Homology between Egg White Sulfhydryl Oxidase and Quiescin Q6 Defines a New Class of Flavin-linked Sulfhydryl Oxidases*

The flavin-dependent sulfhydryl oxidase from chicken egg white catalyzes the oxidation of sulfhydryl groups to disulfides with the reduction of oxygen to hydrogen peroxide. Reduced proteins are the preferred thiol substrates of this secreted enzyme. The egg white oxidase shows an average 64% identity (from randomly distributed peptides comprising more than 30% of the protein sequence) to a human protein, Quiescin Q6, involved in growth regulation. Q6 is strongly expressed when fibroblasts enter reversible quiescence (Coppock, D. L., Cina-Poppe, D., Gilleran, S. (1998)Genomics 54, 460–468). A peptide antibody against Q6 cross-reacts with both the egg white enzyme and a flavin-linked sulfhydryl oxidase isolated from bovine semen. Sequence analyses show that the egg white oxidase joins human Q6, bone-derived growth factor, GEC-3 from guinea pig, and homologs found in a range of multicellular organisms as a member of a new protein family. These proteins are formed from the fusion of thioredoxin and ERV motifs. In contrast, the flavin-linked sulfhydryl oxidase from Aspergillus niger is related to the pyridine nucleotide-dependent disulfide oxidoreductases, and shows no detectable sequence similarity to this newly recognized protein family.

A number of these enzymes have been suggested to catalyze disulfide bond formation in the protein secretory pathway (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11), but these proposals have not been widely adopted (11). This paper shows that the two most recently studied flavinlinked sulfhydryl oxidases isolated from egg white (9 -11) and Aspergillus niger (8,12) appear evolutionarily unrelated. Superficially, there are striking similarities between these two oxidases. Both are dimeric secreted glycoproteins (8,9). Both enzymes contain one FAD and a catalytically essential redoxactive disulfide bridge per subunit (8,9), and both form thiolate to flavin charge-transfer complexes in their 2-electron reduced forms (8,9). Neither enzyme reacts with reduced or oxidized pyridine nucleotides and both enzymes oxidize a range of small molecular weight thiol forms (8,9). There are, however, significant differences in substrate specificity. Reduced glutathione appears to be the best substrate of the fungal enzyme with reduced proteins, such as RNase, being either oxidized poorly (8) or not at all (13). In contrast, a range of reduced proteins and peptides are excellent substrates of the egg white oxidase, with k cat /K m values up to 100-fold higher than glutathione (11).
Here, we show that the egg white flavoprotein belongs to a newly recognized family of proteins (14) formed from the fusion of thioredoxin and ERV/ALR motifs (see later). Quiescin Q6 is the best understood member of this family and is highly induced as human fibroblasts enter reversible quiescence (14,15). In contrast, sequence comparisons show that the fungal sulfhydryl oxidase is a member of the pyridine nucleotidedisulfide oxidoreductases (16), appearing closely related to thioredoxin reductase and alkylhydroperoxide reductase. Our work suggests a role for sulfhydryl oxidase-like proteins in aspects of the cell cycle in fibroblasts and in the formation of the extracellular matrix.

EXPERIMENTAL PROCEDURES
Materials-Materials and general methods were as described previously (9,10). Unless otherwise stated, all buffers contained 0.3 mM EDTA. The egg white sulfhydryl oxidase was obtained as described by Hoober et al. (9,10). Bovine sulfhydryl oxidase was partially purified from 500 ml of centrifuged semen (3,000 ϫ g for 10 min) based on the procedure for the rat seminal vesicle enzyme (17). In brief, the 30 -40% ammonium sulfate fraction was redissolved and dialyzed against 50 mM sodium acetate buffer, pH 5.6. The enzyme bound to a CM-52 column equilibrated with this buffer and eluted approximately midway in a linear gradient to 300 mM NaCl in 50 mM sodium acetate buffer. The yellow oxidase fraction used for Western blots was approximately 20% pure by 280/454 nm absorbance ratio and was assayed with dithiothreitol (9).
Isolation and Characterization of Peptides-Peptides of the chicken oxidase were prepared, purified, and sequenced by standard methods. The legend to Fig. 1 lists the digestion steps used for each peptide sequenced. The redox-active cysteine residues of the egg white oxidase were alkylated with a 9-fold excess of monobromobimane (18) essentially as described for iodoacetamide (9). The alkylated enzyme was freed from excess bimane by ultrafiltration, and the nonredox-active disulfide bridge (9) was reduced for 30 min with an 8-fold excess of dithiothreitol in 6 M guanidine hydrochloride, 100 mM phosphate buffer, pH 7.5. A 2-fold molar excess of N-ethylmaleimide over total thiols was then added, and the mixture was incubated for 1 h. The protein was dialyzed against 25 mM Tris buffer, pH 7.7, containing 1 mM EDTA before digestion for 16 h at 37°C with 2% w/w aliquots of LysC added at 0 and 5 h. The bimane-labeled active site peptide was detected using an HPLC diode-array detector at 390 nm. PTH-bimane-Cys elutes on gas-phase sequencing between PTH-Tyr and PTH-Pro (19).
Antibodies and Western Blots-Immunoblotting was as described previously (20). The antibody was prepared in rabbits against a peptide * This work was supported in part by National Institutes of Health Grant GM26643 (to C. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
EDPQFPKVQWPPRE (residues 494 -507, underlined in Fig. 1) using a commercial vendor (Zymed Laboratories Inc. Inc., San Carlos, CA). Specificity was demonstrated by preincubating the purified antibody (2.5 g/ml) with the peptide (4 g/ml) for 1 h on ice in Tris-buffered saline with 0.05% Tween 20 (TBST) with 1% milk (w/v). Blots were incubated with antibody overnight at 4°C, washed 3 times for 5 min in TBST, incubated with a secondary antibody conjugated to horseradish peroxidase (Amersham Pharmacia Biotech; diluted 1/1,000), and developed using the enhanced chemiluminescence kit from Amersham Pharmacia Biotech. Molecular weights were determined using Rainbow high molecular weight markers (Amersham Pharmacia Biotech).

RESULTS AND DISCUSSION
The pure egg white oxidase (9, 10) was digested by trypsin, LysC, and cyanogen bromide methods, applied singly or in combination (see legend to Fig. 1 and "Experimental Proce-dures"). A total of 13 random peptides and an active site peptide (see later) were purified by HPLC and evaluated by MALDI-TOF and gas-phase sequencing. The sequence of each peptide was used to search the protein data bases and all but one (see legend to Fig. 1) show high levels of identity (from 45 to 100%) with Quiescin Q6 (Ref. 14 and see later). The 13 of 14 peptides are numbered in Fig. 1. Fortuitously, 3 peptides (11-13) are found to comprise a contiguous 51-residue segment toward the C terminus of Q6.
Peptide 10 contains the redox-active disulfide bridge in this flavoprotein. It was obtained by first converting the oxidase to the 4-electron reduced state by dithionite titration (9). The dithiol was then alkylated under anaerobic conditions with monobromobimane (18) (see "Experimental Procedures"). Under these conditions, both active site cysteine residues are labeled, as observed with iodoacetamide (9), yielding a strongly fluorescent peptide modified at Cys-450 and Cys-453 (Q6 numbering).
The egg white oxidase contains a total of about 595 amino acids (9), similar to the 582 residues of Q6 (14). In aggregate, the oxidase peptides shown in Fig. 1 represent 32% of the total sequence with an average identity of 64% (81% similarity). The probability that this degree of sequence identity would arise by chance is negligible. Thus, a sulfhydryl oxidase secreted into the egg white of the laying hen has clear homology to a protein involved in growth regulation in human fibroblast cells (14,15). This conclusion is strengthened by the demonstration that the egg white oxidase strongly cross-reacts (Fig. 2) with a peptide antibody raised against a peptide comprising residues 494 -507 of Q6 (underlined in Fig. 1). This reaction is completely blocked by the addition of competing peptide (Fig. 2). We have also partially purified sulfhydryl oxidase from bovine semen (see "Experimental Procedures"), based on the early work of Ostrowski et al. (17) on the rat seminal vesicle enzyme. This oxidase also contains FAD (6) and appears mechanistically similar to the egg white enzyme (9). Fig. 2 shows a strong specific response to the bovine enzyme. Thus, both vertebrate sulfhydryl oxidases appear related to Quiescin Q6. The differing apparent molecular weights in Fig. 2 may reflect varying carbohydrate contents (9).
The sequence of a third sulfhydryl oxidase, that from A. niger (8), is available from the patent literature (12). Despite markedly similar properties to the egg white protein mentioned earlier, there is undetectable sequence homology between them (not shown). However, data base searches with the A. niger sequence show 30% identity (46% similarity) with alkyl hydroperoxide reductase from Bacillus halodurans, 23% identity (40% similarity) with thioredoxin reductase (TRX B) from My-FIG. 1. Comparison of the sequence of egg white sulfhydryl oxidase peptides with sequence and domain structure of Quiescin Q6. Peptides 1-13 were obtained as described under "Experimental Procedures" and were located in the sequence of Q6 (U97276) as described. Peptide 10 contains the active site disulfide bridge of sulfhydryl oxidase and is boldfaced. Peptide 14 (AGEEAAASAR) could not be placed in the sequence of Q6, or any other protein in the data base, and is not shown. Digestions were as follows: peptides 1, 2, 5, and 7, trypsin; 4, 9, and 12, total CNBr fragments followed by trypsin; 10, 11, and 13, LysC; and 3, 6, and 8, LysC followed by trypsin. The location of the peptide used to prepare antibodies against Q6 (residues 494 -507) is underlined.
FIG. 2. Chicken egg white sulfhydryl oxidase and bovine semen sulfhydryl oxidase are immunologically related to Quiescin Q6. Western blots of egg white sulfhydryl oxidase (SOX; 1 g), bovine semen sulfhydryl oxidase (100 ng, ϳ20% pure), and whole cell extracts from quiescent WI38 human fibroblasts (100 g) were immunoblotted with a purified antibody against Q6 (see "Experimental Procedures"). Duplicate blots were incubated with antibody that had been preincubated with peptide (ϩ) or with the antibody alone (Ϫ). The apparent molecular mass of bovine semen sulfhydryl oxidase is about 66 kDa, similar to the rat enzyme (17), whereas the apparent molecular mass of Q6 and egg white SOX are between 80 and 90 kDa (unglycosylated molecular weights of 64 and 67 kDa, respectively) (9,14). cobacterium tuberculosis, and convincing homology with several other members of the pyridine nucleotide-disulfide oxidoreductase family (16). This similarity was previously predicted (16): divergent evolution of a sulfhydryl oxidase from this enzyme family could easily be envisaged with the loss of a pyridine-nucleotide binding site and substitution of molecular oxygen as electron acceptor.
The sequence of the egg white oxidase reflects a distinctly different lineage. Using the Q6 sequence, no members of the pyridine nucleotide-disulfide oxidoreductase family appear in any search protocols (not shown). However, these searches reveal that the egg white oxidase, like Q6 (14), was probably formed via fusion of at least two domains. First, a single thioredoxin motif is evident from residues ϳ35-125 in Q6. Thioredoxins are small proteins of approximately 95-110 amino acids with a range of redox and nonredox roles (21,22). A conspicuous member of the thioredoxin superfamily is mammalian protein-disulfide isomerase (21,23,24). The role of PDI in shuffling disulfide bonds during the maturation of proteins within the endoplasmic reticulum has been extensively documented (21,23,24). PDI contains two functional thioredoxin domains with the active site sequence WCGHC (21,24). Our peptide sequencing of sulfhydryl oxidase has yet to encounter this sequence, although it is observed between residues 69 and 73 in Q6 (Fig. 1) (14). Interestingly, the redox-active disulfide in the oxidase (9,16) lies toward the C terminus within the second recognizable feature: the ERV/ALR motif (residues 406 -503 of Q6; Fig. 1). ERV1 was the first identified member of an emerging gene family involved in the yeast cell cycle and in the biogenesis of mitochondria (25). The homologous mammalian counterpart, ALR1 is a 125 amino acid protein that has been implicated in a range of cell development processes from spermatogenesis to the regeneration of liver tissue (25,26). Although its precise physiological role is not known, ALR appears to be secreted from hepatocytes in an active form in response to liver damage (27).
Quiescin Q6 homologs containing both thioredoxin and ERV motifs are not found in any prokaryote genome sequence or in Saccharomyces cerevisiae. They are, however, widely distributed in metazoans (e.g. Caenorhabditis elegans, Drosophila melanogaster, guinea pig). In humans, sequences similar to Q6 have been named bone-derived growth factor (GenBank TM L42379) and cell growth inhibitory factor (GenBank TM E12644). All these homologs and the ERV proteins contain a region homologous to the active site disulfide bridge of the egg white sulfhydryl oxidase. Fig. 3 illustrates this homology with representative examples.
These studies suggest that Quiescin Q6 is a flavoprotein sulfhydryl oxidase. Indeed, immunoprecipitates of fibroblast cell extracts show detectable sulfhydryl oxidase activity. 2 Quiescin Q6 does not apparently contain the ADP-binding motif (28) found in a number of FAD-linked flavoproteins including the A. niger sulfhydryl oxidase (not shown). However, searching the Q6 sequence using the program eMOTIF (29) identifies the region 442-451 (Fig. 1) as a pattern found in the ferredoxin-NADP ϩ reductase (FNR) superfamily of flavoproteins (30) with a stringency of at least one in 10 7 . Cytochrome P450 reductase and nitric-oxide synthase contain an FNR domain. It is of interest that this 442-451 region is contiguous with the active site disulfide loop (residues 450 -453; Q6 numbering; see earlier). Obviously, the ultimate significance of these observations must await a crystal structure of the egg white enzyme. Although the precise functions of Q6 and its homologs must await further work, there are a number of observations suggesting a connection with the extracellular matrix (14,15). Q6 lacks the endoplasmic reticulum retention signal and is secreted into the fibroblast culture medium. 3 In addition, a number of the genes expressed at the onset of reversible quiescence, including 4 collagens and decorin, are well known components of the extracellular matrix. These secreted proteins typically contain multiple disulfide bridges essential for structural integrity. Taking one example, disulfide bond formation in many collagens is required to direct the assembly of trimeric proproteins and, in some cases, is involved in the generation of extracellular collagen networks (31)(32)(33). If the specificity of Quiescin Q6 were as broad as the egg white protein (11), Q6 might contribute to disulfide bond formation in a variety of secreted proteins.
In summary, the egg white oxidase has multiple homologs in multicellular organisms. Apart from the rat seminal vesicle enzyme, none of these proteins have been assigned an enzymatic function. It now appears likely that some will be involved in extracellular matrix formation as outlined above. Finally, the A. niger sulfhydryl oxidase and this new sulfhydryl oxidaselike protein family appear to have evolved convergently. Our current work is aimed at the cloning, overexpression, and characterization of Quiescin Q6, and a comparison of its properties with those of sulfhydryl oxidases from egg white and seminal vesicle. We are also exploring the role of the thioredoxin motif in these flavin-dependent oxidases.
FIG. 3. The active site disulfide of egg white sulfhydryl oxidase is homologous to Quiescin Q6 homologs. The sequence of this active site peptide was aligned with representative members of the Quiescin Q6 family: the guinea pig homolog GEC-3 (U82982); three C. elegans homologs (CeQ6r1-3: Z69637, U80848, and U39646, respectively), and a D. melanogaster homolog (AC005655). Alignments were calculated using PileUp (Genetics Computer Group 9.0) and the consensus was calculated with the program Pretty (Genetics Computer Group 9.0) with a plurality of 3.