Biosynthesis and post-translational processing of the precursor to brain-derived neurotrophic factor.
We examined the biosynthesis and post-translational processing of the brain-derived neurotrophic factor precursor (pro-BDNF) in cells infected with a pro-BDNF-encoding vaccinia virus. Metabolic labeling, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis reveal that pro-BDNF is generated as a 32-kDa precursor that is N-glycosylated and glycosulfated on a site, within the pro-domain. Some pro-BDNF is released extracellularly and is biologically active as demonstrated by its ability to mediate TrkB phosphorylation. The precursor undergoes N-terminal cleavage within the trans-Golgi network and/or immature secretory vesicles to generate mature BDNF (14 kDa). Small amounts of a 28-kDa protein that is immunoprecipitated with BDNF antibodies is also evident. This protein is generated in the endoplasmic reticulum through N-terminal cleavage of pro-BDNF at the Arg-Gly-Leu-Thr(57)- downward arrow-Ser-Leu site. Cleavage is abolished when Arg(54) is changed to Ala (R54A) by in vitro mutagenesis. Blocking generation of 28-kDa BDNF has no effect on the level of mature BDNF and blocking generation of mature BDNF with alpha(1)-PDX, an inhibitor of furin-like enzymes, does not lead to accumulation of the 28-kDa form. These data suggest that 28-kDa pro-BDNF is not an obligatory intermediate in the formation of the 14-kDa form in the constitutive secretory pathway.
Mowla
S J
SJ
Salk Institute, La Jolla, California 92037-1099, USA.
Farhadi
H F
HF
Pareek
S
S
Atwal
J K
JK
Morris
S J
SJ
Seidah
N G
NG
Murphy
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D019208:Brain-Derived Neurotrophic Factor / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004622:Embryo, Mammalian; D006026:Glycoside Hydrolases; D006031:Glycosylation; D006624:Hippocampus / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009457:Neuroglia / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D010766:Phosphorylation; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D020813:Receptor, trkB / Q000187:drug effects / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics
D019208:Brain-Derived Neurotrophic Factor; D011498:Protein Precursors; D011994:Recombinant Proteins; C426674:brain-derived neurotrophic factor precursor; D020813:Receptor, trkB; D006026:Glycoside Hydrolases