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(Journal of Nutrition. 1999;129:2143-2146.)
© 1999 The American Society for Nutritional Sciences

Research Communication

Activation of Chick Tendon Lysyl Oxidase in Response to Dietary Copper1

Robert B. Rucker*2, Brian R. Rucker*, Alyson E. Mitchell*,{dagger}, Chang Tai Cui*, Michael Clegg*, Taru Kosonen*, Janet Y. Uriu-Adams*, Eskouhie H. Tchaparian*, Michelle Fishman* and Carl L. Keen*

Departments of * Nutrition and {dagger} Environmental Toxicology, University of California, Davis, Davis, CA 95616

2To whom correspondence should be addressed.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Lysyl oxidase (EC 1.4.3.13), a cuproenzyme, can account for 10–30% of the copper present in connective tissue. Herein, we assess the extent to which tissue copper concentrations and lysyl oxidase activity are related because the functional activity of lysyl oxidase and the copper content of chick tendon are both related to dietary copper intake. Chicks (1-d old) were fed diets (basal copper concentration, 0.4 µg/g diet) to which copper was added from 0 to 16 µg/g diet. Liver and plasma copper levels tended to normalize in chickens that consumed from 1 to 4 µg copper/g of diet, whereas tendon copper concentrations suggested an unusual accumulation of copper in chickens that consumed 16 µg copper/g diet. The molecular weight of lysyl oxidase was also estimated using matrix-assisted laser desorption ionization/time-of-flight/mass spectrometry (MALDI/TOF/MS). A novel aspect of these measurements was estimation of protein mass directly from the surface of chick tendons and aortae. Whether copper deficiency (0 added copper) or copper supplementation (16 µg copper/g of diet) caused changes in the molecular weight of protein(s) in tendon corresponding to lysyl oxidase was addressed. The average molecular weight of the peak corresponding to lysyl oxidase in tendon and aorta from copper-deficient birds was 28,386 Da ± 86, whereas the average molecular weight of corresponding protein in tendon from copper-supplemented birds was 28,639 Da ± 122. We propose that the shift in molecular weight is due in part to copper binding and the formation of lysyl tyrosyl quinone, the cofactor at the active site of lysyl oxidase.

KEY WORDS: • chickens • copper • lysyl oxidase • collagen • elastin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Skeletal and vascular tissue defects are associated with nutritional copper deficiency (Reiser et al. 1992Citation ). The biochemical basis for the defects arises in part from a reduction in lysyl oxidase (EC 1.4.3.13) functional activity (Medeiros and Wildman 1997Citation , Rucker et al. 1996Citation ). Two forms of lysyl oxidase have been identified (intracellular and extracellular); they appear to be the product of four separate genes (Decitre et al. 1998Citation , Jourdan-Le Saux et al. 1998Citation , Smith-Mungo and Kagan, 1998Citation ). The extracellular form of lysyl oxidase catalyzes the oxidative deamination of lysyl and/or hydroxylysyl residues in collagens and elastin to the corresponding peptidyl {delta}-semialdehydes. This process is an initial step in a complex series of reactions that result in covalent cross-links (Reiser et al. 1992Citation ). The intracellular form(s) of lysyl oxidase appear to function as an antioncogene, e.g., a ras recision gene, or as a target for antioncogene transcription factors, e.g., immune response factor-1 (Di Donato et al. 1997Citation , Dimaculangan et al. 1994Citation , Friedman et al. 1997Citation , Kenyon et al. 1993Citation , Li et al. 1997Citation , Tan et al. 1996Citation ).

In tendon, lysyl oxidase (as protein) can amount to 200–300 µg/g of tissue or ~8–10 nmol/g (Rucker et al. 1996Citation ). With respect to dietary copper, the amount of lysyl oxidase protein and the levels of lysyl oxidase mRNA in connective tissue do not appear to be significantly influenced by altered intake (Rucker et al. 1996Citation and 1998Citation ). Although dietary copper deficiency can depress lysyl oxidase functional activity, data are not available that directly link changes in lysyl oxidase activity with changes in tissue copper. Of interest, the steady-state activity of lysyl oxidase increases in response to feeding dietary copper well beyond the theoretical amounts required for cross-link formation (Opsahl et al. 1982Citation ). A proposed mechanism is that lysyl oxidase activation occurs in post-translational steps that are copper dependent and that result in the formation of peptidyl tri[oxo]phenylalanyl derivatives (TOPA) or lysyl tyrosine quinone (Mu et al. 1992Citation , Nakamura et al. 1996Citation , Wang et al. 1996Citation and 1997Citation ). Recently, we observed that the reaction takes place in a postgolgi secretory pathway that brings lysyl oxidase and copper into contact with each other (Kosonen et al. 1997Citation , Rucker et al. 1998Citation ). Herein, data are presented that lysyl oxidase activation in chick tendon and aorta is related to the tissue copper concentration. Moreover, the apparent molecular weight of tendon and aortic protein corresponding to lysyl oxidase was observed to be ~300 Da greater in copper-supplemented vs. copper-deficient chicks. Matrix-assisted laser desorption ionization/time of flight/mass spectrometry (MALDI/TOF/MS) was used to detect such changes directly from the surface of tendon and aortic tissues.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Supplies and diet.

Male white leghorn chicks were purchased from La Mont Hatchery (Coburg, OR). Diets varying in copper content were prepared as described by Opsahl et al. 1982Citation using spray-dried skim milk and CuSO4·3H2O as a source of copper. Chemicals were purchased from Fisher Scientific (Pittsburgh, PA), Pierce Chemical (Rockford, IL), Aldridge Chemical (Milwaukee, WI) or Sigma Biochemical (St. Louis, MO).

Animal model.

Two experiments were conducted. In both experiments, 1-d-old white leghorn cockerals (~40 g) were used. In Experiment 1, copper was added to the basal diet at 0, 1, 2, 4 or 16 µg Cu/g diet. At the end of this experiment (35 d), birds were killed by CO2 inhalation; plasma and liver were removed, weighed, frozen in liquid nitrogen and stored at -20°C until analysis. In Experiment 2, three groups of birds (n = 20) were fed 0, 4 or 16 µg Cu/g diet to validate selected measures obtained in Experiment 1. The animal protocol for these studies was approved by the UC Davis Animal Use Research Committee and complied with NIH and USDA guidelines (NRC 1996Citation ).

Tendon lysyl oxidase extraction and assay.

Lysyl oxidase functional activity was assayed by a modification of the assay first described by Trackman et al. (1981)Citation . Tendon samples (50 mg) were fragmented and ground in liquid nitrogen using a metal mortar and pestle. Next, readily soluble protein was extracted into PBS (0.01 mol/L, pH 7.6) for 1–2 h (2–3 mL). The tissue was centrifuged (10,000 x g for 30 min). The tissue pellet was then homogenized into 3 mL of 6 mol/L urea (buffered with an 0.1 mol/L sodium borate at pH 8.2) using a fritted glass homogenizer. The tissue was extracted (4°C for 8–12 h) with agitation and the supernatant fraction collected (10,000 x g for 30 min). This step was repeated and the supernatant fractions combined.

Assays for lysyl oxidase (3 mL total volume) contained the following: 0.25 mg of sodium homovanillate, 40 µg horseradish peroxidase, tendon urea extract equivalent to ~1 µg of lysyl oxidase (usually 0.5 mL urea extract), 3.33 mmol/L cadaverine and sodium borate buffer (0.05 mol/L, pH 8.2). Assays were performed at 40°C. Fluorescence as a result of hydrogen peroxide production and homovanillate oxidation was monitored continuously at 315 nm (excitation) and 425 nm (emission) for 10 min.3

Copper analyses.

Copper analyses were performed by graphite furnace atomic absorption spectrophotometry (Chung et al. 1988Citation ) or by inductively coupled plasma atomic emission spectrometry (Thermo Jarrell Ash, Trace Scan, Franklin, MA). Samples were digested as described by Clegg et al. (1981)Citation and analyzed in 0.05 mol/L HNO3 at 324.7 mm (slit width 0.5 mm). Standards were obtained from Fisher Chemical (St. Louis, MO).

Mass spectrometric analysis.

MALDI/TOF/MS spectra were obtained using a Hewlett-Packard G2025A mass spectrometer (Palo Alto, CA). Sinnapinic acid was used as the organic acid matrix, with bovine serum albumin and purified fractions of lysyl oxidase as calibrants. Partially purified lysyl oxidase was obtained from chick aorta (~50 g) as described previously (Romero-Chapman et al. 1991Citation ; Rucker et al. 1996Citation ). Spectra were collected using positive polarity in a single-shot mode over a mass range of 105 Da and summed. Laser energy varied from 7.00 to 17.00 µJ.

Spectra were then obtained from chick tendon or aorta directly. Tissue samples were frozen under liquid nitrogen and fragmented into small pieces. The pieces were then rinsed with PBS and nanopure water. The tissue was homogenized in distilled water (1:1, wt/v) and centrifuged. Sinnapinic acid was allowed to penetrate the sample by sonification. The sonicated tissue was layered onto a standard 10-position MALDI probe tip using pressure. A layer of sinnapinic acid was crystallized by vacuum over the top of each sample. MALDI/TOF/MS analysis was conducted using samples of tendon from chicks fed either the basal diet or a diet to which copper was added at 16 µg/g diet. Corresponding aortic samples were used for validation.

Statistics.

Linear regression and estimation of r2 were performed as described by Campbell (1989)Citation . A Dunnett test was used for multiple comparisons (Dunnett 1955Citation and 1970Citation )


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Copper levels in liver and plasma tended to normalize (at 35 d) at dietary copper intakes as low as 1 µg/g/ diet (Table 1Citation ). Tendon copper concentrations in response to increasing dietary copper (Table 1Citation and Fig. 1ACitation ) suggested an unusual accumulation of copper, particularly in chickens fed 16 µg copper/g. Lysyl oxidase activity in tendon was related to copper intake and tendon copper concentration (Figs. 1ACitation and BCitation ).


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  		Table 1. Body weight and plasma, liver and tendon copper concentrations in chickens fed diets varying in copper12

 


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  		Figure 1. Lysyl oxidase activity in response to changes in (A) dietary and (B) tendon copper in chickens. Lysyl oxidase activity is expressed as nmol H2O2 generated/(min·g fresh tissue). In Experiment 1 (Fig. 1BCitation ), copper was fed at 0 ({circ}), 1 (•), 2 ({blacktriangleup}), 4 ({blacksquare}) or 16 ({square}) µg copper/g diet. In this experiment, given copper values were not matched with the corresponding values for lysyl oxidase activity; thus, average value ± SEM are given for the different dietary groups. Matched values for tendon copper and lysyl oxidase activity were obtained in Experiment 2 (•, Fig. 1ACitation ). The correlation between lysyl oxidase activity and tendon copper in Experiment 2 was r 2 = 0.88. Values with differing superscripts are significantly different, P < 0.05.

 
Several recent advances have contributed greatly to our understanding of mechanisms important to the activation for copper-containing amine oxidases (Matsuzaki et al. 1995Citation , Mu et al. 1992Citation , Wang et al. 1996Citation and 1997Citation ). After incorporation into amine oxidase, copper aids in catalyzing the formation of tyrosine-derived quinone cofactors, such as the TOPA derivatives. Regulation of the functional activity therefore involves not only transcriptional and post-transcriptional events, but post-translational steps as well. In the case of lysyl oxidase, lysyl tyrosyl quinone is the eventual cofactor formed after copper-mediated catalysis at the active site of lysyl oxidase (Wang et al. 1996Citation and 1997Citation ). We hypothesized that for lysyl tyrosine quinone generation to occur, lysyl oxidase and copper must share a portion of the pathway(s) for their respective cellular transport (Kosonen et al. 1997Citation , Rucker et al. 1998Citation ). We also suggested that lysyl oxidase plays a primary role as a vehicle for copper export from connective tissue cells (Kosonen et al. 1997Citation ). At steady state, this process can account for a substantial percentage of the total cellular copper. Chick tendon normally contains ~8–10 nmol lysyl oxidase/g tissue (Rucker et al. 1996Citation ); full saturation of the lysyl oxidase active site accounts for ~15% of the total copper in tendon. In addition, lysyl oxidase can bind from 3 to 4 additional mol copper/mol lysyl oxidase (Gacheru et al. 1990Citation ).

That copper deprivation results in modifications in lysyl oxidase was suggested by the lower molecular weight of tendon protein corresponding to lysyl oxidase (Fig. 2Citation ). The molecular weight of protein corresponding to lysyl oxidase from copper-supplemented chicks was consistently ~300 Da greater in mass than corresponding protein from tendon of chicks fed diets with no added copper. Identical results were obtained using corresponding aortic samples (data not shown).



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  		Figure 2. Matrix-assisted laser desorption ionization/time-of-flight/mass spectrometry (MALDI/TOF/MS) spectra (relative abundance vs. m/z) of tendon samples from chicks fed diets to which 0 or 16 µg/g copper was added. Typical spectra from six samples are shown (three with Cu supplement: A, B, C; three copper deficient: D, E, F). Very similar spectra were obtained from corresponding chick aortic samples (data not shown). The lysyl oxidase standard was obtained from chick aorta (Romero-Chapman et al. 1991Citation ). Of potential importance, lysyl oxidase from copper-deficient chicks could not be obtained due to extensive proteolysis during the latter stages of purification. Extraction of tendon samples with 4–6 mol/L urea resulted in complete removal of laser desorbable protein from tissue surfaces. Average molecular weight for protein corresponding to lysyl oxidase from copper-deficient chick tissues (both tendon and aorta) was 28,386 ± 86 Da, and for copper-supplemented chick tissues (tendon and aorta), 28,639 ± 122 Da. The MW of the lysyl oxidase standard was 28,572 Da (average of two determinations, Wu et al. 1992Citation ).

 
Although inferential, the differences in molecular weight of tendon protein corresponding to lysyl oxidase are compatible with possible variations in metal binding (3–4 mol copper/mol protein) and lysyl tyrosine quinone formation. These data provide an additional example of the relationship between nutrient intake and post-translational protein modifications to enzyme activation. Further, the data underscore the potentially novel use of MALDI/TOF/MS at the in situ or tissue level to detect changes in protein mass in response to changing a dietary variable.


   ACKNOWLEDGMENTS
 
The authors thank A. Daniel Jones, T. T. Yip and William Hutchens for advice and use of resources at the Facility for Advanced Instrumentation, University of California, Davis.


   FOOTNOTES
 
1 Supported by Public Health Service National Institutes of Health grants HD 26777, AM 25358 (C.L.K., J.Y.U.-A. and R.B.R), an ARS-U.S. Department of Agriculture grant from the Human Nutrition Research Initiative (R.B.R.), and a grant from the Academy of Finland (T.K.). Back

3 Decreased rates of hydrogen peroxide production were observed when PBS extracts of tendon were added to assays containing functional lysyl oxidase activity. Extraction with PBS is essential to remove the inhibitory factors before extraction with urea. The release of lysyl oxidase from tendon and aorta required extraction into 4–6 mol/L urea (Rucker et al. 1996). Back

Manuscript received December 30, 1998. Revision accepted August 30, 1999.


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