The C-terminal region of human plasma fetuin-B is dispensable for the raised-elephant-trunk mechanism of inhibition of astacin metallopeptidases

© The Author(s) 2019.

Fetuin-B, a liver-derived plasma protein is essential for fertilization.

SummaryThe zona pellucida (ZP) is a glycoprotein matrix surrounding mammalian oocytes. Upon fertilization, ZP hardening prevents sperm from binding to and penetrating the ZP. Here, we report that targeted gene deletion of the liver-derived plasma protein fetuin-B causes premature ZP hardening and, consequently, female infertility. Transplanting fetuin-B-deficient ovaries into wild-type recipients restores fertility, indicating that plasma fetuin-B is necessary and sufficient for fertilization. In vitro fertilization of oocytes from fetuin-B-deficient mice only worked after rendering the ZP penetrable by laser perforation. Mechanistically, fetuin-B sustains fertility by inhibiting ovastacin,…

Structure of mammalian plasma fetuin-B and its mechanism of selective metallopeptidase inhibition

The co-crystal structure of the metallopeptidase astacin with its specific protein inhibitor fetuin-B reveals a novel mechanism of inhibition.

A Wnt-specific astacin proteinase controls head formation inHydra

AbstractTheHydrahead organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood.Here we report the identification of an astacin family proteinase as a Wnt processing factor.Hydraastacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone.HAS-7siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is re…

Additional file 11 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Additional file 11: Fig. S9. Uncropped Western blot and gel images

The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Abstract Background The Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood. Here we report a proteomic analysis of Hydra head tissue leading to the identification of an astacin family proteinase as a Wnt processing factor. Results Hydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteo…

The interaction of recombinant subdomains of the procollagen C-proteinase with procollagen I provides a quantitative explanation for functional differences between the two splice variants, mammalian tolloid and bone morphogenetic protein 1.

The procollagen C-proteinase (PCP) is a zinc peptidase of the astacin family and the metzincin superfamily. The enzyme removes the C-terminal propeptides of fibrillar procollagens and activates other matrix proteins. Besides its catalytic protease domain, the procollagen C-proteinase contains several C-terminal CUB modules (named after complement factors C1r and C1s, the sea urchin UEGF protein, and BMP-1) and EGF-like domains. The two major splice forms of the C-proteinase differ in their overall domain composition. The longer variant, termed mammalian tolloid (mTld, i.e., PCP-2), has the protease- CUB1-CUB2-EGF1-CUB3-EGF2-CUB4-CUB5 composition, whereas the shorter form termed bone morphog…

Astacin

The protease domain of procollagen C-proteinase (BMP1) lacks substrate selectivity, which is conferred by non-proteolytic domains.

Abstract Procollagen C-proteinase (PCP) removes the C-terminal pro-peptides of procollagens and also processes other matrix proteins. The major splice form of the PCP is termed BMP1 (bone morphogenetic protein 1). Active BMP1 is composed of an astacin-like protease domain, three CUB (complement, sea urchin Uegf, BMP1) domains and one EGF-like domain. Here we compare the recombinant human full-length BMP1 with its isolated proteolytic domain to further unravel the functional influence of the CUB and EGF domains. We show that the protease domain alone cleaves truncated procollagen VII within the short telopeptide region into fragments of similar size as the full-length enzyme does. However, u…

Proenzyme Structure and Activation of Astacin Metallopeptidase

Proteolysis is regulated by inactive (latent) zymogens, with a prosegment preventing access of substrates to the active-site cleft of the enzyme. How latency is maintained often depends on the catalytic mechanism of the protease. For example, in several families of the metzincin metallopeptidases, a >cysteine switch> mechanism involves a conserved prosegment motif with a cysteine residue that coordinates the catalytic zinc ion. Another family of metzincins, the astacins, do not possess a cysteine switch, so latency is maintained by other means. We have solved the high resolution crystal structure of proastacin from the European crayfish, Astacus astacus. Its prosegment is the shortest struc…

Sizzled Is Unique among Secreted Frizzled-related Proteins for Its Ability to Specifically Inhibit Bone Morphogenetic Protein-1 (BMP-1)/Tolloid-like Proteinases

BMP-1/tolloid-like proteinases (BTPs) are major enzymes involved in extracellular matrix assembly and activation of bioactive molecules, both growth factors and anti-angiogenic molecules. Although the control of BTP activity by several enhancing molecules is well established, the possibility that regulation also occurs through endogenous inhibitors is still debated. Secreted frizzled-related proteins (sFRPs) have been studied as possible candidates, with highly contradictory results, after the demonstration that sizzled, a sFRP found in Xenopus and zebrafish, was a potent inhibitor of Xenopus and zebrafish tolloid-like proteases. In this study, we demonstrate that mammalian sFRP-1, -2, and …

Fetuin-A and Cystatin C Are Endogenous Inhibitors of Human Meprin Metalloproteases

Meprin α and β, zinc metalloproteinases, play significant roles in inflammation, including inflammatory bowel disease (IBD), possibly by activating cytokines, like interleukin 1β, interleukin 18, or tumor growth factor α. Although a number of potential activators for meprins are known, no endogenous inhibitors have been identified. In this work, we analyzed the inhibitory potential of human plasma and identified bovine fetuin-A as an endogenous meprin inhibitor with a K(i) (inhibition constant) of 4.2 × 10(-5) M for meprin α and a K(i) of 1.1 × 10(-6) M meprin β. This correlated with data obtained for a fetuin-A homologue from carp (nephrosin inhibitor) that revealed a potent meprin α and β…

Additional file 12 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Additional file 12: Table S3. LNA and RNA probe sequences used for WISH.

Mammalian plasma fetuin-B is a selective inhibitor of ovastacin and meprin metalloproteinases

AbstractVertebrate fetuins are multi-domain plasma-proteins of the cystatin-superfamily. Human fetuin-A is also known as AHSG, α2-Heremans-Schmid-glycoprotein. Gene-knockout in mice identified fetuin-A as essential for calcified-matrix-metabolism and bone-mineralization. Fetuin-B deficient mice, on the other hand, are female infertile due to zona pellucida ‘hardening’ caused by the metalloproteinase ovastacin in unfertilized oocytes. In wildtype mice fetuin-B inhibits the activity of ovastacin thus maintaining oocytes fertilizable. Here we asked, if fetuins affect further proteases as might be expected from their evolutionary relation to single-domain-cystatins, known as proteinase-inhibito…

Additional file 13 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Additional file 13: Table S4. siRNA and qPCR primer sequences.

Intracellular activation of ovastacin mediates pre-fertilization hardening of the zona pellucida

Study question How and where is pro-ovastacin activated and how does active ovastacin regulate zona pellucida hardening (ZPH) and successful fertilization? Study finding Ovastacin is partially active before exocytosis and pre-hardens the zona pellucida (ZP) before fertilization. What is known already The metalloproteinase ovastacin is stored in cortical granules, it cleaves zona pellucida protein 2 (ZP2) upon fertilization and thereby destroys the ZP sperm ligand and triggers ZPH. Female mice deficient in the extracellular circulating ovastacin-inhibitor fetuin-B are infertile due to pre-mature ZPH. Study design, samples/materials, methods We isolated oocytes from wild-type and ovastacin-de…

Additional file 2 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Additional file 2: Table S1. (a) Secretome of Hydra HL HyWnt3 (+) fraction. (b) Secretome of Hydra HL HyWnt3 (-) fraction.

Additional file 3 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Additional file 3: Table S2. Complete proteome data of HyWnt3(+) and HyWnt3(-) HL fractions.

Limited proteolysis by acrosin affects sperm-binding and mechanical resilience of the mouse zona pellucida.

Abstract The encounter of oocyte and sperm is the key event initiating embryonic development in mammals. Crucial functions of this existential interaction are determined by proteolytic enzymes, such as acrosin, carried in the sperm head acrosome, and ovastacin, stored in the oocyte cortical granules. Ovastacin is released upon fertilisation to cleave the zona pellucida, a glycoprotein matrix surrounding the oocyte. This limited proteolysis hardens the oocyte envelope, and thereby provides a definitive block against polyspermy and protects the developing embryo. On the other hand, acrosin, the renowned and most abundant acrosomal protease, has been thought to enable sperm to penetrate the oo…

Additional file 10 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Additional file 10: Fig. S8. Developmental balance between ectopic structures. (a) siHAS-7/siNdr electroporation blocks ectopic axis formation after subsequent AZK treatment. (b-c) siHAS-7/Wnt8 electroporation and AZK treatment reduces ectopic tentacle development in double axis animals (b) and leads to multiple secondary axis formation in a fraction of the treated animals (c). Red arrows denote secondary axes. The asterisk denotes the peduncle region. (d) Ectopic tentacle inhibition is clearly evident in animals electroporated with siWnt8 followed by AZK treatment. Note that few residual ectopic tentacles are detectable in c and d mostly on the side not directly hit by the electroporation …

Additional file 5 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Additional file 5: Fig. S3. Expression of HAS genes in the interstitial stem cell cluster. (a) t-SNE representation of interstitial cells with clusters labeled by cell state as presented in [25]. (b) Interstitial cell cluster annotation of HyDkk1/2/4 and ataxin genes identified in HyWnt3(+) head lysate fraction. The cells in the t-SNE plots were colored based on expression levels for the respective gene. The transcript IDs are as follows: HMP1: t1098aep, HAS-1: t20535aep, HAS-2: t18494aep, HAS-3: t22149aep, HAS-4: t11453aep, HAS-5: t596aep, HAS-6: t19593aep, HAS-7: t16296aep, HAS-8: t22154aep, HAS-9: t3416aep, HAS-10: t10258aep, HAS-11: t19316aep. HyDkk1/2/4: t8678aep. Cluster label abbrevi…

Additional file 6 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Additional file 6: Fig. S4. Detection of HAS-7 by Western blot. (a) Antigenic peptide competition demonstrates the specificity of the HAS-7 antibody. A Western blot for tissue lysates as in Fig. 3a was performed using primary antibody solution with (right panel) or without (left panel) 1 mg/ml of the antigenic peptide used for generating the HAS-7 antibody. The HAS-7 peptide effectively reduces the detection of specific bands at ~ 40 and 70 kDa. (b) Ni-NTA affinity purified recombinant HAS-7. Separation by 12% SDS-PAGE was followed by staining with Coomassie brilliant blue (left) or transfer to PVDF and immunodetection (right) using the Penta-His-antibody as described above. For each lane 1…

Additional file 8 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Additional file 8: Fig. S6. Representative images of knockdown and transgenic phenotypes. Representative images of HAS-7 siRNA treated (a-c), HAS-7 siRNA/AZK treated (d-g) or transgenic actin::HyWnt3 (h-i) animals. Scale bars: 200 μm. The inset in Fig. S6a shows an early stage of ectopic axis formation recorded 4 days after electroporation. Red arrows indicate the hypostome areas of the two heads.

Additional file 7 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Additional file 7: Fig. S5. Evidence for normal function and morphology of ectopic heads and tentacles. (a-b) Both heads in a HAS-7 siRNA treated animal with a double axis are able to capture and feed on artemia. The arrow denotes an ectopic foot induced by the secondary head. Scale bars = 500 μm. (c-e) Ectopic tentacles induced by ALP treatment show anatomic and molecular features of functional tentacles as demonstrated by immunocytochemistry using a nematocyst-specific antibody (anti-CPP-1) [53]. CPP-1 is a structural component of mature nematocysts in battery cells of tentacles. (c) Overview of CPP-1-stained hydra with ectopic tentacles. Scale bar = 200 μm. (d-e) Enlargement from boxed a…

Additional file 1 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Additional file 1: Fig. S1. Ion exchange chromatogram of hydra head lysate pool. (a) 7 fractions of 0.5 ml exceeding an absorption unit threshold of 0.175 were collected as indicated. The cut-off was chosen to provide a critical total protein concentration (> 80 μg) for the subsequent proteome analysis. (b) Peak fractions from (a) were re-screened for HyWnt3-His processing activity. A fragment of Hydra cadherin extracellular domain comprising the first two N-terminal cadherin repeats (HmCadherin1-2) was used as control substrate to monitor unspecific matrix metalloproteinase activity. Accordingly, fractions 4-5 were pooled and analyzed by mass spectrometry as HyWnt3-His(+) sample, fracti…

Additional file 14 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Additional file 14: The individual data values for Figs. 3f and 5f.

Additional file 9 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Additional file 9: Fig. S7. Function of HAS-7 in regeneration. Animals bisected after HAS-7 siRNA electroporation do not show axis duplication in head (a-b) or foot (c-d) regenerates. Animals were bisected at 50% of body length at day 6 after electroporation and documented at day 0 (a, c) and day 4 (b, d) after bisection. Representatives of 25 bisected hydras examined. Scale bars: 200 μm. (e) Heat map showing the dynamics of transcript levels for HyWnt3(+) astacin genes compared to HyWnt3 and beta-Catenin. Only components that were significantly differentially expressed (P

Additional file 4 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Additional file 4: Fig. S2. Phylogenetic tree of astacin metalloproteinases established by PhyLM 3.0 (SEAVIEW package) and based on an alignment of the catalytic domains only, omitting pro-sequences and multiple C-terminal domains. Numbers indicate probability values (in %) obtained from 100 bootstrap replications. Protein abbreviations from bottom: fAST, flavastacin (Flavobacterium meningosepticum, i.e. Chryseobacterium meningosepticum, i.e. Elisabethkingia meningoseptica, Q47899, used as outgroup); HEA-1, Hydractinia echinata astacin-1 (Q2MCX9); HEA-3, H. echinata astacin-3 (Q2MCX7); HEA-4, H. echinata astacin-4 (Q2MCX6); HMP1, Hydra vulgaris metalloproteinase-1 (NP_001296695.1), AST, ast…