Identification of amino acid residues responsible for von Willebrand factor binding to sulfatide by charged-to-alanine-scanning mutagenesis.
von Willebrand factor (VWF) performs its hemostatic functions through binding to various proteins. The A1 domain of VWF contains binding sites of not only physiologically important ligands, but also exogenous modulators that induce VWF-platelet aggregation. Sulfatides, 3-sulfated galactosyl ceramides, that are expressed on oligodendrocytes, renal tubular cells, certain tumor cells and platelets, have been suggested to interact with VWF under some pathological conditions. The binding of VWF to sulfatide requires the A1 domain, but its binding sites have not been precisely identified. Here, we report that alanine mutations at Arg1392, Arg1395, Arg1399 and Lys1423 led to decreased VWF-sulfatide binding. These sites have been reported to be the binding sites for platelet membrane glycoprotein (GP) Ib and/or snake venom botrocetin, and, interestingly, are identical to the monoclonal antibody (mAb) NMC4 epitope previously reported to inhibit the VWF-GPIb interaction. We observed that NMC4 also inhibited VWF interaction with sulfatides in a dose-dependent manner. Thus, we conclude that VWF binding sites of sulfatide overlap those of platelet GPIb and botrocetin.
Nakayama
Takayuki
T
Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan.
Matsushita
Tadashi
T
Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan. tmatsu@med.nagoya-u.ac.jp.
Yamamoto
Koji
K
Department of Transfusion Medicine, Nagoya University Hospital, Nagoya, Aichi, 466-8550, Japan.
Mutsuga
Noriko
N
Department of Endocrinology and Diabetes, Nagoya University Hospital, Nagoya, Aichi, 466-8550, Japan.
Kojima
Tetsuhito
T
Department of Medical Technology, Nagoya University School of Health Sciences, Nagoya, Aichi, 461-0047, Japan.
Katsumi
Akira
A
Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan.
Nakao
Norihiko
N
Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan.
Sadler
J Evan
JE
Howard Hughes Medical Institute, Departments of Medicine and Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO, 63110, USA.
Naoe
Tomoki
T
Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan.
Saito
Hidehiko
H
Nagoya Central Hospital, Nagoya, Aichi, 453-0801, Japan.
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D018360:Crystallography, X-Ray; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007477:Ions / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013433:Sulfoglycosphingolipids / Q000737:chemistry*; D014841:von Willebrand Factor / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007477:Ions; D013433:Sulfoglycosphingolipids; D014841:von Willebrand Factor; D000409:Alanine