Purification and characterisation of a brain‐derived neurotrophic factor/ neurotrophin‐3 (BDNF/NT‐3) heterodimer

Abstract

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are known to exist in solution as non-covalently linked homodimeric proteins. The recent elucidation of the crystal structure of the NGF homodimer, as well as the conservation of structural motifs in the neurotrophins, raised the possibility that neurotrophin heterodimers might also occur. The formation of BDNFNT-3 heterodimers was explored using a vaccinia virus expression system. Upon co-infection of cells with viruses expressing BDNF and NT-3, we could identify a BDNFNT-3 heterodimer as a biosynthetic product and separate it from the BDNF and NT-3 homodimers. We could also show that the BDNF/NT-3 heterodimers can be formed irrespective of wild-type or exchanged prosequences, indicating that prosequence specificity does not influence dimer formation. In all neuronal survival assays that were used, the BDNFNT-3 heterodimer was shown to be 10-fold less active compared with a mixture of BDNF and NT-3 homodimers. This lower specific activity was also measured in a neuronal population co-expressing receptors for BDNF and NT-3. The low biological activity of the heterodimer observed with neurons was not paralleled by a reduced ability of the heterodimer to interact with trkB or trkC receptors, as assessed by the induction of tyrosine phosphorylation of these receptors expressed by fibroblast cell lines.

Neurotrophin-4, Alone or Heterodimerized with Brain-derived Neurotrophic Factor, Is Sorted to the Constitutive Secretory Pathway

“…NT-4/BDNF Heterodimers Are Formed within Co-transfected Cells-Neurotrophins are normally released as homodimers, but co-expression of different members of the neurotrophin family can result in the formation of bioactive heterodimers (7,32). Previously, we reported that co-expression of NT-3 with BDNF results in secretion of BDNF/NT-3 heterodimers and that heterodimerizing NT-3 with BDNF redirects NT-3 from the constitutive to the regulated secretory pathway (2).…”

Contributions of the Optic Tectum and the Retina as Sources of Brain-Derived Neurotrophic Factor for Retinal Ganglion Cells in the Chick Embryo

“…The retinal concentration of BDNF after injection was ϳ100 -500 times higher (total BDNF ϭ 5-10 ng) than the concentration of endogenous BDNF at this age (total BDNF/retina Ϸ 50 pg) ; J. E. Johnson, Wake Forest University, personal communication). Alternative explanations of BDNF immunoprecipitation with trkC antibodies include the possibilities that heterodimers of exogenous BDNF and endogenous NT-3 form that bind to trkC (Jungbluth et al, 1994;Philo et al, 1994), that trk and p75 may coprecipitate (Huber and Chao, 1995), or that the trkC antibody may recognize certain isoforms of trkB (Garner et al, 1996) although it does not recognize the predominant form of trkB (Lefcort et al, 1996).…”

“…To determine the localization of BDNF at the protein level, we used a previously characterized affinitypurified serum raised against a peptide (B6) of the N-terminal portion of BDN F. This antibody recognizes BDN F in Western blots (Jungbluth et al, 1994) and does not cross-react with NT-3. When this antibody was applied to the retinae of 8.5-d-old chick embryos, the GCL, but no other layer in the retina, was heavily labeled (Fig.…”

“…A rabbit serum was raised against the N-terminal portion of mouse BDN F (His-Ser-Asp-Pro-Ala-Arg-ArgGly-Glu-Leu-Ser-Val-C ys) coupled to K L H. This serum was affinitypurified by the peptide coupled to Sepharose, as described elsewhere (Jungbluth et al, 1994). For controls the purified serum was incubated at a dilution of 1:200 in the presence of the BDN F peptide and the neurotrophin-3 (N T-3) peptide representing an N T-3 sequence (T yrAla-Glu-His-Lys-Ser-His-Arg-Gly-Glu-T yr-Ser-Val-C ys) coupled to Sepharose beads, respectively, in 0.1% Triton X-100 in PBS.…”

Neurotrophins and B-cell malignancies

“…Neurotrophins naturally form homo-dimers and have been shown to form hetero-dimers experimentally, though the functionality of hetero-dimers is somewhat in question [48,49].…”