Syntheses and activities of the functional structures of a glycolipid

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Syntheses and activities of the functional structures of a glycolipid essential for membrane protein integration Kohki Fujikawa, Sonomi Suzuki, Ryohei Nagase, Shiori Ikeda, Shoko Mori, Kaoru Nomura, Ken-ichi Nishiyama, and Keiko Shimamoto ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00654 • Publication Date (Web): 31 Jul 2018 Downloaded from http://pubs.acs.org on August 1, 2018

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Syntheses and activities of the functional structures of a glycolipid essential for membrane protein

integration.

Kohki Fujikawa1†, Sonomi Suzuki2†, Ryohei Nagase1, Shiori Ikeda2, Shoko Mori1, Kaoru Nomura1, Ken-ichi Nishiyama2*, Keiko Shimamoto1*

1

Bioorganic Research Institute, Suntory Foundation for Life Sciences, 8-1-1 Seikadai, Seika-cho,

Soraku-gun, Kyoto 619-0284, Japan 2

Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka,

Iwate 020-8550, Japan

† These authors contributed equally to this work.

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Abstract MPIase is the first known glycolipid that is essential for membrane protein integration in the inner

membrane of E. coli. Since the amount of natural MPIase available for analysis is limited and it

contains structural heterogeneity, precisely designed synthetic derivatives are promising tools for

further elucidation of its membrane protein integration mechanism. Thus, we synthesized the

minimal unit of MPIase, a trisaccharyl pyrophospholipid termed mini-MPIase-3, and its derivatives.

Integration assays revealed that the chemically synthesized trisaccharyl pyrophospholipid possesses

significant activity, indicating that it includes the essential structure for membrane integration.

Structure-activity relationship studies demonstrated that the number of trisaccharide units and the

6-O-acetyl group on N-acetylglucosamine contribute to efficient integration. Furthermore, anchoring

in the membrane by a lipid moiety was essential for the integration. However, the addition of

phosphorylated glycans devoid of the lipid moiety in the assay solution modulated the integration

activity of MPIase embedded in liposomes, suggesting an interaction between phosphorylated

glycans and substrate proteins in aqueous solutions. The prevention of protein aggregation required

the 6-O-acetyl group on N-acetylglucosamine, a phosphate group at the reducing end of the glycan,

and a long glycan chain. Taken together, we verified the mechanism of the initial step of the

translocon-independent pathway in which a membrane protein is captured by a glycan of MPIase,

which maintains its structure to be competent for integration, and then MPIase integrates it into the

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membrane by hydrophobic interactions with membrane lipids.

Introduction Membrane proteins such as receptors, transporters, ion channels, and enzymes play important

roles in the biomembranes of all organisms. The fundamental molecular basis of membrane protein

integration appears to be conserved among all organisms, as witnessed by the wide distribution of

similar channel complexes, named translocons, from prokaryotes such as Escherichia coli to eukaryotes such as plants and animals.1 Both translocon-dependent2 and -independent pathways3

have been found to act in membrane protein integration (Figure 1a). It has been reported that YidC,

another partner protein of translocons in E. coli, serves as an “insertase” in both pathways. YidC has

a hydrophilic groove in the inner leaflet of the membrane that allows the transmembrane segment of the substrate to slide into the membrane.4, 5 In addition to these known factors, we found that

membrane integration is also regulated by membrane components. In the course of constructing an

in vitro integration system using membrane lipids, we made two discoveries. First, a physiological

concentration (2~3 wt% in E. coli membrane lipids) of diacylglycerol (DAG) inhibited the

integration. Substrate proteins spontaneously integrated into liposomes containing only E. coli

phospholipids (PL), but the integration was completely inhibited by the addition of 5% DAG even if

translocons and/or YidC were present in liposomes. Second, the addition of an extract from the inner

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membrane of E. coli restored the integration, suggesting the presence of an unknown factor, a

“membrane protein integrase.” We named the factor “MPIase” after its function and purified it from

the inner membrane of E. coli. The purified MPIase restored the translocon-independent integration

in DAG-containing liposomes and the translocon-dependent integration in those containing SecYEG.

Recently, we reported that the insertion of the F0c subunit of F0F1 ATPase is dependent on MPIase and is stimulated by YidC.6 We considered that MPIase functions at an initial step of protein

integration into the membrane, while YidC allows complete insertion at a later step. Our structure

determination studies revealed that, surprisingly, MPIase is a glycolipid devoid of proteinaceous components despite its enzyme-like activity (Figure 1b).7-9 It has approximately 10 (n = 9-11)

trisaccharide units, each of which is comprised of 4-acetamido-4-deoxyfucose (Fuc4NAc),

2-acetamido-2-deoxymannuronic acid (ManNAcA), and 2-acetamido-2-deoxyglucose (GlcNAc),

and has a diacylglycerol (DAG) anchor at the reducing end of the glycan through a pyrophosphate

linkage. In addition, approximately 30% of the GlcNAc 6-OH is acetylated. MPIase is the first

known glycolipid to participate in membrane protein integration. Previously, we reported that the

glycan part of MPIase serves like a chaperone by interacting with membrane proteins to prevent their aggregation.7 We also showed that MPIase integrates higher numbers of membrane proteins than their equivalents; that is, it drives the integration reaction like an enzyme.4 Based on these features, we proposed the novel concept of a “glycolipozyme.”7 However, the membrane protein

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integration mechanism aided by MPIase remains unsolved at the molecular and functional group

levels. The problems with determining this mechanism include the following issues. 1) Only a

limited amount of MPIase can be obtained from natural sources. 2) Natural MPIase contains

heterogeneity in the number of O-Ac groups, length of glycan, and types of fatty acid. 3) Chemical

modifications are difficult due to its poor solubility in both water and organic solvents and its

nonselective reactivity among a large number of functional groups. The chemical synthesis of partial

structures of MPIase can supply structurally defined molecules to provide precise information on

structure-activity relationships of MPIase. In this paper, we describe the synthesis of trisaccharyl

pyrophospholipid, a minimal unit of MPIase (termed mini-MPIase-3), and its derivatives and discuss

their activities.

Results and Discussion

Synthetic strategy of mini-MPIase-3 (1) The retrosynthetic strategy for mini-MPIase-3 (1) shown in Figure 2 contained some challenging

steps. It required syntheses of unusual sugars (Fuc4NAc and ManNAcA), construction of a

β-mannosaminide bond, selective O-acetylation on 6-OH of GlcNAc, and introduction of a labile

pyrophospholipid. We already reported the synthesis of the Fuc4NAcα(1→4)ManNAcAβ disaccharide in the course of the structural determination of MPIase.7,

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In our approach, the

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challenging β-mannosaminide was constructed by steric inversion at C2 of β-glucoside,10 and then the 6-OH of mannosamine was oxidized into uronic acid. We adopted this strategy for the synthesis

of the trisaccharide. A similar concept was reported by the Boons group in their synthesis of the glycan part for enterobacterial common antigen (ECA).11 ECA has the same glycan sequences as

MPIase, but with more repeats of the trisaccharide unit (n = 18 to 55) compared to MPIase (n = 9 to 11), and ECA contains a monophospholipid instead of a pyrophospholipid.12-14 Paulsen et al. also reported the synthesis of the trisaccharide unit of ECA;15 however, no one has reported introducing

the phospholipid part. Although ECA has the same glycan sequence as MPIase, it does not show membrane protein integration activity,7 suggesting some sort of necessary role for the

pyrophospholipid part. We planned for a labile pyrophosphate to be constructed near the end of the

synthesis and for benzyl (Bn) type protecting groups to be employed, because they can be cleaved

mildly without affecting the 6-OAc group on GlcNAc, DAG, and pyrophosphate.

Synthesis of trisaccharide units Properly protected monosaccharide units were synthesized under a well-designed strategy (Figure

2). Coupling of the Glc donor (7) and the GlcNAc acceptor (8) gave the desired β-disaccharide (9) in 74% yield (Scheme 1). Then, deprotection of the Bz group at O-2 of 9, introduction of the Tf group

(10), and substitution with NaN3 accompanied by the inversion of the configuration produced

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a β-mannosaminide type structure (11) in 46% yield over 3 steps. Removing the benzylidene group followed by regioselective acetylation at O-6 of 11 afforded the disaccharide acceptor (6).

Glycosylation of 6 and the fucosamine donor (5) yielded the trisaccharide (12) in 86% yield with an

α/β ratio of 7/1. The desired α-isomer was easily separated by a silica gel column. Elimination of the Ac group at O-6 of the mannosamine moiety (12) prior to the oxidation of the resulting 6-OH, and

then protection by the Bn ester provided the mannosaminuronate (13) in good yield. Deprotection of

the TBDPS group, followed by acetylation successfully gave the 6-OAc glucosamine derivative (14). The reduction of both azide groups11, 16, 17 of 14 followed by N-acetylation afforded the acetoamide

(15) in high yield as reported by the Boons group. Selective liberation of the hydroxyl group at the

reducing end provided the trisaccharide hemiacetal (4) in 86% yield over 2 steps.

Introduction of the pyrophospholipid The obtained 4 was reacted with commercially available phosphoramidite bearing allyl groups,

and the subsequent oxidation with tert-butyl hydroperoxide produced the phosphoric ester (16).

Although 4 was a mixture of α and β-isomers, only the desired α-16 was isolated along with the trisaccharide hemiacetal and the oxazoline after silica gel purification.18, 19 When the 2-N3 derivative of glucose was used for phosphorylation, both α- and β-phosphoric esters were isolated. These

results suggested that the β-isomer of the phosphoric ester was unstable owing to the participation of

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the N-acetoamide group and was easily decomposed during the purification step. Removal of allyl

groups on phosphoric acid gave the phosphorylated trisaccharide (3).

When the phospholipid was activated as a pentavalent phosphoramide, a dimerized byproduct was

obtained from the unreacted phospholipid, and the purification step was laborious because of their high polarity and instability.20, 21 Thus, a phosphoramidite (2) bearing a Bn group and DAG was synthesized from phosphorus trichloride,22, 23 which was easily purified by a silica gel column and

could be stored for several months at -20°C.

Condensation of 3 and 2 in the presence of 4,5-dicyanoimidazole in MeCN at room temperature

followed by oxidation with tert-butyl hydroperoxide provided the fully protected trisaccharyl

pyrophospholipid (17) in 52% yield. Finally, deprotection of Bn and PMB groups under

hydrogenolysis conditions afforded trisaccharyl pyrophospholipid (1) possessing the 6-OAc group

on GlcNAc, namely mini-MPIase-3, in 75% yield.

Syntheses of glyco-pyrophospholipids containing dolicol or undecaprenol as the biosynthetic

precursors of oligosaccharide have been reported, where the final deprotection step is accomplished by basic hydrolysis of O-Ac groups.24-26 In our case, synthesis of trisaccharyl pyrophospholipid

containing a base-labile O-Ac group and DAG was achieved by employing Bn-type protecting

groups that are cleavable under neutral conditions. To the best of our knowledge, this is the first

synthesis of glyco-pyrophospholipid bearing DAG as a lipid moiety.

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In addition, trisaccharyl monophosphate [18, Trisac-P], trisaccharyl pyrophospholipid devoid of

the 6-OAc on GlcNAc, [19, mini-MPIase-3 (6-OH)], and trisaccharyl monophosphate devoid of the

6-OAc on GlcNAc [20, Trisac-P (6-OH)] were synthesized in a similar manner (Figure 3).

Furthermore, the glycan moiety of natural MPIase with an attached monophosphate group [21,

Polysac-P] was obtained by pyrophosphatase digestion, and the polysaccharide moiety lacking a

phosphate group [22, Polysac] was gained through the hydrolysis of natural MPIase with AcOH (SI).

Integration activity assay The protein integration activity was evaluated as previously reported (Figure 4, Methods).27-29 We

added 5 wt% of DAG into the liposomes prepared from E. coli PL to inhibit spontaneous integration.30 A designated amount of MPIase derivative was contained in the control liposomes (DAG/PL). A 35S-labeled model substrate, 3L-Pf3 phage coat protein,31, 32 was synthesized by using

a cell-free protein synthesis system in the presence of control liposomes (Figure 4a) or liposomes

containing MPIase derivatives (Figure 4b). The reaction mixture was then divided into two fractions.

One fraction (A) gave the total amount of synthesized proteins via autoradiography of SDS-PAGE.

The other fraction (B) gave the amount of the integrated protein in the liposome membranes after

proteinase K digestion of proteins outside of liposomes. The net integration percentage was

calculated from the total amount of synthesized protein (A) and the amount of the integrated protein

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in the liposome membranes (B). If the incorporated MPIase derivative was active, the integrated

proteins were protected from the digestion and gave a strong band B during autoradiography after

SDS-PAGE, while band B disappeared after digestion if the derivative was inactive. Normalized

integration values were calculated by the subtraction of the control percentage from the net

integration percentages in each experiment.

Integration activity of MPIase derivatives embedded in liposomes We examined membrane protein integration activities by using the above assay system. We added

MPIase derivatives in liposomes to give a concentration of 5 wt% of the total PL amount (Figure 5a).

The net integration percentage into the control liposomes was 0.9±0.5%, while those of natural

MPIase, 1, and 19 were 20.5±2.4%, 5.2±0.8%, and 2.0±0.5%, respectively. Natural MPIase resulted in efficient membrane protein integration as reported in our previous study.7 It should be noted that 1

showed significant activity despite the length of its glycan chain being about only one-tenth that of

natural MPIase. On the other hand, 19, which is devoid of the 6-OAc group on GlcNAc, showed

almost no activity. We also observed that other translocon-independent substrate proteins, such as

wild-type Pf3 phage coat protein and M13H5 phage procoat protein, were also integrated into the

liposomes by natural MPIase or 1, but not by 19 (Figure S1). These results indicated that 1 possesses

an essential structure for membrane integration, and that the Ac group on GlcNAc 6-OH plays an

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important role in membrane protein integration. Next, we compared their integration activities based

on mol concentrations (Figure 5b). Dose-dependency of the normalized integration values showed

that the activity of natural MPIase was much higher than that of 1, The activity of 19, which is

devoid of the 6-OAc group on GlcNAc, was still weak even at a high concentration. A longer glycan

would enable membrane protein integration at lower concentrations. However, when the values were

compared based on the number of trisaccharide units, the activity of 1 was comparable to that of

natural MPIase (Figure 5c). Thus, we concluded that the number of trisaccharide units affects the

potency of activity.

Influence on integration activity by the addition of anchorless MPIase derivatives To investigate the role of anchoring MPIase by a lipid moiety in the membrane, we added

anchorless MPIase derivatives (21, 22, 18, and 20) into assay solutions containing control liposomes

(Figure 6a). None showed integration activity even at a high concentration of 1.0 mg/mL. These

results demonstrated that the anchoring of MPIase by a lipid moiety in the membrane is essential for

its membrane protein integration activity. Interestingly, when we used liposomes containing 5 wt%

of 1 instead of the control liposomes, 21 markedly enhanced the integration activity of 1 (Figure 6b).

In our previous report, we observed that 21 (previously termed PP-MPIase) has potent integration activity.7 Since we prepared 21 by pyrophosphatase digestion of natural MPIase and previously used

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it without purification, the combination of the resulting 21 and unreacted MPIase that might be

embedded into the liposomes could cause strong integration. In this study, chemically defined 21

was used, and thus, the importance of anchoring of MPIase became evident. Unlike 21, 18 decreased

the activity of 1 embedded in liposomes while neither 22 nor 20 exhibited significant synergic

effects. The comparison between 18 and 20 revealed the importance of the acetyl group. A direct

interaction between 21 and the protein in the solution was implied because 21 also enhanced

spontaneous integration into the liposomes, including a trace amount of DAG, even if they did not

contain 1 (Figure S2). Gel filtration experiments revealed that 21 forms a soluble complex with a substrate protein (3L-Pf3 coat) and prevents the protein from aggregating (Figure 6c),7 while neither

22 (lack of phosphate, Figure 6c) nor Polysac-P (6-OH) (lack of Ac group on GlcNAc, Figure S3)

prevented the aggregation. These results demonstrated that the prevention of protein aggregation by

21 leads to the enhancement of integration activity, and that both the phosphate group at the reducing

end of the glycan and the 6-OAc group on GlcNAc of 21 contributed to the interaction with the

substrate membrane protein. Taking these results into consideration, we propose the enhancement

mechanism to be as follows (Figure 7). i) Without additives, hydrophobic proteins rapidly aggregate

and fail to integrate. ii) The negative charge of the phosphate group of 21 binds to the positive

charges of basic amino acids in the protein. Then, the long glycan chain of 21 interacts with the

membrane protein through the 6-OAc group on GlcNAc as well as other functional groups and

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prevents the protein from aggregating. This results in an increase in integration-competent proteins.

iii) Although 18 can also interact with membrane proteins through a phosphate group and a 6-OAc

group, its glycan chain is too short to cover all of the hydrophobic regions, and thus 18 causes the

competitive inhibition of binding with proteins against 1 embedded in liposomes.

Mechanism of membrane protein integration Since we observed an interaction between phosphorylated glycan and membrane proteins in

aqueous solution (Figure 6b, c), similar interactions are expected between proteins and the glycan

chain embedded in the membrane. We presume that MPIase exists in the inner membrane of E. coli

with the glycan chain facing the cytoplasmic side, it captures a hydrophobic membrane protein

released from a ribosome, and it prevents the aggregation of proteins similar to a chaperone (Figure 7 iv).7 The interaction of MPIase with proteins requires a phosphate group at the reducing end of the

glycan and a 6-OAc group on GlcNAc. In addition to the importance of the 6-OAc group on GlcNAc

as demonstrated in this paper, it is considerable that the acetoamide (NHAc) groups are also involved in the integration activity, because digestion of MPIase with proteinase K33 attenuated the

activity, probably due to the cleavage of N-Ac groups. Since natural MPIase includes more than 30

NHAc and OAc groups, their hydrogen bonds and/or hydrophobic interactions should work

cooperatively. Membrane proteins captured by the phosphorylated glycan near membranes should be

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transferred to the membranes by hydrophobic interactions with membrane lipids. Although only one

trisaccharide unit in 18 was not enough to prevent aggregation of proteins in aqueous solution, on

the membrane surface, 1 successfully integrated proteins into membranes (Figure 7 v). It is thus

plausible that proteins captured near membranes can be rapidly transferred into them without

aggregation. Furthermore, 1 in the membrane at high concentration showed significant activity,

suggesting that multiple molecules of 1 might accumulate and cooperate on the membrane to interact with proteins. Since it is known that many glycans serve as clusters,34-39 MPIase as well might

assemble on the membrane to form highly hydrophobic domains by excluding water molecules, and

these domains might serve as platforms for the capture and integration of hydrophobic proteins.

Negative charges of the pyrophosphates of MPIase located on the membrane surface might

contribute to membrane protein integration, as it has been reported that negative charges of acidic

phospholipids (phosphatidylglycerol, cardiolipin) attract basic amino acids in membrane proteins to the membrane surface.40-42 In this study, we focused on the interaction between the glycan with the

substrate protein. Our recent analyses have demonstrated that MPIase/mini-MPIase-3 does not

disturb the morphology of the membrane but alters the physicochemical properties such as the

membrane fluidity, the details of which will be published elsewhere. A combination of the glycan

interacting with the protein and its effects on the membrane facilitate the integration. At this stage,

we have not determined the structure and topology of the 3L-Pf3 coat in the membrane integrated by

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1, but the obtained fragment after protease digestion showed the same size on SDS-PAGE as those

from experiments using liposomes containing natural MPIase or E. coli inner membrane vesicles.

The hydrophilic N-terminus of the 3L-Pf3 coat was certainly embedded in the membrane because the

radiolabeled methionine at the N-terminus remained after digestion, whereas some residues from the

C-terminus were outside of the membrane and cleaved by the protease. However, we cannot rule out

the possibility that the N-terminus did not fully cross the membrane and still stayed inside. We are

now conducting solid-state NMR experiments to determine which residues are exposed to the

aqueous environment. To complete the integration, in which the N-terminus of the transmembrane

region locates on the opposite side of the membrane, the assistance of YidC might be required because YidC enhances the membrane protein integration in the presence of MPIase.6 Modification

of functional groups in natural MPIase is difficult; however, synthetic derivatives designed from 1 or

18 should enable precise structure-activity relationship studies. In addition, such derivatives could

also be promising as solubilizing reagents for membrane proteins.

Conclusions MPIase is an essential factor for membrane protein integration found in the inner membrane of E.

coli. In this study, we synthesized mini-MPIase-3 (1), a trisaccharyl pyrophospholipid, and its

derivatives (18, 19, and 20) as partial structures of MPIase. The synthetic strategy shown in this

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work is applicable to further synthesis of MPIase derivatives. The synthesized MPIase derivatives

and natural MPIase derivatives (21, 22) were subjected to membrane protein integration assays.

Among them, 1, which possesses only one trisaccharide unit, showed significant integration activity,

indicating that this includes the minimal functional structure for the integration activity.

Structure-activity relationship studies demonstrated that the number of trisaccharide units and the

6-OAc group on GlcNAc are important. Furthermore, the anchoring of MPIase in the membrane by

a lipid moiety is essential for membrane protein integration. Although 21 added in the reaction

solution was inactive by itself, it enhanced the integration activity of 1 embedded in the liposomes.

In contrast, 18 decreased the integration activity of 1. These results proved the existence of an

interaction between the glycan part of MPIase and proteins, and revealed that the 6-OAc group on

GlcNAc, the phosphate group at the reducing end of the glycan, and the length of the glycan

contribute to preventing the aggregation of proteins. The necessity of anchoring MPIase in the

membrane suggests the assembly of MPIase in the membrane, the attraction of substrate proteins by

pyrophosphate groups on the membrane surface, and the cooperation of MPIase with a ribosome

and/or translocons. In this study, we verified the mechanism of the initial step of the

translocon-independent pathway. More detailed structure-activity relationships, the characterization

of the complex with a membrane protein, three-dimensional structural determination of MPIase on

the membrane surface, observation of the clustering state of MPIase in the membrane, and detection

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of interactions with other membrane components are currently underway to understand the

membrane protein integration mechanism aided by MPIase.

Methods Reconstitution of MPIase derivatives into liposomes Liposomes were prepared as described27 with slight modifications. Briefly, polar phospholipids of E. coli (Avanti Polarlipids, Inc.) (1 mg), dioleoylglycerol (50 µg), and appropriate amounts of MPIase derivatives dissolved in solvent (solvent C (chloroform/ethanol/water:3/7/4) or chloroform) were mixed well. Solvent was evaporated using a stream of nitrogen gas and then under vacuum. Liposomes were formed by sonicating the dried residues with 100 µl of buffer A (50 mM HEPES-KOH, pH 7.5, 1 mM dithiothreitol). The liposome suspension was frozen and stocked at -80°C.

Protein integration assays The activity of protein integration was determined by a protease protection assay as previously described.27-29 3L-Pf3 coat protein,31, 32 used as a substrate, was in vitro synthesized by means of a pure system27, 43 in the presence of liposomes (1 mg/mL as phospholipids). Liposome suspensions were thawed at room temperature followed by sonication immediately before the in vitro synthesis. The reaction mixture (20 µL) was incubated for 30 min at 37°C and terminated by chilling on ice. An aliquot (3 µL) was immediately precipitated by trichloroacetic acid (5%) and used as a translation control. Another aliquot (15 µL) was mixed with an equal amount of proteinase K (1

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mg/mL) and then incubated for 20 min at 25°C. The residual materials after digestion were then precipitated by trichloroacetic acid (5%). These samples were analyzed by SDS-PAGE and autoradiography as previously described.29 Radioactive bands were visualized by a PhosphorImager (GE) and quantitated by ImageQuant software (GE).

Gel filtration assay 3L-Pf3 coat was synthesized in the presence or absence of additives (21 or 22, 200 µg/mL). The translation mixture of 3L-Pf3 coat (50 µL) in the presence of and absence of additives (21 or 22) was applied to a Superose 12 column, which had been equilibrated with 50 mM HEPES-KOH (pH 7.5). The column was developed at 1.0 mL/min, and 0.5 mL fractions were collected from 5 min after the start. An aliquot of each fraction (100 µL) was precipitated with TCA (5%), followed by analysis of autoradiography after SDS-PAGE. The amount of 3L-Pf3 coat in each fraction was determined and is plotted as a percentage of the total amount.

Associated Content Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: General methods, experimental procedure/NMR/MS data of newly synthesized compounds, supplementary membrane protein integration assays, and supplementary gel filtration experiment (PDF)

Author Information

Corresponding Author

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Keiko Shimamoto, Tel: +81 50-3182-0693; Fax: +81 774-98-6262; e-mail: [email protected] Ken-ichi Nishiyama, Tel: +81 19 621 6471; Fax: +81-19-621-6243; e-mail: [email protected]

ORCID Keiko Shimamoto, 0000-0003-1086-5756

Notes The authors declare no competing financial interest.

Acknowledgments We thank A. Kuhn (University of Hohenheim) for plasmids, T. Ueda (The University of Tokyo) for the pure system, M. Saikudo (Iwate University) for technical help, M. Horikawa (SUNBOR) for the advice regarding HPLC analyses, and S. Nakanishi (SUNBOR) for reading the manuscript. The experiments involving radioisotopes were carried out at the Radioisotope Laboratory of Iwate University. This work was supported by JSPS KAKENHI (Grant Numbers 15H06844, 17K13262 to K.F., 25291009, 26102703, 26119701, 15KT0073, 16H01374, 16K15083, 17H02209 to K.N., and 22310142, 25282235, 25620137, 16H01166, 18H04433 (Middle Molecular Strategy) to K.S.).

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simplest membrane protein, Biochem Biophys Res Commun 394, 733-736. 28. Koch, H. G., Hengelage, T., Neumann-Haefelin, C., MacFarlane, J., Hoffschulte, H. K., Schimz, K. L., Mechler, B., and Muller, M. (1999) In vitro studies with purified components reveal signal recognition particle (SRP) and SecA/SecB as constituents of two independent protein-targeting pathways of Escherichia coli, Mol. Biol. Cell 10, 2163-2173. 29. Nishiyama, K., Ikegami, A., Moser, M., Schiltz, E., Tokuda, H., and Muller, M. (2006) A derivative of lipid A is involved in signal recognition particle/SecYEG-dependent and -independent membrane integrations, J. Biol. Chem. 281, 35667-35676. 30. Kawashima, Y., Miyazaki, E., Muller, M., Tokuda, H., and Nishiyama, K. (2008) Diacylglycerol specifically blocks spontaneous integration of membrane proteins and allows detection of a factor-assisted integration, J Biol Chem 283, 24489-24496. 31. Kiefer, D., and Kuhn, A. (1999) Hydrophobic forces drive spontaneous membrane insertion of the bacteriophage Pf3 coat protein without topological control, EMBO J 18, 6299-6306. 32. Kiefer, D., Hu, X., Dalbey, R., and Kuhn, A. (1997) Negatively charged amino acid residues play an active role in orienting the Sec-independent Pf3 coat protein in the Escherichia coli inner membrane, Embo J 16, 2197-2204. 33. Ebeling, W., Hennrich, N., Klockow, M., Metz, H., Orth, H. D., and Lang, H. (1974) Proteinase K from tritirachium album limber, Eur J Biochem 47, 91-97. 34. Mammen, M., Choi, S. K., and Whitesides, G. M. (1998) Polyvalent interactions in biological systems: implications for design and use multivalent ligands and inhibitors, Angew Chem Int Ed Engl 37, 2754-2794. 35. Sato, S., Yoshimasa, Y., Fujita, D., Yagi-Utsumi, M., Yamaguchi, T., Kato, K., and Fujita, M. (2015) A self-assembled spherical complex displaying a gangliosidic glycan cluster capable of interacting with amyloidogenic proteins, Angew Chem Int Ed Engl 54, 8435-8439. 36. Chen, J., Gao, J., Wu, J., Zhang, M., Cai, M., Xu, H., Jiang, J., Tian, Z., and Wang, H. (2015) Revealing the carbohydrate pattern on a cell surface by super-resolution imaging, Nanoscale 7, 3373-3380. 37. Matsuzaki, K., Kato, K., and Yanagisawa, K. (2010) Abeta polymerization through interaction with membrane gangliosides, Biochim Biophys Acta 1801, 868-877. 38. Simons, K., and Lingwood, D. (2010) Lipid rafts as a membrane-organizing principle, Science 327, 46-50. 39. Simons, K., and Sampaio, J. L. (2011) Membrane organization and lipid rafts, Cold Spring Harb Perspect Biol 3, a004697. 40. Crooke, E. (2001) Escherichia coli DnaA protein--phospholipid interactions: in vitro and in vivo, Biochimie 83, 19-23. 41. Matsumoto, K. (2001) Dispensable nature of phosphatidylglycerol in Escherichia coli: dual roles of

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anionic phospholipids, Molecular Microbiology 39, 1427-1433. 42. Breukink, E., Demel, R. A., de Korte-Kool, G., and de Kruijff, B. (1992) SecA insertion into phospholipids is stimulated by negatively charged lipids and inhibited by ATP: a monolayer study, Biochemistry 31, 1119-1124. 43. Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., and Ueda, T. (2001) Cell-free translation reconstituted with purified components, Nat. Biotechnol. 19, 751-755.

Figure legends

Figure 1. (a) Schematic model for membrane protein integration in E. coli. In the

translocon-dependent pathway, a protein generated from a ribosome targets membranes with a signal

recognition particle (SRP), is embedded into membranes by the translocon SecYEG, and then is

positioned in the membranes by YidC. The translocon-independent pathway was previously believed

to be a spontaneous process facilitated by hydrophobic interactions between transmembrane regions

of the protein and membrane lipids. However, it became evident that MPIase (M) is indispensable in both pathways.7 (b) Structure of MPIase. Fatty acids of DAG moiety (R2) vary with the culture conditions of E. coli.

Figure 2. Synthetic strategy of mini-MPIase-3 (1).

Figure 3. MPIase derivatives.

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Figure 4. Overview of integration activity assay. (a) The control liposomes. (i) The control

liposomes are prepared from DAG and PL (ii) Radioisotope-labeled 3L-Pf3 proteins are synthesized

by an in vitro translation system in the presence of liposomes. (iii) The reaction mixture is divided

into two fractions. One fraction gives the total amount of synthesized proteins by autoradiography

after SDS-PAGE (band A). (iv) When protease is added to the other fraction, proteins outside

liposomes are cleaved, and thus band B is not detected. (b) The liposomes containing the active

factor. If the liposomes contain the active factor, band B reflects the amount of proteins integrated in

the liposomes.

Figure 5. Integration activity of MPIase derivatives. (a) Net percentages of protein integration into

liposomes composed of DAG/PL, MPIase/DAG/PL/, 1/DAG/PL, and 19/DAG/PL. The values were

obtained from at least three independent experiments. A representative autoradiography profile after

SDS-PAGE is shown below. (b) Normalized integration values for the number of molecules (nmol)

in each reaction tube. (c) Normalized integration values for the number of trisaccharide units (nmol)

in each reaction tube.

Figure 6. (a) Normalized integration values by the addition of anchorless MPIase derivatives into a

reaction mixture with control liposomes (DAG/PL). (b) Normalized integration values by the

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addition of anchorless MPIase derivatives into a reaction mixture with liposomes containing

1/DAG/PL. (c) The amount of 3L-Pf3 coat in each fraction after gel filtration chromatography is

plotted as a percentage of the total amount. The hydrophobic substrate proteins (3L-Pf3 coat) rapidly

aggregated and were eluted at the void volume. By the addition of 21, the peak of 3L-Pf3 coat

protein shifted to fractions of lower molecular weight. On the other hand, the addition of 22 did not

alter the elution peak.

Figure 7. Proposed mechanism for the initial step of translocon-independent integration involving

MPIase. i) Without MPIase, hydrophobic proteins rapidly aggregate and fail to integrate. ii) The long

glycan chain of MPIase (21) can bind the protein to prevent aggregation like a chaperone in the

soluble milieu. iii) The short glycan of 18 is not enough to prevent the aggregation of proteins. iv)

MPIase captures proteins on the membrane surface and then transfers them to the membrane. v)

Even if the glycan of 1 is short, the uncovered region of the protein can migrate into the membrane

prior to the aggregation, and/or multiple molecules of 1 can work cooperatively. The oligomeric state

of MPIase derivatives and the topology of the integrated protein remain unclear.

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Figure 1

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AcHN O

HO

HOOC NHAc HO O O O HO HO

1

OAc O O

O O AcHN O P O P O OH OH

O

NHAc PMBO

O OAc BnO BnO O O O O BnO OH BnOOC NHAc AcHN

O BnO

4

N3 PMBO

O C13 H27 C 13 H27

O

O

SPh

OBn

+

OAc O AcHN O O P OH 3 OH

+

P O BnO

O C 13H 27 C 13 H27

O

2

O O

OTBDPS O

BnO HO AcO

O

N

O O BnO NHAc AcHN OAllyl

N P N

+

HO

O O

C 13H 27

C 13 H27

BnO O

Fuc4N donor (5) Ph

O O BnO

6 O OBz

O

CF 3 NPh

Glc donor (7)

+

Diacylglycerol

HO BnO

OTBDPS O AcHN OAllyl

GlcNAc acceptor (8)

Figure 2

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NHAc O

HO

OAc HO HO O O O O HO HOOC NHAc AcHN O O P OH 18 Trisac-P OH NHAc O HO OH HO HO O O O O HO O HOOC NHAc AcHN O O P O P O OH

19

OH

mini-MPIase-3 (6-OH)

O O O

C 13 H 27 O

NHAc O

HO

OH HO HO O O O O HO HOOC NHAc AcHN O O P OH 20 Trisac-P (6-OH) OH NHAc

R1 = Ac or H

O

HO

HOOC NHAc HO O O O HO HO

21

Polysac-P

O

O P OH OH

n = 9-11

NHAc HO

OR 1 O AcHN

O HOOC NHAc HO O O O HO HO

22

Polysac

OR 1 O AcHN O

C 13H 27

H

n = 9-11

Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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7

(a)

8

+

1.0 eq.

OTBDPS O

OBz

BnO O Ph O

O O BnO

(b)

AcHN OAllyl

1.0 eq.

OTBDPS O

OTf

BnO O Ph O

O O BnO

AcHN OAllyl

9

10 N3

(c)

OTBDPS (d) O

BnO O Ph O

O O BnO N3 AcHN OAllyl

(e)

6

O

PMBO

BnO BnO O AcO

11

12

N3

(f)

N3 O

PMBO

(g)

OTBDPS O

O

PMBO

OAc BnO BnO O O O O BnO BnOOC N3 AcHN OAllyl

BnO BnO O O O BnO BnOOC N 3 AcHN OAllyl

13 N P

OAllyl

O

PMBO

OAc O

(i)

15

NHAc

16

4.0 eq.

NHAc

O BnO BnO O O O BnO BnOOCH2 NHAc

(j)

4

BnO BnO O O O BnO BnOOC NHAc AcHN OAllyl

PMBO

14

OAllyl

NHAc

(h)

OTBDPS O O O BnO N3 AcHN OAllyl

O

PMBO

OAc O

(k)

BnO BnO O O O BnO BnOOC NHAc

AcHN O O P OAllyl OAllyl

3

OAc O AcHN O O P OH OH

NHAc

(l)

O

PMBO

BnO BnO O O O BnO BnOOC NHAc

NHAc

(m)

HO

OAc O O

O AcHN O O P O P O OBn O 17 HNEt3

O C13H 27 C 13 H 27

O O

O HO HO O O O HO HOOC NHAc

OAc O O AcHN O O P O P O OH OH

1 mini-MPIase-3

O O C13H 27 C 13H 27

O O

Scheme 1. Synthesis of mini-MPIase-3 (1). (a) TMSOTf, MS4A, CH2Cl2, 0°C to RT, 74% (β). (b) (1) NaOMe, MeOH, RT. (2) Tf2O, Pyr./CH2Cl2, 0°C. (c) NaN3, DMF, 120°C, 46% (3 steps). (d) (1)

p-TsOH, EtOH, 60°C. (2) AcCl, Et3N, CH2Cl2, 0°C, 57% (2 steps). (e) 5 (5 equiv.), NIS, TfOH, 33

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MS4A, CH2Cl2, -20 to 0°C, 86% (α/β=7/1). (f) (1) NaOMe, MeOH, RT. (2) TEMPO, DIB, CH2Cl2/H2O, RT. (3) BnBr, Cs2CO3, DMF, RT, 75% (3 steps). (g) (1) TBAF, THF, RT. (2) Ac2O, Pyr., RT, 77% (2 steps). (h) (1) SnCl2, PhSH, Et3N, MeCN/MeOH/CH2Cl2. (2) Ac2O, MeOH, 89% (2 steps). (i) (1) Ir(COD)(Ph2MeP)2PF6, THF. (2) sat. NaHCO3 aq., I2, THF, 86% (2 steps). (j) (1) Tetrazole, CH2Cl2, -20°C to RT. (2) TBHP, CH2Cl2, -40 to 0°C, 55% (α). (k) Pd(PPh3)4, Et2NH, THF, RT, 95%. (l) (1) 2 (1.5 equiv.), 4,5-dicyanoimidazole, MeCN, RT. (2) TBHP, MeCN, RT, 52%. (m)

Pd-black, H2, MeOH/THF, RT, 75%.

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66x35mm (96 x 96 DPI)

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