Expression and biological activity of Baculovirus generated wild-type human slow alpha tropomyosin and the Met9Arg mutant responsible for a dominant form of nemaline myopathy.
We have previously reported a Met9Arg mutation in the human skeletal muscle alpha tropomyosin gene (TPM3) associated with autosomal dominant nemaline myopathy [Nat. Genet. 9 (1995) 75]. We describe here the generation of wild-type (Wt-tpm3) and Met9Arg (M9R-tpm3) mutant human skeletal muscle slow alpha tropomyosin using the Baculovirus expression vector system (BEVS). This system produces correct posttranslationally modified recombinant tropomyosin proteins in insect cells. We show that the interactions of Wt-tpm3 with actin and tropomyosin are comparable to those of fast alpha tropomyosin isolated from chicken striated muscle. However, the recombinant M9R-tpm3 is at least 100 times less effective at binding actin than Wt-tpm3. This paper represents the first study of this mutation directly on the human isoform of tropomyosin that is involved in nemaline myopathy. It also represents the first time that human tpm3 has been produced using BEVS. This system can now be used to accurately demonstrate the effect of this (and other disease-associated tropomyosin mutations) on the interactions of tpm3 with the other protein components of the muscle thin filament, including those responsible for differing forms of nemaline myopathy.
Akkari
P Anthony
PA
Center for Neuromuscular and Neurological Disorders, The University of Western Australia, 'A' Block 4th floor, QE II Medical Center, Verdun Street, Nedlands, Australia. aakkari@cyllene.uwa.edu.au
Song
Yuhua
Y
Hitchcock-DeGregori
Sarah
S
Blechynden
Lori
L
Laing
Nigel
N
D000199:Actins / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D005822:Genetic Vectors; D006801:Humans; D007526:Isoelectric Point; D013058:Mass Spectrometry; D009154:Mutation; D017696:Myopathies, Nemaline / Q000235:genetics* / Q000378:metabolism; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000378:metabolism; D014335:Tropomyosin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D000199:Actins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C498783:TPM3 protein, human; D014335:Tropomyosin