Cysteine Knot Motif Research Articles

Editing the cystine knot motif of MSTN enhances muscle development of Liang Guang Small Spotted pigs.

Myostatin (MSTN) is a member of the transforming growth factor-β (TGF-β) family, and functions as an inhibitor of muscle growth. Disrupting the inhibitory effect of MSTN on growth can provide an effective way to increase the muscle yield of livestock and poultry. The cysteine knot motif of TGF-β can stabilize the structure of MSTN protein and plays an important regulatory role in the biological function of MSTN. Accordingly, in this study, we used the CRISRP/Cas9 to edit the exon 3 of MSTN in the kidney cells of Liang Guang Small Spotted pig (LPKCs), in order to disrupt the cysteine knot motif of MSTN and remove the inhibitory effect of MSTN on its target genes.MSTN-edited LPKCs were obtained through fluorescence-activated cell sorting (FACS) and used as donor cells for somatic cell nuclear transfer (SCNT) to generate cloned embryos, which were then transferred to surrogate sows to finally obtain eight MSTN-edited Liang Guang Small Spotted piglets. Among them, two survived to 10 days old. Genotyping revealed that these two piglets were gene edited heterozygotes with base deletion and substitution occurred within the coding sequence of C106 and C108 at the cystine knot motif of MSTN. These changes resulted in frameshift mutations, and conversion of C106 and C108 to other amino acids. More developments of muscles were observed at the shoulders and hips of the heterozygotes of MSTN-edited Liang Guang Small Spotted pigs. H&E analysis showed that the cross-sectional area (CSA) of myofiber inMSTN-edited pigs was significantly decreased, and the number of myofiber were significantly increased. Western blot analysis showed that the disruption of C106 and C108 did not affect the expression of MSTN protein, but significantly up-regulated the expression of its target genes such as Myf5, MyoD, Myogenin and other myogenic regulatory factors. In summary, the gene-edited pig model obtained in this study did not cause complete loss of MSTN expression, and could retain other biological functions of MSTN, thereby promoting muscle growth while minimizing the potential adverse effects on complete loss of MSTN in the Liang Guang Small Spotted pigs.

Identification and function analysis of a novel vascular endothelial growth factor, LvVEGF3, in the Pacific whiteleg shrimp Litopenaeus vannamei

VEGF signaling pathway is first discovered in mammals and proved to play important roles in the biological processes of angiogenesis, tumor migration, cell differentiation, apoptosis, host-virus interaction etc. Three members in the VEGF signaling pathway, including LvVEGFR, LvVEGF1 and LvVEGF2 in shrimp have been proved to be related with WSSV infection in our previous studies. Currently, another member of VEGF family, LvVEGF3, was isolated and its function during the WSSV infection of shrimp was studied. The deduced amino acid sequence of LvVEGF3 contained a signal peptide, a typical PDGF/VEGF domain and a cysteine-knot motif (CXCXC). Tissue distribution analysis showed that LvVEGF3 was predominantly expressed in hemocytes. The transcriptional level of LvVEGF3 in hemocytes was apparently up-regulated during WSSV infection. Silencing of LvVEGF3 with double-stranded RNA caused a reduction of the cumulative mortality rate of shrimp during WSSV infection. The expression of LvVEGFR was apparently down-regulated after LvVEGF3 silencing and up-regulated after injection of recombinant LvVEGF3 protein, suggesting an interaction between LvVEGF3 and LvVEGFR. Furthermore, the interaction between LvVEGFR and LvVEGF3 was confirmed using the yeast two-hybrid system. The results provided new insights into understanding the role of VEGF signaling pathway during virus infection.

Sclerosteosis caused by a novel nonsense mutation of SOST in a consanguineous family.

Sclerosteosis, characterized by the hyperostosis of cranial and tubular bones, is a rare autosomal recessive hereditary disorder caused by mutation of SOST gene. Four nonsense mutations of SOST have been identified worldwide. Here, we report two affected siblings who carried a novel nonsense mutation of SOST in a consanguineous family from China. The proband manifested typical symptoms of sclerosteosis, whereas the symptoms were absent in another affected sibling. Two nucleotide substitutions in exon 2 of SOST were identified, c.444_445TC>AA, resulting in a premature stop codon, p.Cys148→Stop. This truncated mutation loses 66 amino acid residues which contain 3 cysteine residues of the cysteine-knot motif, leading to loss of function of SOST. The symptoms of sclerosteosis may be clinically heterogeneous in some patients, even with the same mutation. Our results support the notion that founder effects from the ancestors contribute to the disease onset.

Structure-Function Relationship of Calcins, a Family of High-Affinity Peptide Ligands of Ryanodine Receptors

Calcins is a novel family of scorpion peptides that bind with high affinity and specificity to ryanodine receptors (RyRs) and increase their open probability by inducing the appearance of a long-lasting subconductance state. Here we report two newly-identified calcins (Vejocalcin and Urocalcin) and provide a comprehensive analysis of the structure-function relationship of the eight calcins known to date, based on primary sequence examination, 3D structure modeling, and their effect on RyR1. [3H]Ryanodine binding assays, used as index of the open probability (Po) of RyRs, revealed that all eight calcins (Opicalcin1, Opicalcin2, Imperacalcin, Maurocalcin, Hadrucalcin, Hemicalcin, Vejocalcin, and Urocalcin) activate RyR1 dose-dependently, with EC50s (in nM) 0.35, 5, 8, 12, 37, 71, 91, and 523, respectively. At 1 µM, calcins significantly augmented the bell-shaped [Ca2+]-activity curve of RyR1 with potency Opicalcin1>Opicalcin2>Vejocalcin>Urocalcin. In single channel recordings, the heretofore uncharacterized calcins Opicalcin1, Opicalcin2 and Vejocalcin increased the Po of RyR1 significantly and the fractional conductance was ∼0.45, 0.30, and 0.65, respectively, of the full conductance state. Opicalcin1, Maurocalcin, Hadrucalcin and Vejocalcin induced Ca2+ release from rabbit skeletal SR with ED50s (in nM) 2.8±0.02, 8.3±0.25, 16±0.4, 36±0.5, respectively. A clear Ca2+ release-[3H]ryanodine binding activity correlation (r2=0.99) was obtained. Primary sequence alignment and evolutionary analysis (ClustalW and MEGA5.2) showed high homology among all calcins. An inhibitor cysteine-knot (ICK) motif with βαββ domains stabilized by three disulfide bridges characterizes this peptide family, although Vejocalcin lacks the β2 sheet. Positively-charged residue mutations in the high variable N-terminal region (G1-N14) and negatively-charged residue variations in the conserved C-terminal region (D15-R33) greatly decrease the effect of calcins on RyR1. In conclusion, natural variation in calcin peptides offers a diversified set of RyR ligands with capacity to modulate RyRs with high dynamic range and potency.

High Yield Production and Refolding of the Double-Knot Toxin, an Activator of TRPV1 Channels

A unique peptide toxin, named double-knot toxin (DkTx), was recently purified from the venom of the tarantula Ornithoctonus huwena and was found to stably activate TRPV1 channels by targeting the outer pore domain. DkTx has been shown to consist of two inhibitory cysteine-knot (ICK) motifs, referred to as K1 and K2, each containing six cysteine residues. Beyond this initial characterization, however, the structural and functional details about DkTx remains elusive in large part due to the lack of a high yielding methodology for the synthesis and folding of this cysteine-rich peptide. Here, we overcome this obstacle by generating pure DkTx in quantities sufficient for structural and functional analyses. Our methodology entails expression of DkTx in E. coli followed by oxidative folding of the isolated linear peptide. Upon screening of various oxidative conditions for optimizing the folding yield of the toxin, we observed that detergents were required for efficient folding of the linear peptide. Our synthetic DkTx co-eluted with the native toxin on HPLC, and irreversibly activated TRPV1 in a manner identical to native DkTx. Interestingly, we find that DkTx has two interconvertible conformations present in a 1∶6 ratio at equilibrium. Kinetic analysis of DkTx folding suggests that the K1 and K2 domains influence each other during the folding process. Moreover, the CD spectra of the toxins shows that the secondary structures of K1 and K2 remains intact even after separating the two knots. These findings provide a starting point for detailed studies on the structural and functional characterization of DkTx and utilization of this toxin as a tool to explore the elusive mechanisms underlying the polymodal gating of TRPV1.

Molecular structure of the prothoracicotropic hormone gene in the northern house mosquito, Culex pipiens, and its expression analysis in association with diapause and blood feeding

We cloned the gene that encodes prothoracicotropic hormone (PTTH) in the northern house mosquito, Culex pipiens, and investigated its expression profile in short-day (diapause-destined) and long-day (nondiapause-destined) individuals from the fourth-instar larval stage to 2 months of adulthood, as well as after a blood meal. The deduced C. pipiens PTTH (Cupip-PTTH) amino acid sequence contains seven cysteines with a specific spacing pattern. Sequence alignment suggests that Cupip-PTTH is 23% identical to Drosophila melanogaster PTTH, but is ≥59% identical to the PTTHs of other mosquitoes. Cupip-PTTH has structural characteristics similar to those of Bombyx mori PTTH and some vertebrate nerve growth factors with cysteine-knot motifs. PTTH transcripts exhibit a daily cycling profile during the final (fourth) larval instar, with peak abundance occurring late in the scotophase. The fourth-larval instar stage is one day longer in short-day larvae than in long-day larvae, resulting in larger larvae and adults. This additional day of larval development is associated with one extra PTTH cycle. No cycling was observed in pupae, but PTTH transcripts were slightly higher in short-day pupae than in long-day pupae throughout much of the pupal stage. PTTH expression persisted at a nearly constant level in diapausing adult females for the first month but then dropped by ∼50%, while expression decreased at the beginning of adulthood in nondiapausing females and then remained at a low level as long as the females were denied a blood meal. However, when nondiapausing females were offered a blood meal, PTTH transcripts rose approximately 7 fold in 2 h and remained elevated for 24 h. A few diapausing females (∼10%) will take a blood meal when placed in close proximity to a host, but much of the blood is ejected and such meals do not result in mature eggs. Yet, elevated PTTH mRNA expression was also observed in diapausing females that were force fed. Our results thus point to several distinctions in PTTH expression between short-day and long-day mosquitoes, but both types of females responded to a blood meal by elevating levels of PTTH mRNA.

The comparison of VEGFR-1-binding domain of VEGF-A with modelled VEGF-C sheds light on receptor specificity

Receptor specificity determines the role of vascular endothelial growth factors (VEGFs), which either induce angiogenesis via VEGFR-1 and VEGFR-2 receptors or lymphangiogenesis via the VEGFR-3 receptor. Among the VEGFs, VEGF-A and VEGF-B predominantly induce angiogenesis while VEGF-C and VEGF-D induce lymphangiogenesis. The answer for the question of why VEGF-C and VEGF-D are not able to bind VEGFR-1 and behave as angiogenic growth factors may hide behind the details of the tertiary structures of these proteins. In the present study, the tertiary structure of human VEGF-C protein was modelled and the model was compared with the known human VEGF-A tertiary structure. In overall, the modelled structure highly resembled the structure of VEGF-A. The respective key residues that are involved in cysteine-knot motif formation in VEGF-A are similarly located and identically oriented in VEGF-C, indicating the presence of a VEGF-A-like homodimer. However, a VEGF-C homodimer created via monomer docking did not superimpose well with the VEGF-A homodimer. Rigid docking models of VEGF-C with the VEGFR-1 receptor revealed that in the VEGF-C–VEGFR-1 complex, the receptor–protein-interacting residues were not correctly oriented to induce angiogenesis via VEGFR-1. Mapping the electrostatic surface potentials to the protein surfaces revealed noteworthy number of dissimilarities between VEGF-A and VEGF-C, indicating that overall both proteins differ in their folding properties and stability.

Identification of an Interleukin 17F/17A Heterodimer in Activated Human CD4+ T Cells

IL-17F and IL-17A are members of the IL-17 pro-inflammatory cytokine family. IL-17A has been implicated in the pathogenesis of autoimmune diseases. IL-17F is a disulfide-linked dimer that contains a cysteine-knot motif. We hypothesized that IL-17F and IL-17A could form a heterodimer due to their sequence homology and overlapping pattern of expression. We evaluated the structure of recombinant IL-17F and IL-17A proteins, as well as that of natural IL-17F and IL-17A derived from activated human CD4+ T cells, by enzyme-linked immunosorbent assay, immunoprecipitation followed by Western blotting, and mass spectrometry. We find that both IL-17F and IL-17A can form both homodimeric and heterodimeric proteins when expressed in a recombinant system, and that all forms of the recombinant proteins have in vitro functional activity. Furthermore, we find that in addition to the homodimers of IL-17F and IL-17A, activated human CD4+ T cells also produce the IL-17F/IL-17A heterodimer. These data suggest that the IL-17F/IL-17A heterodimer may contribute to the T cell-mediated immune responses.

Retinal Phenotype–Genotype Correlation of Pediatric Patients Expressing Mutations in the Norrie Disease Gene

To correlate the ophthalmic findings of patients with pediatric vitreoretinopathies with mutations occurring in the Norrie disease gene (NDP). One hundred nine subjects with diverse pediatric vitreoretinopathies and 54 control subjects were enrolled in the study. Diagnoses were based on retinal findings at each patient's first examination. Samples of DNA from each patient underwent polymerase chain reaction amplification and direct sequencing of the NDP gene. Eleven male patients expressing mutations in the NDP gene were identified in the test group, whereas the controls demonstrated wild-type NDP. All patients diagnosed as having Norrie disease had mutations in the NDP gene. Four of the patients with Norrie disease had mutations involving a cysteine residue in the cysteine-knot motif. Four patients diagnosed as having familial exudative vitreoretinopathy were found to have noncysteine mutations. One patient with retinopathy of prematurity had a 14-base deletion in the 5' untranslated region (exon 1), and 1 patient with bilateral persistent fetal vasculature syndrome expressed a noncysteine mutation in the second exon. Mutations disrupting the cysteine-knot motif corresponded to severe retinal dysgenesis, whereas patients with noncysteine mutations had varying degrees of avascular peripheral retina, extraretinal vasculature, and subretinal exudate. Patients exhibiting severe retinal dysgenesis should be suspected of carrying a mutation that disrupts the cysteine-knot motif in the NDP gene.

Two Distinct Pathways for O-Fucosylation of Epidermal Growth Factor-like or Thrombospondin Type 1 Repeats

Epidermal growth factor-like (EGF) repeats and thrombospondin type 1 repeats (TSRs) are both small cysteine-knot motifs known to be O-fucosylated. The enzyme responsible for the addition of O-fucose to EGF repeats, protein O-fucosyltransferase 1 (POFUT1), has been identified and shown to be essential in Notch signaling. Fringe, an O-fucose beta1,3-N-acetylglucosaminyltransferase, elongates O-fucose on specific EGF repeats from Notch to form a disaccharide that can be further elongated to a tetrasaccharide. TSRs are found in many extracellular matrix proteins and are involved in protein-protein interactions. The O-fucose moiety on TSRs can be further elongated with glucose to form a disaccharide. The discovery of O-fucose on TSRs raised the question of whether POFUT1, or a different enzyme, adds O-fucose to TSRs. Here we demonstrate the existence of a TSR-specific O-fucosyltransferase distinct from POFUT1. Similar to POFUT1, the novel TSR-specific O-fucosyltransferase is a soluble enzyme that requires a properly folded TSR as an acceptor substrate. In addition, we found that a previously identified fucose-specific beta1,3-glucosyltransferase adds glucose to O-fucose on TSRs, but it does not modify O-fucose on an EGF repeat. Similarly, Lunatic fringe, Manic fringe, and Radical fringe are all capable of modifying O-fucose on an EGF repeat, but not on a TSR. Taken together, these results suggest that two distinct O-fucosylation pathways exist in cells, one specific for EGF repeat and the other for TSRs.

Crystal Structure of Human Vascular Endothelial Growth Factor-B: Identification of Amino Acids Important for Receptor Binding

The development of blood vessels (angiogenesis) is critical throughout embryogenesis and in some normal postnatal physiological processes. Pathological angiogenesis has a pivotal role in sustaining tumour growth and chronic inflammation. Vascular endothelial growth factor-B (VEGF-B) is a member of the VEGF family of growth factors that regulate blood vessel and lymphatic angiogenesis. VEGF-B is closely related to VEGF-A and placenta growth factor (PlGF), but unlike VEGF-A, which binds to two receptor tyrosine kinases VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), VEGF-B and PlGF bind to VEGFR-1 and not VEGFR-2. There is growing evidence of a role for VEGF-B in physiological and pathological blood vessel angiogenesis. VEGF-B may provide novel therapeutic strategies for the treatment of vascular disease and be a potential therapeutic target in aberrant vessel formation. To help understand at the molecular level the differential receptor binding profile of the VEGF family of growth factors we have determined the crystal structure of human VEGF-B(10-108) at 2.48 Angstroms resolution. The overall structure is very similar to that of the previously determined cysteine-knot motif growth factors: VEGF-A, PlGF and platelet-derived growth factor-B (PDGF-B). We also present a predicted model for the association of VEGF-B with the second domain of its receptor, VEGFR-1. Based on this interaction and the present structural data of the native protein, we have identified several putative residues that could play an important role in receptor recognition and specificity.

Differential effects of alpha subunit Asparagine56 oligosaccharide structure on equine lutropin and follitropin hybrid conformation and receptor-binding activity.

The gonadotropins, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and chorionic gonadotropin (CG), are cysteine-knot growth-factor superfamily glycoproteins composed of a common alpha subunit noncovalently associated with a hormone-specific beta subunit. The cysteine-knot motifs in both subunits create two hairpin loops, designated L1 and L3, on one side of the knot, with the intervening long loop, L2, on the opposite side. As the average alpha-subunit loop 2 oligosaccharide mass increased from 1482 to 2327, LH and FSH receptor-binding affinities of the dual-specificity eLH declined significantly, while the decrease in FSH receptor-binding affinity for eFSH was not significant. In the present study, we characterized hormone-specific glycosylation of alphaL2 oligosaccharides in eLHalpha, eFSHalpha, and eCGalpha preparations. MALDI mass spectrometry revealed 28-57 structures, including high mannose, hybrid, bi-, and triantennary oligosaccharides. The same intact subunit preparations and their alphaL2 loop-deglycosylated derivatives were combined with either eLHbeta or eFSHbeta, and the circular dichroism (CD) spectrum for each preparation was determined. We predicted that hybrid hormone preparations obtained by combining intact eLHalpha, eFSHalpha, and eCGalpha preparations with eLHbeta might exhibit differences in conformation that would disappear when the alphaL2 oligosaccharide attached to alphaAsn(56) was removed by selective peptide-N-glycanase digestion (N(56)dg-alpha). CD data supported the first prediction; however, elimination of alphaL2 oligosaccharide actually increased the conformational differences. The intact alpha subunit:eFSHbeta hybrids had virtually identical CD spectra, as expected. However, the N(56)dg-alpha:eFSHbeta hybrid spectra differed from each other. Oligosaccharide removal altered the conformation of most hybrids, suggesting that alphaAsn(82) oligosaccharide (located in alphaL3) also influenced gonadotropin conformation.

Solution structure of the main alpha-amylase inhibitor from amaranth seeds.

The most abundant alpha-amylase inhibitor (AAI) present in the seeds of Amaranthus hypochondriacus, a variety of the Mexican crop plant amaranth, is the smallest polypeptide (32 residues) known to inhibit alpha-amylase activity of insect larvae while leaving that of mammals unaffected. In solution, 1H NMR reveals that AAI isolated from amaranth seeds adopts a major trans (70%) and minor cis (30%) conformation, resulting from slow cis-trans isomerization of the Val15-Pro16 peptide bond. Both solution structures have been determined using 2D 1H-NMR spectroscopy and XPLOR followed by restrained energy refinement in the consistent-valence force field. For the major isomer, a total of 563 distance restraints, including 55 medium-range and 173 long-range ones, were available from the NOESY spectra. This rather large number of constraints from a protein of such a small size results from a compact fold, imposed through three disulfide bridges arranged in a cysteine-knot motif. The structure of the minor cis isomer has also been determined using a smaller constraint set. It reveals a different backbone conformation in the Pro10-Pro20 segment, while preserving the overall global fold. The energy-refined ensemble of the major isomer, consisting of 20 low-energy conformers with an average backbone rmsd of 0.29 +/- 0.19 A and no violations larger than 0.4 A, represents a considerable improvement in precision over a previously reported and independently performed calculation on AAI obtained through solid-phase synthesis, which was determined with only half the number of medium-range and long-range restraints reported here, and featured the trans isomer only. The resulting differences in ensemble precision have been quantified locally and globally, indicating that, for regions of the backbone and a good fraction of the side chains, the conformation is better defined in the new solution structure. Structural comparison of the solution structure with the X-ray structure of the inhibitor when bound to its alpha-amylase target in Tenebrio molitor shows that the backbone conformation is only slightly adjusted on complexation, while that of the side chains involved in protein-protein contacts is similar to those present in solution. Therefore, the overall conformation of AAI appears to be predisposed to binding to its target alpha-amylase, confirming the view that it acts as a lid on top of the alpha-amylase active site.

Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST).

Sclerosteosis is a progressive sclerosing bone dysplasia with an autosomal recessive mode of inheritance. Radiologically, it is characterized by a generalized hyperostosis and sclerosis leading to a markedly thickened and sclerotic skull, with mandible, ribs, clavicles and all long bones also being affected. Due to narrowing of the foramina of the cranial nerves, facial nerve palsy, hearing loss and atrophy of the optic nerves can occur. Sclerosteosis is clinically and radiologically very similar to van Buchem disease, mainly differentiated by hand malformations and a large stature in sclerosteosis patients. By linkage analysis in one extended van Buchem family and two consanguineous sclerosteosis families we previously mapped both disease genes to the same chromosomal 17q12-q21 region, supporting the hypothesis that both conditions are caused by mutations in the same gene. After reducing the disease critical region to approximately 1 Mb, we used the positional cloning strategy to identify the SOST gene, which is mutated in sclerosteosis patients. This new gene encodes a protein with a signal peptide for secretion and a cysteine-knot motif. Two nonsense mutations and one splice site mutation were identified in sclerosteosis patients, but no mutations were found in a fourth sclerosteosis patient nor in the patients from the van Buchem family. As the three disease-causing mutations lead to loss of function of the SOST protein resulting in the formation of massive amounts of normal bone throughout life, the physiological role of SOST is most likely the suppression of bone formation. Therefore, this gene might become an important tool in the development of therapeutic strategies for osteoporosis.

Left-right asymmetric expression of the TGF beta-family member lefty in mouse embryos.

Examples of lateral asymmetry are often found in vertebrates, such as the heart being on the left side, but the molecular mechanism governing the establishment of this left-right (L-R) handedness is unknown. A diffusible morphogen may determine L-R polarity, but a likely molecule has not so far been identified. Here we report on the gene lefty, a member of the transforming growth factor-beta family, which may encode a morphogen for L-R determination. Lefty protein contains the cysteine-knot motif characteristic of this superfamily and is secreted as a processed form of relative molecular mass 25K-32K. Surprisingly, lefty is expressed in the left half of gastrulating mouse embryos. This asymmetric expression is very transient and occurs just before the first sign of lateral asymmetry appears. In the mouse mutants iv and inv, which cause situs inversus, the sites of lefty expression are inverted, indicating that lefty is downstream of iv and inv. These results suggest that lefty may be involved in setting up L-R asymmetry in the organ systems of mammals.

Androgens promote the ontogenetic development of immune response in immature male CBA mice

FSH is a member of the pituitary glycoprotein hormone (GPH) family and consists of a non-covalently linked GPH α-subunit and a hormone-specific β-subunit. The GPH α- and FSH β-subunits contain a cysteine-knot motif and several N-linked glycosylation sites, both of which are important for biological activity. FSH as well as LH and CG have gonadotropic function and are thus grouped as gonadotropins. FSH binds to FSHR located on the plasma membrane of granulosa cells in the ovary and stimulates follicular growth. In the testis, FSH binds to FSHR in Sertoli cells and stimulates spermatogenesis. The synthesis and release of FSH are regulated by GnRH; gonadal proteins such as activin, inihibin, and follistatin; and gonadal steroids. FSH is implicated in hypergonadotropic ovarian dysgenesis, and its plasma levels are useful in the investigation of menstrual irregularities. Recombinant human FSH is used for treatment of infertility to stimulate follicular development.