The final enzymatic step required for collagen cross-linking is the extracellular oxidative deamination of peptidyl-lysine
and -hydroxylysine residues by lysyl oxidase. A cross-linked collagenous ...extracellular matrix is required for bone formation.
The goals of this study were to compare the transforming growth factor (TGF)-β1 regulation of lysyl oxidase enzyme activity
and steady state mRNA levels to changes in COL1A1 mRNA levels in MC3T3-E1 osteoblastic cells. TGF-β1 increased steady state
lysyl oxidase and COL1A1 mRNA levels in a dose- and time-dependent manner. The increase in lysyl oxidase mRNA levels was transient,
peaking at 12 h and 8.8 times controls in cells treated with 400 pM TGF-β1. COL1A1 steady state mRNA levels increased maximally
to 3.5-fold of controls. Development of increased lysyl oxidase enzyme activity was delayed and was of slightly lower magnitude
than the increase in its mRNA levels. This suggested limiting post-translational processing of lysyl oxidase proenzyme. Pulse-labeling/immunoprecipitation
studies demonstrated slow proenzyme secretion and proteolytic processing. Development and application of an independent assay
for lysyl oxidase proenzyme proteolytic processing activity verified its proportionately lower stimulation by 400 pM TGF-β1.
Thus, lysyl oxidase regulation by TGF-β1 in osteoblastic cell cultures occurs at both pre- and post-translational levels.
This regulation is consistent with increased production of a collagenous extracellular matrix.
Inspiration of CdCl2 results in a focally fibrotic response in rat lungs and markedly increases the activity of lung lysyl oxidase. Western blot analyses of urea-extractable rat lung proteins ...revealed that the levels of an immunoreactive, 32,000-Da protein were markedly increased in the cadmium-exposed rat lung tissue, consistent with the induction of lysyl oxidase protein. Anion exchange chromatography revealed low levels of multiple peaks of catalytically functional lysyl oxidase in control rat lung extracts, while the profile of cadmium-exposed rat lung extracts displayed markedly elevated levels of multiple peaks of enzyme activity indicating that the charge heterogeneity is expressed in the activated enzyme. The cadmium-induced enzyme was purified as a species of 32 kDa, without resolving individual ionic variants. The catalytic and physical properties of the isolated enzyme were very similar to those of previously well characterized basal enzyme of bovine aorta, including the presence of a pyrroloquinoline quinone-like carbonyl cofactor. The copper and cadmium content of the cadmium-induced enzyme indicated little if any replacement of tightly-bound copper by cadmium in the exposed lung.
The cloning of the 3′-untranslated region of rat lysyl oxidase cDNA was completed. cDNA clones were generated by reverse transcriptase PCR from neonatal rat aorta smooth muscle cell RNA, and ...sequenced. Several polyadenylated clones were obtained, providing 2.1 kb of new sequence. Clones were polyadenylated at three different positions. The cDNA clones were verified by PCR-cloning and sequencing of genomic DNA, and by Northern blotting studies. Evidence is presented that the polyadenylation patterns of rat lysyl oxidase mRNAs are similar, but not identical to mouse or human transcripts. Interestingly, the nonconsensus polyadenylations in rat did not occur at the same positions as was found in mouse lysyl oxidase cDNAs. Multiple transcription initiation sites were found by primer extension mapping. Thus, the complex pattern of rat lysyl oxidase mRNAs on Northern blots is principally due to differential use of polyadenylation signals, and to the occurrence of multiple transcription initiation sites. All clones lacked a previously reported 258 by segment nearly identical to a conserved segment of the 3′-untranslated region of elastin cDNA. We conclude that the elastin-like sequence previously reported in rat lysyl oxidase cDNA is not a species-specific sequence, and most probably resulted from spurious ligation reactions during construction of the cDNA library.
Lysyl oxidase had been purified to near homogeneity from bovine aorta and bovine ligamentum nuchae employing a modification of methods described by Harris et al., and Stassen and his colleagues. The ...aortic enzyme gives rise to at least three peaks and the ligament enzyme resolves into at least four peaks upon chromatography on DEAE cellulose. The molecular weight of each peak of both enzymes is approximately 30,000 daltons in sodium dodecyl sulfate. The aortic enzyme aggregates to species with molecular weights varying from approximately 60,000 to 1,000,000 daltons upon dialysis out of urea into phosphate-buffered saline. Temperature studies reveal that lysyl oxidase is stable to temperatures as high as 80 degrees C, although the assay optimum is 52 degrees C. Studies in progress suggest the temperature dependency of assay may reflect conformational changes in the elastin substrate.
PDF
