Calpain is involved in C-terminal truncation of human aggrecan.
Mature aggrecan is generally C-terminally truncated at several sites in the CS (chondroitin sulfate) region. Aggrecanases and MMPs (matrix metalloproteinases) have been suggested to be responsible for this digestion. To identify whether calpain, a common intracellular protease, has a specific role in the proteolysis of aggrecan we developed neoepitope antibodies (anti-PGVA, anti-GDLS and anti-EDLS) against calpain cleavage sites and used Western blot analysis to identify calpain-generated fragments in normal and OA (osteoarthritis) knee cartilage and SF (synovial fluid) samples. Our results showed that human aggrecan contains six calpain cleavage sites: one in the IGD (interglobular domain), one in the KS (keratan sulfate) region, two in the CS1 and two in the CS2 region. Kinetic studies of calpain proteolysis against aggrecan showed that the aggrecan molecule was cleaved in a specific order where cuts in CS1 was the most preferred and cuts in KS region was the second most preferred cleavage. OA and normal cartilage contained low amounts of a calpain-generated G1-PGVA fragment (0.5-2%) compared with aggrecanase-generated G1-TEGE (71-76%) and MMP-generated G1-IPEN (23-29%) fragments. Significant amounts of calpain-generated GDLS and EDLS fragments were found in OA and normal cartilage, and a ARGS-EDLS fragment was detected in arthritic SF samples. The results of the present study indicate that calpains are involved in the C-terminal truncation of aggrecan and might have a minor role in arthritic diseases.
Struglics
André
A
Department of Orthopaedics, Lund University, Sweden. andre.struglics@med.lu.se
Hansson
Maria
M
D000293:Adolescent; D053674:Aggrecans / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002154:Calpain / Q000378:metabolism*; D002358:Cartilage, Articular / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007231:Infant, Newborn; D007700:Kinetics; D008969:Molecular Sequence Data; D010003:Osteoarthritis / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D013379:Substrate Specificity; D013582:Synovial Fluid / Q000378:metabolism
D053674:Aggrecans; D010446:Peptide Fragments; D010450:Endopeptidases; D002154:Calpain; C083641:aggrecanase