Characterization of the bovine neurofilament NF-M protein and cDNA sequence, and identification of in vitro and in vivo calpain cleavage sites.
Neurofilaments are the major components of the neuronal cytoskeleton, and accumulations of these proteins are associated with several important human diseases. Here we report the cloning and sequencing of bovine NF-M, the first NF-M cloned from a large domestic mammal. The bovine sequence proves to be generally more similar to that of human NF-M than the previously described mammalian sequences, suggesting that bovine neurofilaments are a useful model for biochemical studies of application to humans. However, we noted some unusual features within the 16 lys-ser-pro (KSP2) type phosphopeptide repeats and also note that the number of these repeats correlates well with the size of animal. We also characterized two in vitro calpain cleavage sites by direct peptide sequencing, finding that both are located in the glutamic acid rich E segment. Finally, we show biochemically that the more abundant and stable of these calpain fragments can also be detected in vivo.
Shaw
Gerry
G
Department of Neuroscience, University of Florida College of Medicine, McKnight Brain Institute, Gainesville, FL 32610, USA.
Yang
Cui
C
Zhang
Lu
L
Cook
Petra
P
Pike
Brian
B
Hill
William D
WD
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D002417:Cattle; D045744:Cell Line, Tumor; D002645:Chickens; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D018604:Epitope Mapping; D006801:Humans; D016900:Neurofilament Proteins / Q000235:genetics* / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein
D000906:Antibodies; D018076:DNA, Complementary; D016900:Neurofilament Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C061195:neurofilament protein M; D002154:Calpain