46

Noda Institute for Scientiﬁc Research GRANT
2011 Research Grant

Determination of the biosynthetic pathway of a novel
glycolipid involved in preprotein translocation andmembrane protein integration
Ken-ichi NISHIYAMACryobiofrontier Research Center, Iwate University

Research aims
Molecular mechanisms underlying preprotein translocation across, and membrane protein integration into biological membranes are conserved in all organisms at fundamental levels. The understanding of the mechanisms is not only
one of the most important subjects in cell biology, but also
important for applications such as secretory production ofuseful proteins and functional analysis of membrane proteins with unknown function as target of drug designs. We
developed a reconstitution system of protein translocationand integration in E. coli, which reﬂects the in vivo reactions precisely1,2). By means of this system, we found that
not only the known proteinaceous translocation/integration
factors but also a novel complex glycolipid are necessaryfor the reactions. A subset of membrane proteins was foundto be integrated in a manner dependent on only this glycolipid. After this function, we named this glycolipid MPIase(Membrane Protein Integration)2). This research was aimed
to clarify the biosynthetic pathway of MPIase. To achievethis, we determined the structure of MPIase (Fig. 1), studied the structure-activity relationship, and clariﬁed themechanisms for MPIase function3). Based on these results,
we predicted the synthetic pathway of MPIase, and thenisolated factors involved in the pathway.
Methods
Puriﬁcation and detection of MPIase
Inner membrane vesicles, prepared from E. coli
MC4100, were successively extracted using urea, sodium
cholate and trichloroacetic acid. Highly puriﬁed MPIase
was obtained by anion exchange and partition chromatographies. MPIase was detected either by anisaldehyde-H2SO4
on TLC or by silver on SDS-PAGE2).
Reconstitution of liposomes and assaying protein integration
MPIase was mixed with phospholipids supplemented
with diacylglecerol (DAG) in a solvent (chloroform :ethanol : water⫽3/7/4), followed by evaporation. Liposomes
were then formed in aqueous buffer by sonication. The protein integration activity was measured using 3L-Pf3 coat4)
as substrate, which was synthesized in vitro in the presenceof liposomes. The integrated materials were detected asproteinase K-resistant bands.
Anti-MPIase antibody
MPIase was crosslinked to KLH, a carrier protein, bymeans of EDAC, a crosslinker, followed by immunizationin rabbits.
Results
1.

Figure 1. Structure of MPIase. The lipid part possesses fatty acids of
C16 or C18. In the glycan chain, MPIase has a repeating unit consisting of
4-acetamide-fucose (Fuc4NAc), N-acetylmannosaminuronic acid (ManNAnA) and N-acetylglucosamine (GlcNAc), where n⫽9–11. About 1/3 ofR is acetylated.

Structure determination of MPIase
The structure of MPIase was determined by a combination of NMR and MS analyses of natural MPIase and its
acid- or alkaline-hydrolysates. Partially synthetic compounds were used to conﬁrm the structure. As a result, theMPIase structure was concluded to be that shown in Fig. 1.
Surprisingly, the glycan chain was similar to that of ECA(enterobacterial common antigen), an outer membrane constituent. The differences between MPIase and ECA werethat the repeating number in MPIase was n⫽9–11 whilethat in ECA was n⫽18–55, and that the linker to the lipid inMPIase was a pyrophosphate while that in ECA was a47phosphate.
2.

Structure-activity relationship of MPIase
MPIase derivatives were obtained chemically and enzymatically for the analysis of structure-activity relationship.
A pyrophosphatase cleaved at the center of the pyrophosphate to yield PP-MPIase. On the other hand, both the center of pyrophosphate and acetyl groups in GlcNAc(-R) weredigested by NaOH(NaOH-MPIase). PP-MPIase was foundto be more active than authentic MPIase, while NaOHMPIase was inactive as to protein integration. These indicate that the lipid part of MPIase was not essential, but thatacetyl groups in GlcNAc were essential.
Next, we examined whether or not ECA was inactive,
since ECA possesses a glycan similar to that in MPIase.
ECA was found to be completely inactive, suggesting thatthe glycan length is important for activity.
By means of PP-MPIase, which is soluble and active, we
examined whether or not the coupling of protein synthesis
with protein integration is necessary. Protein integration occurred after protein synthesis, if PP-MPIase was present
during translation, indicative of the post-translational integration. Furthermore, PP-MPIase formed a soluble complex with a membrane protein, indicating that MPIase possesses an integration-dedicated chaperon activity.
3.

Isolation of biosynthetic factors of MPIase
A subcellular localization of MPIase was examined using
anti-MPIase antibody. As expected, MPIase was localized
exclusively in inner membrane of E. coli. This antibody
also reacted with mature and precursor forms of ECA, because of the similarity of glycan. On the other hands, in thewecA and wecF mutants, defective in the ECA biosynthesis,no ECA-derived bands were detected, while MPIase wasdetected, indicating that the biosynthetic pathways of bothglycolipids are different.
The ﬁrst step of ECA biosynthesis is the attachment ofGlcNAc to undecaprenyl pyrophosphate. On the other hand,
it is assumed that GlcNAc is directly attached to phosphatidic acid (PA) or DAG. For these reasons, we mixed UDPGlcNAc with either PA, DAG or CDP-DAG in the presenceof E. coli cytosol or inner membranes. We obtained a possible GlcNAc-PP-DAG compound by mixing UDP-GlcNAc,
PA and inner membranes, in a manner dependent on eachfactor. We puriﬁed this activity as the Cdh protein (CDPDAG hydrolase). However, even in the cdh mutant, MPIase
was normally expressed, indicating that there is a factor
other than Cdh.
Conclusion
In this research, we determined the complete structure ofMPIase and studied the structure-activity relationship. We
found that MPIase possesses not only the integration-dedicated chaperone activity but also an integrase activity
which subsequently functions. We have reported thatMPIase functions catalytically2). These observations revealthat MPIase possesses an enzyme-like activity, to be called‘glycolipozyme’.
While the structures of MPIase and ECA are similar, thesubcellular localization and biosynthetic pathways are quite
different. We isolated Cdh as a ﬁrst enzyme involved in
MPIase biosynthesis, however, the overall biosynthesis ofMPIase was not affected by disruption of the cdh gene. It issuggested that multiple enzymes are involved in a same
step of biosynthesis, consistent with the important functionof MPIase in vivo. In near future, it is expected that anotherfactor could be isolated using a cdh mutant.
References1)

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Nishiyama, K. et al. (2006) A derivative of lipid A is involved in signal recognition particle/SecYEG-dependent and -independent membrane integrations. J. Biol. Chem. 281: 35667–35676.
Nishiyama, K. et al. (2010) A novel complete reconstitution system
for membrane integration of the simplest membrane protein.
Biochem. Biophys. Res. Commun. 394: 733–736.
Nishiyama, K. et al. (2012) MPIase is a glycolipozyme essential formembrane protein integration. Nature Communications. 3, 1260.
DOI: 10.1038/ncomms2267
Kawashima, Y., Miyazaki, E., Muller, M., Tokuda, H. & Nishiyama,K. (2008) Diacylglycerol speciﬁcally blocks spontaneous integrationof membrane proteins and allows detection of a factor-assisted integration. J. Biol. Chem. 283: 24489–24496.