The glycophosphatidylinositol anchor affects the conformation of Thy-1 protein.
Thy-1 has the structure of a single variable-type immunoglobulin domain anchored to the external face of the plasma membrane via a glycophosphatidylinositol moiety. When the lipid is removed from this anchor by either phospholipase C or D, the reactivity of the delipidated Thy-1 for a range of antibodies, including those known to be determined by amino acid residues, is impaired. We have investigated in detail the effect of delipidation on the reaction with the OX7 monoclonal antibody, determined by the allelic variant residue Arg 89. Analysis of the kinetics of OX7 binding shows that delipidation affects primarily the dissociation of antibody, increasing the dissociation rate constant kdiss from 0.27 x 10(-3) s-1 to 2.39 x 10(-3) s-1. Addition of phospholipase to preformed antibody-antigen complex causes an immediate change from the slow to the faster dissociation rate, implying that delipidation induces a conformational change in the Thy-1 protein that is sufficiently strong to dissociate bound antibody. This conformational change can be demonstrated directly by the circular dichroism spectrum of human Thy-1 that detects changes in the environment of Tyr residues located near the antigenic epitopes. Molecular dynamics studies suggest that, on delipidation, a conformational change occurs in the glycan chain that affects the protein in the region of the antigenic epitopes. This study thus demonstrates that the glycophosphatidylinositol anchor strongly influences the conformation of Thy-1 protein by a mechanism that could occur generally with membrane proteins of this class.
Barboni
E
E
Laboratory of Neurobiology, National Institute for Medical Research, Mill Hill, London, UK.
Rivero
B P
BP
George
A J
AJ
Martin
S R
SR
Renoup
D V
DV
Hounsell
E F
EF
Barber
P C
PC
Morris
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003902:Detergents; D000939:Epitopes / Q000378:metabolism; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000502:physiology*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D050356:Lipid Metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D018800:Thy-1 Antigens / Q000737:chemistry*
D003902:Detergents; D000939:Epitopes; D017261:Glycosylphosphatidylinositols; D018800:Thy-1 Antigens