Dataset Information

Recombinant production, purification, crystallization, and structure analysis of human transforming growth factor ?2 in a new conformation.

ABSTRACT: Transforming growth factor ? is a disulfide-linked dimeric cytokine that occurs in three highly related isoforms (TGF?1-TGF?3) engaged in signaling functions through binding of cognate TGF? receptors. To regulate this pathway, the cytokines are biosynthesized as inactive pro-TGF?s with an N-terminal latency-associated protein preceding the mature moieties. Due to their pleiotropic implications in physiology and pathology, TGF?s 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-TGF?2 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 TGF?2, which adopted a conformation deviating from previous structures, with a distinct dimeric conformation that would require significant rearrangement for binding of TGF? receptors. This new conformation may be reversibly adopted by a certain fraction of the mature TG?2 population and represent a hitherto undescribed additional level of activity regulation of the mature growth factor once the latency-associated protein has been separated.

SUBMITTER: Del Amo-Maestro L

PROVIDER: S-EPMC6572864 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Recombinant production, purification, crystallization, and structure analysis of human transforming growth factor β2 in a new conformation.

Del Amo-Maestro Laura L Marino-Puertas Laura L Goulas Theodoros T Gomis-Rüth F Xavier FX

Scientific reports 20190617 1

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 ha ...[more]

PMID: 31209258

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Project description:Transforming growth factor-beta (TGF-β), a member of the TGF-β cytokine superfamily, is known to bind to sulfated glycosaminoglycans (GAGs), but the nature of this interaction remains unclear. In a recent study, we found that preterm human milk TGF-β2 is sequestered by chondroitin sulfate (CS) in its proteoglycan form. To understand the molecular basis of the TGF-β2-CS interaction, we utilized the computational combinatorial virtual library screening (CVLS) approach in tandem with molecular dynamics (MD) simulations. All possible CS oligosaccharides were generated in a combinatorial manner to give 24 di- (CS02), 192 tetra- (CS04), and 1536 hexa- (CS06) saccharides. This library of 1752 CS oligosaccharides was first screened against TGF-β2 using the dual filter CVLS algorithm in which the GOLDScore and root-mean-square-difference (RMSD) between the best bound poses were used as surrogate markers for in silico affinity and in silico specificity. CVLS predicted that both the chain length and level of sulfation are critical for the high affinity and high specificity recognition of TGF-β2. Interestingly, CVLS led to identification of two distinct sites of GAG binding on TGF-β2. CVLS also deduced the preferred composition of the high specificity hexasaccharides, which were further assessed in all-atom explicit solvent MD simulations. The MD results confirmed that both sites of binding form stable GAG-protein complexes. More specifically, the highly selective CS chains were found to engage the TGF-β2 monomer with high affinity. Overall, this work present key principles of recognition with regard to the TGF-β2-CS system. In the process, it led to the generation of the in silico library of all possible CS oligosaccharides, which can be used for advanced studies on other protein-CS systems. Finally, the study led to the identification of unique CS sequences that are predicted to selectively recognize TGF-β2 and may out-compete common natural CS biopolymers.

Project description:BackgroundNerve growth factor (NGF) contributes to pain in knee osteoarthritis (KOA) patients. Transforming growth factor-beta (TGF-β) stimulates NGF expression in chondrocytes from KOA patients. However, the correlation between synovial TGF-β and NGF levels has not been sufficiently studied in human KOA patients. Further, the mechanism governing NGF regulation by TGF-β in synovial cells is unclear.MethodsDuring total knee arthroplasty, we extracted the synovial tissue (SYT) of 107 subjects with unilateral Kellgren/Lawrence grade 3-4 KOA confirmed by radiography. We examined the distribution of TGF-β and NGF using immunohistochemistry, and analyzed the relationship between NGF and TGFB mRNA levels. Cultured synovial cells extracted from SYT were exposed to culture medium (control), human recombinant TGF-β (rhTGF-β), rhTGF-β + ALK5 inhibitor SB505124, rhTGF-β + transforming growth factor activating kinase 1 (TAK1) inhibitor (5Z)-7-oxozeaenol, or rhTGF-β + p38 inhibitor SB203580 for 30 min, 6 h and 24 h. NGF mRNA expressed by the cultured cells and NGF protein levels in the cell supernatant were detected by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Phosphorylation of p38 was evaluated by western blotting.ResultsNGF mRNA levels were positively correlated with those of TGFB. Cells expressing TGF-β and NGF protein were observed in the lining layer of SYT. TGF-β stimulated increased NGF mRNA expression and NGF protein production. The ALK5 inhibitor completely suppressed the TGF-β-mediated increase in NGF expression and NGF production in synovial cells. ALK5, TAK1 and p38 inhibitors inhibited the TGF-β-induced phosphorylation of p38, and TAK1 and p38 inhibitors partially inhibited the TGF-β-mediated increase in NGF expression and NGF production in synovial cells.ConclusionTGF-β regulates NGF production via the TGF-β/ALK5 signaling pathway in osteoarthritic synovium. This effect may partially occur through inhibition of the TAK1/p38 pathway in the SYT of KOA patients.

Dataset Information

Recombinant production, purification, crystallization, and structure analysis of human transforming growth factor ?2 in a new conformation.

Publications

Recombinant production, purification, crystallization, and structure analysis of human transforming growth factor β2 in a new conformation.

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