Cathepsin X binds to cell surface heparan sulfate proteoglycans.
Glycosaminoglycans have been shown to be important regulators of activity of several papain-like cathepsins. Binding of glycosaminoglycans to cathepsins thus directly affects catalytic activity, stability or the rate of autocatalytic activation of cathepsins. The interaction between cathepsin X and heparin has been revealed by affinity chromatography using heparin-Sepharose. Conformational changes were observed to accompany heparin-cathepsin X interaction by far UV-circular dichroism at both acidic (4.5) and neutral (7.4) pH. These conformational changes promoted a 4-fold increase in the dissociation constant of the enzyme-substrate interaction and increased 2.6-fold the kcat value also. The interaction between cathepsin X and heparin or heparan sulfate is specific since dermatan sulfate, chondroitin sulfate, and hyaluronic acid had no effect on the cathepsin X activity. Using flow cytometry cathepsin X was shown to bind cell surface heparan sulfate proteoglycans in wild-type CHO cells but not in CHO-745 cells, which are deficient in glycosaminoglycan synthesis. Moreover, fluorescently labeled cathepsin X was shown by confocal microscopy to be endocytosed by wild-type CHO cells, but not by CHO-745 cells. These results demonstrate the existence of an endocytosis mechanism of cathepsin X by the CHO cells dependent on heparan sulfate proteoglycans present at the cell surface, thus strongly suggesting that heparan sulfate proteoglycans can regulate the cellular trafficking and the enzymatic activity of cathepsin X.
Nascimento
Fábio D
FD
Departamento de Bioquímica, Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo, Brazil.
Rizzi
Claudia C A
CC
Nantes
Iseli L
IL
Stefe
Ivica
I
Turk
Boris
B
Carmona
Adriana K
AK
Nader
Helena B
HB
Juliano
Luiz
L
Tersariol
Ivarne L S
IL
D000818:Animals; D016466:CHO Cells; D002384:Catalysis; D056657:Cathepsin K; D002403:Cathepsins / Q000737:chemistry* / Q000378:metabolism; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004396:Coloring Agents / Q000494:pharmacology; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000737:chemistry; D003871:Dermatan Sulfate / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D005434:Flow Cytometry; D019812:Heparan Sulfate Proteoglycans / Q000737:chemistry*; D006497:Heparan Sulfate / Q000737:chemistry; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry; D006834:Hydrazines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008247:Lysosomes / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012492:Salts / Q000494:pharmacology; D013379:Substrate Specificity; D013431:Sulfates / Q000737:chemistry; D014466:Ultraviolet Rays
D056657:Cathepsin K; D002403:Cathepsins; D004396:Coloring Agents; D003546:Cysteine Endopeptidases; D003871:Dermatan Sulfate; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D006820:Hyaluronic Acid; D006834:Hydrazines; D012492:Salts; D013431:Sulfates; C417561:Alexa 488 hydrazide; C534130:CTSK protein, human