Detection of chondroitin sulfate-binding proteins on the membrane.
Chondroitin sulfate is a ubiquitous component of proteoglycans that is present both in the extracellular matrix and at the cell surface of various tissues. Until recently, chondroitin sulfate has attracted less attention than heparan sulfate and dermatan sulfate, owing to the limited number of known chondroitin sulfate-binding proteins. To determine the biological function of chondroitin sulfate, biotinylated probes were prepared and used to search for binding proteins. Chondroitin sulfates A, C, D, and E were biotinylated through either the uronic acid or the residual core peptide. Lysates from mouse Lewis lung carcinoma (3LL) cells were blotted onto a nitrocellulose membrane after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the membrane was treated with the biotinylated chondroitin sulfates. Among the chondroitin sulfate variants, the E type showed the most intense bands upon visualization of the membrane with avidin-conjugated alkaline phosphatase and the appropriate substrates. The binding of chondroitin sulfate E to proteins in the cell lysate was not affected by the A, C or D variants but was reduced by treatment with dermatan sulfate. Lysates from 3LL cells were also treated with biotinylated chondroitin sulfate E and, after two-dimensional (2-D) electrophoresis and blotting, several chondroitin sulfate E-binding proteins including lamins and heterogeneous nuclear ribonucleoproteins were identified by mass spectrometry.
Department of Biochemistry, School of Medicine, Kinki University, Osaka-Sayama, Osaka, Japan. asaito@med.kindai.ac.jp
Akio
A
Saito
Hiroshi
H
Munakata
D000818:Animals; D001360:Avidin / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D002809:Chondroitin Sulfates / Q000378:metabolism*; D003871:Dermatan Sulfate / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D034882:Lamins / Q000378:metabolism*; D008566:Membranes / Q000737:chemistry*; D051379:Mice; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014407:Tumor Cells, Cultured
D034882:Lamins; D001360:Avidin; D003871:Dermatan Sulfate; D001710:Biotin; D002809:Chondroitin Sulfates