Abstract
Transforming growth factor β is a disulfide-linked dimeric cytokine that occurs in three highly related isoforms (TGFβ1–TGFβ3) engaged in signaling functions through binding of cognate TGFβ receptors. To regulate this pathway, the cytokines are biosynthesized as inactive pro-TGFβs with an N-terminal latency-associated protein preceding the mature moieties. Due to their pleiotropic implications in physiology and pathology, TGFβs are privileged objects of in vitro studies. However, such studies have long been limited by the lack of efficient human recombinant expression systems of native, glycosylated, and homogenous proteins. Here, we developed pro-TGFβ2 production systems based on human Expi293F cells, which yielded >2 mg of pure histidine- or Strep-tagged protein per liter of cell culture. We assayed this material biophysically and in crystallization assays and obtained a different crystal form of mature TGFβ2, which adopted a conformation deviating from previous structures, with a distinct dimeric conformation that would require significant rearrangement for binding of TGFβ receptors. This new conformation may be reversibly adopted by a certain fraction of the mature TGβ2 population and represent a hitherto undescribed additional level of activity regulation of the mature growth factor once the latency-associated protein has been separated.
Highlights
Transforming growth factors β (TGFβs) are members of a superfamily of conserved cytokines, which includes bone morphogenetic proteins, activins, and inhibins[1,2,3]
Mature human and mouse TGFβ2 growth-factor moiety (GF) were obtained in high yields (~8–10 mg/liter of cell culture) from Escherichia coli systems[34]
We developed a recombinant human overexpression system based on Expi293F cells grown in suspension, which produces high yields of well-folded human N-terminally histidine- or Strep-tagged pro-TGFβ2 with native post-translational modifications
Summary
Transforming growth factors β (TGFβs) are members of a superfamily of conserved cytokines, which includes bone morphogenetic proteins, activins, and inhibins[1,2,3]. Human TGFβs are produced as latent glycosylated precursors (pro-TGFβs), which consist of an ~250-residue N-terminal pro-domain dubbed latency-associated protein (LAP) and a C-terminal ~110-residue mature growth-factor moiety (GF). Two such precursors are joined through disulfides in the homodimeric small latent www.nature.com/scientificreports/. The large latent complexes are transformed into biologically active GFs by thrombospondin 1, reactive oxygen species, integrins, and/or peptidases such as furin and other related pro-protein convertases Proteolytic cleavage by these enzymes severs the linker between GF and LAP, but the two proteins remain noncovalently associated until physically separated for function[2,10,13,24,25]. We report the X-ray crystal structure of its GF, which was obtained in a different crystallographic space group and deviates from current structures
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