-deficient mice are probably more related to decreased collagen cross-links and the resultant change in tensile strength in the aorta and diaphragm than to changes in elastin content or maturation.
In LOX/
mice, while aortic and diaphragmatic
changes are the most obvious phenotypic abnormalities, other organs and
tissues are certainly also affected. Upon dissection,
LOX
/
mouse skin tears easily, and the ribs are softer
and cut with minimal force. LOX
/
paraffin-infiltrated
embryos deform more readily upon transverse or sagittal cutting.
Furthermore, the elastin in the conducting airways and pulmonary
vessels is altered in less developed in LOX null animals. Similarly,
the heart is probably affected with elastin defects in the heart
valves, but since the animals die at parturition, these physiological
defects are difficult to examine in detail. Thus, LOX-induced
cross-linking is not required for organogenesis but is required for the
development of tensile strength in collagen and elastogenesis needed
for the organism to survive ex utero.
A human disease of primary LOX deficiency is not known. If the phenotype were identical to what we see in LOX-deficient mice, one would expect to find stillborns or newborns unable to meet cardiopulmonary physiological stresses ex utero. Since this has not been described, it is likely that either a LOX deficiency either manifests itself as a more severe deficiency impairing embryonic viability earlier in gestation, or compensation by other LOXLs may lead to a milder phenotype that is unappreciated but could predispose to aortic aneurysms/dissection, diaphragmatic hernias, or other connective tissue phenotypes.
Although primary LOX deficiency has not been described in humans,
secondary LOX deficiency resulting from low enzyme activity is
associated with at least three human conditions: lathyrism, copper
deficiency, and Menke's syndrome. Lathyrism is a condition of
decreased lysyl oxidase activity secondary to ingestion of BAPN, a
naturally occurring and specific inhibitor of lysyl oxidase. The
severity of the phenotype in lathyrism is proportional to the quantity
and timing of BAPN ingestion. In animals, BAPN exposure early in life
leads to aortic aneurysms and death, while later in life it manifests
in bone and tendon abnormalities (44). Primary human copper deficiency
manifesting as anemia, neutropenia, and bone fractures is rarely seen
in developed countries except in some malabsorption syndromes and very
low birth weight infants (45). In developing countries, it is observed
in malnourished children with low copper diets (45). Menke's syndrome
is characterized by a loss of function mutation in the X-linked
ATP7A gene that encodes a copper transporter necessary for
copper absorption to make active cuproenzymes, which includes lysyl
oxidases (46). This results in decreased total lysyl oxidase activity,
likely affecting all active LOX family members. Menke's patients
exhibit developmental delay, loose skin, fragile bones, and aortic
abnormalities. Aortic defects manifest as tortuousity, fragmented
discontinuous elastic lamellae, and consequent aneurysm formation (47,
48). Thus, humans with Menke's syndrome have connective tissue defects that overlap with the phenotype of LOX/
mice.
If gene disruption of the Lox gene results in lethality,
what phenotypes could be expected from the other four-lysyl
oxidase-like genes? The answer to this question requires knowing what
lysine substrates the other four lysyl oxidase-like genes catalyze
in vivo, the spatial and compartmental location of these
enzymes, and the temporal patterns of gene expression. These enzymes
have been notoriously difficult to purify. It has also been difficult to express large quantities of recombinant protein. Thus, gene targeting provides a method to define the roles of each lysyl oxidase
in a biological context. Of the five lysyl oxidases, LOX and LOXL1 are
most homologous, while LOXL2, LOXL3, and LOXL4 represent another
homologous group. Whether either group has a greater affinity toward
collagen versus elastin is unknown. These lysyl oxidases may
have other substrates other than the extracellular matrix proteins,
collagen and elastin. Future research in to the roles of lysyl oxidases
in biology will be revealing.
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FOOTNOTES |
---|
* This work was supported by NIAMS, National Institutes of Health, Grant K08 AR02059; a Barnes-Jewish Hospital Foundation grant (to I. K. H.); NHLRI Grant HL53325 (to R. P. M.); and by NHLBI Grant P01 HL 29594, National Institutes of Health (to S. D. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Medicine,
Division of Dermatology, Washington University, 660 South Euclid Ave.,
Campus Box 8123, St. Louis, MO 63110. Tel.: 314-454-8928; Fax:
314-454-5626; E-mail: ihornstr@im.wustl.edu.
Published, JBC Papers in Press, December 7, 2002, DOI 10.1074/jbc.M210144200
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ABBREVIATIONS |
---|
The abbreviations used are:
LOX, lysyl
oxidase;
BAPN, -aminopropionitrile;
MOPS, 4-morpholinepropanesulfonic acid;
DHLNL, dihydroxylysinonorleucine;
HLNL, hydroxylysinonorleucine.
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