Forschung – Research

Proteolytic networks

ASTACINS are a subfamily of the METZINCIN superfamily of metalloproteinases. The first to be characterized was the crayfish enzyme astacin. To date more than a 1000 members of this family have been identified in organisms ranging from bacteria to humans. Astacins are involved in developmental morphogenesis, matrix assembly, tissue differentiation and digestion. Family members include the procollagen C-proteinase (BMP1, bone morphogenetic protein 1), tolloid and mammalian tolloid-like, HMP (Hydra vulgaris metalloproteinase), sea urchin BP10 (blastula protein) and SPAN (Strongylocentrotus purpuratus astacin), the 'hatching' subfamily comprising alveolin, ovastacin, LCE, HCE ('low' and 'high' choriolytic enzymes), nephrosin (from carp head kidney), UVS.2 from frog, and the meprins.


Structure of crayfish astacin (Bode et al., 1992)


Relations between astacin proteases (Gomis-Rüth et al., 2012)

 


Modular composition of astacins (Gomis-Rüth et al., 2012)

  • Gomis-Rüth FX, Trillo-Muyo S, Stöcker W (2012) Functional and structural insights into astacin metallopeptidases Biol Chem 393, 1027-1041(Review)
  • Becker-Pauly C, Barre O, Schilling O, Auf dem Keller U, Ohler A, Broder C, Schütte A, Kappelhoff R, Stöcker W, Overall CM (2011) Proteomic analyses reveal an acidic prime side specificity for the astacin metalloprotease family reflected by physiological substrates Mol Cell Proteomics DOI 101074/mcpM1111009233
  • Yiallouros I, Kappelhoff R, Schilling O, Wegmann F, Helms MW, Auge A, Brachtendorf G, Große Berkhoff E, Beermann B, Hinz HJ, König S, Peter-Katalinic J, Stöcker W (2002) Activation mechanism of pro-astacin. Role of the pro-peptide, tryptic and autoproteolytic cleavage and importance of precise amino-terminal processing. J Mol Biol 224, 237-246.
  • Yiallouros I, Große Berkhoff E, Stöcker W (2000) The roles of Glu93 and Tyr149 in astacin-like zinc peptidases. FEBS Lett 484, 224-228.
  • Stöcker, W., Grams, F., Baumann, U., Reinemer, P., Gomis-Rüth, F.X., McKay, D.B. & Bode, W. (1995) The metzincins - Topological and sequential relations between the astacins, adamalysins, serralysins, and matrixins (collagenases) define a superfamily of zinc-peptidases. Protein Sci 4, 823-840
  • Bode W, Gomis-Rüth F-X, Huber R, Zwilling R, Stöcker W (1992) Structure of astacin and implications for activation of astacins and zinc ligation of collagenases. Nature 358, 164-167

 

MEPRINS are cell surface bound and/or soluble astacins which catalyze a variety of reactions on and beyond the cell surface.


High resolution X-ray crystal structure of human meprin beta, a membrane bound sheddase, which catalyses the proteolytic processing of other membrane proteins and soluble proteins at the cell surface (Arolas et al. 2012).

  • Sterchi EE, Stöcker W, Bond JS (2008) Meprins, membrane-bound and secreted astacin metalloproteinases. Mol Aspects Med 29, 309-328
  • Arolas JL, Broder C, Jefferson T, Guevara T, Sterchi EE, Bode W, Stöcker W, Becker-Pauly C, Gomis-Rüth FX (2012) Structural basis for the sheddase function of human meprin β metalloproteinase at the plasma membrane. Proc Natl Acad Sci USA109, 16131-16136

 


BTPs - The BMP1/tolloid-proteases (BTPs) of the astacin family are key players in extracellular matrix assembly and growth factor processing.


Model of human BMP1 (bone morphogenetic protein 1), red: catalytic domain.

  • Bijakowski C, Vadon-Le Goff S, Delolme F, Bourhis J-M, Lécorché P, Ruggiero F, Becker-Pauly C, Yiallouros I, Stöcker W Dive, V, et al (2012) Sizzled is unique among secreted frizzled-related proteins for its ability to specifically inhibit BMP-1/tolloid-like proteinases. J Biol Chem 287, 33581-33593
  • Vadon-Le Goff S, Kronenberg D, Bourhis JM, Bijakowski C, Raynal N, Ruggiero F, Farndale RW, Stöcker W, Hulmes DJ, Moali C (2011) Procollagen C-proteinase enhancer stimulates procollagen processing by binding to the C-propeptide region only. J Biol Chem 286, 38932-38938
  • Wermter C, Höwel M, Hintze V, Bombosch B, Aufenvenne K, Yiallouros I, Stöcker W (2007) The Protease Domain of the Procollagen C-Proteinase (BMP1) Lacks Substrate Selectivity, which is Introduced by Non-Proteolytic Domains. Biol Chem 388, 513-521
  • Hintze V, Höwel M, Wermter C, Große Berkhoff E, Becker-Pauly C, Beermann B, Yiallouros I, Stöcker W (2006) 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. Biochemistry 45, 6741-6748

 


OVASTACIN - The function of ovastacin has been elucidated recently by Jurrien Dean's group at NIH (Burkart et al. (2012) J Cell Biol 197, 37-44). Ovastacin is expressed in mammalian oocytes and stored in cortical granules. Upon fertilization by sperm the enzyme is released into the perivitelline space to cleave a distinct protein of the zona pellucida. This causes hardening of the egg envelope and prevents entrance of further sperm cells, thus forming a block against polyspermy. This block appears to be crucial for the viability of many mammalian embryos.
In cooperation with Willi Jahnen-Dechent's group from RWTH Aachen we found an intriguing mechanism for the control of zona hardening. The Aachen group had observed that female mice lacking the liver derived plasma protein fetuin-B were infertile, although their ovaries developed normally. The reason was that these mice had undergone premature zona pellucida hardening in the absence of sperm. As it turned out, fetuin-B inactivates as a competitive inhibitor small amounts of ovastacin seeping from unfertilized eggs. In the absence of fetuin-B this tiny amount of protease is sufficient to harden the zona and to render the eggs infertile. It seems there is a fine tuned protelytic web controlling the fertilization in mammals.

 

X-ray-crystal structure of the complex of astacin (gray ribbon) and fetuin-B (coloured ribbon) Cuppari et al., 2019)

  • Cuppari, A., Körschgen, H., Fahrenkamp, D., Schmitz, C., Guevara, T., Karmilin, K., Kuske, M., Olf, M., Dietzel, E., Yiallouros, I., de Sanctis, D., Goulas, T., Weiskirchen, R., Jahnen-Dechent, W., Floehr, J., Stöcker, W., Jovine, L. & Gomis-Rüth, F.-X. (2019) Structure of mammalian plasma fetuin-B and its mechanism of selective metallopeptidase inhibition. IUCrJ 6, 317-330
  • Karmilin K, Schmitz C, Kuske M, Körschgen H, Olf M, Meyer K, Hildebrand A, Felten M, Fridrich S, Yiallouros I, Becker-Pauly C, Weiskirchen R, Jahnen-Dechent W, Floehr J, Stöcker W (2019) Mammalian plasma fetuin-B is a selective inhibitor of ovastacin and meprin metalloproteinases. Scientific Reports 9, 546
  • Körschgen H, Kuske M, Karmilin K, Yiallouros I, Balbach M, Floehr J, Jahnen-Dechent W, Stöcker W (2017) Intracellular activation of ovastacin mediates pre-fertilization hardening of the zona pellucida.
  • Stöcker W, Karmilin K, Hildebrand A, Westphal H, Yiallouros I, Weiskirchen R, Dietzel E, Floehr J, and Jahnen-Dechent W (2014). Mammalian gamete fusion depends on the inhibition of ovastacin by fetuin-B. Biol Chem 395, 1195-1199.
  • Dietzel E, Wessling J, Floehr J, Schäfer C, Ensslen S, Denecke B, Rösing B, Neulen J, Veitinger T, Spehr M, Tropartz T, Tolba R, Renné T, Egert A, Schorle H, Gottenbusch Y, Hildebrand A, Yiallouros I, Stöcker W, Weiskirchen R, and Jahnen-Dechent W (2013) Fetuin-B, a Liver-Derived Plasma Protein Is Essential for Fertilization. Dev Cell 25, 1-7
  • Hedrich, J., Lottaz, D., Meyer, K., Yiallouros, I., Jahnen-Dechent, W., Stöcker, W. and Becker-Pauly C. (2010) Fetuin-A and Cystatin C Are Endogenous Inhibitors of Human Meprin Metalloproteases. Biochemistry 49, 8599-8607