RESUMEN
In contrast to autoantibodies that are functionally silenced or deleted, IgG Abs that react with autologous insulin routinely follow hormone administration and arise spontaneously in autoimmune (type I) diabetes mellitus. To understand Ab interactions with autologous insulin, rat proinsulin I and 32 alanine substituted analogues were expressed as fusion proteins and used to examine 16 anti-insulin mAb in ELISA. The results identify several amino acid residues that contribute to binding by a large majority (>75%) of mAb, although no single residue is uniformly required for binding by all mAb. Replacements at charged or polar residues on the insulin surface including A4 (Asp), A5 (Gln), A9 (Ser) A12 (Ser), A17 (Gln), A18 (Asn), B13 (Glu), and B21 (Glu) consistently decreased mAb binding. Single alanine substitutions at positions A16 (Leu), A11 (Cys), B8 (Gly), and B15 (Leu) that are predicted to alter the core structure or chain folding vary widely in their impact on Ab binding. mAb that bind insulin preferentially on solid phase (i.e., ELISA) are highly sensitive to replacement of single residues, and substitutions that alter conformation abolish binding. In contrast, high affinity mAb that bind insulin in solution are relatively insensitive to substitutions at single residues, and they maintain binding to all mutants, including those with disrupted conformation. For such high affinity mAb, replacement of long hydrophobic side chains can augment binding, suggesting mAb interactions with insulin include an induced fit. Thus, the ability of insulin to function as a "molten globule" may contribute to the diversity and autoreactivity of the anti-insulin repertoire.
Asunto(s)
Alanina/genética , Anticuerpos Monoclonales/química , Mutagénesis Sitio-Dirigida , Proinsulina/genética , Proinsulina/inmunología , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos/genética , Reacciones Antígeno-Anticuerpo , Humanos , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Proinsulina/administración & dosificación , Proinsulina/metabolismo , Ratas , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , PorcinosRESUMEN
The distribution of a soluble form of a cell adhesion molecule, P-selectin, in human platelets and cultivated endothelial cells has been studied by enzyme-linked immunosorbent assay (ELISA). The concentration of soluble P-selectin in the blood plasma of healthy donors and patients with abnormal platelet count has also been determined. P-selectin was measured in the Triton X-100 lysate of platelets and endothelial cells (total P-selectin), in the 100,000g supernatant obtained after sedimentation of the membrane fraction from the homogenate of sonicated platelets and endothelial cells (intracellular soluble P-selectin), in the supernatant of activated and nonactivated platelets, and in the culture medium of endothelial cells. A soluble form of P-selectin which did not coprecipitate with the membrane fraction was detected in platelets and accounted for approximately 10% of the total P-selectin. Platelet activation by thrombin, ADP, or a thromboxane A2 analog resulted in the secretion of 30-50% of the intracellular soluble P-selectin. Measurements of P-selectin in endothelial cell culture revealed that endothelium from aorta contained about twofold more P-selectin than endothelium from umbilical vein. Intracellular soluble P-selectin was identified in both types of endothelial cells. In endothelial cells from the umbilical vein this form made up approximately 10% of the total P-selectin. Soluble P-selectin was also detected in the medium of cultivated endothelial cells, where its content correlated with the total cellular P-selectin. Concentration of P-selectin in blood plasma strongly correlated with the platelet count in the blood of healthy donors and patients with thrombocytosis and thrombocytopenia. These data indicate that platelets serve as one of the main source of plasma P-selectin. However, the presence of P-selectin in the plasma of patients with severe thrombocytopenia suggests that endothelium can also be involved in plasma P-selectin production. Thus, in vitro experiments as well as measurements of plasma P-selectin have shown that both platelets and endothelial cells can produce a soluble form of the protein. Platelet-derived soluble P-selectin and plasma P-selectin were shown to react with antibodies against the cytoplasmic domain of P-selectin. These data prove that at least part of soluble P-selectin is produced by synthesis employing special mRNA which lacks the sequence encoding the transmembrane domain, but not by the proteolytic shedding of the extracellular portion of membrane P-selectin.
Asunto(s)
Plaquetas/metabolismo , Endotelio Vascular/metabolismo , Selectina-P/biosíntesis , Anticuerpos/inmunología , Membrana Celular/metabolismo , Citoplasma/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Hidrólisis , Selectina-P/inmunología , Selectina-P/metabolismo , SolubilidadRESUMEN
Studies on insulin autoantibodies often show a lack of correlation between enzyme-linked immunosorbent assays (ELISAs) and fluid phase radioimmunoassays (RIAs). Similarly, a set of IgG anti-insulin monoclonal antibodies (mAb) from BALB/c mice are found to differ in their binding in ELISAs and RIAs. To understand the structural basis for differential insulin binding, soluble and complexed biotinylated insulin is used to confirm binding properties independent of insulin-coated plastic and radioiodination. The binding properties of intact mAb are also present in Fab fragments, indicating ligand preference is not related to valence or to the Fc portion of Ig. Analysis of binding to soluble or bound ligand in relationship to antibody variable (V) region structures indicates that differential binding in the two assays is a property of heavy chain variable region structure. Studies also show that limited amino acid replacements arising during maturation of the immune response may change the binding preference for an individual mAb. These findings indicate that differences in detection of insulin binding in solid phase and fluid phase are not artefactual but reflect intrinsic structural features of immunoglobulin interaction with insulin.