Structural and functional study of FK domain of Fstl1.

Fstl1 is a TGF-ß superfamily binding protein which involved in many pathological processes. The function of Fstl1 has been widely elucidated, but its structural characterization has not been explored. Here we solved the high-resolution crystal structure of FK domain of murine Fstl1, analyzed its unique characteristics, and investigated its contribution to the function of full-length Fstl1. We found that Fstl1-FK forms a stable dimer in both solution and crystal, which suggest that this protein may function as a dimer during its interaction with TGF-ß, a molecule known to form dimer during activation process. We also found this FK domain is indispensable for the proper function of Fstl1 during the transduction of TGF-ß signaling. These observations provide important insights into the understanding of Fstl1 and may facilitate the exploration of this molecule in clinical study.

Crystal structure of the WFIKKN2 follistatin domain reveals insight into how it inhibits growth differentiation factor 8 (GDF8) and GDF11.

Growth differentiation factor 8 (GDF8; also known as myostatin) and GDF11 are closely related members of the transforming growth factor ß (TGF-ß) family. GDF8 strongly and negatively regulates skeletal muscle growth, and GDF11 has been implicated in various age-related pathologies such as cardiac hypertrophy. GDF8 and GDF11 signaling activities are controlled by the extracellular protein antagonists follistatin; follistatin-like 3 (FSTL3); and WAP, follistatin/kazal, immunoglobulin, Kunitz, and netrin domain-containing (WFIKKN). All of these proteins contain a follistatin domain (FSD) important for ligand binding and antagonism. Here, we investigated the structure and function of the FSD from murine WFIKKN2 and compared it with the FSDs of follistatin and FSTL3. Using native gel shift and surface plasmon resonance analyses, we determined that the WFIKKN2 FSD can interact with both GDF8 and GDF11 and block their interactions with the type II receptor activin A receptor type 2B (ActRIIB). Further, we solved the crystal structure of the WFIKKN2 FSD to 1.39 Å resolution and identified surface-exposed residues that, when substituted with alanine, reduce antagonism of GDF8 in full-length WFIKKN2. Comparison of the WFIKKN2 FSD with those of follistatin and FSTL3 revealed differences in both the FSD structure and position of residues within the domain that are important for ligand antagonism. Taken together, our results indicate that both WFIKKN and follistatin utilize their FSDs to block the type II receptor but do so via different binding interactions.

Gene structure, recombinant expression and function characterization of Siniperca chuatsi Fsrp-3.

A full-length complementary (c)DNA sequence encoding follistatin-related protein 3 (fsrp-3) was determined from skeletal muscle in Chinese mandarin fish Siniperca chuatsi, its molecular structure was characterised and its function suggested. The putative structure of S. chuatsi Fsrp-3 contains an N-terminal domain and two follistatin domains. Quantitative reverse-transcription (qRT)-PCR assays revealed that fsrp-3 messenger (m)RNA was differentially expressed among assayed tissues and was highly expressed in heart and intestine. fsrp-3 mRNA exhibited increasing expression from the larval to the juvenile stage (500 g). To investigate the potential function of S. chuatsi fsrp-3 in muscle growth, we constructed a Fsrp-3 prokaryotic expression system and injected the purified Fsrp-3 fusion protein into the dorsal muscle. Fsrp-3 administration significantly influenced cross-section area, satellite cell activation frequency and nuclear density of S. chuatsi muscle fibres. Following Fsrp-3 treatment, the expression of myogenic regulatory factors was up-regulated and decline in the expression of myostatin was observed. The study revealed that Fsrp-3 may affect muscle growth by regulating myogenic regulatory factor expression and antagonizing myostatin function to initiate satellite cell activation and differentiation in S. chuatsi.

Deficient Follistatin-like 3 Secretion by Asthmatic Airway Epithelium Impairs Fibroblast Regulation and Fibroblast-to-Myofibroblast Transition.

Bronchial epithelial cells (BECs) from healthy children inhibit human lung fibroblast (HLF) expression of collagen and fibroblast-to-myofibroblast transition (FMT), whereas asthmatic BECs do so less effectively, suggesting that diminished epithelial-derived regulatory factors contribute to airway remodeling. Preliminary data demonstrated that secretion of the activin A inhibitor follistatin-like 3 (FSTL3) by healthy BECs was greater than that by asthmatic BECs. We sought to determine the relative secretion of FSTL3 and activin A by asthmatic and healthy BECs, and whether FSTL3 inhibits FMT. To quantify the abundance of the total proteome FSTL3 and activin A in supernatants of differentiated BEC cultures from healthy children and children with asthma, we performed mass spectrometry and ELISA. HLFs were cocultured with primary BECs and then HLF expression of collagen I and α-smooth muscle actin (α-SMA) was quantified by qPCR, and FMT was quantified by flow cytometry. Loss-of-function studies were conducted using lentivirus-delivered shRNA. Using mass spectrometry and ELISA results from larger cohorts, we found that FSTL3 concentrations were greater in media conditioned by healthy BECs compared with asthmatic BECs (4,012 vs. 2,553 pg/ml; P = 0.002), and in media conditioned by asthmatic BECs from children with normal lung function relative to those with airflow obstruction (FEV1/FVC ratio < 0.8; n = 9; 3,026 vs. 1,922 pg/ml; P = 0.04). shRNA depletion of FSTL3 in BECs (n = 8) increased HLF collagen I expression by 92% (P = 0.001) and α-SMA expression by 88% (P = 0.02), and increased FMT by flow cytometry in cocultured HLFs, whereas shRNA depletion of activin A (n = 6) resulted in decreased α-SMA (22%; P = 0.01) expression and decreased FMT. Together, these results indicate that deficient FSTL3 expression by asthmatic BECs impairs epithelial regulation of HLFs and FMT.

Effects of FSTL1 on the proliferation and motility of breast cancer cells and vascular endothelial cells.

BACKGROUND: Treatments that prevent the motility of breast cancer cells and inhibit formation of new capillary vessels are urgently needed. FSTL1 is a secreted protein that has been implicated in maintaining the normal physiological function of the cardiovascular system, in addition to a variety of other biological functions. We investigated the role of FSTL1 in the proliferation and migration of breast cancer and vascular endothelial cells. METHODS: Human umbilical vein endothelial cells and human breast cancer BT-549 cells were used to test the effects of FSTL1 and the N-terminal domain of FSTL1. Immunofluorescence microscopy and 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide, transwell invasion, and wound healing assays were conducted. RESULTS: Different doses of the N-terminal fragment of FSTL1 (FSTL-N) have variable effects on the migration of these cells. However, FSTL1 does not significantly affect tube formation in vitro from vascular endothelial cells. FSTL1-FL and FSTL1-N have modest effects on the invasion of breast cancer and vascular endothelial cells. Interestingly, FSTL1-FL, but not FSTL-N, modulates vascular endothelial cell polarization. CONCLUSION: FSTL1 modestly affects the proliferation of breast cancer cells and vascular endothelial cells. Our findings improve our understanding of the functions of FSTL1 in breast cancer development and angiogenesis.

Expression, characterization, and preliminary X-ray crystallographic analysis of recombinant murine Follistatin-like 1 expressed in Drosophila S2 cells.

The matricellular protein Follistatin-like 1 (FSTL1) has been shown to negatively regulate bone morphogenetic protein (BMP)/Smad1/5/8 signaling by functioning as an antagonist and has been implicated in physiological and pathological events including organogenesis, immunity and cardiovascular disease. It is therefore an attractive target for potential therapeutic intervention studies. In this study, we established a high-level expression system in Drosophila S2 cells which could produce about 12.5 mg of recombinant murine Follistatin-like 1 protein (rFSTL1) per liter of culture medium. The recombinant protein was then purified to greater than 95% purity using Ni-NTA agarose affinity chromatography followed by HiLoad 16/60 Superdex 200 gel filtration. The biological activity of rFSTL1 was evaluated by its ability to negatively regulate BMP/Smad1/5/8 signaling in cultured mink lung epithelial cells. Furthermore, we crystallized a truncated form of rFSTL1 containing the follistatin-like domain using the sitting drop vapor diffusion method. In conclusion, we have generated and purified biologically active recombinant FSTL1 protein, which will be important for further protein structure and drug discovery studies.

Characterization of follistatin-type domains and their contribution to myostatin and activin A antagonism.

Follistatin (FST)-type proteins are important antagonists of some members of the large TGF-ß family of cytokines. These include myostatin, an important negative regulator of muscle growth, and the closely related activin A, which is involved in many physiological functions, including maintenance of a normal reproductive axis. FST-type proteins, including FST and FST-like 3 (FSTL3), differentially inhibit various TGF-ß family ligands by binding each ligand with two FST-type molecules. In this study, we sought to examine features that are important for ligand antagonism by FST-type proteins. Previous work has shown that a modified construct consisting of the FST N-terminal domain (ND) followed by two repeating follistatin domains (FSD), herein called FST ND-FSD1-FSD1, exhibits strong specificity for myostatin over activin A. Using cell-based assays, we show that FST ND-FSD1-FSD1 is unique in its specificity for myostatin as compared with similar constructs containing domains from FSTL3 and that the ND is critical to its activity. Furthermore, we demonstrate that FSD3 of FST provides affinity to ligand inhibition and confers resistance to perturbations in the ND and FSD2, likely through the interaction of FSD3 of one FST molecule with the ND of the other FST molecule. Additionally, our data suggest that this contact provides cooperativity to ligand antagonism. Cross-linking studies show that this interaction also potentiates formation of 1:2 ligand-FST complexes, whereas lack of FSD3 allows formation of 1:1 complexes. Altogether, these studies support that domain differences generate FST-type molecules that are each uniquely suited ligand antagonists.

Structure of myostatin·follistatin-like 3: N-terminal domains of follistatin-type molecules exhibit alternate modes of binding.

TGF-ß family ligands are involved in a variety of critical physiological processes. For instance, the TGF-ß ligand myostatin is a staunch negative regulator of muscle growth and a therapeutic target for muscle-wasting disorders. Therefore, it is important to understand the molecular mechanisms of TGF-ß family regulation. One form of regulation is through inhibition by extracellular antagonists such as the follistatin (Fst)-type proteins. Myostatin is tightly controlled by Fst-like 3 (Fstl3), which is the only Fst-type molecule that has been identified in the serum bound to myostatin. Here, we present the crystal structure of myostatin in complex with Fstl3. The structure reveals that the N-terminal domain (ND) of Fstl3 interacts uniquely with myostatin as compared with activin A, because it utilizes different surfaces on the ligand. This results in conformational differences in the ND of Fstl3 that alter its position in the type I receptor-binding site of the ligand. We also show that single point mutations in the ND of Fstl3 are detrimental to ligand binding, whereas corresponding mutations in Fst have little effect. Overall, we have shown that the NDs of Fst-type molecules exhibit distinctive modes of ligand binding, which may affect overall affinity of ligand·Fst-type protein complexes.

The structure of FSTL3.activin A complex. Differential binding of N-terminal domains influences follistatin-type antagonist specificity.

Transforming growth factor beta family ligands are neutralized by a number of structurally divergent antagonists. Follistatin-type antagonists, which include splice variants of follistatin (FS288 and FS315) and follistatin-like 3 (FSTL3), have high affinity for activin A but differ in their affinity for other ligands, particularly bone morphogenetic proteins. To understand the structural basis for ligand specificity within FS-type antagonists, we determined the x-ray structure of activin A in complex with FSTL3 to a resolution of 2.5 A. Similar to the previously resolved FS.activin A structures, the ligand is encircled by two antagonist molecules blocking all ligand receptor-binding sites. Recently, the significance of the FS N-terminal domain interaction at the ligand type I receptor site has been questioned; however, our data show that for FSTL3, the N-terminal domain forms a more intimate contact with activin A, implying that this interaction is stronger than that for FS. Furthermore, binding studies revealed that replacing the FSTL3 N-terminal domain with the corresponding FS domain considerably lowers activin A affinity. Therefore, both structural and biochemical evidence support a significant interaction of the N-terminal domain of FSTL3 with activin A. In addition, structural comparisons with bone morphogenetic proteins suggest that the interface where the N-terminal domain binds may be the key site for determining FS-type antagonist specificity.

Identification of a follistatin-related protein from the tick Haemaphysalis longicornis and its effect on tick oviposition.

The identification of ovary-associated molecules will lead to a better understanding of the physiology of tick reproduction and vector-pathogen interactions. A gene encoding a follistatin-related protein (FRP) was obtained by random sequencing from the ovary cDNA library of the tick Haemaphysalis longicornis. The full-length cDNA is 1157 bp, including an intact ORF encoding an expected protein with 289 amino acids. Three distinct domains were present in the deduced amino acids, namely, the follistatin-like domain, KAZAL, and two calcium-binding motifs, EFh. The sequence shows homology with the follistatin-related protein (FRP), which was thought to play some roles in the negative regulation of cellular growth. RT-PCR showed that the gene was expressed throughout the developing stages and mainly in the ovary as well as in fat body, hemocytes, salivary glands, and midgut. This gene was expressed in GST-fused recombinant protein with an expected size. The mouse antiserum against the recombinant protein recognized a 56-kDa native protein in both tick ovary and hemolymph. The recombinant proteins were found to have binding activity for both activin A and bone morphogenetic protein-2 (BMP-2). Silencing of FRP by RNAi showed a decrease in tick oviposition, which is consistent with the effect of a recombinant protein vaccine on the adult tick. These results showed that the tick FRP might be involved in tick oviposition. This is the first report of a member of follistatin family proteins in Chelicerata, which include ticks, spiders, and scorpions.

Differential actions of follistatin and follistatin-like 3.

Follistatin (FS) is an important physiological regulator of activin and other TGFbeta superfamily members. The recently discovered follistatin-like 3 (FSTL3; a.k.a. FLRG; FSRP) shares significant structural and functional homology with FS, but also has some interesting differences, including a prominent nuclear localization. The existence of these two related proteins allows detailed molecular and biochemical comparisons of the biologic roles of their individual structural elements. Current studies indicate that the heparin binding sequence is essential for the ability of FS to inhibit autocrine activin but is not sufficient to confer this activity on FSTL3. Preliminary analysis of FSTL3 transgenic mice suggests that FSTL3 regulates gonadal development and function through inhibition of the paracrine activity of activin and/or other related factors. These studies have identified important structural elements necessary for biological activity of FS and FSTL3 and potential roles for FSTL3 in vivo.

Structural characterization of TSC-36/Flik: analysis of two charge isoforms.

Recombinant forms of the glycoprotein TSC-36/Flik were expressed in human cells and used to compare their structural and functional properties with those described for other members of the BM-40/SPARC/osteonectin protein family. TSC-36 was found to occur in two charge isoforms that differ in the extent of sialylation of otherwise identical N-linked, complex type oligosaccharides. Conformational analysis with both circular dichroism and intrinsic fluorescence spectroscopy showed a lack of significant structural changes upon calcium addition or depletion. This finding is in contrast to results obtained for several other BM-40 family members and indicates that the extracellular calcium-binding domain in TSC-36 is non-functional. The lack of conservation of important functional features common to several other members of the BM-40 family indicates that TSC-36, despite its sequence homology to BM-40, has evolved clearly distinct properties.

Characterization of rat follistatin-related gene: effects of estrous cycle stage and pregnancy on its messenger RNA expression in rat reproductive tissues.

Follistatin-related gene (FLRG) was first identified as a target of a chromosomal translocation in a human B-cell leukemia. Because FLRG protein binds to activins and bone morphogenetic proteins, FLRG is postulated to be a regulator of these growth factors. However, physiological aspects of FLRG are unclear. To elucidate the physiology of FLRG, we examined expression of FLRG in reproductive tissues of the rat. FLRG mRNA was abundantly expressed in the placenta. FLRG mRNA was also expressed in the ovary, uterus, testis, lung, adrenal gland, pituitary, kidney, small intestine, and heart. During the second half of pregnancy, expression of FLRG in the placenta continuously increased, whereas follistatin mRNA levels decreased from Day 12 to Day 14 and remained low thereafter. FLRG was also expressed in decidua. Levels of decidual FLRG mRNA remained low from Day 12 to Day 16 and then noticeably increased until Day 20. In contrast, follistatin mRNA was highly expressed in the decidua on Day 12, continuously decreased until Day 16, and then remained at relatively low levels thereafter. During the rat estrous cycle, levels of ovarian FLRG mRNA fluctuated diurnally, with highest levels during daytime, and did not change relative to the day of the estrous cycle. The present results suggest that FLRG may play a role in the regulation of reproductive events.