Fig. 1

Cathepsin L proteolytically processes intracellular PGRN, and in a manner distinct from elastase. a HEK293 stable cell lines stably overexpressing pCDNA4 (pCD4), Cat B, Cat L and Cat D were analyzed for levels of PGRN and its fragments by western blot using Grn-A antibody. The expression levels of different forms (pro-, intermediate- and mature- [40, 41]) of Cat B, Cat L and Cat D were validated in the stable cell lines. b A second, full-length PGRN polyclonal goat antibody was applied to detect PGRN and its proteolytic fragments in HEK-Cat L cells in comparison to HEK-pCDNA4 cells (Con). c Recombinant PGRN was incubated with same amount of elastase (E), Cat B (B), Cat L (L) or Cat D (D) optimal reaction conditions. Cat L, but not Cat B or Cat D, processed PGRN into protein fragments after the reaction. d Recombinant PGRN protein products were detected after the addition of increasing concentrations of recombinant Cat L (left panel) or elastase (right panel). e Cleavage sites used by Cat L and elastase on PGRN were mapped by LC/MS analysis of the small peptides produced from the in vitro proteolytic reaction. All detected peptides are listed in Tables 1 and 2. Annotated MS/MS spectra of the identified granulin peptides were included in Additional file 1: Figure S2. Cat L and elastase cleavage sites are labeled by blue triangle and red triangle, respectively. Common cleavage sites are highlighted by the asterisk sign. Previously published elastase cleavage sites are labeled by green triangle. Elastase cleavage sites replicated from the current study are highlighted by plus sign. N-glycosylation sites were highlighted in red