Characterization of proteoglycan degradation by calpain.
Degradation of cartilage proteoglycans was investigated under neutral conditions (pH 7.5) by using pig kidney calpain II (EC 3.4.22.17; Ca(2+)-dependent cysteine proteinase). Aggregate and monomer degradation reached a maximum in 5 min at 30 degrees C when the substrate/enzyme ratio was less than 1000:1. The mode of degradation was limited proteolysis of the core protein; the size of the products was larger than that of papain-digested products and comparable with that of trypsin-digested products. The hyaluronic acid-binding region was lost from the major glycosaminoglycan-bearing region after incubation with calpain II. Calpains thus may affect the form of proteoglycans in connective tissue. Ca(2+)-dependent proteoglycan degradation was unique in that proteoglycans adsorb large amounts of Ca2+ ions rapidly before activation of calpain II: 1 mg of pig cartilage proteoglycan monomer adsorbed 1.3-1.6 mu equiv. of Ca2+ ions before activation of calpain II, which corresponds to half the sum of anion groups in glycosaminoglycan side chains. This adsorption of Ca2+ was lost after solvolysis of proteoglycan monomer with methanol/50 mM-HCl, which was used to desulphate glycosaminoglycans. Therefore cartilage proteoglycans are not merely the substrates of proteolysis, but they may regulate the activation of Ca(2+)-dependent enzymes including calpains through tight chelation of Ca2+ ions between glycosaminoglycan side chains.
Suzuki
K
K
Department of Orthopedic Surgery, Faculty of Medicine, Kyoto University, Japan.
Shimizu
K
K
Hamamoto
T
T
Nakagawa
Y
Y
Murachi
T
T
Yamamuro
T
T
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002154:Calpain / Q000378:metabolism*; D002809:Chondroitin Sulfates / Q000494:pharmacology; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D010206:Papain / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism*; D013552:Swine; D014357:Trypsin / Q000378:metabolism
D046911:Macromolecular Substances; D011509:Proteoglycans; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates; D014357:Trypsin; D002154:Calpain; D010206:Papain; D002118:Calcium