Rodney M. Hewick

The Myostatin Propeptide and the Follistatin-related Gene Are Inhibitory Binding Proteins of Myostatin in Normal Serum

Myostatin, also known as growth and differentiation factor 8, is a member of the transforming growth factor β superfamily that negatively regulates skeletal muscle mass (1). Recent experiments have shown that myostatin activity is detected in serum by a reporter gene assay only after activation by acid, suggesting that native myostatin circulates as a latent complex (2). We have used a monoclonal myostatin antibody, JA16, to isolate the native myostatin complex from normal mouse and human serum. Analysis by mass spectrometry and Western blot shows that circulating myostatin is bound to at least two major proteins, the myostatin propeptide and the follistatin-related gene (FLRG). The myostatin propeptide is known to bind and inhibit myostatin in vitro (3). Here we show that this interaction is relevant in vivo, with a majority (>70%) of myostatin in serum bound to its propeptide. Studies with recombinant V5-His-tagged FLRG protein confirm a direct interaction between mature myostatin and FLRG. Functional studies show that FLRG inhibits myostatin activity in a reporter gene assay. These experiments suggest that the myostatin propeptide and FLRG are major negative regulators of myostatin in vivo.

Molecular characterization and expression of the gene encoding human erythroid-potentiating activity.

Erythropoietin is the primary physiological regulator of erythropoiesis; however, in vitro studies have identified another class of mediators which appear to be important in stimulating erythroid progenitors. These factors have generally been referred to as burst-promoting activities (BPA), because they stimulate the growth of early erythroid progenitors referred to as burst-forming units-erythroid (BFU-E) which give rise to colonies of up to thousands of haemoglobinized cells1,2. We recently reported purification of a burst-promoting activity from medium conditioned by the Mo T-lymphoblast cell line infected with human T-cell lym-photropic virus type II (HTLV-II)3,4. This purified glycoprotein of relative molecular mass (Mr) 28,000 also stimulates colony formation by more mature erythroid precursors (CFU-E) and is therefore referred to as erythroid-potentiating activity (EPA)5. Purified EPA specifically stimulates human and murine cells of the erythroid lineage, unlike murine interleukin-3 (IL-3) which stimulates precursor cells from all haematopoietic lineages6. We report here the isolation of a complementary DNA molecular clone encoding EPA and its use in producing EPA in COS (monkey) cells and CHO (Chinese hamster ovary) cells. We also define the organization of the EPA gene in human DNA.

Proteomics in drug discovery.

The promise of genomics has dramatically altered the way drug discovery is now viewed. Overshadowed by the exuberance for genomics are the observations that most disease processes and treatments are manifest at the protein level and that there may not be a good correlation between gene expression and protein expression. An alternative and complementary approach to genomics is protein expression profiling - proteomics. The authors describe the technology, its advantages and some applications.