Effect of Intracellular Interactions on the Processing and Secretion of Bone Morphogenetic Protein-15 (BMP-15) and Growth and Differentiation Factor-9: IMPLICATION OF THE ABERRANT OVARIAN PHENOTYPE OF BMP-15 MUTANT SHEEP

Wu Xiang Liao,R Kelly Moore,Fumio Otsuka,Shunichi Shimasaki

doi:10.1074/jbc.m210598200

Abstract

Bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) are members of the transforming growth factor-beta 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 co-expressed 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.

Highlights

Bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) are members of the transforming growth factor-␤ superfamily
Both cell lines that were transfected with phBMP-15F or with phBMP-15F/hGDF-9M secreted BMP-15 which was detected with the anti-FLAG Ab as three bands: the proprotein detected at ϳ50 kDa and two bands of the mature protein detected at 16 and 17 kDa as previously reported (12)
Cross-linking of BMP-15 revealed a homodimer of mature BMP-15 that migrated at ϳ34 kDa and cross-linking of GDF-9 revealed a homodimer of mature GDF-9, which migrated at ϳ40 kDa (Fig. 2, lanes 2 and 6)

Summary

Introduction

Bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) are members of the transforming growth factor-␤ superfamily. When GDF-9 is co-expressed, the processing and secretion of InvBMP-15 is abolished, and the processing of GDF-9 is severely impaired, suggesting that the heterodimers of InvBMP-15/GDF-9 proproteins are not susceptible to proteolytic cleavage and 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. Residue 23 of the mature domain of BMP-15, resulting in the synthesis, if any, of a very short peptide, which is highly unlikely to be biologically active Both mutant ewes exhibit increased ovulation and lambing rates in the heterozygotes, whereas the homozygous mutant ewes are infertile with a phenotype that closely resembles that of GDF-9 knockout mice (10)

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Results

Conclusion