Expression and binding activity of the carboxyl-terminal portion of the core protein of PG-M, a large chondroitin sulfate proteoglycan.
PG-M is a large chondroitin sulfate proteoglycan that has been shown to be expressed in the prechondrogenic condensation area of the developing chick limb buds. We previously isolated cDNA clones encoding the core protein of PG-M (Shinomura, T., Nishida, Y., Ito, K., and Kimata, K. (1993) J. Biol. Chem. 268, 14461-14469). The amino acid sequence deduced from the cDNA analysis revealed the presence of two epidermal growth factor-like domains, a C-type lectin-like domain, and a complement regulatory protein (CRP)-like domain at the COOH terminus. The COOH-terminal portion has been expressed as a fusion protein with glutathione S-transferase in Escherichia coli to test its carbohydrate binding activity using affinity chromatography. The purified fusion protein binds to immobilized D-mannose, D-galactose, L-fucose, and N-acetyl-D-glucosamine in a calcium-dependent manner. Furthermore, the fusion protein binds to heparin- or heparan sulfate-Sepharose. To investigate roles of each COOH-terminal domain, we have made a truncated construct which lacks the CRP-like domain and determined if the CRP-like domain is involved in the binding activity. The removal of this domain resulted in the complete loss of both C-type lectin-like and heparin binding activities. The results suggest that a whole set of epidermal growth factor-, lectin-, and CRP-like domains may serve a functional structure for these bindings.
Institute for Molecular Science of Medicine, Aichi Medical University, Japan.
M
M
Ujita
T
T
Shinomura
K
K
Ito
Y
Y
Kitagawa
K
K
Kimata
D053674:Aggrecans; D000818:Animals; D050260:Carbohydrate Metabolism; D002642:Chick Embryo; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism*; D002846:Chromatography, Affinity; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism; D066298:In Vitro Techniques; D037102:Lectins; D037181:Lectins, C-Type; D011509:Proteoglycans; D011993:Recombinant Fusion Proteins
D053674:Aggrecans; D011508:Chondroitin Sulfate Proteoglycans; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D006493:Heparin; D006497:Heparan Sulfate; D037102:Lectins; D037181:Lectins, C-Type; D011509:Proteoglycans; D011993:Recombinant Fusion Proteins