Figure 3 | Scientific Reports

Figure 3

From: The serine proteinase hepsin is an activator of pro-matrix metalloproteinases: molecular mechanisms and implications for extracellular matrix turnover

Figure 3

Hepsin degrades aggrecan. (A) Hepsin cleaves within the interglobular domain of aggrecan. Left panel: Deglycosylated bovine aggrecan (400 nM) was incubated with hepsin, matriptase or trypsin (all 10 nM) for 16 hours at 37 °C. Incubation products were separated on a 7.5% SDS-PAGE gel and western blotted using an antibody recognising the aggrecan G1 domain. Results are representative of 3 independent experiments. Right panel: Hepsin generates a novel cleavage within the aggrecan IGD. Recombinant human G1-IGD-G2 was incubated with matriptase or hepsin in a 1:20 enzyme to substrate ratio (156 nM G1-IGD-G2 and 7.8 nM proteinase) for 16 h at 37 °C then resolved by SDS-PAGE and stained with silver. (B) Left panel: In a similar experiment, products were western blotted using BC-3 and BC-14 neoepitope antibodies. Incubation of G1-IGD-G2 with ADAMTS-5 (1.6 μg/ml) and MMP-13 (6 μg/ml) acted as positive controls for aggrecanase and MMP cleavage, respectively. Right panel: G1-IGD-G2 (156 nM) was incubated with hepsin (7.8 nM) for 8 h at 37 °C. Products (Ia, Ib, IIa and IIb) were separated by SDS-PAGE and stained with silver, western blotted using an anti-G1 aggrecan antibody or blotted onto PVDF and stained with Coomassie brilliant blue R250, after which the desired band (IIb) was excised and subjected to N-terminal sequencing. Where images have been cropped for clarity, full-length gels/blots are presented in supplementary information. The bands on each figure are taken from the same gels/blots under the same exposure conditions. (C) N-terminal sequencing identifies novel cleavage site. Left panel: Schematic representation of the hepsin-cleaved aggrecan fragments based on data from B. The desired product (IIb; red line), corresponding to the new N-terminus following cleavage (red dashed box in B), was excised from a Coomassie-stained blot. Right panel: Known MMP and aggrecanase cleavage sites within the aggrecan IGD including the novel hepsin cleavage at Arg375↓Ser376 confirmed by N-terminal sequencing (highlighted by red arrow).