Production of a recombinant single-chain variable-fragment (scFv) antibody against sulfoglycolipid.
Mammalian sulfoglycolipids are comprised of two major classes of compounds, sulfatide (SO(3)-3Gal-ceramide) and seminolipid (SO(3)-3Gal-alkylacylglycerol). Sulfatide is present in relatively high levels in myelin, and seminolipid is present in testis. The sulfation of these sulfoglycolipids is catalyzed by a common enzyme, cerebroside sulfotransferase (CST). Disruption of the Cst gene in mice revealed that sulfatide and seminolipid are essential for, respectively, myelin formation and spermatogenesis. The present study describes the generation of a recombinant single-chain variable fragment (scFv) antibody against sulfoglycolipid, for use in the functional analysis of sulfoglycolipids in living cells. A positive hybridoma producing anti-sulfoglycolipid IgG3, referred to as DI8, was initially obtained by immunizing CST-null mice with an isolated sulfatide. The DI8 monoclonal antibody was found to bind specifically to sulfoglycolipids with the terminal 3-O-sulfated galactose structure, as evidenced by ELISA and thin-layer chromatogram-immunostaining. The antibody stained seminolipid on the cell surface of spermatogenic cells of wild-type testis, but it did not react with any cells in the seminiferous tubules of CST-null testis. Total RNA was extracted from this hybridoma, and cDNAs that encode the variable regions of the heavy and light chains of IgG3 were obtained by RT-PCR. These DNA fragments were linked through a DNA linker coding (Gly(4)Ser)(3), and the recombinant scFv fragment was then inserted into a phagemid vector pCANTAB 5E. The scFv antibody that was displayed at the tip of the M13 phage in the form of a g3p fusion protein bound to sulfatide. Furthermore, a soluble form of the scFv antibody was also found to bind to the sulfoglycolipids in ELISA.
Cheng
Xinyao
X
Department of Biochemistry, Osaka University Medical School, Osaka 565-0871.
Zhang
Yanglong
Y
Kotani
Norihiro
N
Watanabe
Tae
T
Lee
Seungho
S
Wang
Xiangchun
X
Kawashima
Ikuo
I
Tai
Tadashi
T
Taniguchi
Naoyuki
N
Honke
Koichi
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006017:Glycolipids / Q000276:immunology*; D006825:Hybridomas / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology; D013433:Sulfoglycosphingolipids / Q000276:immunology*; D015238:Sulfotransferases / Q000235:genetics; D013737:Testis / Q000276:immunology
D006017:Glycolipids; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D013433:Sulfoglycosphingolipids; C009233:seminolipid; D015238:Sulfotransferases; C020556:galactosylceramide sulfotransferase