Prediction of heparin binding of mutated short sequences of rat thyroglobulin.

BACKGROUND: Binding of thyroglobulin (Tg) to heparin is involved in Tg transcytosis via megalin. Rat Tg (rTg) binds to heparin through an exposed carboxyl terminal region (RELPSRRLKRPLPVK, Arg2489-Lys2503) rich in positively charged residues. This region is not entirely conserved in human Tg (hTg) (Arg2489-Glu2503, REPPARALKRSLWVE), resulting in lower affinity binding. Here, we developed a score to predict to what extent secondary structure modifications affect the heparin-binding ability of rTg. METHODS: We designed eight synthetic peptides, including one with the Arg2489-Lys2503 sequence of rTg (rTgP), one with the corresponding sequence of hTg (hTgP), and six "mutant" peptides, each carrying a point mutation obtained by replacing one amino acid residue of rTgP with the corresponding residue of hTgP. Heparin binding was assessed in solid-phase assays. The Bmax and the constants of dissociation (Kd) were calculated. RESULTS: Using a no-fee online service, we obtained predictions of peptide secondary structures and developed a scoring system to estimate to what extent mutations are expected to modify rTg secondary structure. The score was designated as Probability of Secondary Structure Change (PSSC) and it significantly correlated with the BMax (R = 0.942, P < 0.001) and the Kds (R = - 0.744, P < 0.01) of heparin binding of hTgP and of the "mutant" peptides. CONCLUSIONS: The PSSC score allows predicting to what extent point mutations are likely to affect the heparin-binding ability of short sequences of proteins: in this case rTg, regardless of whether mutations affect charge of the sequence. The secondary structure of Tg is likely to play a role in heparin binding.

Toward bifunctional antibody catalysis.

Antibodies that catalyze the deprotonation of unactivated benzisoxazoles to give the corresponding salicylonitriles were prepared using as antigen a 2-aminobenzimidazolium derivative coupled to a carrier protein via its benzene ring. The hapten was designed to induce an antibody binding site with both a base and an acid, in position to initiate proton transfer and stabilize developing negative charge at the phenoxide leaving group, respectively. Consistent with this design, the catalysts exhibit bell-shaped pH-rate profiles, while chemical modification identified several functional groups that could participate in bifunctional catalysis. One of the antibodies, 13G5, is particularly notable in catalyzing the elimination of 6-glutaramidebenzisoxazole with a > 10(5)-fold rate acceleration over background and an effective molarity of > 10(4) M for its catalytic base. These properties compare favorably to the efficiencies achieved by the best previously characterized antibodies with substantially more reactive substrates.

A common T-cell epitope between human thyroglobulin and human thyroid peroxidase is related to murine experimental autoimmune thyroiditis.

We have investigated functional common T-cell epitopes between human thyroglobulin (hTg) and human thyroid peroxidase (hTPO) in mice. Four hTg peptides, Tg-P1, Tg-P2, Tg-P3 and Tg-P4, in which 5 amino acid residues are identical to those of hTPO, and 1 hTPO peptide, TPO-P4 relevant to Tg-P4, were prepared. Among these peptides, only Tg-P4 (residues 2730-2743) and TPO-P4 (residues 118-131) were highly antigenic and both peptides shared the common T-cell epitope. In addition, when the spleen cells from mice immunized with mouse Tg (mTg) were restimulated in vitro by Tg-P4 or TPO-P4 as well as by mTg, these cells transferred thyroiditis to naive recipient mice. These findings indicate that this common T-cell epitope between hTg and hTPO is immunogenic and related to the development of murine experimental autoimmune thyroiditis.

Synthesis and examination of antigenicity of four hormonogenic sites and two non-hormonogenic sites of human thyroglobulin.

Four peptides (HTg-1, 1-10; HTg-2, 2547-2558; HTg-4, 2592-2603; HTg-6, 2737-2748) which contain hormonogenic acceptor tyrosine (Tyr) residues and two control peptides (HTg-3, 2582-2591; HTg-5, 2687-2694) of human thyroglobulin (Tg) were synthesized, radioiodinated and their binding with serial anti-human Tg antisera which had been raised in two rabbits (TG-1, TG-2) tested. Although increased binding of each of the six peptides was observed, HTg-4 and HTg-2 had higher binding whereas HTg-1 and HTg-6 showed lower binding with the immune gamma globulin from both rabbits. Each of the six peptides was iodinated with inorganic iodine (127I) using the chloramine-T method and the inhibitory activity of each peptide on the interaction between 125I-T4 and anti-Tg antibodies was tested. At the same time, Tg obtained from a normal thyroid tissue (NTg, iodine content 0.38%) and from a Hürthle cell adenoma (CTg, iodine content 0.000%) were also tested for inhibition of 125I-T4 binding. 125I-T4 binding with rabbit anti-Tg antisera was displaced not only by NTg and CTg but also by three out of four hormonogenic peptides. Among the three peptides, HTg-2 had the highest inhibitory activity, inhibiting 125I-T4 binding to the extent of 21.5% (TG-1) and 16.0% (TG-2). Two control peptides (HTg-3, HTg-5) did not inhibit 125I-T4 binding with anti-Tg antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

A facile synthesis of an aldehydic analog of platelet activating factor and its use in the production of specific antibodies.

The multistep synthesis of a platelet activating factor (PAF) analog having a reactive aldehyde group at the omega-end of the sn-1 position is described. A novel ozonolysis of a double bond was employed to generate the aldehyde group in high yield under mild conditions. The aldehyde group was generated at the last step of the synthesis to avoid any reactions of protection and deprotection. The natural chiral center at the sn-2 position was introduced at the first step so that no steric resolution of the final product was needed. This analog of PAF was conjugated to thyroglobulin via reductive amination and then used to immunize rabbits for production of specific antibodies. The purified antibodies bind stereospecifically to tritiated PAF and crossreact minimally with lyso-PAF, plasmalogens and other phospholipids. The solid-phase radioimmunoassay thus developed detects as low as 20 pg of PAF per assay tube and should be applicable to the quantitation of PAF in biological systems.