Isolation of a Germin-like Protein with Manganese Superoxide Dismutase Activity from Cells of a Moss, Barbula unguiculata *

A novel extracellular Mn-superoxide dismutase (SOD) was isolated from a moss, Barbula unguiculata. The SOD was a glycoprotein; the apparent molecular mass of its native form was 120 kDa, as estimated by gel filtration chromatography, and that of its monomer was 22,072 Da, as estimated by time of flight mass spectroscopy. The protein had manganese with a stoichiometry of 0.80 Mn/monomer. The cDNA clone for a gene encoding the extracellular Mn-SOD was isolated. Sequence analysis showed that it has a strong similarity to germin (oxalate oxidase) and germin-like proteins (GLPs) of several plant species and possesses all the characteristic features of members of the germin family. The clone encoding this extracellular Mn-SOD was therefore designated B. unguiculata GLP (BuGLP). BuGLP had no oxalate oxidase activity. In addition, the cDNA for a gene encoding the moss mitochondrial Mn-SOD was isolated. Its amino acid sequence had little similarity to that of BuGLP, even though a close similarity was observed among the mitochondrial Mn-SODs of various organisms. BuGLP was the first germin-like protein that was really demonstrated to be a metalloprotein with Mn-SOD activity but no oxalate oxidase activity.

Superoxide dismutase (SOD) 1 (EC 1.15.1.1) is of major importance in protecting living cells against superoxide anion toxicity produced under oxidatively stressed circumstances. Phylogenetic distribution of the three types of SOD, CuZn-SOD, Mn-SOD, and Fe-SOD, in plants and microorganisms has been of general interest (1). Fe-SOD is found in prokaryotes, eukaryotic algae (2,3), and bryophytes (4), but in higher plants it is detected only in a number of unrelated plant families (1,5). CuZn-SOD is not generally found in prokaryotes (6) except for the periplasm of a few Gram-negative bacteria (7). Most eukaryotic algae lack CuZn-SOD (8), except for some green algae (9). All of the land plants examined have both cytosolic and chloroplast CuZn-SODs. As for Mn-SOD, it is found in the cytosol of all prokaryotes and in the mitochondria of all eukaryotes examined.
Germin is a water-soluble glycoprotein with a monomer molecular mass of about 25 kDa and forms oligomers that are highly resistant to proteolysis (18,19). Germins are expressed during seed germination of wheat and barley (18,19) and in mature leaves in response to pathogen attack (20 -22), and they are identified as oxalate oxidase from the amino acid sequence homology (18). Thus, germins are suggested to catalyze cellwall reinforcement by oxidative cross-linking (23) or to protect leaves against infectious microorganisms by generating H 2 O 2 (22,24). In plants other than wheat and barley, proteins closely related to germins are identified and termed germin-like proteins (GLPs) (25). Some of these GLPs have no oxalate oxidase activity, and the biological function of some GLPs is not comparable to germins.
Our interest in this study has been focused on determining how land plants evolved SOD molecules to adapt cells to the oxygen-accumulated atmosphere. We have taken notice of SODs of the bryophytes, which are considered to be the first land plants and to occupy a critical position in the evolution of land plants. We first studied SODs of liverwort, Marcantia paleacea var. diptera, which is evolutionarily the most basal lineage among bryophytes, and found that it has a cytosolic CuZn-SOD with properties similar to CuZn-SODs found in choloroplasts in higher plants (26,27). Then, we expanded the study to the SODs of a moss, Barbula unguiculata, that is considered morphologically to be more closely related to vascular plants. During the study, we found that cells of the moss excreted Mn-SOD in the medium. The extracellular Mn-SOD was found, quite interestingly, a germin-like protein with Mn-SOD activity but no oxalate oxidase activity. The protein is the first germin-like protein with SOD activity carrying manganese as a prosthetic group.

EXPERIMENTAL PROCEDURES
Plant Material and Culture Conditions-Cells of B. unguiculata (AS cell line) were propagated by shaking on a gyratory shaker at 110 rpm at 25 Ϯ 1°C in the light as described previously (28).
Preparation of Intracellular Crude Extracts and Extracellular Supernatant-Intracellular crude extracts were prepared as described previously (29) except that the concentration of EDTA was 0.1 mM instead of 1 mM in the grinding medium and that 15 cycles of sonication were carried out for disruption of cells. Extracelluar supernatant was ob-* 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AB028454 and AB028460 for BuGLP and the moss mitochondrial Mn-SOD, respectively.
Identification of Three SOD Isozymes-Identification of SOD isozymes was carried out as described previously (4) by nondenaturing polyacrylamide gel electrophoresis with SOD activity staining.
Purification of Extracellular Mn-SOD-Ammonium sulfate was added to the extracellular supernatant up to 90% saturation. The precipitate was dissolved in a minimum amount of 20 mM Tris-HCl (pH 8.0), dialyzed against the same buffer, concentrated by ultrafiltration (Centriflo CF25, Amicon), and applied to a column (2.5 ϫ 100 cm) of Sephadex G-100 (Amersham Pharmacia Biotech). The fractions containing SOD activity were pooled and applied to a column (1.6 ϫ 5 cm) of concanavalin A-agarose (Honen Co.) equilibrated with the same buffer. The column was washed with 500 ml of the buffer, and then the adsorbed proteins were eluted with 100 ml of the buffer containing 0.5 M ␣-methyl-D-glucoside.
Characterization of Extracellular Mn-SOD-Superoxide dismutase assay was carried out essentially as described by McCord and Fridovich (31) using cytochrome c as the detector and xanthine-xanthine oxidase as the superoxide generator.
Oxalate oxidase activity was assayed by two methods. One was essentially as described by Dumas et al. (32) in which the activity was detected with gel electrophoresis followed by transfer onto a nitrocellulose sheet and activity staining. The other was the spectrophotometric assay, as described by Zhang et al. (33) Molecular mass of the native form of the Mn-SOD was estimated by gel filtration with Sephadex G-100. The molecular mass of the monomer form was estimated by a REFLEX TM matrix-assisted laser desorption ionization time of flight mass spectrophotometer (Bruker). Data were acquired in the positive linear mode at 30 kV. A saturated solution of sinapinic acid in a mixture of 0.1% trifluoroacetic acid and 30% acetonitrile was used as a matrix. Singly charged (MϩH) ϩ and doubly charged (Mϩ2H) 2ϩ ions of bovine serum albumin were used as standards to calibrate the mass spectrum (m/z 66,431 and 33,216, respectively).
Manganese in the purified enzyme was determined by a Z-9000 Zeeman atomic absorption spectrometer (Hitachi).
The amino-terminal sequence and an inner amino acid sequence were analyzed using a gas-phase protein-peptide sequencer (model 473A, Applied Biosystems).
Detection of glycoprotein was carried out as described by Zacharius et al. (34) using the periodic acid-Shiff technique on the electrophoretic gel.
Isolation of cDNA for the Moss Extracellular Mn-SOD-Total RNA was isolated from 3-day-cultured cells (2 g) by the standard guanidine isothiocyanate extraction and cesium chloride ultracentrifugation method (35). First strand cDNA was synthesized from total RNA with a system for rapid amplification of cDNA 3Ј ends (Life Technologies, Inc.). The sense primer used was 5Ј-GA(T/C)GA(A/G)GA(T/C)GGI(T/ C)T(C/G)CA(A/G)GA(T/C)TT(T/C)TG-3Ј, which was synthesized to the amino-terminal amino acid sequence of the moss extracellular Mn-SOD and in reference to the conserved amino acid sequences of plant germins. Reaction conditions of PCR amplification were as follows: 30 cycles of 30 s of denaturing at 94°C, 30 s of annealing at 50°C, and 1 min of elongation at 72°C. The PCR products (about 800 base pairs) were amplified, cloned into pCRII vector with a TA-cloning kit (Invitrogen, San Diego, CA) and sequenced on both strands.
Isolation of cDNA for the Moss Mitochondrial Mn-SOD-The moss mRNA was purified with Oligotex TM -dT30 (Super) (Takara Shuzo, Kyoto, Japan), and a cDNA library was constructed as described previously (27). To prepare the probes for isolation of cDNAs, reverse transcription-PCR was first performed with Superscript TM II RNase H Ϫ reverse transcriptase (Life Technologies, Inc.). The pair of primers used were as the sense primer, 5Ј-AAICA(T/C)CA(T/C)(G/C)(A/C)(G/ C)(A/G)C(T/C)TA(T/C)G-3Ј, and the antisense primer, 5Ј-TAIGCATG(T/ C)TCCCAIAC(A/G)TC-3Ј, which were synthesized in reference to the most conserved region of the known sequences of plant Mn-SODs. Reaction conditions were as follows: 40 cycles of 30 s of denaturing at 94°C, 30 s of annealing at 40°C, and 1 min of elongation at 70°C. A PCR product (about 400 base pairs) was amplified, subcloned into pCRII vector with the TA-cloning kit (Invitrogen, San Diego, CA), and sequenced to confirm that it was part of the cDNA for the moss mitochondrial Mn-SOD. The cDNA library was screened with the PCR product, which was labeled with digoxigenin using a digoxigenin DNA labeling and detection kit (Roche Molecular Biochemicals). Inserts from the moss cDNA clones that hybridized to digoxigenin-labeled DNA probes were extracted from recombinant phage, subcloned into the pUC 118 vector (TaKaRa Shuzo, Kyoto, Japan), and sequenced on both strands.
Nucleotide Sequence Analysis-Sequencing was carried out by the dideoxy chain termination method using Thermo Sequenase dye terminator sequencing premix kit, version 2.0 (Amersham Pharmacia Biotech) and with a DNA sequencer (model 373A, Applied Biosystems, CA).
Protein Assay-Total protein concentration was measured using bicinchoninic acid kit for protein determination (Sigma).

Isolation of an Extracellular
Mn-SOD-Intracellular crude extracts and extracellular supernatant were subjected to nondenaturing gel electrophoresis, and SOD activity was stained (Fig. 1). There existed three kinds of SOD isozyme in the intracellular extracts (Fig. 1A): the upper band, which was insensitive to either cyanide or H 2 O 2 , indicates Mn-SOD; the two bands sensitive to either cyanide or H 2 O 2 indicates CuZn-SOD; and the band insensitive to cyanide but sensitive to H 2 O 2 indicates Fe-SOD. In the extracellular supernatant (Fig. 1B), there was a broad SOD activity band with very slow migration on native PAGE that is sensitive to neither cyanide nor H 2 O 2 , suggesting the presence of an EC Mn-SOD.
EC Mn-SOD was purified by the procedure summarized in Table I. After ammonium sulfate precipitation, Sephadex G-100 chromatography was adopted. The apparent molecular mass of EC Mn-SOD estimated from the chromatography was about 120 kDa. The fractions containing SOD activity were pooled and applied to the concanavalin A-agarose affinity column. EC Mn-SOD was eluted in the buffer containing 0.5 M ␣-methyl-D-glucoside with a 12-fold purification and a yield of 17%.
Characterization of EC Mn-SOD-Although the SOD activity staining of purified EC Mn-SOD showed a broad band with a slow migration on native PAGE, it migrated on SDS-PAGE as a single sharp band with an apparent molecular mass of 28 kDa ( Fig. 2A). When the SDS gel was stained with periodic acid-Schiff reagent, the protein band turned visible (Fig. 2B), indicating clearly that EC Mn-SOD is a glycoprotein, as shown in all examples of germin and GLPs (25). Presence of the carbohydrate chain in the protein was also confirmed by effective purification of EC Mn-SOD by binding it to concanavalin Aagarose followed by displacing it with ␣-methyl-D-glucoside. The affinity binding to concanavalin A shows that the carbohydrate chain is a N-glycan containing mannose core structure.
The molecular mass of monomer of EC Mn-SOD, estimated by time of flight mass spectroscopy, was 22,072 Da, and that of the doubly charged ion (Mϩ2H) 2ϩ was 10,980 Da. Involvement of manganese ion in the mass number was not clear because it could be removed during measurement. There exists the discrepancy of the molecular mass of monomer obtained from SDS-PAGE, mass spectrometry and amino acid sequence. SDS-PAGE is known not always to show the exact molecular masses, and the most reliable value are from mass spectrometry. However, it is difficult in this situation to determine the extent of oligomerization of the molecular mass of 120 kDa of the active form of EC Mn-SOD, estimated from gel filtration. Although native forms of germins of GLPs were reported to be pentamer or hexamer, it is possible that the native BuGLP could be tetramer or higher.
The presence of manganese in purified EC Mn-SOD was confirmed by atomic absorption spectrometry. Purified EC Mn-SOD had manganese with a stoichiometry of 0.80 Mn/SOD subunit. No copper, zinc, or iron was detected.
Primary Structure of EC Mn-SOD-The amino-terminal amino acid sequence of EC Mn-SOD indicated that it was a germin-like protein. To isolate cDNA for the SOD, degenerate oligonucleotide primers were designed and used for reverse transcription PCR. A PCR-amplified DNA fragment of approximately 800 base pairs was found to encode a peptide very similar to the original peptide sequence. The PCR fragment was used to screen EC Mn-SOD cDNA clones from the moss cDNA library. One clone encoding a protein very similar to the original peptide sequence was further analyzed. In comparison with entries in the data base, the deduced peptide sequence revealed strong similarity to GLPs and germin (oxalate oxidase) of several plant species; the percentage of residue identities for matched residues between EC Mn-SOD and germins and between EC Mn-SOD and GLPs were around 48 and 53%, respectively. Fig. 3 shows the deduced sequence alignment of EC Mn-SOD in comparison with other germins and a GLP. The alignment indicates that EC Mn-SOD also possesses two conserved histidine-containing motifs, the "germin box." The germin box (25) is part of a longer 20-or 21-amino acid motif of GX 5 HXHX 11 G (Fig. 3, residues 82-102), and is followed (usually after 15 residues) by a second motif of 16 amino acids, GX 5 PX 4 HX 3 N (Fig. 3, residues 123-138). These two histidinecontaining motifs are parts of the ␤-strands within the ␤-barrel elements (25). The fact clearly showed that EC Mn-SOD belongs to a member of germins or GLPs. The clone was therefore designated BuGLP.
The deduced BuGLP consists of 194 amino acids and has a molecular mass of 20,766 Da. Time of flight mass spectrometry gave a larger value, 22,072 Da. This difference, 1306 Da, could be mainly ascribed to attachment of a saccharide chain composed of around seven monosaccharides. However, the exact content of saccharide chain could not be deduced because the composition of the saccharide was not identified yet, and the contribution of manganese ion to the monomer mass number in mass spectrometry was ambiguous. The fact that the array of 54 amino acids from the amino-terminal was sequenced, but Asn-55 was not, and the fact that the consensus sequence for the asparagine-type sugar chain, NX(T/S), appears once in residues 55-57 in the BuGLP sequence clearly indicated that Asn-55 could be only the site for the saccharide chain.
BuGLP Has No Oxalate Oxidase Activity-Wheat and barley germins are identified as oxalate oxidase (18). As for GLPs in plants, some have no oxalate oxidase activity, and their biological functions are not comparable to those of germins. Whether BuGLP has oxalate oxidase activity or not was examined by two methods, the direct staining of the nitrocellulose sheet blotted from the electrophoretic gel and the spectrophotometric method. No activity was detected by these methods.
Primary Structure of the Moss Mitochondrial Mn-SOD-Most of the mitochondrial Mn-SODs from various organisms have to be dimeric to function. Each subunit contains around 200 amino acid residues and has a molecular mass of about 23 kDa. The residues that ligate the metal ion are the same in all  forms of the enzyme, namely three histidines and an aspartate. (36). The moss B. unguiculata also has a Mn-SOD in mitochondria (Fig. 1). The isolation of BuGLP that has Mn-SOD activity has raised a further interest in the sequence similarity between BuGLP and the mitochondrial Mn-SOD of the moss, especially the similarity in the manganese ligating residues. Therefore, we isolated the single, full-length cDNA encoding the mitochondrial Mn-SOD. Fig. 4 shows the sequence alignment of the mitchondrial Mn-SODs from the moss and other organisms. A close sequence identity (42-64%) was observed between the moss Mn-SOD and others. The amino acid residues conserved in all forms of mitchondrial Mn-SOD also exist in the moss mitchondrial Mn-SOD, especially all of the metal binding ligands; the first, His-28, in the motif 27 LHHXKHHXTYV; the second, His-74, in the motif running from residue 66 to the end of the helix base, FNGGGHXNHSIFWK; and the third and fourth, Asp-161 and His-165, in the long motif running from residue 156 in the last ␤-strands through to the fifth main helix, 156 PLLGIDVWEHAYYLQYKNVRPDYLKNIW. Thus, the moss mitchondrial Mn-SOD is a typical mitochondrial Mn-SOD.
There is little sequence similarity between the moss mitochondrial Mn-SOD and BuGLP. The percentage of residue identity for matched residues, if any, was calculated to be only 15%. From a standpoint of the sequence similarity, it is therefore clear that the present EC Mn-SOD belongs to a family of germin-like protein rather than the Mn-SOD commonly found in mitochondria.

DISCUSSION
The present report deals with distinctive nature of an extracellular protein newly isolated from a moss, B. unguiculata. The sequence of the protein showed clearly the similarity to germin or GLPs of plant origins but not to the mitochondrial Mn-SOD of the same moss. As for activity, on the contrary, it had SOD activity with manganese as a prosthetic group but none of the oxalate oxidase activity commonly shown in germins. Although there are no reports that germins or GLPs possess SOD activity, the present results on the sequence similarity, together with the in vivo oligomeric form (120 kDa) with glycosylated monomers (22,072 Da), indicates that the EC Mn-SOD is a member of GLPs.
Although there is little sequence similarity between BuGLP with Mn-SOD activity and the moss mitochondrial Mn-SOD, one interesting question is whether the amino acid residues, which are highly indicative of a role in manganese binding and enzyme activity, are similar between them. Gane et al. (37) showed the germin/oxalate oxidase three-dimensional model and described that the three histidine residues (His-88, His-90, and His-134, numbered as for BuGLP in Fig. 3) lie on neigh-boring antiparallel ␤-strands and form a cluster of adjacent side-chain imidazole groups. They predicted that the histidine cluster is the metal-binding site, the oxalate oxidase active site, although the metal cofactor of germin/oxalate oxidase has never been demonstrated. Kotsira and Clonis (30) employed chemical techniques to modify barley root germin to identify amino acid residues essential for enzyme activity and suggested that germin is a metalloenzyme, the metal(s) of which is involved in the oxidative mechanism, although the metal has not yet been identified. BuGLP has Mn-SOD activity, and manganese was actually identified with a stoichiometry of 0.80 atom/monomer. This is the first identification of metal in germins and GLPs. The primary sequence of BuGLP indicates that the protein has the germin box, including three histidine residues (His-88, His-90, and His-134). This, together with the fact that three histidines and an aspartate in mitochondrial Mn-SOD have been shown to ligate manganese (36), indicates that the histidine cluster in BuGLP could be assigned as the manganese-binding site. More detailed experiments are being conducted to prove the hypothesis.
The specific role of germins is considered to be generation of Ca 2ϩ and H 2 O 2 by degradation of oxalate, both of which are required for peroxidase-mediated reactions, such as cross-linking reaction of cell wall polysaccharides and lignification during germination (23). GLPs are isolated in gymnosperms, such as pines, and in dicot species (25), but some of GLPs have no oxalate oxidase activity. Thus, it is still unclear whether their biological function is comparable to germins despite numerous studies on GLPs and their expression. In plants, extracellular CuZn-SODs are identified (14,15), the physiological function of which is considered to be the production of H 2 O 2 from superoxide, which also facilitates the biosynthesis of lignin. The fact that BuGLP is extracted readily by 1 M NaCl from the moss cells suggests that BuGLP is localized outside of the cell and probably associated with the cell wall with no covalent crosslink. The evidence, together with the lack of oxalate oxidase activity in BuGLP, makes it possible to speculate that BuGLP is also involved in supplying H 2 O 2 by disproportionation of superoxide for the cross-linking of cell wall. The residues that are identical to those of the moss mitochondrial Mn-SOD are shown as white letters on black. Asterisks indicate the amino acid residues that ligate manganese and are critical for enzyme activity (see text). Sources of sequences deduced from the cDNAs and accession numbers are as follows: moss, this work; wheat, GenBank TM accession number U73172; Chlamydomonas, GenBank TM accession number U24500; Escherichia coli, GenBank TM accession number M94879.