title: Identification of amino acid residues responsible for von Willebrand factor binding to sulfatide by charged-to-alanine-scanning mutagenesis.
abstract: 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.
authors: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan.
authors: Takayuki
authors: T
authors: Nakayama
authors: 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.
authors: Tadashi
authors: T
authors: Matsushita
authors: Department of Transfusion Medicine, Nagoya University Hospital, Nagoya, Aichi, 466-8550, Japan.
authors: Koji
authors: K
authors: Yamamoto
authors: Department of Endocrinology and Diabetes, Nagoya University Hospital, Nagoya, Aichi, 466-8550, Japan.
authors: Noriko
authors: N
authors: Mutsuga
authors: Department of Medical Technology, Nagoya University School of Health Sciences, Nagoya, Aichi, 461-0047, Japan.
authors: Tetsuhito
authors: T
authors: Kojima
authors: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan.
authors: Akira
authors: A
authors: Katsumi
authors: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan.
authors: Norihiko
authors: N
authors: Nakao
authors: Howard Hughes Medical Institute, Departments of Medicine and Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO, 63110, USA.
authors: J Evan
authors: JE
authors: Sadler
authors: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan.
authors: Tomoki
authors: T
authors: Naoe
authors: Nagoya Central Hospital, Nagoya, Aichi, 453-0801, Japan.
authors: Hidehiko
authors: H
authors: Saito
mesh_terms: 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*
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D007477:Ions; D013433:Sulfoglycosphingolipids; D014841:von Willebrand Factor; D000409:Alanine