Expression and Characterization of Bone Morphogenetic Protein-2 in Chinese Hamster Ovary Cells

Israel, David I.; Nove, John; Kerns, Kelvin M.; Moutsatsos, Ioannis K.; Kaufman, Randal J.

doi:10.3109/08977199209046403

Cited by 181 publications

(134 citation statements)

References 33 publications

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“…Additionally, there were also high molecular mass complexes detectable in cross-linked samples, which migrated at 65ϳ180 kDa. These molecules most likely represent complexes of the mature domains bound to the prodomains, a phenomenon that is common in the TGF-␤ superfamily (13)(14)(15).…”

Section: Resultsmentioning

confidence: 99%

Effect of Intracellular Interactions on the Processing and Secretion of Bone Morphogenetic Protein-15 (BMP-15) and Growth and Differentiation Factor-9

Moore²,

Otsuka³

et al. 2003

Journal of Biological Chemistry

144

138

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Bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) are members of the transforming growth factor-␤ superfamily. Both molecules are closely related in their primary structures and share a nearly identical spatiotemporal expression pattern in the oocyte during folliculogenesis in mammals. Here we have established a series of cell lines, which express recombinant BMP-15, GDF-9, or both, and investigated whether they form homodimers and/or heterodimers. We demonstrate the first evidence that both BMP-15 and GDF-9 can form non-covalent homodimers when expressed individually, while when both are coexpressed BMP-15/GDF-9 heterodimers are produced. Interestingly, when GDF-9 and BMP-15 are co-expressed the processing of both proproteins are significantly impaired as compared with that of the singly expressed proproteins, suggesting that the proprotein heterodimer is less susceptible to proteolytic cleavage than the individual homodimers. Since BMP-15 mutant sheep, called Inverdale, exhibit severe defects in ovarian function we have also established stable transformants expressing the mutant BMP-15 (InvBMP-15) alone or together with GDF-9. Although InvBMP-15 was previously predicted to be unable to form homodimers, we show here that it does form non-covalent dimers; however, the processing efficiency of InvBMP-15 proprotein is significantly lower than wild-type BMP-15. Surprisingly, when GDF-9 is co-expressed, the processing and secretion of InvBMP-15 is abolished, and the processing of GDF-9 is also severely impaired, suggesting that the heterodimers of InvBMP-15/GDF-9 proproteins are not susceptible to proteolytic cleavage and thus degrade in the cells. Based on these findings we propose a novel hypothesis that a decrease in GDF-9 secretion may be involved in causing infertility in homozygous Inverdale ewes.

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Section: Resultsmentioning

confidence: 99%

Effect of Intracellular Interactions on the Processing and Secretion of Bone Morphogenetic Protein-15 (BMP-15) and Growth and Differentiation Factor-9

Moore²,

Otsuka³

et al. 2003

Journal of Biological Chemistry

144

138

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“…It is known that BMP-2 binds strongly to extracellular matrix and cell surfaces (see, for example, Israel et al, 1992). BMP-2 is eluted only at high salt (0.7 M NaCI) and in the presence of 6 M urea from a heparin-Sepharose column .…”

Section: Discussionmentioning

confidence: 99%

“…Similarly, a BMP-2 has been recovered from Xenopus Zaevis embryos in partially purified form (Shoda et al, 1994). Recently, by means of recombinant BMP-2 from CHO cells Israel et al, 1992), biological activity and physical binding constants could be determined in a variety of systems. A puzzling range of specific activities have been reported for the purified BMP-2.…”

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confidence: 99%

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Human Bone Morphogenetic Protein 2 Contains a Heparin‐Binding Site which Modifies Its Biological Activity

Ruppert

Hoffmann

Sebald

1996

European Journal of Biochemistry

521

444

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Bone morphogenetic protein 2 (BMP-2) plays a decisive role during bone regeneration and repair as well as during various stages of embryonal development. A cDNA encoding mature human BMP-2 could be efficiently expressed in Esclzerichia coli, and after renaturation a dimeric BMP-2 protein of M , 26000 was prepared with a purity greater 98 %. The recombinant BMP-2 was functionally active as demonstrated by the induction of alkaline phosphatase activity in the C3HlOT1/2 fibroblast cell line (EC,, of 70 nM) and proteoglycan synthesis in embryonic chicken limb bud cells (EC,,(15)(16)(17)(18)(19)(20). A peptide 1-17 representing the N-terminal basic part of BMP-2 as well as heparin increased the specific activity of the protein about fivefold in the limb bud assay. These observations suggested that the N-termini reduce the specific activity of BMP-2, probably by interacting with heparinic sites in the extracellular matrix. This conclusion was supported by a variant EHBMP-2, where the N-terminal residues 1-12 of BMP-2 had been substituted by a dummy sequence of equal length and which showed an EC,, value of around 1 nM which was affected neither by heparin nor by peptide 1 -17. A physical interaction between BMP-2 and heparin could be seen in biosensor experiments, where BMP-2 bound to immobilized heparin with a dissociation constant, Kd, of approximately 20 nM, whereas the heparin-binding of variant EHBMP-2 was negligible. These results identify the basic N-terminal domains of dimeric BMP-2 as heparin-binding sites that are not obligatory for receptor activation but modulate its biological activity.Keywords: human bone morphogenetic protein ; bacterial expression ; heparin binding; limb bud; proteoglycan synthesis ; variant bone morphogenetic protein 2.Bone morphogenetic protein 2 (BMP-2) is one of the proteins originally identified as factors extractable from bone and triggering ectopic bone formation at non-skeletal sites in vivo (Urist, 1965;Sampath and Reddi, 1981;Urist et al., 1982; for review see Wozney, 1992;Rosen and Thies, 1992;Reddi, 1994). After cloning and expression in Chinese hamster ovary (CHO) cells Wang et al., 1990) purified BMP-2 has been established to induce de novo bone formation and bone repair in adult animals (see, for example, Toriumi et al., 1991), and chondrogenic and osteogenic differentiation in various cellular systems in vitro. In addition, BMP-2 regulates, similarly to its nearest homologue BMP-4, diverse fundamental processes during embryonic development (see, for example, Kingsley, 1994). The mature BMP-2 protein is closely related to the Drosophila dpp protein (Padgett et al., 1993) and the Xenopus BMP-2 (Plessow et al., 1991). The human BMP-2 can substitute the Drosophila homologue dpp during dorsalventral patterning, and, vice versa, a recombinant dpp protein induces ectopic bone formation in rats (Padgett et al., 1993;Sampath et al., 1993). BMP-2 and the other BMP have great potential for medical therapeutic applications, in particular beCorrespondence to W. Sebald, Physiologisc...

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“…The PPARg2 gene Pro12Ala polymorphism, consisting of a C to G base change, leads to the substitution of proline with alanine in codon 12 of exon 2. Proline is able to inhibit the alpha helix formation that alanine encourages, thus amino acid changes lead to altered PPARg2 isoform function (Israel et al, 1992). This Pro12Ala functional variation has been correlated with coronary heart disease (CHD), type 2 diabetes, obesity, and lipid disorders, all of which are important risk factors for cerebrovascular disease.…”

Section: Introductionmentioning

confidence: 99%

Correlation between PPARg2 gene Pro12Ala polymorphism and cerebral infarction in an Inner Mongolian Han Chinese population

Zhang

et al. 2016

Genet. Mol. Res.

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ABSTRACT. The variant of PPAR-g2 has been shown to promote the increase of carotid IMT in patients suffering from cerebral infarction and the Pro12Ala polymorphism in the peroxisome proliferatoractivated receptorg2 (PPARg2) gene may be associated with cerebral infarction. However, due to the different genetic background, race, and regional variations of cerebral infarction patient, the results of investigations into this subject differ. The aim of this study was to investigate this polymorphism in relation to cerebral infarction among the Inner Mongolian Han Chinese population. A total of 574 Han Chinese individuals from Inner Mongolian were selected randomly, including 302 patients with cerebral infarction and 272 healthy controls. Polymerase chain reaction-restriction fragment length polymorphism was used to determine genotypes of the PPARg2 Pro12Ala variant and results were confirmed by direct sequencing. Genotype frequencies were found to be 90.7 and 91.9% for P/P, 8.6 and 7.7% for P/A, and 0.7 and 0.4 for A/A in the cerebral infarction and control groups, respectively. No statistically significant differences in genotype distribution were observed between the two groups (P > 0.05). Moreover, PPARg2 Pro12Ala genotype was not significantly associated with altered fasting blood glucose, blood pressure, or serum lipid profiles. After adjustment for gender, body mass index, and smoking habit, logistic regression was used to analyze the relationship between the Pro12Ala polymorphism and cerebral infarction (odds ratio = 0.888, 95% confidence interval = 0.106-7.460, P > 0.05), revealing that this variant was not the main pathogenic factor involved. Therefore, the Pro12Ala mutation of PPARg2 may not be associated with cerebral infarction in the Inner Mongolian Han Chinese population.

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Expression and Characterization of Bone Morphogenetic Protein-2 in Chinese Hamster Ovary Cells

Cited by 181 publications

References 33 publications

Effect of Intracellular Interactions on the Processing and Secretion of Bone Morphogenetic Protein-15 (BMP-15) and Growth and Differentiation Factor-9

Effect of Intracellular Interactions on the Processing and Secretion of Bone Morphogenetic Protein-15 (BMP-15) and Growth and Differentiation Factor-9

Human Bone Morphogenetic Protein 2 Contains a Heparin‐Binding Site which Modifies Its Biological Activity

Correlation between PPARg2 gene Pro12Ala polymorphism and cerebral infarction in an Inner Mongolian Han Chinese population

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