Structural and enzymatic analysis of ulilysin metalloprotease, the prototype of the catalytic domain of the human igfbp protease papp-a
- Autores: Cynthia Tallant Blanco
- Directores de la Tesis: Francesc Xavier Gomis-Rüth (dir. tes.), María Isabel Vázquez Baanante (tut. tes.)
- Lectura: En la Universitat de Barcelona ( España ) en 2010
- Idioma: español
- Tribunal Calificador de la Tesis: Ulrich Baumann (presid.), Isabel Usón Finkenzeller (secret.), Francesc Canals Suris (voc.)
- Materias:
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- Resumen
The metzincin clan encompasses several families of zinc-dependent metalloproteases with proven function in physiology and pathology. They act as broad spectrum protein degraders or as sheddases, operating through limited proteolysis. Among the structurally uncharacterized metzincin families are the pappalysins, of which the most thoroughly studied member is human pregnancy-associated plasma protein A (PAPP-A), a heavily glycosylated 170-kDa multidomain protein specifically cleaving insulin-like growth factor (IGF)-binding proteins (IGFBPs). Proulilysin is a 38-kDa archaeal protein that shares sequence similarity with PAPP-A but encompasses only the pro-domain and the catalytic domain. It undergoes calcium-mediated autolytic activation.
The mature protein has been solved and it adopts a three-dimensional structure with two subdomains separated by an active site cleft containing the catalytic zinc ion. This structure is reminiscent of human members of the adamalysin/ADAMs (a disintegrin and a metalloprotease) family of metzincins. Ulilysin has been crystallized in a product complex with an argininevaline dipeptide occupying the active-site S1' and S2' positions and in a complex with the broad-spectrum hydroxamic acid-based metalloprotease inhibitor, batimastat. This molecule inhibits mature ulilysin with an IC50 value of 61 M. The binding of batimastat to ulilysin evokes binding to vertebrate MMPs but is much weaker. These data give insight into substrate specificity and mechanism of action and inhibition of the novel pappalysin family. Once ulilysin is activated, it hydrolyses IGFBP-2 to -6 and insulin chain ß in vitro but not IGFBP-I or IGF-II. We report that ulilysin is also active against several other substrates, viz (azo)casein, azoalbumin, and extracellular matrix components. Ulilysin has gelatinolytic but not collagenolytic activity. Moreover, the proteolysis-resistant skeletal proteins actin and elastin are also cleaved, as is fibrinogen, but not plasmin and 1-antitrypsin from the blood coagulation cascade. Ulilysin develops optimal activity at pH 7.5 and strictly requires peptide bonds preceding an arginine residue, as determined by means of a novel fluorescence resonance energy transfer assay, thus pointing to biotechnological applications as an enzyme complementary to trypsin.
A methionine-1,4-turn is found directly below the zinc-binding site, which is structurally and spatially conserved in all metzincins. We have studied this structural element in ulilysin, by generating ten mutants that replaced methionine with proteogenic amino acids. We compared recombinant overexpression yields, autolytic and tryptic activation, proteolytic activity, thermal stability, and three-dimensional structure with those of the wild type. All forms were soluble and could be purified, although with varying yields, and three variants underwent autolysis, could be activated by trypsin, and displayed significant proteolytic activity. Both bulky and small side chains, as well as hydrophilic ones, showed diminished thermal stability. Leucine and cysteine mutants crystallized and showed structures that were indistinguishable from the wild type. These studies reveal the Met-turn acts as a plug that snugly inserts laterally into a core structure created by the protein segment engaged in zinc binding and thus contributes to its structural integrity, which is indispensable for function.
