Dissociation of the carbohydrate-binding and splicing activities of galectin-1.
Galectin-1 (Gal1) and galectin-3 (Gal3) are two members of a family of carbohydrate-binding proteins that are found in the nucleus and that participate in pre-mRNA splicing assayed in a cell-free system. When nuclear extracts (NE) of HeLa cells were subjected to adsorption on a fusion protein containing glutathione S-transferase (GST) and Gal3, the general transcription factor II-I (TFII-I) was identified by mass spectrometry as one of the polypeptides specifically bound. Lactose and other saccharide ligands of the galectins inhibited GST-Gal3 pull-down of TFII-I while non-binding carbohydrates failed to yield the same effect. Similar results were also obtained using GST-Gal1. Site-directed mutants of Gal1, expressed and purified as GST fusion proteins, were compared with the wild-type (WT) in three assays: (a) binding to asialofetuin-Sepharose as a measure of the carbohydrate-binding activity; (b) pull-down of TFII-I from NE; and (c) reconstitution of splicing in NE depleted of galectins as a test of the in vitro splicing activity. The binding of GST-Gal1(N46D) to asialofetuin-Sepharose was less than 10% of that observed for GST-Gal1(WT), indicating that the mutant was deficient in carbohydrate-binding activity. In contrast, both GST-Gal1(WT) and GST-Gal1(N46D) were equally efficient in pull-down of TFII-I and in reconstitution of splicing activity in the galectin-depleted NE. Moreover, while the splicing activity of the wild-type protein can be inhibited by saccharide ligands, the carbohydrate-binding deficient mutant was insensitive to such inhibition. Together, all of the results suggest that the carbohydrate-binding and the splicing activities of Gal1 can be dissociated and therefore, saccharide-binding, per se, is not required for the splicing activity.
Voss
Patricia G
PG
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Gray
Richard M
RM
Dickey
Seth W
SW
Wang
Weizhong
W
Park
Jung W
JW
Kasai
Ken-Ichi
K
Hirabayashi
Jun
J
Patterson
Ronald J
RJ
Wang
John L
JL
D017398:Alternative Splicing; D002241:Carbohydrates / Q000737:chemistry*; D002467:Cell Nucleus / Q000378:metabolism; D037483:Galectin 1 / Q000737:chemistry*; D037502:Galectin 3 / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D011485:Protein Binding; D040901:Proteomics / Q000379:methods; D012313:RNA / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017381:Spliceosomes / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
D002241:Carbohydrates; D037483:Galectin 1; D037502:Galectin 3; D011994:Recombinant Proteins; D014157:Transcription Factors; D012313:RNA; D005982:Glutathione Transferase