Distinct effects on splicing of two monoclonal antibodies directed against the amino-terminal domain of galectin-3

Previous experiments had established that galectin-3 (Gal3) is a factor involved in cell-free splicing of pre-mRNA. Addition of monoclonal antibody NCL-GAL3, whose epitope maps to the NH 2-terminal 14 amino acids of Gal3, to a splicing-competent nuclear extract inhibited the splicing reaction. In co...

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Bibliographic Details
Published in:Archives of biochemistry and biophysics 2008-07, Vol.475 (2), p.100-108
Main Authors: Gray, Richard M., Davis, Michael J., Ruby, Katherine M., Voss, Patricia G., Patterson, Ronald J., Wang, John L.
Format: Article
Language:English
Subjects:
Amino Acid Motifs
Amino Acid Sequence
Animals
Antibodies, Monoclonal - immunology
Carbohydrate-binding proteins
Epitope Mapping
Galectin 3 - chemistry
Galectin 3 - genetics
Galectin 3 - isolation & purification
Galectin 3 - metabolism
Glutathione Transferase - metabolism
HeLa Cells
Humans
Hybridomas
Lectins
Molecular Sequence Data
Monoclonal antibodies
Protein Structure, Tertiary
Rats
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - isolation & purification
Recombinant Fusion Proteins - metabolism
RNA Precursors - genetics
RNA Precursors - metabolism
RNA processing
RNA Splicing
Spliceosome
Spliceosomes
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Summary:Previous experiments had established that galectin-3 (Gal3) is a factor involved in cell-free splicing of pre-mRNA. Addition of monoclonal antibody NCL-GAL3, whose epitope maps to the NH 2-terminal 14 amino acids of Gal3, to a splicing-competent nuclear extract inhibited the splicing reaction. In contrast, monoclonal antibody anti-Mac-2, whose epitope maps to residues 48–100 containing multiple repeats of a 9-residue motif PGAYPGXXX, had no effect on splicing. Consistent with the notion that this region bearing the PGAYPGXXX repeats is sequestered through interaction with the splicing machinery and is inaccessible to the anti-Mac-2 antibody, a synthetic peptide containing three perfect repeats of the sequence PGAYPGQAP (27-mer) inhibited the splicing reaction, mimicking a dominant-negative mutant. Addition of a peptide corresponding to a scrambled sequence of the same composition (27-mer-S) failed to yield the same effect. Finally, GST–hGal3(1–100), a fusion protein containing glutathione- S-transferase and a portion of the Gal3 polypeptide including the PGAYPGXXX repeats, also exhibited a dominant-negative effect on splicing.
ISSN:0003-9861
1096-0384
DOI:10.1016/j.abb.2008.04.010