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

Open AccessPublished:June 29, 2011DOI:https://doi.org/10.1074/jbc.M111.251157
      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.

      Introduction

      Chondroitin sulfate (CS)
      The abbreviations used are: CS
      chondroitin sulfate
      CS-A
      CS from whale cartilage
      CS-C
      CS from shark cartilage
      CS-AC
      CS from bovine trachea cartilage
      CS-D
      CS from shark fin cartilage
      CS-E
      CS from squid skin
      CH
      chondroitin
      DS
      dermatan sulfate
      HA
      hyaluronan
      HPR
      N-acetylheparosan
      cABC
      chondroitin ABC lyase
      cABCI
      chondroitin ABC lyase I
      cABCII
      chondroitin ABC lyase II
      cACI
      chondroitin AC lyase I
      cACII
      chondroitin AC lyase II
      0S
      GlcUA-GalNAc
      4S
      GlcUA-GalNAc (4-sulfate)
      6S
      GlcUA-GalNAc (6-sulfate)
      diSD
      GlcUA (2-sulfate)-GalNAc (6-sulfate)
      diSE
      GlcUA-GalNAc (4-, 6-sulfates)
      sCH
      chemo-enzymatically synthesized CH
      sC4S
      chemo-enzymatically synthesized CS with 4S units
      sC6S
      chemo-enzymatically synthesized CS with 6S units
      ODV
      occlusion-derived virus
      AcMNPV
      A. californica multicapsid nucleopolyhedrovirus
      CAPS
      3-(cyclohexylamino)propanesulfonic acid.
      is a linear polysaccharide of (-4 d-glucuronic acid (GlcUA) β1–3 N-acetyl-d-galactosamine (GalNAc) β1-)n repeating disaccharide units carrying sulfate groups at various positions of the sugar residues (
      • Esko J.D.
      • Kimata K.
      • Lindahl U.
      ,
      • Habuchi O.
      ). 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 (
      • Iozzo R.V.
      • Schaefer L.
      ). These functions are mainly ascribed to the sulfate groups, building structural specificity and diversity into the polysaccharide framework.
      Major disaccharide structures of CS are the non-sulfated unit (GlcUA-GalNAc, 0S), monosulfated unit at the C-4 position of the GalNAc residue (GlcUA-GalNAc (4-sulfate), 4S), monosulfated unit at the C-6 position of the GalNAc residue (GlcUA-GalNAc (6-sulfate), 6S), disulfated unit at the C-4 and C-6 positions of the GalNAc residue (GlcUA-GalNAc (4-, 6-sulfates), diSE), and disulfated unit at the C-2 position of GlcUA and the C-6 position of GalNAc residues (GlcUA (2-sulfate)-GalNAc (6-sulfate), diSD). During biosynthesis of CS chains, some GlcUA residues are epimerized into l-iduronic acid (IdoUA). The chain containing IdoUA residues is designated as a subfamily member of CS, dermatan sulfate (DS).
      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 β-elimination reaction, which generates an unsaturated 4,5-bond on the uronic acid at the site of cleavage. Chondroitin ABC lyase I (cABCI) (
      • Yamagata T.
      • Saito H.
      • Habuchi O.
      • Suzuki S.
      ) and II (cABCII) (
      • Hamai A.
      • Hashimoto N.
      • Mochizuki H.
      • Kato F.
      • Makiguchi Y.
      • Horie K.
      • Suzuki S.
      ) are prepared from Proteus vulgaris. Chondroitin AC lyase I (cACI) (
      • Yamagata T.
      • Saito H.
      • Habuchi O.
      • Suzuki S.
      ) and II (cACII) (
      • Hiyama K.
      • Okada S.
      ) are from Flavobacterium heparinum and Arthrobacter aurescens, respectively. Chondroitinase B (
      • Michelacci Y.M.
      • Dietrich C.P.
      ) 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 non-reducing 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 (
      • Kato F.
      • Iwata H.
      • Mimatsu K.
      • Miura T.
      ) and spinal cord injury (
      • Bradbury E.J.
      • Moon L.D.
      • Popat R.J.
      • King V.R.
      • Bennett G.S.
      • Patel P.N.
      • Fawcett J.W.
      • McMahon S.B.
      ).
      In our experiments, wherein chondroitin sulfotransferases were expressed in a baculovirus system, we found chondroitinase activity in the conditioned medium of baculovirus-infected insect cells. We purified the enzyme and identified it as a truncated molecule of occlusion-derived virus envelope protein 66 (ODV-E66) (
      • Hong T.
      • Braunagel S.C.
      • Summers M.D.
      ) of Autographa californica nucleopolyhedrovirus (AcMNPV). The truncated molecule has chondroitinase activity distinct from that already reported.

      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 (
      • Yamagata T.
      • Saito H.
      • Habuchi O.
      • Suzuki S.
      ,
      • Hamai A.
      • Hashimoto N.
      • Mochizuki H.
      • Kato F.
      • Makiguchi Y.
      • Horie K.
      • Suzuki S.
      ,
      • Hiyama K.
      • Okada S.
      ,
      • Michelacci Y.M.
      • Dietrich C.P.
      ) and hyaluronidases (
      • Ohya T.
      • Kaneko Y.
      ,
      • Baker J.R.
      • Yu H.
      • Morrison K.
      • Averett W.F.
      • Pritchard D.G.
      ) 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 (
      • Hamai A.
      • Hashimoto N.
      • Mochizuki H.
      • Kato F.
      • Makiguchi Y.
      • Horie K.
      • Suzuki S.
      ,
      • Prabhakar V.
      • Capila I.
      • Soundararajan V.
      • Raman R.
      • Sasisekharan R.
      ). HA lyase (
      • Ohya T.
      • Kaneko Y.
      ) from Streptomyces hyalurolyticus does not digest CS/DS, whereas that from Streptococcus sp. digests CH to a lesser extent than HA (
      • Baker J.R.
      • Yu H.
      • Morrison K.
      • Averett W.F.
      • Pritchard D.G.
      ). 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 (
      • Michel G.
      • Pojasek K.
      • Li Y.
      • Sulea T.
      • Linhardt R.J.
      • Raman R.
      • Prabhakar V.
      • Sasisekharan R.
      • Cygler M.
      ) and cABC from Bacteroides thetaiotaomicron (
      • Shaya D.
      • Hahn B.S.
      • Bjerkan T.M.
      • Kim W.S.
      • Park N.Y.
      • Sim J.S.
      • Kim Y.S.
      • Cygler M.
      ) are affected by divalent metal ions such as Ca2+ ion and are inhibited by chelators such as EDTA and EGTA. Ca2+ and Mg2+ ions preferentially increase the activity of cABCI toward DS versus CS (
      • Prabhakar V.
      • Capila I.
      • Raman R.
      • Srinivasan A.
      • Bosques C.J.
      • Pojasek K.
      • Wrick M.A.
      • Sasisekharan R.
      ). These chondroitinases have a Ca2+ 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 (Km, kcat, and kcat/Km) of E66 (67–704) for CH were 67.2 μmol/min/mg of protein, 421 μm, 6510 min−1, and 15.5 min−1μm−1, respectively. Those of cABCI (
      • Prabhakar V.
      • Capila I.
      • Raman R.
      • Srinivasan A.
      • Bosques C.J.
      • Pojasek K.
      • Wrick M.A.
      • Sasisekharan R.
      ,
      • Prabhakar V.
      • Capila I.
      • Bosques C.J.
      • Pojasek K.
      • Sasisekharan R.
      ) were reported as 170–230 μmol/min/mg of protein, 1.2–2.4 μm, 2200–3700 min−1, and 3100–9100 min−1μm−1. Those of cABCII (
      • Prabhakar V.
      • Capila I.
      • Soundararajan V.
      • Raman R.
      • Sasisekharan R.
      ) were 29–32 μmol/min/mg of protein, 9.8 μm, 1300 min−1, and 132 min−1μm−1. Specific activities of other bacterial lyases were 40–320 μmol/min/mg of protein. The high Km of E66 (67–704) indicates its low substrate binding affinity in comparison with bacterial chondroitin lyases, although the order of the specific activity of ODV-E66 appears similar.
      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 (
      • Hong T.
      • Braunagel S.C.
      • Summers M.D.
      ). 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 (
      • Toyoda H.
      • Kinoshita-Toyoda A.
      • Selleck S.B.
      ,
      • Toyoda H.
      • Kinoshita-Toyoda A.
      • Fox B.
      • Selleck S.B.
      ) and Anopheles salivary glands, midguts, and ovaries (
      • Sinnis P.
      • Coppi A.
      • Toida T.
      • Toyoda H.
      • Kinoshita-Toyoda A.
      • Xie J.
      • Kemp M.M.
      • Linhardt R.J.
      ). By histological analysis, the CS proteoglycans were found in the apical microvilli of the mosquito midgut (
      • Dinglasan R.R.
      • Alaganan A.
      • Ghosh A.K.
      • Saito A.
      • van Kuppevelt T.H.
      • Jacobs-Lorena M.
      ). 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 (
      • Pinto D.O.
      • Ferreira P.L.
      • Andrade L.R.
      • Petrs-Silva H.
      • Linden R.
      • Abdelhay E.
      • Araújo H.M.
      • Alonso C.E.
      • Pavão M.S.
      ). Therefore, ODV-E66, which degrades CH and C6S, may be beneficial for CS digestion in insects.
      AcMNPV (
      • Rohrmann G.F.
      ) 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 and moves to the midgut. Lepidopteran larvae generate the peritrophic matrix in the luminal side of the midgut, which protects the mucosa from virulence (
      • Hegedus D.
      • Erlandson M.
      • Gillott C.
      • Toprak U.
      ). 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. (
      • Vigdorovich V.
      • Miller A.D.
      • Strong R.K.
      ) previously reported that ODV-E66 had hyaluronidase activity, which was confirmed in this study. However, its activity on HA was ∼600 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 (
      • Takeo S.
      • Fujise M.
      • Akiyama T.
      • Habuchi H.
      • Itano N.
      • Matsuo T.
      • Aigaki T.
      • Kimata K.
      • Nakato H.
      ).
      The N-terminal 23 amino acids of ODV-E66 were reported to traffic viral proteins to the inner nuclear membrane (
      • Hong T.
      • Summers M.D.
      • Braunagel S.C.
      ,
      • Braunagel S.C.
      • Williamson S.T.
      • Saksena S.
      • Zhong Z.
      • Russell W.K.
      • Russell D.H.
      • Summers M.D.
      ). 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 (
      • Kato F.
      • Iwata H.
      • Mimatsu K.
      • Miura T.
      ,
      • Bradbury E.J.
      • Moon L.D.
      • Popat R.J.
      • King V.R.
      • Bennett G.S.
      • Patel P.N.
      • Fawcett J.W.
      • McMahon S.B.
      ) 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.

      Acknowledgments

      We thank Dr. Takashi Sato and Prof. Hisashi Narimatsu, Research Center for Medical Glycoscience, Advanced Industrial Science and Technology (AIST) for donating the C4ST-1 and C6ST-1 expression plasmids, Kiyoshi Suzuki and Hiroshi Maeda, Central Research Laboratories, Seikagaku Corp. for providing desulfated CH and HPR, and Minoru Fukayama, Aichi Medical University, for assistance in MALDI-TOF MS and N-terminal amino acid sequence analyses.

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