Competitive binding of heparin with hyaluronan to a specific motif in SPACR.
The critical hyaluronan binding motif (HABM) in sialoprotein associated with cones and rods (SPACR) has already been determined. As sialoproteoglycan associated with cones and rods, another interphotoreceptor matrix molecule, binds to chondroitin sulfate and heparin with or without the employment of HABMs, respectively, we evaluated and compared the binding of these glycosaminoglycans to SPACR. A western blotting study in combination with inhibition assays showed that heparin bound specifically to SPACR. A series of GST fusion proteins covering the whole SPACR molecule narrowed down the region responsible for the binding. Finally, a site-directed mutagenesis assay demonstrated that the critical HABM also acts as a specific binding site for heparin. These results were supported with mutual inhibitions by hyaluronan and heparin in analyses using GST fusion proteins and native SPACR derived from retina. Thus, these glycosaminoglycans bind to SPACR in a different manner than to sialoproteoglycan associated with cones and rods. The competitive binding between hyaluronan and heparin to SPACR, mediated through the identical HABM, may dominate the functions of SPACR, in turn involving physiological and pathological processes involved in retinal development, aging and other related disorders.
Zhao
Jinsong
J
Department of Ophthalmology, Aichi Medical University, Nagakute, Aichi, Japan.
Yoneda
Masahiko
M
Takeyama
Masayuki
M
Inoue
Yoko
Y
Kataoka
Takuya
T
Ohno-Jinno
Akiko
A
Isogai
Zenzo
Z
Iwaki
Masayoshi
M
Zako
Masahiro
M
D020816:Amino Acid Motifs; D000818:Animals; D001667:Binding, Competitive / Q000502:physiology*; D015153:Blotting, Western; D002645:Chickens; D015180:Electrophoresis, Gel, Two-Dimensional; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000378:metabolism*
D005136:Eye Proteins; D011509:Proteoglycans; D011993:Recombinant Fusion Proteins; D006820:Hyaluronic Acid; D006493:Heparin