High levels of active quiescin Q6 sulfhydryl oxidase (QSOX) are selectively present in fetal serum

The participation of thiol-oxidoreductases such as thioredoxin during implantation, embryogenesis and fetal development has been extensively studied. Here, we analyzed the expression of the thioredoxin superfamily enzyme quiescin Q6/sulfhydryl oxidase (QSOX) during development. Results show that QSOX is present in fetal bovine serum (4 months' gestation), but its levels decrease with time after birth (from P1 to P60). We also demonstrate that a sulfhydryl oxidase activity correlates with QSOX expression in such sera, suggesting a putative role in the redox modulation of developmental programs.     

High levels of active quiescin Q6 sulfhydryl oxidase (QSOX) are selectively present in fetal serum

Abstract

The participation of thiol-oxidoreductases such as thioredoxin during implantation, embryogenesis and fetal development has been extensively studied. Here, we analyzed the expression of the thioredoxin superfamily enzyme quiescin Q6/sulfhydryl oxidase (QSOX) during development. Results show that QSOX is present in fetal bovine serum (4 months' gestation), but its levels decrease with time after birth (from P1 to P60). We also demonstrate that a sulfhydryl oxidase activity correlates with QSOX expression in such sera, suggesting a putative role in the redox modulation of developmental programs.     

A flavin-dependent sulfhydryl oxidase in bovine milk

Both metal and flavin-dependent sulfhydryl oxidases catalyze the net generation of disulfide bonds with the reduction of oxygen to hydrogen peroxide. The first mammalian sulfhydryl oxidase to be described was an iron-dependent enzyme isolated from bovine milk whey (Janolino, V.G., and Swaisgood, H.E. (1975) J. Biol. Chem. 250, 2532-2537). This protein was reported to contain 0.5 atoms of iron per 89 kDa subunit and to be completely inhibited by ethylenediaminetetraacetate (EDTA). However the present work shows that a soluble 62 kDa FAD-linked and EDTA-insensitive sulfhydryl oxidase apparently constitutes the dominant disulfide bond-generating activity in skim milk. Unlike the metalloenzyme, the flavoprotein is not associated tightly with skim milk membranes. Sequencing of the purified bovine enzyme (>70% coverage) showed it to be a member of the Quiescin-sulfhydryl oxidase (QSOX) family. Consistent with its solubility, this bovine QSOX1 paralogue lacks the C-terminal transmembrane span of the long form of these proteins. Bovine milk QSOX1 is highly active toward reduced RNase and with the model substrate dithiothreitol. The significance of these new findings is discussed in relation to the earlier reports of metal-dependent sulfhydryl oxidases.     

Quiescin sulfhydryl oxidase 1 promotes invasion of pancreatic tumor cells mediated by matrix metalloproteinases

Quiescin sulfhydryl oxidase 1 (QSOX1) oxidizes sulfhydryl groups to form disulfide bonds in proteins. We previously mapped a peptide in plasma from pancreatic ductal adenocarcinoma (PDA) patients back to an overexpressed QSOX1 parent protein. In addition to overexpression in pancreatic cancer cell lines, 29 of 37 patients diagnosed with PDA expressed QSOX1 protein in tumor cells, but QSOX1 was not detected in normal adjacent tissues or in a transformed, but nontumorigenic cell line. To begin to evaluate the advantage QSOX1 might provide to tumors, we suppressed QSOX1 protein expression using short hairpin (sh) RNA in two pancreatic cancer cell lines. Growth, cell cycle, apoptosis, invasion, and matrix metalloproteinase (MMP) activity were evaluated. QSOX1 shRNA suppressed both short and long isoforms of the protein, showing a significant effect on cell growth, cell cycle, and apoptosis. However, QSOX1 shRNA dramatically inhibited the abilities of BxPC-3 and Panc-1 pancreatic tumor cells to invade through Matrigel in a modified Boyden chamber assay. Mechanistically, gelatin zymography indicated that QSOX1 plays an important role in activation of MMP-2 and MMP-9. Taken together, our results suggest that the mechanism of QSOX1-mediated tumor cell invasion is by activation of MMP-2 and MMP-9.     

The effect of polyamines on cell culture cells

Growth of KB cells was inhibited by both spermine and spermidine, but the inhibition is reduced in conditioned medium. The amount of spermine required for 50% inhibition of plating varied according to the type of serum used with medium 199 (calf, fetal bovine, and horse; 0.55, 0.9, and 24 μg/ml respectively). The spermine oxidase activity of the three sera was calf > horse > fetal bovine, which is not the same ordering as was obtained for the inhibition. When the concentration of sera in the media was varied, the inhibition decreased as calf and fetal bovine sera concentration increased, whereas, with horse serum, an increase in serum concentration increased the inhibition. The opposite effects of increasing concentrations of the sera on the inhibition suggest that at least two factors are involved in the inhibition. A scheme which involves three factors (spermine oxidase, another enzyme and its activator) is postulated to account for the inhibitions and reversals observed. Spermine oxidase alone cannot account for the action of polyamine on cells.     

QSOX contains a pseudo-dimer of functional and degenerate sulfhydryl oxidase domains

Quiescin sulfhydryl oxidase (QSOX) catalyzes formation of disulfide bonds between cysteine residues in substrate proteins. Human QSOX1 is a multi-domain, monomeric enzyme containing a module related to the single-domain sulfhydryl oxidases of the Erv family. A partial QSOX1 crystal structure reveals a single-chain pseudo-dimer mimicking the quaternary structure of Erv enzymes. However, one pseudo-dimer “subunit” has lost its cofactor and catalytic activity. In QSOX evolution, a further concatenation to a member of the protein disulfide isomerase family resulted in an enzyme capable of both disulfide formation and efficient transfer to substrate proteins.     

Differential reactivity of filarial antigens with human sera from bancroftian filariasis endemic zone

The reacting pattern of circulating filarial antigen fraction-2 fromWuchereria bancrofti and soluble antigen from adultBrugia malayi with bancroftian filarial sera were analysed by immunoblotting technique and enzyme linked immunosorbent assay. Microfilaraemic sera reacted specifically with proteins of molecular weight 200, 120, 97, 56, 54, 43, 26 and 17 kDa of circulating Filarial antigen fraction-2 and 44, 40, 38, 31, 22 and 18 kDa ofBrugia malayi adult soluble antigen. Clinical filarial sera identified protein molecules of 56, 54 and 42 kDa of circulating filarial antigen fraction-2 and 19, 16 and 14 kDa ofBrugia malayi adult soluble antigen. Some components of both the antigen preparation were also identified by endemic normal serai.e.proteins 120, 97, 62, 43 and 33 kDa of circulating filarial antigen fraction-2 and 170, 120, 43, 31 and 12 kDa ofBrugia malayi adult soluble antigen. One of the sodium dodecyl sulphate-polyacrylamide gel electropherosis fractions of circulating filarial antigen fraction-2 (CFA₂-8) andBrugia malayi adult soluble antigen fraction-6 when used in enzyme linked immunosorbent assay could differentiate microfilaraemic sera from endemic normal and clinical filarial sera. The other antigen fractions (CFA₂-2, 6 and 7 andBmA-2) showed a high geometric mean titre of filarial immunoglobulin G antibodies in endemic normal sera when compared to microfilaraemia and clinical filarial sera. These proteins need to be further studied to assess their involvement in protecting from filarial infection in an endemic area.     

An Inhibitory Antibody Blocks the First Step in the Dithiol/Disulfide Relay Mechanism of the Enzyme QSOX1

Quiescin sulfhydryl oxidase 1 (QSOX1) is a catalyst of disulfide bond formation that undergoes regulated secretion from fibroblasts and is over-produced in adenocarcinomas and other cancers. We have recently shown that QSOX1 is required for incorporation of particular laminin isoforms into the extracellular matrix (ECM) of cultured fibroblasts and, as a consequence, for tumor cell adhesion to and penetration of the ECM. The known role of laminins in integrin-mediated cell survival and motility suggests that controlling QSOX1 activity may provide a novel means of combating metastatic disease. With this motivation, we developed a monoclonal antibody that inhibits the activity of human QSOX1. Here, we present the biochemical and structural characterization of this antibody and demonstrate that it is a tight-binding inhibitor that blocks one of the redox-active sites in the enzyme, but not the site at which de novo disulfides are generated catalytically. Sulfhydryl oxidase activity is thus prevented without direct binding of the sulfhydryl oxidase domain, confirming the model for the interdomain QSOX1 electron transfer mechanism originally surmised based on mutagenesis and protein dissection. In addition, we developed a single-chain variant of the antibody and show that it is a potent QSOX1 inhibitor. The QSOX1 inhibitory antibody will be a valuable tool in studying the role of ECM composition and architecture in cell migration, and the recombinant version may be further developed for potential therapeutic applications based on manipulation of the tumor microenvironment.     

Crucial effect of the first CXXC motif of human QSOX 1b on the activity to different substrates

Among all sulphhydryl oxidases involved in disulphide formation, quiescin-sulphhydryl oxidase (QSOX) is unique for its multidomain structure, protein thiol oxidation activity and highly efficient catalysis. In this study, site-directed mutagenesis and molecular modelling methods were integrated to investigate the structural and functional characteristics of QSOX, especially the importance of the three CXXC motifs. Site-directed mutagenesis suggested that the C449-C452 motif was essential for the activity of human QSOX 1b; the C70-C73 motif was fundamental in electron transfer from thiol-containing substrate including reduced proteins, DTT, GSH rather than the phosphine-based thiol reductant TCEP, to the C449-C452 motif; and the C509-C512 motif was not involved in electron transfer during disulphide formation. The different roles of the CXXC motifs indicated that there were discrepant electron transfer pathways for the oxidation of thiol-containing substrates and non-thiol disulphide reductants. Molecular modelling method was then used to draw a reasonable picture for the electron transfer process and to elucidate the mechanism of electron transfer when different substrates were oxidized, which will greatly enhance our understanding of the action mechanism of QSOX.     

Separation of putrescine oxidase and spermidine oxidase in foetal bovine serum with the aid of a specific radioactive assay of spermidine oxidase.

1. A sensitive and specific assay for spermidine oxidase is described. The method involves the separation of [14C]spermidine (substrate) from [14C]putrescine (product) and other 14C-labelled products on a Dowex 50 cation-exchange column: 92% of the putrescine applied to the column was eluted by 2.3 M-HCl, but this treatment left 96% of the spermidine bound to the column. Unchanged spermidine could be removed from the column by elution with 6 M-HCl. 2. By means of this assay, foetal and adult bovine serum were each shown to contain spermidine oxidase activity, putrescine being a major product of the oxidation of spermidine by the serum enzymes. 3. In foetal bovine serum, spermidine oxidase activity is separable from putrescine oxidase activity by chromatography on a cadaverine-Sephadex column, by gel filtration and by ion-exchange column chromatography. Putrescine oxidase was purified 1900-fold and spermidine oxidase 130-fold by these procedures. The former oxidized putrescine but not spermidine, and spermidine oxidase exhibited no activity with putrescine as substrate.     

Avian sulfhydryl oxidase is not a metalloenzyme: adventitious binding of divalent metal ions to the enzyme

Metal- and flavin-dependent sulfhydryl oxidases catalyze the generation of disulfide bonds with reduction of oxygen to hydrogen peroxide. The mammalian skin enzyme has been reported to be copper-dependent, but a recent protein sequence shows it belongs to the Quiescin/sulfhydryl oxidase (QSOX) flavoprotein family. This work demonstrates that avian QSOX is not a metalloenzyme, and that copper and zinc ions inhibit the oxidation of reduced pancreatic ribonuclease by the enzyme. Studies with Zn(2+), as a redox inactive surrogate for copper, show that one Zn(2+) binds to four-electron-reduced QSOX by diverting electrons away from the flavin and into two of the three redox active disulfide bridges in the enzyme. The resulting zinc complex is modestly air-stable, reverting to a spectrum of the native protein with a t(1/2) of 40 min, whereas the four-electron-reduced native QSOX is reoxidized in less than a second under comparable conditions. Using tris(2-carboxyethyl)phosphine hydrochloride (TCEP), an alternate substrate of QSOX that binds Zn(2+) relatively weakly (unlike dithiothreitol), allows rapid inhibition of oxidase activity to be demonstrated at low micromolar metal levels. Zinc binding was followed by rapid-scanning spectrophotometry. Copper also binds the four-electron-reduced form of QSOX with a visible spectrum suggestive of active site occupancy. In addition to interactions with the reduced enzyme, dialysis experiments show that multiple copper and zinc ions can bind to the oxidized enzyme without the perturbation of the flavin spectrum seen earlier. These data suggest that a reinvestigation of the metal content of skin sulfhydryl oxidases is warranted. The redox-modulated binding of zinc to QSOX is considered in light of evidence for a role of zinc-thiolate interactions in redox signaling and zinc mobilization.     

Polyamine degradation in foetal and adult bovine serum.

1. Using protein-separative chromatographic procedures and assays specific for putrescine oxidase and spermidine oxidase, adult bovine serum was found to contain a single polyamine-degrading enzyme with substrate preferences for spermidine and spermine. Apparent Km values for these substrates were approx. 40 microM. The apparent Km for putrescine was 2 mM. With spermidine as substrate, the Ki values for aminoguanidine (AM) and methylglyoxal bis(guanylhydrazone) (MGBG) were 70 microM and 20 microM respectively. 2. Bovine serum spermidine oxidase degraded spermine to spermidine to putrescine and N8-acetylspermidine to N-acetylputrescine. Acrolein was produced in all these reactions and recovered in quantities equivalent to H2O2 recovery. 3. Spermidine oxidase activity was present in foetal bovine serum, but increased markedly after birth to levels in adult serum that were almost 100 times the activity in foetal bovine serum. 4. Putrescine oxidase, shown to be a separate enzyme from bovine serum spermidine oxidase, was present in foetal bovine serum but absent from bovine serum after birth. This enzyme displayed an apparent Km for putrescine of 2.6 microM. The enzyme was inhibited by AM and MGBG with Ki values of 20 nM. Putrescine, cadaverine and 1,3-diaminopropane proved excellent substrates for the enzyme compared with spermidine and spermine, and N-acetylputrescine was a superior substrate to N1- or N8-acetylspermidine.     

Human quiescin-sulfhydryl oxidase, QSOX1: probing internal redox steps by mutagenesis

The flavoprotein quiescin-sulfhydryl oxidase (QSOX) rapidly inserts disulfide bonds into unfolded, reduced proteins with the concomitant reduction of oxygen to hydrogen peroxide. This study reports the first heterologous expression and enzymological characterization of a human QSOX1 isoform. Like QSOX isolated from avian egg white, recombinant HsQSOX1 is highly active toward reduced ribonuclease A (RNase) and dithiothreitol but shows a >100-fold lower k cat/ K m for reduced glutathione. Previous studies on avian QSOX led to a model in which reducing equivalents were proposed to relay through the enzyme from the first thioredoxin domain (C70-C73) to a distal disulfide (C509-C512), then across the dimer interface to the FAD-proximal disulfide (C449-C452), and finally to the FAD. The present work shows that, unlike the native avian enzyme, HsQSOX1 is monomeric. The recombinant expression system enabled construction of the first cysteine mutants for mechanistic dissection of this enzyme family. Activity assays with mutant HsQSOX1 indicated that the conserved distal C509-C512 disulfide is dispensable for the oxidation of reduced RNase or dithiothreitol. The four other cysteine residues chosen for mutagenesis, C70, C73, C449, and C452, are all crucial for efficient oxidation of reduced RNase. C452, of the proximal disulfide, is shown to be the charge-transfer donor to the flavin ring of QSOX, and its partner, C449, is expected to be the interchange thiol, forming a mixed disulfide with C70 in the thioredoxin domain. These data demonstrate that all the internal redox steps occur within the same polypeptide chain of mammalian QSOX and commence with a direct interaction between the reduced thioredoxin domain and the proximal disulfide of the Erv/ALR domain.     

Inhibition of lymphocyte growth by spermidine in medium containing fetal bovine serum

The effect of spermidine and fetal bovine serum on DNA, RNA, and protein synthesis in phytohemagglutinin-stimulated human lymphocytes was investigated. At 10(-4) M spermidine, DNA, RNA, and protein synthesis ceased and 70% of the original cell population died within 62 hr. Lower spermidine concentrations had no significant effect on DNA and protein synthesis, but caused an early, unexplained increase in the rate of RNA synthesis. Heating at 56 degrees C for 30 min had no effect on the plasma amine oxidase activity in fetal bovine and horse sera but abolished the activity in human plasma. It is concluded that low amounts of aminoaldehydes and acrolein produced by plasma amine oxidase at spermidine concentrations below 10(-4) M do not noticeably alter lymphocyte metabolism. However, the aminoaldehydes and acrolein produced become abruptly cytotoxic at 10(-4) M spermidine.     

Protein substrate discrimination in the quiescin sulfhydryl oxidase (QSOX) family

This work explores the substrate specificity of the quiescin sulfhydryl oxidase (QSOX) family of disulfide-generating flavoenzymes to provide enzymological context for investigation of the physiological roles of these facile catalysts of oxidative protein folding. QSOX enzymes are generally unable to form disulfide bonds within well-structured proteins. Use of a temperature-sensitive mutant of ubiquitin-conjugating enzyme 4 (Ubc4') as a model substrate shows that QSOX activity correlates with the unfolding of Ubc4' monitored by circular dichroism. Fusion of Ubc4' with the more stable glutathione-S-transferase domain demonstrates that QSOX can selectively introduce disulfides into the less stable domain of the fusion protein. In terms of intermolecular disulfide bond generation, QSOX is unable to cross-link well-folded globular proteins via their surface thiols. However, the construction of a septuple mutant of RNase A, retaining a single cysteine residue, demonstrates that flexible protein monomers can be directly coupled by the oxidase. Steady- and pre-steady-state kinetic experiments, combined with static fluorescence approaches, indicate that while QSOX is an efficient catalyst for disulfide bond formation between mobile elements of structure, it does not appear to have a significant binding site for unfolded proteins. These aspects of protein substrate discrimination by QSOX family members are rationalized in terms of the stringent steric requirements for disulfide exchange reactions.     

Characterization and Partial Purification of AIM: A Plasma Protein That Induces Rat Cerebral Type 2 Astroglia from Bipotential Glial Progenitors

Studies on glial cultures have demonstrated that fetal bovine serum contains a factor that induces bipotential glial precursors known as oligodendrocyte-type 2 astrocyte (O-2A) progenitors to become type 2 astroglia rather than oligodendroglia. The goal of this research project was to characterize and purify this factor, which we refer to as the astroglia-inducing molecule (AIM). Using cultures enriched in O-2A progenitors, we determined that AIM is present in human and bovine sera and that fetal bovine serum qualified as the best serum for purifying AIM. AIM is heat and trypsin labile and may be a plasma glycoprotein. A 240-fold enriched AIM preparation was produced by applying an ammonium sulfate precipitate of fetal bovine serum to heparin and then lentil lectin-agarose, followed by gel filtration chromatography. In crude preparations, AIM activity migrated at 50 kDa by gel filtration. With enrichment, activity was seen at several molecular masses, all of which were approximate multiples of 50 kDa. Treatment with 6 M guanidine hydrochloride generated an AIM with a molecular mass between 12 and 18 kDa, a result suggesting that AIM aggregates. On a preparative isoelectric focusing gel, AIM activity most frequently migrated between pH values of 3 and 4; however, proteins with isoelectric points of greater than 9 or at 6 also had activity in several experiments. These data suggest that either multiple AIMs exist or that a single AIM exists that associates with other proteins. Immunofluorescence for ganglioside GD3 and glial fibrillary acidic protein confirmed that AIM preparations induce type 2 astroglia from O-2A progenitors and suggests that AIM has little effect on type 1 astroglia. Because none of the known growth factors that have been tested to date mimics its effects. AIM may be a novel differentiation factor.     

Inhibition of lymphocyte growth by spermidine in medium containing fetal bovine serum

Summary The effect of spermidine and fetal bovine serum on DNA, RNA, and protein synthesis in phytohemagglutinin-stimulated human lymphocytes was investigated. At 10⁻⁴ M spermidine, DNA, RNA, and protein synthesis ceased and 70% of the original cell population died within 62 hr. Lower spermidine concentrations had no significant effect on DNA and protein synthesis, but caused an early, unexplained increase in the rate of RNA synthesis. Heating at 56°C for 30 min had no effect on the plasma amine oxidase activity in fetal bovine and horse sera but abolished the activity in human plasma. It is concluded that low amounts of aminoaldehydes and acrolein produced by plasma amine oxidase at spermidine concentrations below 10⁻⁴ M do not noticeably alter lymphocyte metabolism. However, the aminoaldehydes and acrolein produced become abruptly cytotoxic at 10⁻⁴ M spermidine. This work was supported in part by the Cystic Fibrosis Foundation.     

Putrescine-oxidase activity in adult bovine serum and fetal bovine serum.

Putrescine-oxidase activity was found in fetal bovine serum (FBS) with a pH optimum of 8.0 and in adult bovine serum (ABS) with a pH optimum of 9.8. The crude FBS enzyme had a KM for putrescine of 2.58 x 10(-6) M and a Vmax of 0.53 nmol per hr per 50 microliter serum. Aminoguanidine competitively inhibited the enzyme with a KI of 1.8 x 10(-8) M. Spermidine and spermine proved competitive inhibitors of putrescine for both the FBS and the crude ABS putrescine oxidases. The Vmax for the ABS putrescine oxidase was 2.10 nmol per hr per 50 microliter serum, and the KM for putrescine, 50.3 x 10(-6) M. The K1 of the ABS putrescine oxidase for aminoguanidine was 41 x 10(-6) M. On the basis of both the KM and KI values, the adult serum enzyme, at its optimal pH of 9.8, bound spermidine and spermine more avidly than the smaller putrescine and aminoguanidine; whereas the FBS enzyme, at pH 8.0, bound aminoguanidine and putrescine more tightly than the larger polyamines. Each of the enzymes retained over 80% of its activity after heating at 56 degrees C for 30 min. Applications of these data to the study of polyamines in tissue culture and to the purification of diamine oxidases are discussed.     

The effect of soluble rat liver proteins on the activity of microsomal stearoyl-CoA and linoleoyl-CoA desaturase.

1. The influence of bovine serum albumin and soluble rat liver proteins on the activity of rat liver microsomal delta9 and delta6 desaturases has been studied. 2. In the absence of bovine serum albumin, the delta9 desaturase which converts stearoyl-CoA into oleoyl-CoA, shows a non-linear correlation between enzyme activity and protein concentration. 3. Optimum concentrations of bovine serum albumin have three main effects on the enzyme activity: (i) establishes a linear relationship between enzyme activity and protein concentration, (ii) stimulates the enzyme activity 2--3-fold and (iii) raises the optimum substrate concentration from 10 to 100 muM. 4. A highly purified soluble liver protein of molecular weight 24 000 also stimulated the enzyme activity and brought about a linear relationship between enzyme activity and protein concentration. 5. It was concluded that the non-linear kinetics were due to limiting amounts of substrate binding protein in the microsomal preparations. 6. The delta6 desaturase which converts linoleoyl-CoA into gamma-linolenoyl-CoA was also stimulated by bovine serum albumin and soluble liver proteins. 7. The significance of the fatty acid-binding proteins is discussed.     

Effects of fetal versus postnatal sera upon adipose tissue stromal-vascular cells in primary culture

Summary This experiment was conducted to determine if serum factors are responsible for differences in cellularity of prenatal and postnatal pig adipose tissue as determined by in vitro measurement of cellular proliferation and enzyme-histochemical metabolic development. Cellular proliferation of stromal-vascular cells derived from rat inguinal adipose tissue was measured by [³H]-thymidine incorporation. Coverslip cultures were used for analysis of histochemical differentiation. Cells were incubated in media containing 10% fetal bovine, fetal pig, mature pig, or various combinations of these sera. Fetal bovine serum promoted more [³H]-thymidine incorporation than fetal or postnatal pig sera. Fetal pig sera also stimulated more [³H]-thymidine incorporation than mature pig sera. Sera from adult pigs promoted differentiation and lipid filling of adipocytes. Fetal pig sera stimulated histochemical expression of enzymes, but did not induce lipid filling. Fetal bovine serum produced histochemically undifferentiated cells. Addition of fetal bovine serum to media containing mature pig sera reduced lipid accumulation and histochemical reactivity of cells. This effect of fetal serum was thus due to specific inhibition of lipid deposition and not substrate restriction. These experiments demonstrated that serum factors have a major influence on morphological development of fetal and postnatal adipose tissue.