Nebulin is a full-length template of actin filaments in the skeletal muscle sarcomere: an immunoelectron microscopic study of its orientation and span with site-specific monoclonal antibodies

1993 ◽  
Vol 14 (5) ◽  
pp. 476-483 ◽  
Author(s):  
John Wright ◽  
Qi-Quan Huang ◽  
Kuan Wang
Keyword(s):  
Skeletal Muscle ◽  
Monoclonal Antibodies ◽  
Microscopic Study ◽  
Actin Filaments ◽  
Full Length ◽  
Site Specific

scholarly journals Isolation of Nebulin from Rabbit Skeletal Muscle and Its Interaction with Actin

2010 ◽  
Vol 2010 ◽  
pp. 1-8 ◽  
Author(s):  
Ryo Chitose ◽  
Atsushi Watanabe ◽  
Masato Asano ◽  
Akira Hanashima ◽  
Kouhei Sasano ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Column Chromatography ◽  
Salt Solution ◽  
Actin Filaments ◽  
Thin Filament ◽  
Full Length ◽  
Rabbit Skeletal Muscle ◽  
Native Form ◽  
20 Nm ◽  
Vertebrate Skeletal Muscle

Nebulin is about 800 kDa filamentous protein that binds the entire thin filament of vertebrate skeletal muscle sarcomeres. Nebulin cannot be isolated from muscle except in a completely denatured form by direct solubilization of myofibrils with SDS because nebulin is hardly soluble under salt conditions. In the present study, nebulin was solubilized by a salt solution containing 1 M urea and purified by DEAE-Toyopearl column chromatography via 4 M urea elution. Rotary-shadowed images of nebulin showed entangled knit-like particles, about 20 nm in diameter. The purified nebulin bound to actin filaments to form loose bundles. Nebulin was confirmed to bind actin,α-actinin,β-actinin, and tropomodulin, but not troponin or tropomyosin. The data shows that full-length nebulin can be also obtained in a functional and presumably native form, verified by data from experiments using recombinant subfragments.

Monoclonal Antibodies as Probes of Triad Structure and Excitation-Contraction Coupling in Skeletal Muscle

1990 ◽  
pp. 371-382 ◽  
Author(s):  
Mario S. Rosemblatt ◽  
Gonzalo Pérez ◽  
Bojena Antoniu ◽  
Evelyn Reilley ◽  
Noriaki Ikemoto
Keyword(s):  
Skeletal Muscle ◽  
Monoclonal Antibodies ◽  
Excitation Contraction Coupling ◽  
Contraction Coupling

scholarly journals The Instability of Dimeric Fc-Fusions Expressed in Plants Can Be Solved by Monomeric Fc Technology

2021 ◽  
Vol 12 ◽  
Author(s):  
Pia Gattinger ◽  
Shiva Izadi ◽  
Clemens Grünwald-Gruber ◽  
Somanath Kallolimath ◽  
Alexandra Castilho
Keyword(s):  
Monoclonal Antibodies ◽  
Fusion Proteins ◽  
Degradation Products ◽  
Therapeutic Proteins ◽  
Cost Effective ◽  
Full Length ◽  
Peptide Sequence ◽  
Reporter Protein ◽  
The Stability

The potential therapeutic value of many proteins is ultimately limited by their rapid in vivo clearance. One strategy to limit clearance by metabolism and excretion, and improving the stability of therapeutic proteins, is their fusion to the immunoglobulin fragment crystallizable region (Fc). The Fc region plays multiple roles in (i) dimerization for the formation of “Y”-shaped structure of Ig, (ii) Fc-mediated effector functions, (iii) extension of serum half-life, and (iv) a cost-effective purification tag. Plants and in particular Nicotiana benthamiana have proven to be suitable expression platforms for several recombinant therapeutic proteins. Despite the enormous success of their use for the production of full-length monoclonal antibodies, the expression of Fc-fused therapeutic proteins in plants has shown limitations. Many Fc-fusion proteins expressed in plants show different degrees of instability resulting in high amounts of Fc-derived degradation products. To address this issue, we used erythropoietin (EPO) as a reporter protein and evaluated the efforts to enhance the expression of full-length EPO-Fc targeted to the apoplast of N. benthamiana. Our results show that the instability of the fusion protein is independent from the Fc origin or IgG subclass and from the peptide sequence used to link the two domains. We also show that a similar instability occurs upon the expression of individual heavy chains of monoclonal antibodies and ScFv-Fc that mimic the “Y”-shape of antibodies but lack the light chain. We propose that in this configuration, steric hindrance between the protein domains leads to physical instability. Indeed, mutations of critical residues located on the Fc dimerization interface allowed the expression of fully stable EPO monomeric Fc-fusion proteins. We discuss the limitations of Fc-fusion technology in N. benthamiana transient expression systems and suggest strategies to optimize the Fc-based scaffolds on their folding and aggregation resistance in order to improve the stability.

Discrimination of rat Brunner's gland carbohydrate antigens by site-specific monoclonal antibodies

2016 ◽  
Vol 432 ◽  
pp. 76-82 ◽  
Author(s):  
Tomoyuki Chimuro ◽  
Hiroyuki Kuroyama ◽  
Yukinobu Goso ◽  
Kazuhiko Ishihara ◽  
Makoto Kurihara
Keyword(s):  
Monoclonal Antibodies ◽  
Carbohydrate Antigens ◽  
Site Specific ◽  
Brunner’S Gland ◽  
Brunner's Gland

Complete sequence and in vitro expression of a tissue-specific phosphatidylinositol-linked N-CAM isoform from skeletal muscle

Development ◽  
1988 ◽  
Vol 104 (1) ◽  
pp. 165-173 ◽  
Author(s):  
C.H. Barton ◽  
G. Dickson ◽  
H.J. Gower ◽  
L.H. Rowett ◽  
W. Putt ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Cell Surface ◽  
Protein Sequence ◽  
Single Copy ◽  
In Vitro Transcription ◽  
Full Length ◽  
Covalent Attachment ◽  
Size Difference

Neural cell adhesion molecules (N-CAMs) are a family of cell surface sialoglycoproteins encoded by a single copy gene. A full-length cDNA clone that encodes a nontransmembrane phosphatidylinositol (PI) linked N-CAM of Mr 125 × 10(3) has been isolated from a human skeletal muscle cDNA library. The deduced protein sequence encodes a polypeptide of 761 amino acids and is highly homologous to the N-CAM isoform in brain of Mr 120 × 10(3). The size difference between the 125 × 10(3). The size difference between the 125 × 10(3) Mr skeletal muscle form and the 120 × 10(3) Mr N-CAM form from brain is accounted for by the insertion of a block of 37 amino acids called MSD1, in the extracellular domain of the muscle form. Transient expression of the human cDNA in COS cells results in cell surface N-CAM expression via a putative covalent attachment to PI-containing phospholipid. Linked in vitro transcription and translation experiments followed by immunoprecipitation with anti-N-CAM antibodies demonstrate that the full-length clone of 761 amino acid coding potential produces a core polypeptide of Mr 110 × 10(3) which is processed by microsomal membranes to yield a 122 × 10(3) Mr species. Taken together, these results demonstrate that the cloned cDNA sequence encodes a lipid-linked, PI-specific phospholipase C releasable surface isoform of N-CAM with core glycopeptide molecular weight corresponding to the authentic muscle 125 × 10(3) Mr N-CAM isoform. This is the first direct correlation of cDNA and deduced protein sequence with a known PI-linked N-CAM isoform from skeletal muscle.

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