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

Abstract

Vertebrate 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-inhibitors. We show that fetuin-A is not an inhibitor of any tested protease. In stark contrast, the closely related fetuin-B selectively inhibits astacin-metalloproteinases such as meprins and ovastacin, but not astacins of the tolloid-subfamily, nor any other proteinase. The analysis of fetuin-B expressed in various mammalian cell types, insect cells, and truncated fish-fetuin expressed in bacteria, showed that the cystatin-like domains alone are necessary and sufficient for inhibition. This report highlights fetuin-B as a specific antagonist of ovastacin and meprin-metalloproteinases. Control of ovastacin was shown to be indispensable for female fertility. Meprin inhibition, on the other hand, renders fetuin-B a potential key-player in proteolytic networks controlling angiogenesis, immune-defense, extracellular-matrix-assembly and general cell-signaling, with implications for inflammation, fibrosis, neurodegenerative disorders and cancer.

A Wnt-specific astacin proteinase controls head formation inHydra

“…We concluded from these results that metalloproteinases, in particular astacin-type proteinases, are likely candidates for the observed HyWnt3-His processing activity. To confirm this notion, we tested the proteolytic activity of HL on HyWnt3 in the presence of recombinant mammalian Fetuin-B, which was recently shown to function as a highly specific physiological inhibitor of astacin-type proteinases like ovastacin [19]. As shown in Fig.…”

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

“…Protein hFB with a C-terminal hexahistidine-tag, thus spanning residues C 16 -P 382 -IEGRHHHHHH, was expressed and secreted to the extracellular medium by mammalian ExpiCHO-S cells according to the manufacturer’s instructions (ThermoFisher Scientific) as previously reported 35 . Human meprin β was obtained as a zymogen as described 51 and activated with trypsin 52 .…”

“…To complement these studies, we here report the crystal structure of the complex between the human ortholog of fetuin-B (hFB), which is the physiologically relevant species for studying human fertility 42 , and 202-residue mature crayfish astacin, which is a useful model for the 197-residue catalytic domain of human ovastacin (35% sequence identity; 48% similarity; see also 35 ). These studies revealed unexpected differences with mFB in terms of proteolytic susceptibility and the spatial arrangement of the cystatin domains, which enabled us to identify dispensable structural elements for inhibition.…”

Mammalian egg coat modifications and the block to polyspermy

“…First identified as a putative mammalian hatching enzyme (Quesada, Sánchez, Alvarez, & López‐Otín, 2004), ovastacin contains a unique heptapeptide motif that ensures its localization in the CGs as a proenzyme of 44 kDa (Burkart et al, 2012; Xiong, Zhao, Beall, Sadusky, & Dean, 2017); following oocyte activation, the protein is released as an active enzyme of 29 kDa that lacks the propeptide as well as a C‐terminal fragment (Körschgen et al, 2017). On the basis of in vitro cleavage studies with plasminogen that was either activated by trypsin or combined with tissue plasminogen activator (t‐PA; Karmilin et al, 2019)—a molecule that is also found in the oocyte CGs (Huarte, Belin, & Vassalli, 1985)—it is thought that in vivo maturation of ovastacin depends on the action of one or more serine proteases (Karmilin et al, 2019; Figure 2).…”