Purification and identification of monoubiquitin-phosphoglycerate mutase Bcomplex from human colorectal cancer tissues

Ubiquitin-conjugated proteins in human colorectal cancer tissues were analy zed by the immunoprecipitation with the antibody FK2 against conjugated ubi quitin followed with SDS-PAGE. In these immunoprecipitable proteins, a 38-k Da protein was abundant in the tumor regions but almost absent in the adjac ent normal regions in 17/26 patients, thus we attempted to purify it. Using immunoaffinity chromatography with the antibody FK2 followed by gel filtra tion 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, resu lting 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 monoubiqu itinated phosphoglycerate mutase-B, whose calculated mass is 37,369 Da. Wes tern blot using an antibody against phosphoglycerate mutase-B revealed the presence of the 38-kDa protein in the anti-ubiquitin immunoprecipitates der ived from the tumor regions, but not from normal counterparts. In addition, part of non-ubiquitinated phosphoglycerate mutase-B (29 kDa) was also foun d in the anti-ubiquitin immunoprecipitates, whose levels were higher in the tumor regions than in the adjacent normal regions. These results suggest t hat monoubiquitination of phosphoglycerate mutase-B as well as formation of a noncovalent complex containing ubiquitin and phosphoglycerate mutase-B i ncreases in colorectal cancer and novel modification of phosphoglycerate mu tase-B might have a pathophysiological role. (C) 2001 Wiley-Liss, lnc.