Immunological determinants of nerve growth factor involved in p140^trk (Trk) receptor binding

Abstract

Monoclonal anti-NGF antibodies that specifically inhibit the biological activity of mouse beta-NGF were used to study the structural determinants involved in the interaction of NGF with its receptors gp75LNGFR and Trk. None of the three antibodies--N60, M15, and 27/21--showed any reactivity toward denatured NGF. Three experimental methods--radioimmunoassay (RIA), enzyme-linked immunoassay (ELISA), and slot blots--detected no significant cross reactivity between the antibodies and BDNF or NT-3. RIA showed that M15 and N60 recognize the same or an overlapping antigenic site, but 27/21 recognizes a different epitope. Only 27/21, and not N60 or M15, immunoprecipitated beta-NGF crosslinked to LNGFR receptor. Thus, the epitope recognized by 27/21 does not overlap the LNGFR receptor binding site. N60, M15, and 27/21 all block binding of NGF to Trk in a manner consistent with competitive inhibition. Purified Fab fragments of N60 and M15 gave similar results to the intact antibodies. The other subunits present in the 7S complex of NGF, i.e. the alpha and gamma subunits, competitively inhibited binding of antibodies to beta-NGF. Only the gamma subunit inhibited phosphorylation of Trk and biological activity of beta-NGF. These findings suggest that the M15, N60, and 27/21 antibodies bind to a specific site on the surface of NGF where they competitively inhibit binding to the Trk NGF receptor. The region encompassing the N-terminus, the C-terminus, and the loop on the surface of beta-NGF containing residues 60-80 is proposed as important for binding to the Trk receptor.

Construction of a mutated pro-nerve growth factor resistant to degradation and suitable for biophysical and cellular utilization

“…This step purified and concentrated proNGF123 Ϸ30-fold. This partially purified proNGF123 then was purified further to Ϸ99% purity and 300-fold enriched by using an immunoaffinity column made with the N-60 monoclonal antibody (30) (Fig. 3 B and C).…”

A nerve growth factor peptide retards seizure development and inhibits neuronal sprouting in a rat model of epilepsy.

“…In these experiments, we used a conformationally constrained peptide that mimics both the conformation and sequence of the variable L3 region of NGF (26), which is thought to be part of a trkA-binding domain of the NGF molecule (29). The peptide R-1 1 was designed from two noncontiguous elements of NGF making up the loop region L3 (CRASNPVESGC, residues 58-68) and part of the cysteine knot region (CVC, residues 108-110) of NGF.…”

“…Although the receptor-binding domains of the neurotrophin molecules have yet to be fully elucidated, a variety of data including a 2.3-A x-ray crystal solution of NGF (25) implicate the variable regions within the molecule in receptor binding (26)(27)(28)(29). Although intraventricularly administered antibodies against NGF have already been shown to inhibit amygdaloid kindling (22,23) and kindlinginduced mossy fiber sprouting (23), it has not yet been demonstrated directly that these in vivo effects are receptor mediated.…”

A TrkA-selective, Fast Internalizing Nerve Growth Factor-Antibody Complex Induces Trophic but Not Neuritogenic Signals

“…If both NGF epitopes are occupied with the capture mAb, no subsequent binding of mAb NGF30-biotin would occur. This sandwich ELISA was used previously to study anti-NGF mAb 27/21 which binds one epitope of an NGF protomer and leaves the epitope of the other NGF protomer exposed and available for binding (38). mAb NGF30 with captured NGF did not allow substantial binding of NGF30-biotin (Table II, (Table II, row 2), or to nonspecific binding to BSA (Table II, row 4).…”