Baculovirus Envelope Protein ODV-E66 Is a Novel Chondroitinase with Distinct Substrate Specificity*

Chondroitin sulfate is a linear polysaccharide of alternating d-glucuronic acid and N-acetyl-d-galactosamine residues with sulfate groups at various positions of the sugars. It interacts with and regulates cytokine and growth factor signal transduction, thus influencing development, organ morphogenesis, inflammation, and infection. We found chondroitinase activity in medium conditioned by baculovirus-infected insect cells and identified a novel chondroitinase. Sequence analysis revealed that the enzyme was a truncated form of occlusion-derived virus envelope protein 66 (ODV-E66) of Autographa californica nucleopolyhedrovirus. The enzyme was a novel chondroitin lyase with distinct substrate specificity. The enzyme was active over a wide range of pH (pH 4–9) and temperature (30–60 °C) and was unaffected by divalent metal ions. The ODV-E66 truncated protein digested chondroitin most efficiently followed by chondroitin 6-sulfate. It degraded hyaluronan to a minimal extent but did not degrade dermatan sulfate, heparin, and N-acetylheparosan. Further analysis using chemo-enzymatically synthesized substrates revealed that the enzyme specifically acted on glucuronate residues in non-sulfated and chondroitin 6-sulfate structures but not in chondroitin 4-sulfate structures. These results suggest that this chondroitinase is useful for detailed structural and compositional analysis of chondroitin sulfate, preparation of specific chondroitin oligosaccharides, and study of baculovirus infection mechanism.

groups at various positions of the sugar residues (1,2). CS chains are covalently attached to a core protein, in the form of proteoglycans, and are present ubiquitously in extracellular matrices and on cell surfaces. CS chains play various biological roles in development, organ morphogenesis, inflammation, and infection by interacting with cytokines and growth factors, regulating their signal transduction (3). These functions are mainly ascribed to the sulfate groups, building structural specificity and diversity into the polysaccharide framework.
Studies of CS/DS chains have been developed by extensive use of enzymes that specifically digest these chains. These enzymes depolymerize the substrates via a ␤-elimination reaction, which generates an unsaturated 4,5-bond on the uronic acid at the site of cleavage. Chondroitin ABC lyase I (cABCI) (4) and II (cABCII) (5) are prepared from Proteus vulgaris. Chondroitin AC lyase I (cACI) (4) and II (cACII) (6) are from Flavobacterium heparinum and Arthrobacter aurescens, respectively. Chondroitinase B (7) is from F. heparinum. Each enzyme exhibits a unique specificity. cABCI is an endoeliminase that depolymerizes chondroitin-4-sulfate (C4S), 6-sulfate (C6S), and DS to their respective unsaturated di-and tetrasaccharides. cABCII is an exoeliminase that cleaves CS and DS at the nonreducing end and digests short oligosaccharides more efficiently than cABCI. Both cACI and cACII depolymerize C4S and C6S, but not DS. Chondroitinase B cleaves the 1-4 glycosidic bond of the DS IdoUA residue and does not cleave that of the GlcUA residue of CS and DS. These chondroitin lyases have been widely used for quantification and structural analysis of CS/DS and production of anti-CS/DS monoclonal antibodies that recognize neo-epitopes generated by these chondroitinases. In addition, they have been applied to treatment of intervertebral disc protrusion (8) and spinal cord injury (9).
In our experiments, wherein chondroitin sulfotransferases were expressed in a baculovirus system, we found chondroitinase activity in the conditioned medium of baculovirus-in-* This work was supported in part by the New Energy and Industrial Technology Development Organization (NEDO) and by special research funding from Seikagaku Corporation. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1
Enzyme Purification from Virus-infected Insect Cell Culture-AcMNPV baculovirus stock solution was prepared with sf9 cells infected by using the Bac-to-Bac baculovirus expression system. Conditioned medium (Sf-900 II serum-free medium, 100 ml) after 6 days of culture at 27°C was collected and centrifuged at 1000 ϫ g for 10 min. The supernatant was ultracentrifuged at 120,000 ϫ g for 30 min, and then the supernatant was concentrated by ultrafiltration (Amicon Stirred Cell 8200, 10,000 molecular weight cutoff filter; Millipore) to ϳ10 ml. The concentrated medium was applied to a DEAE-Sepharose column (1.6 ϫ 12 cm) equilibrated with 50 mM Tris-HCl, pH 7.2, containing 50 mM NaCl, and the bound molecules were eluted with a linear gradient of 50 -500 mM NaCl. Chondroitinase activity in the fractions (2 ml/tube) was measured, and the fractions with activity were pooled and rechromatographed on a Q Sepharose column (1.2 ϫ 10 cm) equilibrated with 50 mM Tris-HCl, pH 7.2. Elution was performed with a linear gradient of 50 -500 mM NaCl. The fractions (1.0 ml/tube) containing chondroitinase activity were collected and concentrated by ultrafiltration (Amicon Ultra-15, 10,000 molecular weight cutoff filter, Millipore). The concentrated solution (200 l) was applied to a column of Superdex 75 equilibrated with 50 mM Tris-HCl, pH 7.2, containing 0.15 M NaCl. The fractions (0.5 ml/tube) con-taining chondroitinase activity were collected. The sample was precipitated by adding 100% trichloroacetic acid to a final concentration of 5%. The precipitate was washed with acetone and applied to SDS-polyacrylamide gel electrophoresis.
Identification of the Enzyme-The proteins in the polyacrylamide gel were detected with a silver staining kit for mass spectrometry (Wako, Japan). The protein band corresponding to chondroitinase activity was excised from the gel and digested with trypsin. The extracted solution containing digested peptides was concentrated in vacuo, desalted with Zip-Tip C18 (Millipore), and extracted with 0.1% (v/v) trifluoroacetic acid in 50% (v/v) acetonitrile containing ␣-cyano-4-hydroxycinnamic acid matrix solution. The extracted solution was subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with an AutoFlex mass spectrometer (Bruker), and the corresponding protein was identified with the MASCOT search system (Matrix Science) (18).
The proteins in the polyacrylamide gel were transferred to a PVDF membrane (Immobilon P, Millipore) for Western blotting. The membrane was stained with Coomassie Brilliant Blue, and the protein band corresponding to chondroitinase activity was excised from the membrane. The N-terminal amino acid sequence of the protein in the membrane strips was determined with a Procise LC 492 protein sequencer (Applied Biosystems).
The sample was applied to a DEAE-Sepharose column equilibrated with 50 mM Tris-HCl, pH 7.2, containing 50 mM NaCl, and the expressed proteins were eluted with a linear gradient of 50 -500 mM NaCl. The fractions with chondroitinase activity were collected and then applied to a nickel-Sepharose column and washed with 50 mM Tris-HCl, pH 7.2, containing 500 mM NaCl and 20 mM imidazole. His-tagged proteins were eluted with 50 mM Tris-HCl, pH 7.2, containing 500 mM NaCl and 500 mM imidazole and dialyzed against 50 mM Tris-HCl, pH 7.2 containing 150 mM NaCl.
Enzyme Activity and Kinetic Analysis-Chondroitin lyase activity was measured by absorbance at 232 nm (A 232 ) essentially as described by Yamagata et al. (4). A typical incubation mixture contained a measured quantity of enzyme protein and 100 g of substrate in 50 l of 50 mM phosphate buffer, pH 6.8. Control medium was a blank mixture of heat-inactivated enzyme. After incubation at 37°C for 30 min, the reaction was stopped by heating for 1 min in a boiling water bath. The reaction mixture was then diluted with 450 l of distilled water, and UV absorption was measured at 232 nm against the corresponding blank mixture. One unit of enzyme was defined as activity required for the eliminative cleavage of substrate, yielding UV-absorbing materials corresponding to 1 mol of unsaturated GlcUA residues per min, as calculated with a value of 5500 for its molar absorption coefficient (⑀) (5). Protein concentrations were determined by the Bradford method (Bio-Rad) using bovine serum albumin as a standard.
For kinetic analysis, chondroitin lyase activities of recombinant E66 proteins (2 ng/ml) were measured with various concentrations (0.04 -1.2 mM) of CH as substrate in the reaction solutions (50 l) at 37°C for 10 min. Kinetic parameters (K m and V max ) were determined with double-reciprocal plots (Lineweaver-Burk plots) of chondroitin lyase activities (v, nmol/ min) and substrate concentrations (S, M) from the Michaelis-Menten equation. The k cat was calculated by dividing V max by the concentration of enzyme in the reaction. The analyses were performed in at least three experiments.
Compositional Analysis of Depolymerized Products-The digests of CH and CS (20 g) were treated with purified E66 (67-704) protein (0.5 g) at 37°C for 24 h and divided into di-, tetra-, and longer saccharides by gel filtration chromatography on the Superdex peptide column (1.0 ϫ 300 mm). The flow rate was 1.0 ml/min using 0.2 M NaCl developing buffer. The elution was determined by UV absorption at 220 nm to detect the unsaturated GlcUA and N-acetyl group of GalNAc residues. The divided fractions, except the disaccharide fraction and original substrates, were digested completely with cABC to the unsaturated disaccharides, whose compositions were measured with fluorometric post-column high performance liquid chromatography (HPLC) as described previously (19).

Purification and Identification of Putative Chondroitinase-
In a series of experiments in which CH was sulfated with recombinant chondroitin sulfotransferases expressed with baculovirus systems, we noticed that some recombinant fractions from the conditioned medium of baculovirus-infected insect cells generated short CS chains. The conditioned medium of the baculovirus-infected sf9 cells, even without sulfotransferase cDNA, showed significant chondroitinase activity, whereas that obtained from non-infected host cells did not (data not shown), suggesting that the activity was present in the baculovirus. The solution containing CH-degrading activity yielded digestion products mainly composed of di-and tetrasaccharides that absorbed UV light at 232 nm. The prepared disaccharides showed the same elution patterns as authentic unsaturated disaccharides in the HPLC assay system. The tetrasaccharides represented molecular masses corresponding to unsaturated tetrasaccharides in MALDI-TOF MS analysis (data not shown). Thus, the enzyme expressed by baculovirus is a chondroitin lyase.
The lyase-active protein in the virus-infected cell culture medium was partially purified by sequential steps of column chromatography with DEAE-Sepharose, Q Sepharose, and Superdex 75 columns. SDS-PAGE analysis of the chondroitinase fractions of Superdex 75 showed some proteins containing a major band of 61 kDa (supplemental Fig. 1). The major band was excised from the polyacrylamide gel, digested with trypsin, and analyzed by MALDI-TOF MS and the MASCOT search system. The protein was identified as ODV-E66 of AcMNPV. In parallel, the 61-kDa band in the gel was transferred to a PVDF membrane, excised, and analyzed. Its N-terminal amino acid sequence was AFRQNNIQEL, which corresponds to amino acid residues 67-76 of ODV-E66. These results strongly suggested that ODV-E66 was responsible for the chondroitin lyase activity in the conditioned medium.
Next, we examined the effects of divalent metal ions, chelating reagent, and sodium chloride concentration. The chondroi-tin lyase activity of E66 (67-704) was unaffected by 20 mM divalent metal salts (CaCl 2 , MgCl 2 , MnCl 2 , and CoCl 2 ) and 20 mM EDTA when compared with reaction in 50 mM potassium sodium phosphate buffer. Activity was unaffected at 0.15 M NaCl and decreased to ϳ63% in 0.5 M NaCl (Fig. 2B).
We also examined the effect of reaction temperature. The activity of E66 (67-704) increased linearly at 4 -37°C and was highest at 37°C. The activity was maintained up to 60°C and decreased rapidly at temperatures above 65°C (Fig. 2C). The activity of E66 (70 -704) showed essentially the same patterns with variations in pH, metal ions, and temperature (data not shown).  AUGUST 19, 2011 • VOLUME 286 • NUMBER 33

JOURNAL OF BIOLOGICAL CHEMISTRY 29029
Substrate Specificity-Next, we investigated the substrate specificity of ODV-E66 using various glycosaminoglycans. When these substrates (100 g) were treated with 0.05 g of recombinant enzyme E66 (67-704) at 37°C for 30 min, the substrates were digested as summarized in Table 2. CH was the best substrate. All the CS substrates prepared from different animals were digested to lesser extents (16 -30%) than CH. HA was minimally digested by ODV-E66 protein (0.16%) in a range of pH 5-8. The enzyme did not digest DS, heparin, heparan sulfate, and HPR even when the enzyme was present at 10-fold excess (0.5 g).
We analyzed the digestion products of individual reactions using 20 g of substrate and 0.5 g of E66 (67-704) at 37°C for 24 h by separation on a Superdex peptide column (Fig. 3). CH was completely digested to tetra-and disaccharides (Fig. 3A), whereas the reaction products of other CS substrates revealed partial depolymerization. Both CS-A and CS-E (Fig. 3, B and F) showed stronger resistance to depolymerization by ODV-E66 than did CS-C, CS-AC, and CS-D (Fig. 3, C-E). Large proportions of long oligo-and polysaccharides remained in the reactions of CS-E (94.2%) and CS-A (78.1%) after 24 h. In contrast, 33-42% of CS-C, CS-AC, and CS-D was digested to di-or tetrasaccharides (Table 3).
We analyzed the disaccharide composition of the digestion products using a fluorescent post-column HPLC system. Interestingly, the disaccharide fractions obtained from all the CS digestions consisted mainly of 0S and 6S disaccharides, and only a small amount of 4S disaccharide was detected in the CS-C and CS-AC digests. We further analyzed the disaccharide composition of the tetrasaccharide fractions of the ODV-E66 digests by treating them with cABC. More than half the disaccharide residues of the tetrasaccharide fractions consisted of 0S and 6S, regardless of the disaccharide composition of the CS sources (Table 3). These results indicate that the enzyme preferentially digests polysaccharides having CH and C6S structures.
To characterize the substrate specificity of ODV-E66 chondroitinase activity, we used sCH, sC4S, and sC6S substrates, which had an average molecular weight of 12,000 and consisted of 100% non-sulfated, 91.9% 4-sulfated, and 93.9% 6-sulfated disaccharides, respectively. We treated these substrates with E66 (67-704) at 37°C for 24 h and separated the digests by gel filtration chromatography (Fig. 4, A-C). Most of the sCH and sC6S substrates were degraded to di-and tetrasaccharides. In contrast, sC4S was minimally digested, and both long oligosaccharides and polysaccharides remained in the reactions. The yields of di-and tetrasaccha-rides in the sCH and sC6S reactions were ϳ60 and 40%, and their disaccharide compositions were similar to the source polysaccharides (Table 4). A further reaction for an additional 24 h, by adding the same amount of the enzyme, did not alter the compositions of di-and tetrasaccharides (data not shown). Therefore, they were limited digests. The ratios of the di-and tetrasaccharides digested from sC4S were 4.8% and 2.6%, respectively, and 92.5% of the reaction remained as polysaccharides. The separated di-and tetrasaccharide fractions from sC4S contained large proportions of nonsulfated units, distinct from the source polysaccharide, whereas  the long saccharide fraction contained a proportion of the 4-sulfated unit, similar to the source polysaccharide (92.4% versus 91.9%, Table 4).

DISCUSSION
We found CS-degrading activity in a baculovirus expression system and identified the enzyme as a truncated form of ODV-E66, one of the envelope proteins of AcMNPV baculovirus. ODV-E66 truncated protein is a novel chondroitin lyase with distinct substrate specificity. It selectively cleaves 0S and 6S units of CS chains. This is the first report of a CS-degrading enzyme in viruses. The enzyme is a new chondroitinase with unique substrate specificity and utility in CS analysis.
To date, various chondroitinases (4 -7) and hyaluronidases (21,22) have been identified in many types of bacteria. They have unique substrate specificities but often share substrates. For example, cABCI and cABCII degrade C4S, C6S, and DS. cACI and cACII degrade C4S and C6S, but not DS. cABCI, cACI, and cACII degrade both CS and HA, to a greater or less extent. There are two opposing studies on whether cABCII digests HA (5,23). HA lyase (21) from Streptomyces hyalurolyticus does not digest CS/DS, whereas that from Streptococcus sp. digests CH to a lesser extent than HA (22). When compared with bacteria chondroitinases, ODV-E66 showed narrow substrate specificity with regard to CS structure. It cleaves both 0S and 6S units of CS chains but does not cleave the other units (4S, diSD, and diSE). It degrades HA with quite low activity when compared with CH and does not degrade DS. ODV-E66 is an endoeliminase as various oligosaccharides were generated as intermediates from CS substrates containing undigestible disaccharide units in the reaction.  cABCI, cABCII, cACI, and chondroitinase B are most active around pH 8, whereas cACII and HA lyases act under acidic conditions (pH 5-6). In contrast, ODV-E66 exhibited high activity over a wide pH range. Optimum temperatures for cABCI, cABCII, cACI, cACII, and chondroitinase B activity are 37, 37, 40, 50, and 30°C, and their activity decreases immediately at higher temperatures. In contrast, ODV-E66 exhibited high activity over a wide range of temperatures. These observations suggest that ODV-E66 maintains a stable structure under different conditions.
The enzyme activities of both chondroitinase B from F. heparinum (24) and cABC from Bacteroides thetaiotaomicron (25) are affected by divalent metal ions such as Ca 2ϩ ion and are inhibited by chelators such as EDTA and EGTA. Ca 2ϩ and Mg 2ϩ ions preferentially increase the activity of cABCI toward DS versus CS (26). These chondroitinases have a Ca 2ϩ binding region at the active site. In contrast, the chondroitinase activity of ODV-E66 was unaffected by divalent ions and chelating reagent, suggesting that the structure of its catalytic site may differ from those of bacterial chondroitinases. Structural analysis of the ODV-E66 catalytic sites remains to be performed to characterize its mechanism of action.
Specific activity and kinetic parameters (K m , k cat , and k cat / K m ) of E66 (67-704) for CH were 67.2 mol/min/mg of protein, 421 M, 6510 min Ϫ1 , and 15.5 min Ϫ1 M Ϫ1 , respectively. Those of cABCI (26,27)  Full-length ODV-E66 contains a 23-amino acid signal sequence at the N terminus, a polysaccharide lyase family sequence (amino acids 83-291), and a baculovirus E66 superfamily sequence at the C terminus (residues 316 -704) (NCBI Conserved Domain Search). Mature protein isolated from the conditioned medium of baculovirus-infected cells was a truncated form starting at amino acid 67. A truncated form starting at amino acid 70 was previously reported as a mature protein of ODV-E66 (10). Recombinant proteins E66 (67-704) and E66 (70 -704) showed high chondroitin lyase activity, whereas E66 (24 -704), which lacked the N-terminal signal sequence, showed significantly lower activity than the mature proteins. Thus, it is likely that ODV-E66 is processed by certain proteinases that remove the N-terminal fragments of amino acids 66 and 69 to achieve high CH/CS-degrading activity.
Insects such as fruit fly (Drosophila) and mosquito (Anopheles) have low sulfated CS proteoglycans. CS proteoglycans have been detected in Drosophila ovaries, embryos, larvae, and adults (28,29) and Anopheles salivary glands, midguts, and ovaries (30). By histological analysis, the CS proteoglycans were found in the apical microvilli of the mosquito midgut (31). CS of fruit fly and mosquito is composed mainly of 0S disaccharide units and only a small amount of 4S disaccharide units. Drosophila larvae in the L3 stage contain significant amounts of 6S (32). Therefore, ODV-E66, which degrades CH and C6S, may be beneficial for CS digestion in insects.
AcMNPV (33) is one of the occluded baculoviruses, which are double-stranded DNA viruses that infect lepidopteran insect larvae. The ODV of AcMNPV coated with envelope proteins (occlusion body) is ingested with plants by an insect larva

compositions of various chondroitin sulfates and the digested fractions and molar ratios of the disaccharide units of the fractions
The substrates were treated with E66 (67-704) at 37°C for 24 h as described under ЉExperimental ProceduresЉ (Fig.3). The abbreviations used are: di, disaccharide fraction; tetra, tetrasaccharide fraction; long, longer saccharides than tetrasaccharide fraction; origin, CS substrate before digestion; ND, not detected.  and moves to the midgut. Lepidopteran larvae generate the peritrophic matrix in the luminal side of the midgut, which protects the mucosa from virulence (34). The peritrophic matrix of lepidopteran larvae may contain CS glycosaminoglycans as in other insects. ODV-E66 may be released with other envelope proteins from the midgut occlusion body with the high pH and proteinases secreted from the midgut epithelia. The truncated ODV-E66 that acquires CH-degrading activity may digest the CS barrier of the peritrophic matrix of the host midgut and facilitate viral infection to the epithelial cells. Vigdorovich et al. (35) previously reported that ODV-E66 had hyaluronidase activity, which was confirmed in this study. However, its activity on HA was ϳ600 times lower than its CH-degrading activity. It is not necessary to have HA-degrading enzymes for baculovirus pathogenesis of insects because insects have neither HA nor HA synthase genes (36).

Disaccharide composition
The N-terminal 23 amino acids of ODV-E66 were reported to traffic viral proteins to the inner nuclear membrane (20,37). Baculovirus envelope proteins are transported to the inner nuclear membrane with the N-terminal sequence of ODV-E66. The occlusion bodies are formed in the nucleus and then released to the outside environment. At that time, the active ODV-E66 protein may be expressed, degrading the extracellular matrix containing CS and facilitating the disintegration of the infected host.
ODV-E66 will be included as a new member of the chondroitinases with unique substrate specificity and utility in CS analysis. The enzyme may be useful to prepare highly sulfated oligosaccharides, including diSD and diSE units, which are known to possess various biological activities. Moreover, ODV-E66 may facilitate the virus infection to insect host and be useful for study of baculovirus infection mechanism. Recent studies (8,9) have revealed that chondroitinases may be useful in treatment of spinal cord injuries and intervertebral disc herniation. As ODV-E66 digests CS over a wide range of temperature and pH, it may be another candidate for such medical applications.  E66 (67-704). A-C, the digests (solid lines) with E66 (67-704) and the original substrates (broken lines) of sCH (A), sC4S (B), and sC6S (C) were separated by Superdex peptide column chromatography. The elution was monitored by UV absorption at 220 nm. V 0 and V t indicate the position of void and total volume of the column, respectively. The peaks numbered 2 and 4 indicate di-and tetrasaccharides.

TABLE 4 Disaccharide compositions of chemo-enzymatically synthesized substrates and their digested fractions and molar ratios of the disaccharide units of the fractions
The substrates were treated with E66 (67-704) at 37°C for 24 h as described under ЉExperimental ProceduresЉ (Fig. 4). The abbreviations used are': di, disaccharide fraction; tetra, tetrasaccharide fraction; long, longer saccharides than tetrasaccharide fraction; origin, CS substrate before digestion.