Unsusceptibility of recombinant human Fc fragments of immunoglobulin E to thrombin.
Human immunoglobulin E (IgE) contains a potential recognition sequence for thrombin protease cleavage at N-terminal end of C epsilon 3 domain responsible for binding to alpha chain of high affinity Fc receptor for IgE (Fc epsilon RI alpha), but it remains unknown for the enzyme susceptibility. The human Fc fragments of IgE (IgE-Fc), consisting of C epsilon 2' (Ala329)-C epsilon 3-C epsilon 4 chains (Fc') and of C epsilon 2' (Thr315-Ala329)-C epsilon 3-C epsilon 4 chains (F(c')2), were expressed in the mammalian COS and Chinese hamster ovary (CHO) cells by placing the IgE-Fc cDNA under the control of the cytomegalovirus (CMV) promoter. Under nonreducing condition F(c')2 was not cleaved by thrombin protease as well as native IgE. Neither treatment of F(c')2 with final 0.1% 2-mercaptoethanol at boiling point for 5 min, with a sulfhydryl-reactive biotin derivative, by which monomers in COS-derived F(c')2 preparations were biotinylated at Cys328, nor with neuraminidase, affected the accessibility to the enzyme. These results suggested that F(c')2, either dimeric or monomeric, had compact conformations.
Kamiya
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Central Research Laboratories, Green Cross Corporation, Osaka, Japan.
D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D006801:Humans; D007073:Immunoglobulin E / Q000737:chemistry*; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D008623:Mercaptoethanol; D009439:Neuraminidase; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
D007141:Immunoglobulin Fc Fragments; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D008623:Mercaptoethanol; D001710:Biotin; D009439:Neuraminidase; D013917:Thrombin