Demonstration of thyroactive smaller components released from TSAb-IgG by protease digestion.
Thyroid stimulating (TS) activity (cAMP production in thyroid cells) and TSH binding inhibition (TBI) activity (determined by TSH receptor assay) in fragments released from TSAb-IgG by protease digestion were examined. The unbound fraction (UF) and the bound fraction (BF) were separated using a protein A-Sepharose column after papain hydrolysis (more hydrolysis at pH 5.0 than pH 7.5) of TSAb-IgG. When both fractions were gel filtrated on a Sephadex G-100 column, the TS and TBI activity were found in both Fab fraction (Mr 50 kDa) and the retarded fraction (between Mr 50 and 20 kDa) in the UF, and also in the first fraction (undigested IgG, Mr 160 kDa), the second fraction (Fc with tracer amounts of Fab, Mr 50 kDa), and the retarded fraction (between Mr 50 and 20 kDa) of the BF. The biological activity in the second fraction was suggested as being derived from Fab, because the activity bound to the anti-F(ab')2 column but did not bind to the anti-Fc column. Anti-Tg and anti-TPO activities were found in Fab, but were not found in the retarded fraction that consisted of Mr 20-30 kDa. In pepsin hydrolysis the UF from the protein A column consisted of both F(ab')2 (Mr 100 kDa) and pF'c (CH3) (Mr 25 kDa), and the BF consisted of only the undigested IgG. The biological activities were found in both the F(ab')2 fraction and the retarded fraction (between Mr 100 and 25 kDa). Anti-Tg and anti-TPO activities were found in F(ab')2, but no activity was observed in the Mr 25-kDa fraction. The present study showed that the biological activity of TSAb is distributed in not only the Fab or F(ab')2 fragment, but also in thyroactive smaller components (TSC) (Mr 20-30 kDa) without antigen-binding activity such as anti-Tg and anti-TPO. We suggest that TSC may be released from the Fab fragment region of TSAb-IgG by protease hydrolysis.
Ogura
M
M
2nd Department of Internal Medicine, Kobe University, Hyogo, Japan.
Kouki
T
T
Inui
T
T
Hachiya
T
T
Ochi
Y
Y
Kajita
Y
Y
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D010450:Endopeptidases; D006111:Graves Disease / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000494:pharmacology; D018828:Immunoglobulins, Thyroid-Stimulating / Q000737:chemistry* / Q000494:pharmacology*; D007453:Iodide Peroxidase / Q000378:metabolism; D008135:Long-Acting Thyroid Stimulator / Q000737:chemistry / Q000494:pharmacology; D010206:Papain / Q000737:chemistry; D010434:Pepsin A / Q000737:chemistry; D012685:Sepharose; D013205:Staphylococcal Protein A / Q000378:metabolism; D013552:Swine; D013954:Thyroglobulin / Q000378:metabolism; D013972:Thyrotropin / Q000378:metabolism
D007140:Immunoglobulin Fab Fragments; D018828:Immunoglobulins, Thyroid-Stimulating; D013205:Staphylococcal Protein A; D013972:Thyrotropin; D013954:Thyroglobulin; D012685:Sepharose; D008135:Long-Acting Thyroid Stimulator; D007453:Iodide Peroxidase; D010450:Endopeptidases; D010206:Papain; D010434:Pepsin A