Pseudoproteoglycan (pseudoPG) probes that simulate PG macromolecular structure for screening and isolation of PG-binding proteins.
A proteoglycan (PG) monomer is a macromolecule consisting of one or more glycosaminoglycan (GAG) chains attached to a core protein. PGs have signaling roles and modulatory functions in the extracellular matrix and at the cell surface. To elucidate the functions of higher-order PG structures, pseudoPGs that imitate the PG structure were prepared to develop probes and affinity adsorbents. Poly-L: -lysine (PLL) or polyacrylamide (PAA) was coupled with various GAGs, then biotinylated, and the remaining amino groups were blocked to obtain the pseudoPG probes, biotinyl PLL (BPL)- or PAA (BPA)-GAGs. Lactoferrin exhibited 30-times higher affinity toward BPL-heparin than the conventional single-strand probe, biotin-hydrazide-heparin. Heparin-PLL was immobilized on a formyl-Sepharose and compared with the Hep-Sepharose in which heparin was directly immobilized to amino-Sepharose. Screening for ligands in normal rat brain revealed several proteins that specifically bound to either of the two adsorbents, indicating that the heparin-binding proteins exhibit specific recognition depending on the higher-order structure of the PG.
Nakagawa
Keiko
K
Graduate School of Humanities and Sciences, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan.
Nakamura
Kosuke
K
Haishima
Yuji
Y
Yamagami
Makiko
M
Saito
Kana
K
Sakagami
Hiromi
H
Ogawa
Haruko
H
D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D007700:Kinetics; D008027:Light; D008567:Membranes, Artificial; D015335:Molecular Probes / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011509:Proteoglycans / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D012542:Scattering, Radiation; D012685:Sepharose / Q000737:chemistry; D020349:Surface Plasmon Resonance; D034421:Sus scrofa
D008567:Membranes, Artificial; D015335:Molecular Probes; D011506:Proteins; D011509:Proteoglycans; D001710:Biotin; D012685:Sepharose