|Budget Amount *help
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1998: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1997: ¥1,600,000 (Direct Cost: ¥1,600,000)
The results obtained in this projects concerning the interaction between mouse soluble FcgammaRII (sFcgammaRII) and mouse IgG2b-Fc are summarized as follows :
1. The stable-isotope-assisted NMR analyses revealed that sFcgammaRII binds to the lower hinge and its spatial proximity of the Fc region, which are negatively charged and interact with some positively charged area on the sFcgammaRII surface via electrostatic interactions.
2. An aglycosylated Fc mutant, in which Asn297 is substituted with Ala, has completely lost the ability to bind to sFcgammaRII.We have investigated binding to sFcgammaRII of a variety of Fc analogs prepared by the treatment with endoglycosidases. It has been shown that the endoglycosidase D-treated Fc analog, which contains the carbohydrate chains composed of one GlcNAc and one Fuc residues, has remarkably reduced affinity for sFcgammaRII.The NMR data indicate that the polypeptide-carbohydrate interactions in the Fc region become less extensive upon treatment by endoglycosidase D, rendering the carbohydrate chains more mobile and the conformation of parts of the CH2 domain containing the sFcgammaRII-binding site perturbed.
3. We have identified the glycosylation sites on sFcgammaRII, determined the major glycoforms, and established the protocol for trimming of the carbohydrate chains.
4. By gel-filtration and ultracentrifugation technique, we have shown that stoichiometry of the Fc-sFcgammaRII interaction is 1 : 1. By use of NMR spectroscopy, it has also been demonstrated that the binding of sFcgammaRII onto one of the two symmetrically related sites on Fc induces conformational change in the other site, which preclude the binding of second sFcgammaRII molecule to Fc. We suggest that, by this conformational regulation, spontaneous cross-linking of two FcgammaRII molecules by one IgG molecule in the absence of multivalent antigens, which might trigger undesirable cellular responses, is prevented.