Analysis of plasma proteins that bind to glycosaminoglycans.
Glycosaminoglycan-binding proteins, with specific emphasis on dermatan sulfate, have been investigated in human plasma by affinity chromatography, mass spectrometry and Western blotting. Diluted plasma was applied to affinity columns and bound protein was eluted with 500 mM NaCl. Dermatan sulfate and heparan sulfate bound 7% of the total protein. Heparin bound 22% of the total protein, but chondroitin sulfate A bound only 0.23%. Mass spectrometric analysis identified 20 proteins as dermatan-sulfate-binding proteins, most of which were confirmed by Western blotting. Some of these binding proteins, such as fibrinogen, fibronectin, apolipoprotein B, LMW kininogen, inter-alpha-trypsin inhibitor, and factor H, were degraded to various extents during the chromatography step, but this degradation could be prevented by the inclusion of a serine protease inhibitor. The protein fraction binding to the dermatan sulfate column showed amidase activity, whereas that binding to the heparan sulfate and heparin columns showed 1/2 and 1/20, respectively, of the activity of the dermatan sulfate binding fraction. Dermatan sulfate was similar to heparan sulfate with respect to its capacity to bind plasma proteins and its activation of protease, but differed from chondroitin sulfate and heparin in these properties.
Saito
Akio
A
Department of Biochemistry, School of Medicine, Kinki University, 377-2 Ohno-Higashi, Osaka-Sayama, Osaka 589-8511, Japan. asaito@med.kindai.ac.jp
Munakata
Hiroshi
H
D000581:Amidohydrolases / Q000097:blood / Q000302:isolation & purification; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D006025:Glycosaminoglycans / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding
D001798:Blood Proteins; D002352:Carrier Proteins; D006025:Glycosaminoglycans; D011480:Protease Inhibitors; D000581:Amidohydrolases