Eukaryotic Expression of Recombinant Biglycan

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124

Bone morphogenetic protein-1 (BMP-1) is a metalloprotease that plays important roles in regulating the deposition of fibrous extracellular matrix in vertebrates, including provision of the procollagen C-proteinase activity that processes the major fibrillar collagens I-III. Biglycan, a small leucine-rich proteoglycan, is a nonfibrillar extracellular matrix component with functions that include the positive regulation of bone formation. Biglycan is synthesized as a precursor with an NH 2 -terminal propeptide that is cleaved to yield the mature form found in vertebrate tissues. Here, we show that BMP-1 cleaves probiglycan at a single site, removing the propeptide and producing a biglycan molecule with an NH 2 terminus identical to that of the mature form found in tissues. BMP-1-related proteases mammalian Tolloid and mammalian Tolloid-like 1 (mTLL-1) are shown to have low but detectable levels of probiglycancleaving activity. Comparison shows that wild type mouse embryo fibroblasts (MEFs) produce only fully processed biglycan, whereas MEFs derived from embryos homozygous null for the Bmp1 gene, which encodes both BMP-1 and mammalian Tolloid, produce predominantly unprocessed probiglycan, and MEFs homozygous null for both the Bmp1 gene and the mTLL-1 gene Tll1 produce only unprocessed probiglycan. Thus, all detectable probiglycan-processing activity in MEFs is accounted for by the products of these two genes.Bone morphogenetic protein-1 (BMP-1) 1 is the prototype of a family of metalloproteases involved in morphogenesis in a broad range of species (1). BMP-1 and mammalian Tolloid (mTLD), a somewhat larger protein encoded by alternatively spliced RNAs of the Bmp1 gene (2), affect morphogenesis, at least in part, by providing the procollagen C-proteinase (PCP) activity that cleaves the C-propeptides of procollagens I-III to yield the major fibrous components of extracellular matrix (ECM) (3-6). These two proteases also contribute to the net deposition of insoluble ECM through proteolytic activation of lysyl oxidase (7), 2 an enzyme necessary to the formation of covalent cross-links in collagen and elastic fibers. Two additional, genetically distinct, BMP-1/mTLD-related mammalian proteases have been described and designated mammalian Tolloid-like 1 (mTLL-1) and mTLL-2, due to domain structures identical to that of mTLD (5, 8). Although mTLL-1 has some PCP activity in in vitro assays (5), the significance of this activity is unclear, as procollagen processing appears unaffected in mTLL-1-deficient mice (9). PCP activity was not detected in in vitro assays of mTLL-2 (5).Recently, BMP-1/mTLD-related proteases Xenopus Xolloid (10) and zebrafish Tolloid (11) were shown to exert ventralizing effects during vertebrate embryogenesis by cleaving the secreted protein Chordin, which forms latent complexes with ventralizing TGF-␤-like molecules, such as BMPs 2 and 4 (12). BMP-1 and mTLL-1 are also capable of affecting dorsal-ventral patterning through cleavage of Chordin, whereas mTLD and mTLL-2 do not have detectable levels of t...

Section: Bmp-1 As a Candidate Enzyme For The Proteolytic Processing Omentioning

confidence: 99%

Section: Bmp-1 As a Candidate Enzyme For The Proteolytic Processing Omentioning

confidence: 99%

Section: Overlapping Expression Domains Of Bmp-1/mtld and Biglycan Rnmentioning

confidence: 99%

See 1 more Smart Citation

Bone Morphogenetic Protein-1 Processes Probiglycan

Scott

Imamura²,

Pappano

et al. 2000

130

124

“…This molecule was not secreted and was found instead in the detergent-insoluble fraction of these cells. The lack of modification is not surprising since published reports state that "it is unlikely that insect cells have the post-translational machinery required for effective processing of a proteoglycan" (47).…”

Section: Identification Of the Type XV Amino-terminal Domain Exhibitimentioning

confidence: 99%

Basement Membrane Zone Type XV Collagen Is a Disulfide-bonded Chondroitin Sulfate Proteoglycan in Human Tissues and Cultured Cells

Li¹,

Clark²,

Myers³

2000

Type XV collagen has a widespread distribution in human tissues, but a nearly restricted localization in basement membrane zones. The ␣1(XV) chain contains a highly interrupted collagenous region of 577 residues, and noncollagenous amino-and carboxyl-terminal domains of 530 and 256 residues, respectively. Cysteines are present in each domain and consensus sequences for O-linked glycosaminoglycans are situated in the amino terminus and in two large, noncollagenous interruptions. We now report that type XV collagen is a chondroitin sulfate proteoglycan in human tissues and cultured cells, and that the ␣ chains are covalently linked by interchain disulfide bonds only between the two cysteines in the collagenous region. Western blotting of tissue extracts revealed a diffuse smear with a mean size >400 kDa, which after chondroitinase digestion resolved into a 250-kDa band in umbilical cord, and 250-and 225-kDa bands in placenta, lung, colon, and skeletal muscle. The latter two bands were also directly visualized by alcian blue/silver staining of a purified placenta extract. In a human rhabdomyosarcoma cell line, almost all of the newly synthesized type XV collagen was secreted into the medium and upon chondroitinase digestion just the 250-kDa ␣ chain was generated. Chondroitinase plus collagenase digestion of tissue and medium proteins followed by Western blotting using domain-specific antibodies revealed a 135-kDa amino-terminal fragment containing glycosaminoglycan chains and a 27-kDa fragment representing the intact carboxyl terminus. However, a truncated carboxyl peptide of ϳ8-kDa was also evident in tissue extracts containing the 225-kDa form. Our data suggest that the 225-kDa form arises from differential carboxyl cleavage of the 250-kDa form, and could explain the ϳ19-kDa endostatin-related fragments (John, H., Preissner, K. T., Forssmann, W.-G., and Stä ndker, L. (1999) Biochemistry 38, 10217-10224), which may be liberated from the ␣1(XV) chain. Basement membrane zones (BMZs)1 can be operationally defined as morphological entities consisting of a basement membrane plus its closely associated matrix components, which extend into or originate from the sub-basal lamina (1). The BMZ contains the molecules responsible for attaching basement membranes to their contiguous stroma and/or epithelium. Those components integral to basement membranes include type IV collagen, laminin, entactin/nidogen, and perlecan (for review, see Ref.2), whereas a plethora of other matrix proteins, glycoproteins, and proteoglycans have been assigned to the BMZ using different methods. Many of these constituents have been studied in depth biochemically and ultrastructurally, while several newer and less abundant ones are not yet well characterized. Within this latter category are three more recently discovered nonfibrillar collagens: types XV, XVIII, and XIX (3-6). Independently identified from DNA clone isolation, they are considered members of a unique collagen subclass because of their widespread distribution in BMZs of many tissues (...

“…Highly purified human recombinant decorin and biglycan synthesized by human fibrosarcoma HT1080 cells using the vaccinia virus/T7 phage expression system were purified as described previously (16,17). Fura-2 and Pluronic F-127 acid were from Teflabs, ionomycin was from Calbiochem, and human recombinant EGF was from Life Technologies, Inc. All other reagents were from Sigma or Fisher.…”

Section: Methodsmentioning

confidence: 99%

Decorin Activates the Epidermal Growth Factor Receptor and Elevates Cytosolic Ca2+ in A431 Carcinoma Cells

Patel¹,

Santra²,

McQuillan³

et al. 1998

Self Cite

121

Several independent lines of evidence have implicated decorin, a small leucine-rich proteoglycan, in the inhibition of cell proliferation. However, the mechanism by which decorin mediates its effect on cell proliferation is unclear. Here we report, for the first time, decorinmediated increases in intracellular Ca 2؉ levels of single A431 cells. The effects of decorin persisted in the absence of extracellular Ca 2؉ but were blocked by AG1478, an epidermal growth factor (EGF)-specific tyrosine kinase inhibitor, and by down-regulation of the EGF receptor. The effects of decorin were not mimicked by the structurally homologous protein, biglycan. Our results indicate a novel action of decorin on the EGF receptor, which results in mobilization of intracellular Ca 2؉ providing a possible mechanism by which decorin causes growth suppression.