Purification and identification of monoubiquitin-phosphoglycerate mutase B complex from human colorectal cancer tissues.
Ubiquitin-conjugated proteins in human colorectal cancer tissues were analyzed by the immunoprecipitation with the antibody FK2 against conjugated ubiquitin followed with SDS-PAGE. In these immunoprecipitable proteins, a 38-kDa protein was abundant in the tumor regions but almost absent in the adjacent normal regions in 17/26 patients, thus we attempted to purify it. Using immunoaffinity chromatography with the antibody FK2 followed by gel filtration and SDS-PAGE, approximately 10 pmol of this protein was separated from 34 g of the pooled cancerous tissue and transferred onto a PVDF membrane. The 38-kDa protein was further digested with Achromobacter protease I, resulting in several peptide fragments. Amino acid sequences of these peptides showed complete sequence identity to those derived from either ubiquitin or phosphoglycerate mutase-B, suggesting that the 38-kDa protein is monoubiquitinated phosphoglycerate mutase-B, whose calculated mass is 37,369 Da. Western blot using an antibody against phosphoglycerate mutase-B revealed the presence of the 38-kDa protein in the anti-ubiquitin immunoprecipitates derived from the tumor regions, but not from normal counterparts. In addition, part of non-ubiquitinated phosphoglycerate mutase-B (29 kDa) was also found in the anti-ubiquitin immunoprecipitates, whose levels were higher in the tumor regions than in the adjacent normal regions. These results suggest that monoubiquitination of phosphoglycerate mutase-B as well as formation of a noncovalent complex containing ubiquitin and phosphoglycerate mutase-B increases in colorectal cancer and novel modification of phosphoglycerate mutase-B might have a pathophysiological role.
Usuba
T
T
Department of Surgery, Jikei University School of Medicine, Nishishinbashi, Minato-ku, Tokyo, Japan.
Ishibashi
Y
Y
Okawa
Y
Y
Hirakawa
T
T
Takada
K
K
Ohkawa
K
K
D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D015179:Colorectal Neoplasms / Q000201:enzymology*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010736:Phosphoglycerate Mutase / Q000737:chemistry / Q000302:isolation & purification*; D025801:Ubiquitin / Q000378:metabolism*
D025801:Ubiquitin; D010736:Phosphoglycerate Mutase