Relationship between the antigenic topography and the structure of human growth hormone.
Monoclonal antibodies (MAb) to human GH (hGH) were used to correlate the antigenic topography of the hormone with its structure. Competition experiments performed in a solid phase RIA system allowed us to measure the reactivity toward the MAb of the following hGH derivatives: hGH 20K (a natural variant lacking residues 32-46), hGH selectively modified in His or Met residues, hGH with the C and/or N-terminal disulfide bond reduced and carbamidomethylated, and hGH cleaved between residues 142-143. Results indicated that fragment 32-46 participates in the structure of epitopes EB1/EB3 and that the C-terminal bridge is located in epitope 10D6, whereas opening of both disulfide bridges alters the entire hGH antigenic surface. His-151 and Met-170 were placed in epitopes NA71 and AC8, respectively, whereas His-18 and Met-14 would be involved in the hGH antigenic domain formed by overlapping epitopes 3C11, 10C1, and HG3. MAb AE5, AE12, and AC3 define a flexible hGH region related to sequence 134-150; the respective epitopes show high conformational mobility induced by modifications in other regions of the molecule. Binding of the different hGH derivatives to lactogenic receptors from female rat liver gave some insights on the localization of the hormone-binding site. Epitopes EB1/EB3 and 10D6 were discarded because there was not a direct correlation between their drastic immunological alterations and the binding properties of the respective hGH derivatives. In the same way, epitopes AE5, AE12, and AC3 were excluded from the hGH-binding domain because a disruption in those sites did not affect the hGH interaction with receptors. We conclude that the hGH structure defined by epitopes 3C11, 10C1, and HG3 is probably related to the binding properties of the hormone.
Mazza
M M
MM
Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
Gobet
M G
MG
Biscoglio
M J
MJ
Mihajlovich
V
V
Guillemot
J C
JC
Vita
N
N
Ferrara
P
P
Retegui
L A
LA
D000818:Animals; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000276:immunology; D001667:Binding, Competitive; D055598:Chemical Phenomena; D002621:Chemistry; D002700:Chloramines; D004047:Diethyl Pyrocarbonate; D000939:Epitopes / Q000276:immunology; D005260:Female; D013006:Growth Hormone / Q000276:immunology*; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism; D015394:Molecular Structure; D010446:Peptide Fragments / Q000276:immunology; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D018000:Receptors, Peptide; D014105:Tosyl Compounds
D000911:Antibodies, Monoclonal; D000941:Antigens; D002700:Chloramines; D000939:Epitopes; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; D014105:Tosyl Compounds; C016300:chloramine-T; C028202:placental lactogen receptor; D013006:Growth Hormone; D004047:Diethyl Pyrocarbonate