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

Abstract

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. Chondroitin sulfate (CS)2 is a linear polysaccharide of (-4groups 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.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 (GlcUAGalNAc (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 ␤-elimination reaction, which generates an unsaturated 4,5-bond on the ...

Comparative Proteomics Reveal Fundamental Structural and Functional Differences between the Two Progeny Phenotypes of a Baculovirus

“…4) (21, 22, 29-33). Recently, the truncated ODV-E66 protein was identified as a chondroitin lyase that can efficiently digest chondroitin and chondroitin 6-sulfate (34). The remaining envelope proteins were VP91, HA72 (AC78), and superoxide dismutase (SOD).…”

“…The homologue of HA68, another BV-specific membrane protein, was found to play a role in BV production (61). Among 11 ODV-specific envelope proteins, 7 proteins are essential for oral infectivity (P74, PIF1, PIF2, PIF3, PIF4, PIF5, and PIF6), and 1 protein plays an important role in infection (ODV-E66) (21,22,(30)(31)(32)(33)(34). In addition to the proteins discussed above, VP91 was also found to be a component of the PIF complex (62).…”

Construction of a Chondroitin Sulfate Library with Defined Structures and Analysis of Molecular Interactions

“…Regarding their expression, bacterial expression systems have failed to produce sufficiently active enzymes except in a few cases (26 -28), and baculoviral infection systems were unsuitable because of chondroitinase activity in the virus-infected medium (29). CS sulfotransferases obtained from mammalian expression systems efficiently modified the CH and CS chains.…”

Autographa californicaNucleopolyhedrovirus Ac76: a Dimeric Type II Integral Membrane Protein That Contains an Inner Nuclear Membrane-Sorting Motif

“…ODV-E66 has been shown to have chondroitinase activity (60) and plays an important role in the oral infection process (61). This protein also ought to be on the ODV surface to digest the chondroitin sulfate barrier of the peritrophic matrix of the host midgut, thus facilitating viral infection of epithelial cells (60). Ac83 is the only component with a chitin-binding domain in the PIF complex on the ODV envelope (46,62).…”

“…PIF1 and PIF2, as well as P74, play fundamental roles in ODV binding to insect midgut cells (59); therefore, they should be on the ODV surface. ODV-E66 has been shown to have chondroitinase activity (60) and plays an important role in the oral infection process (61). This protein also ought to be on the ODV surface to digest the chondroitin sulfate barrier of the peritrophic matrix of the host midgut, thus facilitating viral infection of epithelial cells (60).…”