Nephronectin binds to heparan sulfate proteoglycans via its MAM domain.
Nephronectin is a basement membrane protein comprising five N-terminal epidermal growth factor (EGF)-like repeats, a central linker segment containing an Arg-Gly-Asp (RGD) motif and a C-terminal meprin-A5 protein-receptor protein tyrosine phosphatase μ (MAM) domain. Nephronectin has been shown to interact with α8β1 integrin through the central linker segment, but its interactions with other molecules remain to be elucidated. Here, we examined the binding of nephronectin to a panel of glycosaminoglycan (GAG) chains. Nephronectin bound strongly to heparin and chondroitin sulfate (CS)-E and moderately to heparan sulfate (HS), but failed to bind to CS-A, CS-C, CS-D, dermatan sulfate and hyaluronic acid. Deletion of the MAM domain severely impaired the binding of nephronectin to heparin but not CS-E, whereas deletion of the EGF-like repeats reduced its binding to CS-E but not heparin, suggesting that nephronectin interacts with CS-E and heparin through the EGF-like repeats and MAM domain, respectively. Consistent with these results, nephronectin bound to agrin and perlecan, which are heparan sulfate proteoglycans (HSPGs) in basement membranes, in HS-dependent manners. Site-directed mutagenesis of the MAM domain revealed that multiple basic amino acid residues in the putative loop regions were involved in the binding of the MAM domain to agrin. The binding of nephronectin to basement membrane HSPGs was further confirmed by in situ nephronectin overlay assays using mouse frozen tissue sections. Taken together, these findings indicate that nephronectin is capable of binding to HSPGs in basement membranes via the MAM domain, and thereby raise the possibility that interactions with basement membrane HSPGs may be involved in the deposition of nephronectin onto basement membranes.
Laboratory of Extracellular Matrix Biochemistry, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Yuya
Y
Sato
Chisei
C
Shimono
Shaoliang
S
Li
Itsuko
I
Nakano
Naoko
N
Norioka
Nobuo
N
Sugiura
Koji
K
Kimata
Masashi
M
Yamada
Kiyotoshi
K
Sekiguchi
D018171:Agrin / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001485:Basement Membrane; D004622:Embryo, Mammalian / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D019812:Heparan Sulfate Proteoglycans / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary
D018171:Agrin; D016326:Extracellular Matrix Proteins; D019812:Heparan Sulfate Proteoglycans; C432915:nephronectin; C071927:perlecan