Age-related NADH oxidase (arNOX = ENOX3) proteins are superoxide-generating cell surface oxidases that increase in activity with age beginning at about 30 y. A soluble and truncated exfoliated form of the activity is present in blood and other body fluids. The activity was purified to apparent homogeneity from human urine and resolved by 2-D gel electrophoresis into a series of 24 to 32 kDa components of low isoelectric point. The purified proteins were resistant both to N-terminal sequencing and trypsin cleavage. Cleavage with pepsin revealed peptides corresponding to the TM9 family of transmembrane proteins. Peptide antisera raised to all five members of the human TM9 family sequentially blocked the arNOX activity of human saliva and sera. The soluble truncated N-terminus of the human homolog TM9SF4 was expressed in bacteria. The recombinant protein was characterized biochemically and exhibited ar-NOX activity. The findings identify five arNOX isoforms each of which correspond to one of the five known TM9 family members. The exfoliated soluble arNOX forms are derived from the 24 to 32 kDa N-termini exposed to the cell’s exterior at the cell surface. Each of the shed forms contain putative functional motifs characteristic of ECTO-NOX (ENOX) proteins despite only minimal sequence identity. Our findings identify arNOX as having functional characteristics of ENOX proteins and the TM9 superfamily of proteins as the genetic origins of the five known arNOX isoforms present in human sera, plasma and other body fluids1 .
Our work has identified a family of superoxide-generating oxidases that increases in activity with age beginning at about age 30 [
Cell associated arNOX is membrane anchored with its catalytic terminus directed toward the cells’ exterior [
In this report, we identify the five circulating human arNOX isoforms as exfoliated N-termini of the five members of the TM9 family of transmembrane proteins (GenBank NP_006396, NP_001014842, NP_004791, NP_064508, NP_055557). The identification is based on cloned TM9SF4 and TM9SF2, two of the superfamily members and their expression in bacteria and inhibition by specific peptide antisera for the three additional superfamily members, TM9SF1a, TM9SF1b and TM9SF3.
pET11b vector and BL21 (DE3) competent cells were purchased from Novagen (Madison, WI). I.M.A.G.E. Full length cDNA IRAUp969E0589D was from ima-Genes (Berlin, Germany).
Plasmids carrying TM9SF4 sequence were prepared by inserting the TM9SF4 sequence into the pET11b vector (between NheI and BamHI sites). The TM9SF4 sequence was amplified from cloned I.M.A.G.E. full length cDNA IRAUp969E9580D by PCR. The primers used were 5’-GATATACATATGGCTAGCATGGCGACGGCGATGGAT-3’ (forward) and 5’-TTGTTAGCAGCCGGATCCTCAGTCTATCTTCACAGC-3’ (reverse). The PCR products then were doubly digested with NheI and BamHI and were ligated into pET11b vector.
DNA sequences of the ligation products (pET11bTM9SF4) were confirmed by DNA sequencing. Then pET11b-TM9SF4 was transformed into BL21 (DE3) competent cells. A single colony was picked and inoculated into the 5 ml LB/AMP medium. The overnight culture (1 ml) was diluted into 100 ml LB/AMP media (1:100 dilution). The cells were grown with vigorous shaking (250 rpm) at 37˚C to an OD600 of 0.4 - 0.6 and IPTG (0.5 mM) was added for induction. Cultures were collected after 5 h shaking (250 rpm) at 37˚C. Expression of the TM9SF4 was confirmed by SDS-PAGE with silver staining.
Cultures were centrifuged at 6000 g for 20 min. Pellets were resuspended in 20 mM Tris-HCl, pH 8.0 (0.5 mM PMSF added). Cells were broken by French Press at 20,000 psi 3 times.
Plasmids carrying the TM9SF2 (S33-C250 of TM9SF2 with 6× His-tag at the N-terminus) sequence were prepared by inserting the TM9SF2 sequence into the pET11b vector (between NheI and BamHI sites). The TM9SF2 sequence was synthesized by GenScript USA, Inc. (Piscataway, NJ). DNA sequences of the ligation products (pET11b-TM9SF2) were confirmed by DNA sequencing. Expression of recombinant TM9SF2 was the same as that for TM9SF4.
Exfoliated arNOX was purified to apparent homogeneity from a 20% ammonium sulfate fraction from human urine (72-y male) followed by dialysis, YM-10 filtration and preparative SDS-PAGE. For sequencing, the sample was further purified on a Capto Q Imp Res HPLC column and a major fraction collected at 8 min with arNOX activity cleaved with 20 ng/µl trypsin, pH 7.8 or with 20 ng/µl pepsin, pH 3 (with HCl) at 37˚C for 2 h. For trypsin digestion, the sample was first suspended in 50 ml Tris-HCl, pH 8 containing 5 mM DTT and 8 M urea at 60˚C for 1 h. After cooling, 50 mM NH4HCO3 was added to reduce the urea concentration to 1 M before adding trypsin. Peptides were identified by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.
Stock E. coli cells [BL21 (DE3)] containing plasmid pET11b-TM9SF4/-TM9SF2 were stored at −80˚C in glycerol stock solution. Small amount of cells were picked by using a sterile yellow pipette tip and added to 1 ml of LB medium containing ampicillin (100 µg/ml) (LB/amp medium) and mixed well. Twenty µl of the cell resuspension was plated on LB/amp. Cells were grown for more than 20 h until well-rounded colonies were formed. One colony was picked and mixed in 10 ml of LB/amp medium (inoculum). Cells were grown for 8 h and stored at 4˚C until use. The inoculum was centrifuged at 6000 rpm for 6 min. Supernatant was discarded and pellet was resuspended in 4 ml of LB/amp medium. Aliquots of 100 ml of LB/amp media were prepared in four 250 ml—Erlenmeyer flasks. One ml of cell resuspension was added to each flask and cells were grown for 8 h with shaking. IPTG (0.5 mM) was added for induction when the OD600 reached 0.4 to 0.6.
Cells were harvested by centrifugation at 6000 rpm for 6 min, washed with 20 mM Tris-HCI, pH 8.0 and resuspended in 20 mM Tris-HCI, pH 8.0 containing protease inhibitors (0.75 mM PMSF, 3 mM 6-aminocaproic acid and 1.5 mM benzamidine HCl).
Proteins were extracted by French Press (3 passages at 20,000 psi). The extracts were centrifuged at 10,000 rpm for 20 min. Supernatant was discarded and pellets (inclusion bodies) were resuspended in 20 ml of Tris buffer. Two ml of 20% Triton X-100 was added to each tube and sample volume was adjusted to 40 ml with Tris buffer. Tubes were incubated at room temperature for 1 h with shaking and centrifuged at 10,000 rpm for 20 min. Supernatants were discarded and pellets were washed two times with Tris buffer by resuspending in 25 ml of Tris buffer and centrifuged one time with 25 ml of deionized water.
Pellets were resuspended in 20 ml of water and 4 ml of 0.5 M CAPS buffer, pH 11 (50 mM final concentration), 40 µl of 1 M DTT (1 mM final concentration) and 0.4 ml of 30% sodium lauroyl sarcosine (0.3 % final concentration) were added. Sample volumes were adjusted to 40 ml with water. Samples were incubated at room temperature for 17 h.
After solubilization, samples were centrifuged at 10,000 rpm for 20 min and supernatants were collected. The supernatants were filtered through a 0.45 µm syringe filter. The filtrates were poured into two dialysis bags (3500 MWCO, flat width 45 mm and diameter 29 mm, Spectra Por) and dialyzed against cold dialysis buffer A (25 mM Tris-HCl, pH 8.5, 1 mM cysteamine, 0.1 mM cystamine, 1 mM 6-aminocaproic acid and 0.5 mM benzamidine HCl) with 3 changes, against cold dialysis buffer B (25 mM Tris-HCl, pH 8.0, 1 mM 6-aminocaproic acid and 0.5 mM benzamidine HCl) with one change and against dialysis buffer C (50 mM Tris-HCl, pH 8.0, 1 mM 6-aminocaproic acid and 0.5 mM benzamidine HCl) with one change. Dialysis was at least 17 h for each change. After dialysis, PMSF was added to a final concentration of 0.5 mM and samples were centrifuged at 10,000 rpm for 20 min. Supernatant was collected and concentrated to about 16 ml by using a Centriplus Concentrator (Amicon, MWCO 10,000, 470 rpm, 2800× g). Refolded TM9SF4/TM9SF2 was aliquoted to 0.5 ml into microcentrifuge tubes and stored at −80˚C. After refolding, the proteins were purified further by isoelectric focusing.
The refolded recombinant proteins were fucosed on Criterion IEF gels (Bio-Rad, Hercules, CA). The IEF gels were cut at pIs of 7.0, 6.0, and 4.5 based on IEF standards (Bio-Rad). The slices were soaked in 50 mM Tris-Mes buffer, pH 7 at 4˚C overnight to elute the proteins.
Measurements of superoxide production were based on a standard method where reduction of ferricytochrome c by superoxide was monitored from the increase in absorbance at 550 nm with reference at 540 nm [
Oxidation of reduced coenzyme Q10 (CoQ10H2) was measured as the disappearance of reduced CoQ at both 290 and 410 nm [
Protein disulfide-thiol interchange was determined spectrophotometrically from the increase in absorbance at 340 nm resulting from the cleavage of dithiodipyridine (DTDP). Specific activities were calculated using a millimolar extinction coefficient of 6.21 [
Proteins were estimated by the bicinchoninic acid method [
Purified recombinant TM9SF4 was concentrated to 0.7 mg/mL by using a Centricon concentrator (Millipore Corp., Danvers, MA) fitted with a 10,000 nominal molecular weight limit ultracel YM membrane. Samples (500 µL) were combined with 1 µL of trifluoroacetic acid (TFA) in the presence or absence of 15 µL of 10 mM bathocuproine and assayed for superoxide production and NADH oxidation.
Rabbits were immunized with low molecular weight arNOX fragments purified from human urine (
Peptide antibodies were generated in rabbits to the N-terminal sequences of the exfoliated proteins. A cysteine residue was added to the N-terminus of each peptide to facilitate coupling to the carrier protein KLH.
TM9SF1A&1B (aa 72 - 87) IRHKSKSLGEVLDGDR TM9SF2 (aa 89 - 104) GKESENLGQVLFGER TM9SF3 (aa 70 - 88) KKSISHYHETLGEALQGVE TM9SF4 (aa 69 - 84) ITYKAENLGEVLRGDR
For immunoassay, the arNOX source was coated on each of 5 replicated wells of a 96 well ELISA plate and after appropriate washing and blocking, the isoformspecific antibodies were added singly to each of the wells or as an equal mixture if the objective was to quantitate total arNOX. A peroxidase second antibody was added followed by a colorimetric substrate and the resultant absorbance was determined in an automated plate reader. The log of absorbance readings was proportional to the amount of arNOX present and quantitated by means of a standard curve using recombinant TM9SF4.
Amino acids indicated were replaced by alanines by site-directed mutagenesis according to Braman et al. [
Means and standard deviations were analyzed for statistical significance using a two-tailed test.
Based on specific activity measurements and SDS-PAGE, a soluble preparation of arNOX proteins was isolated to apparent homogeneity from human urine (
To determine if the human arNOX proteins might similarly derive from TM9 superfamily members [5,11], recombinant human TM9SF4 was generated from cDNA corresponding to full length TM9SF4. We were unable to express full length TM9SF4 in E. coli. Moreover, the bacteria transfected with cDNA to full length TM9SF4 grew at only 20% to 30% of the rate of non-transfected bacteria. Therefore, since we were primarily interested in the exfoliated form of the protein, cDNA corresponding to the ca. 30 kDa N-terminus of TM9SF4 was prepared and expressed. The truncated form exhibited the marked oscillating activity characteristic of other ENOX proteins family members [12; Figures 2-7].
Crude lysates of bacteria expressing the truncated TM9SF4 exhibited single maxima of arNOX activity separated at intervals of approximately 26 min (
with a burst of superoxide suggestive of not one but as many as five arNOX proteins being present each contributing to a single superoxide burst every 26 min.
The activity pattern exhibited by the expressed truncated TM9SF4 proteins purified by isoelectric focusing exhibited five activity maxima within a time span of 26 min based on NADH oxidation where two of the maxima were separated by about 6 min and the remaining three maxima were separated from each other and from the two maxima separated by 6 min by intervals of about 5 min to generate the patterns shown in Figures 4(a) and (c). What is most characteristic of the arNOX activity patterns is that the maxima labeled exhibited a burst of superoxide production 2 to 4 times that of the other maxima (Figures 4(b) and (d)) and is the predominant activity observed in diluted preparations of the protein (
When protein disulfide thiol interchange activity, a second enzymatic activity associated with ENOX pro-
teins, was measured, a five-maxima pattern similar to that generated for NADH oxidation was obtained (
Results with recombinant TM9SF2 and TM9SF4 proteins expressed as N-terminal (ca. 15 kDa or 30 kDa) fragments were nearly identical.
Superoxide dismutase was added at the end of each assay to ascertain that activity based on superoxide production returned to base line. Both a specific arNOX inhibitor mixture of dormin + Schizandra + salicin [to 2.5 ml of assay volume were added 60 µl of an aqueous mixture of 4 mg/ml Schizandra (Schizandra chinensis extract, 9% schizandrins, Draco, San Jose, CA) plus 1 mg/ml salicin (Sigma, St. Louis, MO) and 20 µL of IBR Dormin (Israli Biotechnology Research, Ramat-Gan, Israel) = AgeLoc (NuSkin Enterprises, Provo, UT)] and an equal mixture of peptide antibodies to each of the 5 arNOX superfamily members at a titer of 1:10,000 inhibited the activity by >90%. Also inhibitory were gallic acid (EC50 = 2 µM), tyrosol (EC50 = 1 µM) and coenzyme Q10 (EC50 = 80 nM).
Peptide antibodies to each of the isoforms along with corresponding DNA probes to each of the isoforms were developed for the soluble forms of each of the five isoforms. The antibodies were used to systematically identify the 5 isoforms in human sera and saliva and to verify that they corresponded to the known sequences of the TM9 superfamily members. DNA sequence information was used to generate RT-PCR probes for each of the
isoforms and demonstrate their expression in both human lymphocytes and human skin explants (not shown) to confirm the genetic origins of the five known arNOX isoforms of human sera, plasma and other body fluids as TM9 superfamily proteins.
Sequential addition of peptide antibodies to sera to associate specific maxima unaffected in the pre-bleed with specific TM9 family members SF1 to SF4 (
The ca. 30 kDa N-terminal regions of the TM9SF proteins, which are exposed at the external surface of the plasma membrane are shed into the blood and other body fluids (saliva, perspiration, urine). They are present in sera and plasma and were measured collectively as arNOX (
*By comparison, values minus blank for a younger volunteer (21-y) were 0.01 ± 0.01.
1 MATAMDWLPWSLLLFSLMCETSAFYVPGVAPINFHQNDPVEIKAVKLTSSRTQLPYEYYS
61 LPFCQPSKITYKAENLGEVLRGDRIVNTPFQVLMNSEKKCEVLCSQSNKPVTLTVEQSRL 121 VAERITEDYYVHLIADNLPVATRLELYSNRDSDDKKKEKDVQFEHGYRLGFTDVNKIYLH 181 NHLSFILYYHREDMEEDQEHTYRVVRFEVIPQSIRLEDLKADEKSSCTLPEGTNSSPQEI 241 DPTKENOLYFTYSVHWEE
Adenine nucleotide binding site (GXVXXG)
Putative copper sites (YVH, HGY)
Putative protein disulfide interchange site (CXXXC)
Conserved CQ/CE
Confirmation of functional assignments of motifs common to arNOX proteins of
Melatonin (100 µM) did not shift the phase of the period (not shown). However, after 1.5 periods, 39 min after melatonin addition, a maximum was skipped.
To determine if the TM9SF forms required copper for activity, recombinant TM9SF4 was assayed in the presence of trifluoroacetic acid (to unfold the protein) with or without the copper chelator, bathocuproine, to remove the copper. The activity, measured either based on NADH oxidation or superoxide production, was diminished when bathocuproine was present (not shown). TFA alone did not reduce activity and activity could be re
*Significant (p = 0.02 - 0.04); **Very Significant (p = 0.003 - 0.004).
stored to TFA and bathocuproine treated preparations by additions of copper.
The age-related NADH oxidase of cells and body fluids [
Based on DNA sequence, TM9SF4 protein was expressed in E. coli and shown to have functional characteristics of an ENOX protein. The pattern of activity oscillations consisting of 5 unequally spaced maxima was the characteristic, yet unique, periodicity (26 min period instead of the 24 min period for ENOX1 or 22 min period for ENOX2). A further defining characteristic of the arNOX proteins is their ability to generate superoxide. Superoxide generation is evidenced by the superoxide dismutase-inhibited reduction of ferricytochrome c. ENOX1 and ENOX2 proteins carry out four electron transfers from NAD(P)H to molecular oxygen. Superoxide is not a reaction product of either ENOX1 or ENOX2 as ferricytochrome c is not reduced by these proteins.
Prior to our analysis of the expressed recombinant arNOX, the pattern of oscillations of human samples was poorly understood. Patterns observed consisted of 4 to 7 maxima with the average being about five. The origins of the maxima, recurring every 26 min, was eventually correctly attributed to individual arNOX forms based on 2- D gel separations (
The human arNOX cDNA encodes a polypeptide having a highly hydrophobic C-terminal portion organized into nine transmembrane domains with a very similar structure and sequence to members of a novel family of multispanning domain proteins designated “TM9SF” (transmembrane protein 9 superfamily) by the Human Gene Nomenclature Committee. The leader member of the TM9SF family is the Saccharomyces cerevisiae EMP70 gene product, a 70 kDa precursor that is processed into a 24 kDa protein (p24a) located in the endosomes [
Hydropathy analysis [
Full length members of the TM9 protein superfamily are all characterized as cell surface proteins (as are arNOX proteins) having a characteristic series of 9 membrane spanning hydrophobic helices that criss-cross the plasma membrane and also are present on endosomes [
We thank Connie Phung for conduct of isoelectric focusing experiments, Dr. Christiaan Meadows, for ELISA results of
arNOX: age-related NADH oxidase;
CAPS: 3 (cyclohexylamino)-1-propane sulfonic acid;
DTDP: dithiodipyridine;
ELISA: enzyme-linked immunosorbent assay;
ENOX: ECTO-NOX;
IPTG: isopropyl-beta-D-thiogalactopyranoside;
MWCO: molecular weight cutoff;
PMSF: phenylmethylsulfonyl fluoride;
SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis;
SF: superfamily;
TFA: trifluoroacetic acid;
TM: transmembrane