Determination of cleavage site of Reelin between its sixth and seventh repeat and contribution of meprin metalloproteases to the cleavage.
Reelin is a secreted glycoprotein whose function is regulated by proteolysis. One of the specific cleavage sites of Reelin, called C-t, is located approximately between the sixth and seventh Reelin repeat but its exact site was unknown. We here show that a metalloprotease present in the culture supernatant of cerebellar granular neurons (CGN) cleaves Reelin between Ala2688 and Asp2689. A Reelin mutant in which Asp2689 is replaced by Lys (Reelin-DK) is resistant to C-t cleavage by culture supernatant of CGN. From biochemical characteristics and the cleavage site preference, meprin α and meprin β were suggested candidate proteases and both were confirmed to cleave Reelin at the C-t site. Meprin α cleaved Reelin-DK but meprin β did not. Actinonin, a meprin α and meprin β inhibitor, did not inhibit the Reelin-cleaving activity of CGN and the amount of Reelin fragments in brains of meprin β knock-out mice was not significantly different from that of the wild-type, indicating that meprin β does not play a major role in Reelin cleavage under basal conditions. We propose that meprin α and meprin β join the modulators of Reelin signalling as they cleave Reelin at a specific site and are upregulated under specific pathological conditions.
Department of Biomedical Science, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan and mhattori@phar.nagoya-cu.ac.jp.
Yoshitaka
Y
Sato
Department of Biomedical Science, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan and mhattori@phar.nagoya-cu.ac.jp.
Daichi
D
Kobayashi
Department of Biomedical Science, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan and.
Takao
T
Kohno
Department of Biomedical Science, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan and.
Yujiro
Y
Kidani
Department of Biomedical Science, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan and.
Johannes
J
Prox
Unit for Degradomics of the Protease Web, Institute of Biochemistry, University of Kiel, Germany.
Christoph
C
Becker-Pauly
Department of Biomedical Science, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan and mhattori@phar.nagoya-cu.ac.jp.
Mitsuharu
M
Hattori
D000818:Animals; D019556:COS Cells; D015816:Cell Adhesion Molecules, Neuronal / Q000378:metabolism*; D018929:Cell Culture Techniques; D002540:Cerebral Cortex / Q000166:cytology*; D002522:Chlorocebus aethiops; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000201:enzymology*; D059748:Proteolysis; D000091042:Reelin Protein; D012697:Serine Endopeptidases / Q000378:metabolism*; D015398:Signal Transduction
D015816:Cell Adhesion Molecules, Neuronal; D016326:Extracellular Matrix Proteins; D006877:Hydroxamic Acids; D009419:Nerve Tissue Proteins; D000091042:Reelin Protein; C000717127:RELN protein, human; C000717128:Reln protein, mouse; D012697:Serine Endopeptidases; D008666:Metalloendopeptidases; C053833:meprin A; C010531:actinonin
Reelin; meprin; neuron; protease; protein degradation