FEBS Letters 583 (2009) 3285–3290

journal homepage: www.FEBSLetters .org

Site-specific N-glycosylation regulates the GPS auto-proteolysis of CD97

Cheng-Chih Hsiao a,b, Kai-Fong Cheng b, Hsin-Yi Chen b, Yi-Hua Chou b, Martin Stacey c,Gin-Wen Chang b, Hsi-Hsien Lin b,*

a Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-San, Tao-Yuan, Taiwanb Department of Microbiology and Immunology, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-San, Tao-Yuan, Taiwanc Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK

a r t i c l e i n f o a b s t r a c t

Article history:Received 2 July 2009Revised 14 August 2009Accepted 1 September 2009Available online 6 September 2009

Edited by Lukas Huber

Keywords:Adhesion-G protein-coupled receptorAuto-proteolysisG protein-coupled receptor proteolytic siteN-Glycosylation

0014-5793/$36.00 � 2009 Federation of European Biodoi:10.1016/j.febslet.2009.09.001

Abbreviations: 7TM, seven-transmembrane; DOCextracellular domain; EGF-TM7, epidermal growth factransmembrane receptor; EMR2, epidermal growth fmucin-like hormone receptor 2; Fc, fragment crystallisareceptor; GPS, GPCR proteolytic site; PARs, protease-atype

* Corresponding author. Fax: +886 32118700.E-mail address: hhlin@mail.cgu.edu.tw (H.-H. Lin)

Auto-proteolysis at the G protein-coupled receptor (GPCR) proteolytic site (GPS) is a hallmark ofadhesion-GPCRs. Although defects in GPS auto-proteolysis have been linked to genetic disorders,information on its regulation remains elusive. Here, we investigated the GPS proteolysis of CD97,a human leukocyte-restricted and tumor-associated adhesion-GPCR. We found that CD97 is incom-pletely processed, unlike its close homolog, epidermal growth factor-like module-containingmucin-like hormone receptor 2. A unique pattern of N-glycosylation within the GPS motif of relatedadhesion-GPCRs was identified. The use of N-glycosylation inhibitors and mutants confirm site-spe-cific N-glycosylation is an important determinant of GPS proteolysis in CD97. Our results suggestthat N-glycosylation may regulate the processing of adhesion-GPCRs leading to the production ofeither cleaved or uncleaved molecules.� 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

Post-translational modification by proteolytic cleavage hasbeen recognized as an important mechanism for the functionalmodulation of a diverse array of cell surface proteins, such asNotch, protease-activated receptors (PARs) and many cell adhesionmolecules [1–3]. Proteolytic modification of these molecules usu-ally takes place in the secretory pathway or on the cell surface,mediated by proprotein convertases or sheddases such as furin,MMPs and ADAMs [4].

In addition to protease-mediated modifications, protease-inde-pendent processing catalyzed by intramolecular cleavage (auto-proteolysis) has recently gained attention as an alternative mech-anism for receptor activation. Examples of these auto-proteolyticmodifications include the SEA module-mediated cleavage ofMuc-1 and dystroglycan [5–7], and the G protein-coupled receptorproteolytic site (GPS) domain-mediated cleavage of adhesion-Gprotein-coupled receptors (GPCRs) [8,9].

chemical Societies. Published by E

, degree of cleavage; ECD,tor module-containing seven-actor-like module-containingble; GPCR, G protein-coupled

ctivated receptors; WT, wild-

.

Based upon the GRAFS phylogenetic classification system pro-posed by Fredriksson et al., the 33 adhesion-GPCR molecules con-stitute the second largest GPCR sub-family in the human proteome[10]. The most distinct feature of adhesion-GPCRs is the chimericcomposition of a long extracellular domain (ECD) and the seven-span transmembrane (7TM) region [9,11]. Unusually large amongGPCRs, the ECD of adhesion-GPCRs typically consists of multipleprotein modules, a stalk region and a novel Cys-rich GPS domain.Specifically, the N-terminus often contains common protein motifsknown to be involved in protein–protein interactions, while thestalk region is rich in potential N- and O-glycosylation residues.The heavily glycosylated receptor was then dissected at the GPSdomain into extracellular a-subunit and 7TM b-subunit, whichwere then expressed on the membrane as a non-covalent hetero-dimer [9,11].

Our previous experiments on epidermal growth factor-likemodule-containing mucin-like hormone receptor 2 (EMR2), an epi-dermal growth factor module-containing seven-transmembranereceptor (EGF-TM7) member of adhesion-GPCR family, have shownthat the GPS proteolysis is mediated by a self-catalytic reactionsimilar to that of N-terminal nucleophile hydrolases [8]. We foundthat the GPS cleavage, which occurs in the endoplasmic reticulum(ER), is critically dependent on the intact stalk region and isachieved via an intramolecular reaction involving the generationand subsequent hydrolysis of a (thio)ester intermediate [8,12]. Ear-lier studies have suggested that proper cleavage at GPS might be a

lsevier B.V. All rights reserved.

3286 C.-C. Hsiao et al. / FEBS Letters 583 (2009) 3285–3290

prerequisite for efficient receptor trafficking to cell surface [13].More recently, deficiency in GPS cleavage has been linked to hu-man genetic disorders. As such, point mutations affecting theGPS cleavage of GPR56 and polycystin-1 have been implicated asthe causes of bilateral frontoparietal polymicrogyria (BFPP) andautosomal dominant polycystic kidney disease (ADPKD), respec-tively [14,15]. Therefore, GPS cleavage appears to be an inherentand essential post-translational modification for the expression,presentation and function of adhesion-GPCRs.

Despite the direct link to diseases, little is known about the reg-ulation of the novel self-catalytic GPS proteolytic reaction. In thisreport, the GPS auto-proteolysis of CD97 was investigated. CD97is a leukocyte-restricted and tumor-associated EGF-TM7 moleculehighly homologous to EMR2 [16,17]. The two molecules shared al-most identical EGF-like domains and �50% homology in the stalkand the 7TM regions. As well as functional roles in leukocytemigration, recruitment and activation [18–21], CD97 is known tobe involved in angiogenesis and the migration and invasion of tu-mor cells [22–24]. By analyzing the GPS cleavage of CD97-mFc-fu-sion protein, we show herein that CD97 and EMR2 have verydifferent GPS cleavage efficiencies. Site-specific N-glycosylationin the stalk was found to play a critical role in regulating GPSauto-proteolysis of CD97. Our results suggest the adhesion-GPCRsmight adopt two different conformations, leading to the produc-tion of either cleaved or uncleaved molecules.

2. Materials and methods

2.1. Reagents and cell culture

General chemicals were of analytical grade and obtained fromSigma (Dorset, UK), unless otherwise stated. CLB/CD97-1 mAb(mouse IgG2a) and EMR2-specific 2A1 mAb (mouse IgG1) werepurchased from AbD Serotec (Kidlington, UK). Horseradish peroxi-dase (HRP)-conjugated 2nd Ab was purchased from Sigma. All cul-ture media were from Invitrogen (Carlsbad, CA) and weresupplemented with 10% heat inactivated fetal calf serum (FCS),2 mM L-glutamine, 50 IU/ml penicillin and 50 lg/ml streptomycin.HEK-293T and COS-7 were cultured in Dulbecco’s Modified EagleMedium (DMEM). CHO-K1 cells were grown in Ham’s F-12 med-ium. Lec1 cells were maintained in a-minimum essential medium(a-MEM). Transient transfection of expression constructs by Lipo-fectamineTM (Invitrogen) was performed as previously described[8]. For the use of N-glycosylation inhibitors, cells were incubatedwith the following inhibitors: tunicamycin at 0.1 lg/ml, 1 lg/mland 10 lg/ml, castenospermine (CAS) at 50 lg/ml, 1-deoxynojiri-mycin (DNJ) at 100 mM, swainsonine (SW) at 10 lg/ml. In all cases,cells were pre-treated with the inhibitors for 12 h, replaced withfresh OPTI-MEM I medium containing inhibitors for a further 24 h.

2.2. Construction of expression vectors

All expression vectors were constructed using pcDNA3.1(+) orpcDNA3.1/myc-His vectors (Invitrogen) unless otherwise specified.EMR2- and CD97-mFc expression constructs have been describedpreviously [8,25]. The chimeric and N-glycosylation site mutantswere generated using polymerase chain reaction (PCR) amplifica-tion by Advantage� HF 2 PCR Kit (BD) using site-specific mutantprimers (Supplementary Table S1).

2.3. Production, purification and glycosidase treatment of mFc-fusionproteins

The production and purification of mFc-fusion proteins werecarried out as described previously [8]. Purified proteins were dia-

lyzed in 50 mM Tris–HCl, pH 7.4, buffer containing 10 mM CaCl2

and 150 mM NaCl at 4 �C. The purified proteins were subjectedto glycosidase treatment by incubation with 1 U of PNGase F, 1 Uof Neuraminidase (Roche Applied Science) in 20 mM sodium phos-phate buffer, pH 7.0, at 37 �C for 20 h prior to SDS–PAGE analysis.

2.4. Western blotting and other protein analysis

For Western blotting, proteins were separated in 8% or 10%SDS–PAGE gels, blotted and probed with anti-mFc or CLB/CD97-1mAbs. For the in vitro cleavage of mFc-fusion proteins, affinity-purified CD97-mFc, EMR2(125)-H516S-mFc, or EMR2(125)-S518A-mFc-fusion proteins were incubated in cleavage buffer(50 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA) with or without250 mM NH2OH (HA) at 37 �C. At various time points, sampleswere withdrawn and analyzed by Western blotting. For the quan-titative analysis of protein bands, the intensity of the uncleavedand cleaved protein bands on Western blots was quantified usingGel-Pro Analyzer 3.1 (Media Cybernetics, MD, USA) according tothe manufacturer’s instructions.

3. Results

3.1. Detection of different GPS auto-proteolytic activities in CD97 andEMR2

While investigating the GPS cleavage of EMR2-mFc in previousexperiments, CD97-mFc was included for comparison. Surpris-ingly, we detected very different degree of cleavage (DOC) in thetwo molecules; EMR2-mFc is almost entirely cleaved (>95% DOC)as reported, but the cleavage of CD97-mFc is only partial (�50%DOC) (Fig. 1A). The same results were observed in all cell linestested including CHO-K1, COS-7 and HEK-293T (data not shown).Most importantly, similar results were seen in the membrane formof CD97, indicating that the cleavage of CD97-mFc mimics that ofCD97 receptor (Supplementary Fig. S1). A point mutation at theGPS site (CD97-S531A) completely inhibits the cleavage, suggest-ing the cleavage mechanism of CD97 is highly similar, if not iden-tical, to that of EMR2 (Supplementary Fig. S1). To further confirmthe partial cleavage of CD97-mFc, fusion proteins were affinitypurified and analyzed by SDS–PAGE. As shown in Fig. 1B, twobroad bands of �95–125 kDa and �50–75 kDa were identified,representing the unprocessed molecule and the cleaved extracellu-lar subunit, respectively. A third distinct band of �35 kDa is that ofcleaved mFc fragment. Subsequent de-glycosylation reaction con-firmed the two broad bands are indeed due to heavy glycosylationas reported (Fig. 1B and Supplementary Fig. S2) [26].

We have previously shown that strong nucleophiles such ashydroxylamine (HA) can greatly enhance the processing rate ofan incomplete GPS proteolytic reaction [8]. This is achieved byfacilitating the hydrolysis of the (thio)ester intermediate, therate-limiting step of the GPS proteolytic reaction. To investigatewhether the uncleaved CD97-mFc identified here is the result ofan incomplete proteolytic reaction (i.e. a stalled intermediate) ora truly unprocessed product, purified proteins were treated withHA in an in vitro cleavage reaction (Fig. 1C). As expected, enhancedcleavage was observed in a ‘‘cleavage-incomplete” protein, EMR2H516S-mFc, in the presence of HA. On the contrary, no cleavagewas detected in cleavage-deficient EMR2 S518A-mFc. Similar tothe cleavage-deficient EMR2 molecule, CD97-mFc showed no en-hanced proteolysis upon HA treatment. This indicated that theuncleaved CD97-mFc is a cleavage-incompetent molecule(Fig. 1C). Altogether, these results suggest that CD97-mFc canadopt two different conformations; one capable of auto-proteolysisand the other not.

A B C

Fig. 1. Detection of different GPS auto-proteolytic activities in CD97 and EMR2. (A) The EGF-like modules and the stalk region are represented by triangles and a black line,respectively. The GPS cleavage site is indicated by an arrow. Western blot analysis of the CD97-mFc (lane 4) and EMR2-mFc (lane 3) is shown. Lanes 1 and 2 are mock andmFc-only control, respectively. Blots were probed with HRP-conjugated anti-mouse Fc-specific mAb that detects the uncleaved protein (�100 kDa) and the mFc subunit(�36 kDa). (B) Affinity purified CD97-mFc proteins were digested without (lane 1) or with PNGase and neuraminidase (lane 2) and visualized by Coomassie brilliant bluestaining. M represents the protein marker (in kDa). The uncleaved protein, the cleaved ECD and mFc subunits are represented by black arrowhead, white arrowhead andasterisk, respectively. (C) Western blot analysis of CD97-mFc and mutant EMR2-mFc-fusion proteins subjected to in vitro cleavage reaction in the presence of NH2OH (HA) at37 �C as indicated.

A

B

C

Fig. 2. GPS auto-proteolysis is mainly regulated by the stalk region. (A) Diagrams depict the EMR2-mFc, CD97-mFc and domain-swapping fusion proteins (indicated bynumbers 1–6, respectively). Western blot analysis of these fusion proteins is shown in the right panel. (B) Sequence alignments of the GPS motifs that are known to becleaved. The conserved residues are highlighted in grey background. The arrow and the boxes indicate the cleavage site and the potential N-glycosylation sites, respectively.The abbreviations represent animal species (h, human; m, mouse; r, rat; d, Drosophila). (C) Western blot analysis of cleavage of the mFc-fusion proteins. Samples includemock-transfected cells (lane 1), cells transfected with constructs expressing mFc (lane 2), EMR2-mFc (lane 3), CD97-mFc (lane 4), EMR3-mFc (lane 5) and EMR4-mFc (lane 6)proteins.

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3.2. Auto-proteolysis of CD97-mFc is regulated predominantly by thestalk region

To locate the region involved in the regulation of GPS auto-proteolysis, we established and compared domain-swappingchimeras of EMR2-mFc and CD97-mFc because of their strong sim-ilarity (Fig. 2). As expected, Fig. 2A shows the auto-proteolyticactivity is mainly determined by the less homologous stalk region

(also see Supplementary Fig. S3A). A closer examination of the stalkregion of related adhesion-GPCRs identified an interesting patternof potential N-glycosylation sites. A minimum of one N-glycosyla-tion site is present in the GPS motif of all adhesion-GPCRs sur-veyed, except EMR2 (Fig. 2B). Furthermore, �50% and �75% DOCwas identified in EMR3 and EMR4, two EGF-TM7 members thatcontain two and one N-glycosylation sites in the GPS motif, respec-tively (Fig. 2C and Supplementary Fig. S3B) [27,28]. These results

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suggest that N-glycosylation in the stalk region (and the GPSdomain) might play a role in determining the conformation ofEGF-TM7-mFc-fusion proteins and hence the DOC.

3.3. Regulation of GPS proteolysis in CD97-mFc by N-glycosylation

N-Linked glycans play an essential role in ER quality control as avital determinant for the proper folding of proteins. To examine therole of N-glycosylation in regulating GPS cleavage, CD97-mFc andEMR2-mFc transfected cells were treated with various N-glycosyl-ation inhibitors (Fig. 3A and B). Tunicamycin interferes with thefirst step of glycoprotein synthesis by inhibiting the enzymaticreaction catalyzing the transfer of core oligosaccharides to the nas-cent peptide chain. Because N-glycosylation influenced proteinsecretion, we could not detect CD97-mFc and EMR2-mFc proteinsin the conditioned media of tunicamycin-treated cells. However,we noticed the DOC of newly synthesized proteins within thetunicamycin-treated cells was greatly reduced (Fig. 3A). The reduc-tion is observed for both CD97-mFc and EMR2-mFc, but is moreprominent in CD97-mFc, rendering it almost entirely uncleaved.

Unlike the tunicamycin treatment however, no significant effecton the DOC of CD97-mFc and EMR2-mFc was observed in cellstreated with castanospermine (CAS) and 1-deoxynojirimycin(DNJ), both glucosidases I and II inhibitors (Fig. 3B). The same istrue for cells treated with swainsonine (SW), a Golgi alpha-man-nosidase II inhibitor. Likewise, when analyzed in Lec1 cells, whichlacks N-acetylglucosaminyltransferase I activity, the DOC of bothEMR2-mFc and CD97-mFc was not altered (Fig. 3C). These resultsare consistent with the consensus that proteolysis takes placewithin ER. Our data further indicate that GPS cleavage occurs afterthe attachment of the oligosaccharide precursor, but does not de-pend on its subsequent processing.

Fig. 3. Regulation of GPS proteolysis of CD97-mFc molecule by N-glycosylation. (A) WesEMR2-mFc proteins. (B) Western blot analysis of the effect of various N-glycosylation inhThe expression patterns of CD97-mFc (lane 2) and EMR2-mFc (lane 1) in Lec 1 cells.

3.4. The role of site-specific N-glycosylation in the regulation of GPSproteolysis in CD97-mFc

A total of 5 potential N-glycosylation (G) sites were identified inthe stalk regions of CD97 (Fig. 4A). To investigate the role of site-specific N-glycosylation in GPS auto-proteolysis, we created andevaluated the DOC of single and multi-N-glycosylation site mu-tants of CD97-mFc proteins. In comparison to the wild-type (WT)protein, all single N-glycosylation site mutants showed slight ef-fects on the DOC either positively or negatively (Fig. 4B and Sup-plementary Fig. S4A). To show this effect is specific and notsimply due to residue changes, we established single mutantsreplacing the three individual residues of the first N-glycosylationsite (G1) (N371Q, V372A and T373A). The DOC of V372A mutant,which should preserve N-glycosylation, was similar to the WT pro-tein. On the other hand, both N371Q and T373A mutants allshowed similar enhanced DOC (Fig. 4B).

Intriguingly, significant modulation of GPS proteolysis was ob-served in multi-N glycosylation site mutants. The cleavage ofCD97(G2,3), CD97(G1-3), CD97(G2-4), CD97(G2-5), and CD97(G1-5) mutants was greatly reduced (only �1–10% DOC), whereas theprocessing of CD97(G4,5) and CD97 (G1,4,5) mutants was effi-ciently enhanced with the DOC of CD97(G4,5) reaching �95%(Fig. 4B and Supplementary Fig. S4B). These results indicate thatsite-specific N-glycosylation in the stalk region is critically in-volved in the GPS auto-proteolysis of CD97-mFc. Specifically, com-bined mutation at the 2nd and 3rd glycosylation sites, G2(N406) andG3(N413), seem to reduce GPS auto-proteolysis. Conversely, muta-tion involving the 4th and 5th sites (G4,5) greatly enhances prote-olysis. Interestingly, the effect of G2,3 mutations on the DOC seemsto dominate over that of G4,5 mutations, as exemplified by themostly uncleaved CD97(G2-5) and CD97(G1-5) mutants (Fig. 4B).

tern blot analysis of the effect of tunicamycin on the GPS cleavage of CD97-mFc andibitors (CAS, DNJ, SW) on the GPS cleavage of CD97-mFc and EMR2-mFc proteins. (C)

A B

Fig. 4. Regulation of GPS proteolysis of CD97-mFc molecule by site-specific N-glycosylation. (A) Diagram depicting the wild-type (WT) CD97-mFc molecule, with a specialattention on the potential N-glycosylation sites. The 5N-glycosylation sites in the stalk were denoted as G1, G2, G3, G4, and G5, respectively. (B) Western blot analysis ofvarious CD97-mFc proteins including the WT and N-glycosylation site mutants.

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4. Discussion

As evidenced by genetic diseases caused by mutations that af-fect GPS cleavage, the self-catalytic proteolysis at the GPS domainis no doubt an essential post-translational modification of the GPSdomain-containing receptors. However, several recent studieshave reported the detection of both cleaved and uncleaved adhe-sion-GPCRs, including GPR56 and flamingo, in vivo [29,30]. Theseresults strongly suggest that the GPS auto-proteolytic reaction isregulated and that the cleaved and uncleaved adhesion-GPCRscould potentially have different cellular functions. This is consis-tent with the recent finding by Yu et al. on the functions of polycy-stin-1, a GPS motif-containing 11-TM molecule associated withADPKD [31]. By comparing the phenotypes of the Pkd1 null and aspecific Pkd1 knock-in animal model expressing only the noncleav-able polycystin-1, Yu and colleagues demonstrated that thecleaved polycystin-1 is critical for kidney tubular integrity, whilethe uncleaved polycystin-1 is required for embryonic development[31]. The fact that both cleaved and uncleaved polycystin-1 mole-cules have distinct biological functions is also reflected by theexpression of the respective protein products in vivo [31,32].

These observations indicated that although the GPS proteolysisis self-catalytic, the reaction is probably not 100% efficient. Alter-natively, it is possible that the adhesion-GPCRs can adopt differentconformations and proceed with or without GPS cleavage. Our dataon the regulation of GPS cleavage in CD97 seem to support the lat-ter mechanism. Likewise, a similar conclusion was reached by Weiet al. who demonstrated that endogenous polycystin-1 can maturevia two different pathways leading to the production of uncleavedfull-length molecule as well as cleaved products [32]. The degree ofcleavage (DOC) shown in this report was determined by comparingthe ratio of the uncleaved to cleaved protein bands on Westernblots. This was based upon the assumption that all mutant proteinshave similar protein stability. As N-glycans are important for pro-tein stability, it should be cautioned that it is also likely that the re-moval of N-glycans at certain positions can influence the overallprotein stability and turn-over. This effect, if occurred, will affectthe measurement of DOC, even though it might not really affectGPS cleavage. Detailed structural analysis of these individual pro-teins by other means such as the circular dichroism spectroscopyor conformational unfolding studies in the future should help dis-tinguish these potential effects.

Based upon available experimental evidence, we suggest amodel where the folding of adhesion-GPCRs in ER can lead toeither a cleavable or uncleavable conformation. In CD97-mFc, thestructural determinant for the two distinct conformations wasfound to include site-specific N-glycosylation in the stalk region

(Figs. 2–4). Consistent with the consensus that GPS cleavage takesplace in the ER lumen, we hypothesize that N-glycosylation at spe-cific sites of CD97 stalk promotes the folding of CD97-mFc precur-sor to a favorable conformation to allow GPS cleavage reaction. Ifthe favorable condition is not met due to differential N-glycosyla-tion, the precursor molecule will adopt a different conformationthat is cleavage-incompetent and thus is expressed as an unc-leaved single-chain protein.

Because of the heterogenous nature of the N-glycosylation siteoccupancy [33], it is possible to produce receptors containing var-iable degrees of N-glycosylation leading to cleavable and uncleav-able conformations. This implies a regulatory mechanism wherebythe eventual cellular function of a specific adhesion-GPCR couldpossibly be determined by the efficiency of site-specific N-glyco-sylation, and hence the relative amounts of the cleaved and unc-leaved receptors. From this point of view, it is interesting to notethat all GPS domain-containing receptors, including adhesion-GPCRs and polycystin-1, contain an extracellular stalk rich in po-tential N- and O-glycosylation sites that are in close proximity ofthe GPS motif. It will be interesting in the future to analyzewhether different factors other than N-glycosylation are involvedin the regulation of GPS auto-proteolytic reaction. Such informa-tion should in turn help shed lights on the generation and the bio-logical functions of the cleaved and uncleaved adhesion-GPCRs.

Acknowledgements

We thank Dr. J.Q. Davies for his critical comments. This work wassupported by grants from National Science Council (NSC96-2320-B-182-005) and Chang Gung Memorial Hospital (CMRPD170012) toH.-H. Lin.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.febslet.2009.09.001.

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