Characterization of a motif for specific binding to hyaluronan in chicken SPACR.
The chicken sialoprotein associated with cones and rods (SPACR) binds to hyaluronan (HA) in the interphotoreceptor matrix space, but the motif for HA binding is still unknown. This study was conducted to determine the critical site required for specific binding to HA. Western blotting study showed that SPACR binds biotinylated HA, and this interaction was specifically inhibited by unlabeled HA. A series of GST fusion proteins covering whole SPACR was prepared, and reactivity with HA was individually screened to narrow down the region for the binding. Further, putative HA-binding motif found near the carboxyl-terminus of SPACR was mutated by site-directed mutagenesis to identify the critical binding site. Finally, we showed that native SPACR derived from retina similarly binds to HA-affinity column under both reducing and non-reducing conditions. These results revealed that the specific putative HA-binding motif is located near the carboxyl-terminus of chicken SPACR, and suggested that a structural integrity such as folded structure is not largely involved in the HA binding.
Zhao
Jinsong
J
Department of Ophthalmology, Aichi Medical University, Nagakute, Aichi, Japan.
Yoneda
Masahiko
M
Takeyama
Masayuki
M
Miyaishi
Osamu
O
Inoue
Yoko
Y
Kataoka
Takuya
T
Ohno-Jinno
Akiko
A
Isogai
Zenzo
Z
Kimata
Koji
K
Iwaki
Masayoshi
M
Zako
Masahiro
M
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002645:Chickens; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism*; D012160:Retina / Q000378:metabolism; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism*
D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; C486428:IMPG1 protein, human; D011509:Proteoglycans; D012795:Sialoglycoproteins; D006820:Hyaluronic Acid