The molecular process of membrane-protein integration is strictly regulated by so-called translocon, a channel composed of a protein complex. Recently, we found a novel integration-stimulating factor termed MPIase (Membrane Protein Integrase) in this integration process on Escherichia coli cells. Surprisingly, this factor was not a proteinous molecule but a sort of glycolipid. In this study, we succeeded in the extraction and purification of MPIase from E. coli strain MC4100 cells and determined its total structure. MPIase was purified from inner-membrane components of the cells, using MonoQ ion-exchange column chromatography and subsequent liquid-liquid partition column chromatography on sephadex LH-20 gel. The constituents of MPIase and its hydrolyzed products were elucidated by 1D and 2D NMR, MALDI-TOF MS, GC-MS and Q-TOF MS. These analyses demonstrated that MPIase contains trisaccharide repeating units composed of G1cNAc, ManNAcA and Fuc4NAc, a pyrophosphate diester linkage, and a diacylglycerol moiety. Furthermore, we compared 13C NMR chemical shift values of MPIase with those of synthetic disaccharide substructures to conclude the glycosidic linkages and sequence of the repeating unit as a-Fuc4NAc-(1→4)-P-ManNAcA-(1→4)-a-GlcNAc(1→3). Consequently, the total structure of MPIase was determined as shown in Figure 1.
