The vaccinia virus/T7 bacteriophage expression system was used to express human decorin in HT-1080 cells by co-infection with vTF7-3, encoding T7 RNA polymerase, and vDCN1, encoding the decorin core protein fused to a polyhistidine-insulin signal sequence fusion-protein cassette. Overexpression using the vaccinia virus/T7 phage system resulted in secretion of approximately 30 mg of decorin/10 9 cells per 24 h which enabled purification and separation of multiple glycoforms under native conditions. Cells were cultured in the presence of [ 35 S]methionine or a mixture of [ H]glucosamine and [35 S]sulfate, and recombinant glycoprotein purified by metal affinity chromatography which resolved the secreted decorin into two classes, a proteoglycan form and a core protein form. About 25% of the recombinant protein was secreted into the culture medium as core protein devoid of glycosaminoglycan chains. The decorin core protein was resolved into two forms (ϳ49 and ϳ53 kDa) that differed in the extent of N-linked oligosaccharide substitution (2 and 3 N-linked oligosaccharides, respectively). Deglycosylation of the recombinant proteoglycans and core proteins resulted in a single band migrating with an apparent molecular mass ϳ 43 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. Far-UV circular dichroism spectra of native decorin proteoglycan showed a minima at 218 nm, consistent with a secondary structure that is predominantly ␤-sheet. Circular dichroism spectra of bovine decorin extracted from articular cartilage and recombinant decorin similarly treated revealed a minima of 205 nm indicating a loss of secondary structure. The affinity of decorin proteoglycan and core protein for collagenlike molecules was demonstrated, with the complement component C1q exhibiting the most striking affinity for decorin, although adherence to collagen types I and V was also observed. The extensive secondary structure maintained in the purified recombinant protein is likely to be important for the biological function of decorin.

This paper identifies two structural proteins of the occluded derived viral envelope of Autographa californica nuclear polyhedrosis virus (AcMNPV): ODV-E18 and ODV-E35. In addition, we identify a protein, ODV-EC27, that is incorporated into the capsid of occluded virus, which is not detected in budded virus. The genes for these proteins reside within the IE0 intron. The intron was sequenced, and five open reading frames (ORF) were identified. ORF 3 (genomic ORF 143) codes for the ODV envelope protein, ODV-E18. ORF 4 (genomic ORF 144) codes for ODV-EC27, and Western blot analyses locate this protein to both the ODV capsid and envelope. Transcripts for both ODV-E18 and ODV-EC27 initiate from conserved TAAG motifs, and transcripts are detected from 16 through 72 hr p.i. Antiserum to ODV-E18 recognizes a band of 18 kDa on Western blots of extracts from infected cells and bands of 18 and 35 kDa on Western blots of proteins from purified ODV envelope. N-terminal amino acid sequencing reveals that both ODV-E18 and ODV-E35 contain the same N-terminus. Antiserum to ODV-EC27 recognizes a protein of 27 kDa on Western blots of extracts from infected cells and bands of 27 and 35 kDa on Western blots of proteins from purified ODV. Using immunogold labeling techniques, ODV-E18 and/or ODV-E35 are detected in viral induced intranuclear microvesicles and are not detected in the plasma membrane, cytoplasmic membranes, or the nuclear envelope. Immunogold labeling using antisera to ODV-EC27 detects this protein on both the ODV envelope and capsid.

Biglycan is a small chondroitin sulfate proteoglycan found in many tissues and is structurally related to decorin, fibromodulin, and lumican. The biological function of biglycan is poorly understood, although several studies have indicated interaction with other extracellular matrix components. We have initiated studies of structural and functional domains of biglycan by transient eukaryotic expression using the vaccinia virus/T7 bacteriophage expression system. A recombinant vaccinia virus, vBGN4 encoding the mature biglycan core protein as a polyhistidine fusion protein under control of the T7 phage promoter was expressed in HT-1080 cells and UMR106 cells. The structure of the recombinant biglycan secreted by these cells was defined by analyzing molecules labeled in the presence of [ Biglycan belongs to a family of small proteoglycans that are characterized by a core protein that consists of a leucine-rich repeat (LRR) 1 domain. Other members of this family include decorin, fibromodulin, lumican, and chicken PG Lb. The LRR is a consensus sequence of 20 -29 residues containing leucine in conserved positions, and is often present as a tandem repeat. The LRR domain of human biglycan consists of 8 repeats of 24 residues. The small proteoglycans belong to a subset of the LRR protein superfamily due to the presence of four similarly spaced cysteine residues at the amino-terminal of the LRR domain. The LRR has been identified in an increasing number of proteins derived from yeast, plant, insect, and mammalian species (reviewed in Ref. 1) and is likely to play a role in proteinprotein interactions (2, 3).Biglycan consists of a core protein of about 42 kDa subjected to extensive post-translational modifications including substitution with two glycosaminoglycan chains. Biglycan extracted from bone or cartilage is devoid of signal and propeptide sequences. Glycosylation of the core protein involves addition of glycosaminoglycan chains and N-linked oligosaccharides. The composition of the glycosaminoglycan polymer is tissue specific; in skin and cartilage, biglycan is substituted with dermatan sulfate chains, however, in bone it is substituted with chondroitin sulfate chains. Biglycan core protein contains two asparagine consensus tripeptides for substitution with N-linked oligosaccharides, which are conserved in the core protein of the structurally related decorin.Numerous studies investigating the interactions of the small chondroitin sulfate proteoglycans, biglycan and decorin, with other matrix components have commonly used proteoglycan extracted from tissue. These reports include descriptions of binding to collagen type I and II (4, 5), collagen type V (6), collagen type VI (7), collagen type XIV (8), transforming growth factor-␤ (9, 10), complement component C1q (11), and divalent cation-dependent self-association (12). The biological significance of these interactions remains unresolved since efficient extraction of biglycan and decorin from cartilage and bone requires the use of chaotropic solvents and detergents ...

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