The C2 domain of calpain 5 contributes to enzyme activation and membrane localization

Vimala Bondada, Jozsef Gal, Charles Mashburn, David W. Rodgers, Katherine E. Larochelle, Dorothy E. Croall, James W. Geddes

Research output: Contribution to journalArticlepeer-review

8 Scopus citations


The enzymatic characteristics of the ubiquitous calpain 5 (CAPN5) remain undescribed despite its high expression in the central nervous system and links to eye development and disease. CAPN5 contains the typical protease core domains but lacks the C terminal penta-EF hand domain of classical calpains, and instead contains a putative C2 domain. This study used the SH-SY5Y neuroblastoma cell line stably transfected with CAPN5-3xFLAG variants to assess the potential roles of the CAPN5 C2 domain in Ca2+ regulated enzyme activity and intracellular localization. Calcium dependent autoproteolysis of CAPN5 was documented and characterized. Mutation of the catalytic Cys81 to Ala or addition of EGTA prevented autolysis. Eighty μM Ca2+ was sufficient to stimulate half-maximal CAPN5 autolysis in cellular lysates. CAPN5 autolysis was inhibited by tri-leucine peptidyl aldehydes, but less effectively by di-Leu aldehydes, consistent with a more open conformation of the protease core relative to classical calpains. In silico modeling revealed a type II topology C2 domain including loops with the potential to bind calcium. Mutation of the acidic amino acid residues predicted to participate in Ca2+ binding, particularly Asp531 and Asp589, resulted in a decrease of CAPN5 membrane association. These residues were also found to be invariant in several genomes. The autolytic fragment of CAPN5 was prevalent in membrane-enriched fractions, but not in cytosolic fractions, suggesting that membrane association facilitates the autoproteolytic activity of CAPN5. Together, these results demonstrate that CAPN5 undergoes Ca2+-activated autoproteolytic processing and suggest that CAPN5 association with membranes enhances CAPN5 autolysis.

Original languageEnglish
Article number119019
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Issue number7
StatePublished - Jun 2021

Bibliographical note

Publisher Copyright:
© 2021 Elsevier B.V.


  • Calcium
  • Calpain
  • Cell culture
  • Immunofluorescence
  • Membrane
  • Plasma membrane
  • Protease
  • Protein-lipid interaction

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology


Dive into the research topics of 'The C2 domain of calpain 5 contributes to enzyme activation and membrane localization'. Together they form a unique fingerprint.

Cite this