ABSTRACT
We have characterized the expression and processing of Osteogenic Protein-1 (hOP-1), a bone morphogenic protein of the TGF-beta family, in Chinese hamster ovary cells. The hOP-1 is initially synthesized as a monomeric 50 kDa pro-protein that is dimerized, glycosylated, and then proteolytically cleaved at the Arg-Xaa-Xaa-Arg maturation site in an acidic cellular compartment before secretion into the medium. Of the four potential N-linked glycosylation sites two are used, one in the mature domain and one in the pro-domain. Gel permeation chromatography of secreted hOP-1 in physiological buffers yields an apparent molecular weight of 110-120 k, indicating that after proteolytic processing the two pro-domains remain non-covalently associated with the disulfide linked mature dimer in a complex termed soluble hOP-1. Purified soluble hOP-1 is significantly more soluble in physiological buffers than the purified mature OP-1.
Subject(s)
Bone Morphogenetic Proteins , Protein Biosynthesis , Protein Processing, Post-Translational , Proteins/isolation & purification , Transforming Growth Factor beta/metabolism , Amino Acid Sequence , Animals , Bone Morphogenetic Protein 7 , CHO Cells , Chromatography, Gel , Cricetinae , Glycosylation , Molecular Sequence Data , Molecular Weight , Precipitin Tests , Protein Conformation , Protein Processing, Post-Translational/genetics , Proteins/chemistry , Transfection/geneticsABSTRACT
We reported previously that a 32-36-kDa osteogenic protein purified from bovine bone matrix is composed of dimers of two members of the transforming growth factor (TGF)-beta superfamily: the bovine equivalent of human osteogenic protein-1 (OP-1) and bone morphogenetic protein-2a, BMP-2a (BMP-2). In the present study, we produced the recombinant human OP-1 (hOP-1) in mammalian cells as a processed mature disulfide-linked homodimer with an apparent molecular weight of 36,000. Examination of hOP-1 in the rat subcutaneous bone induction model demonstrated that hOP-1 was capable of inducing new bone formation with a specific activity comparable with that exhibited by highly purified bovine osteogenic protein preparations. The half-maximal bone-inducing activity of hOP-1 in combination with a rat collagen matrix preparation was 50-100 ng/25 mg of matrix as determined by the calcium content of day 12 implants. Evaluation of hOP-1 effects on cell growth and collagen synthesis in rat osteoblast-enriched bone cell cultures showed that both cell proliferation and collagen synthesis were stimulated in a dose-dependent manner and increased 3-fold in response to 40 ng of hOP-1/ml. Examination of the expression of markers characteristic of the osteoblast phenotype showed that hOP-1 specifically stimulated the induction of alkaline phosphatase (4-fold increase at 40 ng of hOP-1/ml), parathyroid hormone-mediated intracellular cAMP production (4-fold increase at 40 ng of hOP-1/ml), and osteocalcin synthesis (5-fold increase at 25 ng of hOP-1/ml). In long-term (11-17 day) cultures of osteoblasts in the presence of beta-glycerophosphate and L(+)-ascorbate, hOP-1 markedly increased the rate of mineralization as measured by the number of mineral nodules per well (20-fold increase at 20 ng of hOP-1/ml). Direct comparison of TGF-beta 1 and hOP-1 in these bone cell cultures indicated that, although both hOP-1 and TGF-beta 1 promoted cell proliferation and collagen synthesis, only hOP-1 was effective in specifically stimulating markers of the osteoblast phenotype.
Subject(s)
Bone Morphogenetic Proteins , Osteoblasts/drug effects , Osteogenesis/drug effects , Proteins/pharmacology , Alkaline Phosphatase/biosynthesis , Amino Acid Sequence , Animals , Blotting, Western , Bone Morphogenetic Protein 7 , CHO Cells , Cattle , Cricetinae , Cyclic AMP/biosynthesis , Electrophoresis, Polyacrylamide Gel , Enzyme Induction , Humans , Molecular Sequence Data , Osteoblasts/cytology , Osteocalcin/biosynthesis , Parathyroid Hormone/physiology , Rats , Recombinant Proteins/pharmacology , Transforming Growth Factor beta/geneticsABSTRACT
Human tissue-type plasminogen activator (t-PA) cDNA was cloned from uterine tissue and engineered in expression vectors for production in mouse C127 cells. The vectors consisted of the bovine papilloma virus-1 (BPV-1) genome and t-PA transcriptional unit with a mouse metallothionein (MT-1) promoter at the 5' end and MT-1 genomic sequences or SV40 early introns and polyadenylation signals at the 3' end. Analysis of the expression vectors transfected into cells revealed that t-PA is expressed 100- to 200-fold more with an intronless vector utilizing the SV40 polyadenylation signal than with other, intron-containing vectors. RNA analysis of stable cell lines indicated that t-PA expression levels correlated with mRNA abundance. DNA copy number and transcriptional rate of the MT-1 promoter remained constant in cell lines transformed by different BPV expression vectors. Uterine t-PA produced by recombinant DNA means was enzymatically active and similar in properties to Bowes melanoma t-PA.