Unexpected subcellular distribution of a specific isoform of the Coxsackie and adenovirus receptor, CAR-SIV, in human pancreatic beta cells

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1 Diaetologia (2018) 61: ARTICLE Unexpeted suellular distriution of a speifi isoform of the Coxsakie and adenovirus reeptor, CAR-SIV, in human panreati eta ells Eseoghene Ifie 1 & Mark A. Russell 1 & Shalinee Dhayal 1 & Pia Leete 1 & Guido Seastiani 2 & Laura Nigi 2 & Franeso Dotta 2 & Varpu Marjomäki 3 & Deio L. Eizirik 4 & Noel G. Morgan 1 & Sarah J. Rihardson 1 Reeived: 6 Deemer 2017 /Aepted: 2 July 2018 /Pulished online: 3 August 2018 # The Author(s) 2018 Astrat Aims/hypothesis The Coxsakie and adenovirus reeptor (CAR) is a transmemrane ell-adhesion protein that serves as an entry reeptor for enteroviruses and may e essential for their aility to infet ells. Sine enteroviral infetion of eta ells has een impliated as a fator that ould ontriute to the development of type 1 diaetes, it is often assumed that CAR is displayed on the surfae of human eta ells. However, CAR exists as multiple isoforms and it is not known whether all isoforms suserve similar physiologial funtions. In the present study, we have determined the profile of CAR isoforms present in human eta ells and monitored the suellular loalisation of the prinipal isoform within the ells. Methods Formalin-fixed, paraffin-emedded panreati setions from non-diaeti individuals and those with type 1 diaetes were studied. Immunohistohemistry, onfoal immunofluoresene, eletron mirosopy and western lotting with isoformspeifi antisera were employed to examine the expression and ellular loalisation of the five known CAR isoforms. Isoformspeifi qrt-pcr and RNA sequening (RNAseq) were performed on RNA extrated from isolated human islets. Results An isoform of CAR with a terminal SIV motif and a unique PDZ-inding domain was expressed at high levels in human eta ells at the protein level. A seond isoform, CAR-TVV, was also present. Both forms were readily deteted y qrt-pcr and RNAseq analysis in isolated human islets. Immunoytohemial studies indiated that CAR-SIV was the prinipal isoform in islets and was loalised mainly within the ytoplasm of eta ells, rather than at the plasma memrane. Within the ells it displayed a puntate pattern of immunolaelling, onsistent with its retention within a speifi memrane-ound ompartment. Co-immunofluoresene analysis revealed signifiant o-loalisation of CAR- SIV with zin transporter protein 8 (ZnT8), prohormone onvertase 1/3 (PC1/3) and insulin, ut not proinsulin. This suggests that CAR-SIV may e resident mainly in the memranes of insulin seretory granules. Immunogold laelling and eletron mirosopi analysis onfirmed that CAR-SIV was loalised to dense-ore (insulin) seretory granules in human islets, whereas no immunolaelling of the protein was deteted on the seretory granules of adjaent exorine ells. Importantly, CAR-SIV was also found to o-loalise with protein interating with C-kinase 1 (PICK1), a protein reently demonstrated to play a role in insulin granule maturation and traffiking. Eletroni supplementary material The online version of this artile ( ontains peer-reviewed ut unedited supplementary material, whih is availale to authorised users. * Sarah J. Rihardson s.rihardson@exeter.a.uk 1 2 Islet Biology Exeter (IBEx), Institute of Biomedial and Clinial Sienes, University of Exeter Medial Shool, RILD Building (Level 4), Barrak Road, Exeter EX2 5DW, UK Department of Mediine, Surgery and Neurosienes, University of Siena and Fondazione Umerto Di Mario ONLUS Tosana Life Sienes, Siena, Italy 3 4 Department of Biologial and Environmental Siene/Nanosiene Center, University of Jyväskylä, Jyväskylä, Finland Université Lire de Bruxelles (ULB) Center for Diaetes Researh and Welio, Medial Faulty, Université Lire de Bruxelles, Brussels, Belgium

2 Diaetologia (2018) 61: Conlusions/interpretation The SIV isoform of CAR is aundant in human eta ells and is loalised mainly to insulin seretory granules, implying that it may e involved in granule traffiking and maturation. We propose that this suellular loalisation of CAR-SIV ontriutes to the unique sensitivity of human eta ells to enteroviral infetion. Keywords Beta ells. Coxsakie and adenovirus reeptor. Coxsakievirus B. Enterovirus. Insulin granule. Panreas. Protein interating with C-kinase 1 (PICK1) Areviations CAR Coxsakie and adenovirus reeptor CAR-CT Coxsakie and adenovirus reeptor C-terminus CVB Coxsakievirus B ECD Extraellular domain FFPE Formalin-fixed, paraffin-emedded LCM Laser apture mirodisseted MCC Manders o-loalisation oeffiient PC1/3 Prohormone onvertase 1/3 PICK1 Protein interating with C-kinase 1 RNAseq RNA sequening ZnT8 Zin transporter protein 8 Introdution Epidemiologial and pathologial studies have linked enteroviral infetions with the development of type 1 diaetes mellitus [1], ut the mehanisms y whih this might promote islet autoimmunity remain unertain. One possiility is that islet ells are partiularly sensitive to infetion y enteroviruses and, in aord with this, enterovirus serotypes that are most often assoiated with type 1 diaetes (e.g. Coxsakievirus B [CVB]) have a lear tropism for human eta ells [2 4]. Additional support omes from evidene that enterovirus proteins are present in the eta ells of individuals with type 1 diaetes [5, 6] and that speifi viral response pathways are ativated in suh ells [7, 8]. In order for enteroviruses to infet ells, they must ind to speifi ell surfae proteins that serve as vehiles to mediate their entry. Suh inding is thought to alter the onformation of the viral apsid to failitate the entry of viral RNA into the target ell. An endogenous ellular protein known as the Coxsakie and adenovirus reeptor (CAR) is one suh viral reeptor. CAR is a transmemrane protein involved in homotypi ell adhesion and tight juntional integrity [9] ut also serves as a primary ellular attahment protein for CVBs

3 2346 Diaetologia (2018) 61: and adenoviruses [10], presumaly in a suversion of its normal physiologial role. Full-length CAR is enoded y the CXADR gene, whih omprises eight exons and yields a protein with an extraellular domain (ECD) linked y a single transmemrane region to a ytoplasmi tail. Differential spliing yields at least five different isoforms (Fig. 1a,), ut only two of these ontain the transmemrane domain and are likely to e retained within ells. Strutural studies have suggested that enteroviruses ind to the D1 domain in the extraellular region of the protein [11, 12] and, aordingly, four of the isoforms (designated CAR- SIV, CAR-TVV, CAR4/7 and CAR3/7) have een shown to ind enterovirus. However, only CAR-SIV and CAR-TVV retain the transmemrane domain and are thus ale to mediate a produtive infetion in ells. The two solule isoforms are released from ells and may protet from infetion y sequestering ative virus in the extraellular fluid [11]. Interestingly, the two isoforms earing a transmemrane domain vary only in the sequene of the final 26 (CAR-SIV) or 13 (CAR-TVV) amino aids, ut are differentially loalised within ells and may fulfil different funtions [13]. The CAR-SIV isoform (also known as CAR Ex7 [13]) is enoded y the first seven exons of the human CXADR gene and is expressed preferentially on the asolateral surfae of polarised ells. In ontrast, the C-terminal region of the CAR-TVV isoform (also known as CAR Ex8 )isprodued from a rypti splie site within the seventh exon linked to exon 8, and is expressed apially [13]. The C-terminal region enodes a onsensus PDZ-inding domain whose amino aid sequene varies etween the CAR-SIV and the CAR-TVV forms, whih proaly aounts for the differential loalisation of the two variants within polarised ells [13 16]. In support of this, oth the SIV and TVV forms of CAR an interat with proteins suh as memraneassoiated guanylate kinase, WW and PDZ domainontaining protein 1 (MAGI-I), postsynapti density protein 95 (PSD-95) [13, 15, 16] and zonula oludens-1 (ZO- 1) [14], ut only the CAR-SIV isoform interats with protein interating with C-kinase 1 (PICK1) [13]. Strikingly, PICK1 is aundantly expressed in eta ells and has reently een shown to ontrol insulin granule traffiking. Indeed, loss of PICK1 results in impaired gluose tolerane and redued serum insulin onentrations in experimental animals [17, 18]. Thus, an interation etween PICK1 and the SIV isoform of CAR in eta ells ould e of funtional importane. To date, few studies have examined the expression and distriution of CAR isoforms in human eta ells [3, 19]. Ylipaasto et al [3] onfirmed a role for CAR y demonstrating that pre-treatment of isolated human islets with a loking antiody (lone RmB) proteted the ells from CVB infetion [3]. More reently, CAR expression was onfirmed in human panreas, aleit using an antiserum that does not disriminate etween the various isoforms [20]. We have thus haraterised and mapped the expression and distriution of CAR isoforms in human panreas. Methods Tissue and ell lines Formalin-fixed, paraffin-emedded (FFPE) panreati setions from the Exeter Arhival Diaetes Bioank ( and from the Network for Panreati Organ donors with Diaetes (npod; Gainesville, FL, USA) were studied. Samples were from 21 non-diaeti individuals (age range 4 47 years) and ten individuals with type 1 diaetes (age range 6 47 years; see eletroni supplementary material [ESM] Tale 1). Isolated human islets were otained from the Oxford Centre for Islet Transplantation (Oxford, UK) or purhased from Lonza (Basel, Switzerland). On arrival, islets were ultured for 24 h at 37 C and then fixed and proessed for immunostaining y standard immunofluoresene (FFPE) tehniques, or stored at 80 C for RNA extration. Ethis approval (West of Sotland Researh Committee 4 [WoSREC4]; 15/WS/0258) was gained for all the samples studied. The panreati tissue samples from the Exeter Arhival Diaetes Bioank and npod were seleted ased on age of demise, and in the ase of type 1 diaetes, duration of disease; tissue quality; sample availaility (whih is restrited within these ioanks) and appropriateness of tissue for immunohistohemistry/immunofluoresene approahes. Samples with extensive autolysis or evidene of other unrelated panreati diseases were exluded. Blinding of donor type and immunofluoresene stain was performed for the o-loalisation studies. The human eta ell lines EndoC-βH1 and 1.1B4 were ultured as desried in ESM Methods. Antiodies Three different anti-car sera were employed (ESM Tale 2) ased on their seletive immunoreativity against the C-terminus (CAR-CT) or regions within the ECD (CAR-ECD and CAR-RmB) of the protein, respetively, and validated as desried elow and in ESM Fig. 1. These allowed the various isoforms of CAR to e distinguished, as illustrated in Fig. 1. All other antisera are desried in ESM Tale 2. Mutagenesis Site-direted mutagenesis was performed to remove the final three amino aids (SIV) of CAR-SIV to examine the speifiity of the CAR-CT antiserum (ESM Methods). Western lotting The prodution of CAR in human islets and EndoC-βH1 ells and the speifiity of the CAR-CT and CAR-RmB antisera were assessed using western lotting (ESM Methods).

4 Diaetologia (2018) 61: a 1 19 Signal peptide N Extraellular domain Transmemrane domain 259 and 260 Palmitoylated site Cytoplasmi domain C Ig-like C2 type Ig-like C2 type PDZ inding d CAR-CT A. aa CAR-SIV, hcar1, CAR Ex7 ; 40 kda CAR-ECD A. aa ; CAR-RmB Exorine Exorine a 8 CAR-TVV, hcar2, CAR Ex8 ; 39 kda Endorine Endorine CAR4/7; 28 kda CAR3/7; 22 kda CAR isoforms CAR-SIV CAR-TVV CAR 4/7 CAR 3/7 CAR 2/7 NCBI nuleotide ID UniProt ID NM_ P NM_ P NM_ P NM_ P NM_ P CAR2/7; 10 kda Fig. 1 A desription of the CAR isoforms and the seletive expression of CAR-SIV in human islets. (a) CAR protein struture. The signal peptide (red) is leaved to yield a mature protein with an ECD omprising two immunogloulin (Ig)-like domains, type 1 (lue) and type 2 (green). The transmemrane domain (yellow) ridges the extraellular and ytoplasmi regions (pink), whih terminates with a PDZ-inding domain (red). () CXADR exon maps of the five differentially splied isoforms. The type 1 Ig domain is enoded y exons 2 and 3, while type 2 Ig-like domain is enoded y exons 4 and 5. Isoforms 1 and 2 ontain a transmemrane domain and are named CAR-SIV (or hcar1, CAR Ex7 )and CAR-TVV (or hcar2, CAR Ex8 ), respetively (denoted y the three terminal amino aids on their C-termini). The solule isoforms 3, 4 and 5 are named CAR4/7, CAR3/7 and CAR2/7, respetively, refleting exon inlusion or exlusion and lak of the transmemrane domain. The inding regions of the different CAR antiodies are also shown. The CAR-CT antiserum reognises amino aids loated at the C-terminus of the CAR-SIV isoform ut does not reognise the other CAR isoforms. Both the CAR-ECD and CAR-RmB antisera reognise epitopes within the ECD, and are predited to reognise the majority of the longer CAR isoforms (CAR-SIV, CAR-TVV, CAR4/7 and potentially CAR3/7). (, d) Representative immunoytohemial analysis of the expression of different CAR isoforms in normal ontrol panreas tissue stained with () CAR-CT and (d) CAR-ECD antisera. Sale ars, 20 μm. The insets elow represent higher magnifiation images (of the areas outlined y the lak oxes) demonstrating the differential staining profile of the CAR-CT antisera in the endorine (+++) and exorine panreas ( ), and the CAR-ECD antisera in the endorine (+++) and exorine panreas (+); +++, strong; +, weak;, negative. These results are representative of findings in panreas from eight non-diaeti individuals; aa, amino aids, A., antiody Flow ytometry The speifiity of the CAR-CT and CAR- RmB antisera was assessed using flow ytometry (ESM Methods). Immunohistohemistry This was performed using a standard immunoperoxidase approah [21]. Bright-field image aquisition was performed using a Nikon 50i Mirosope fitted with a DS-Fi amera and a DSL2 amera ontrol unit (Nikon, Kingston Upon Thames, UK). Antiody details and onditions are desried in ESM Tale 2. Immunofluoresene To examine multiple antigens within the same setion, FFPE samples were proed in a sequential manner with up to three different antiodies (ESM Tale 2) [22]. Panreas setions were initially sujeted to heat-indued epitope retrieval in 10 mmol/l itrate uffer (ph 6), and relevant antigen antiody omplexes were deteted using seondary antiodies onjugated with fluoresent dyes (Alexa Fluor antimouse 488, anti-rait 555, and anti-guinea pig 568 or 647; Invitrogen, Paisley, UK). Cell nulei were stained with DAPI. After mounting, images were aptured either with a Leia AF6000 mirosope (Leia, Milton Keynes, UK), then were proessed using the standard LAS X Leia software platform (Version ), or with a Leia SP8 onfoal mirosope and Hyvolution2 deonvolution software (Sientifi Volume Imaging, Hilversum, the Netherlands). Coloalisation analysis was undertaken with a JACoP plugin from Image J, version 1.48 Java _20 ( nih.gov/ij/plugins/trak/jaop2.html). Quantitative RT-PCR/semi-quantitative RT-PCR The relative expression levels of the CAR-SIV, CAR-TVV, CAR4/7, CAR3/7 and CAR2/7 isoforms in isolated human islets and laser apture mirodisseted (LCM) islets were determined using quantitative (q)rt-pcr (ESM Methods). RNAseq RNA sequening (RNAseq) was performed using islets otained from normoglyaemi human islet donors or

5 2348 Diaetologia (2018) 61: EndoC-βH1 ells as previously desried [23]. Genes and transripts were assigned a relative overage rate as measured in reads per kiloase of exon model per million mapped reads and ompared with 15 other normal human tissues, analysed y RNAseq and deposited at the Illumina BodyMap 2.0 dataset (GEO aession numer GSE30611), aessed on 9 August Cryo-immune eletron mirosopy We used ryoimmune eletron mirosopy to assess and quantify the loalisation of CAR-SIV, zin transporter protein 8 (ZnT8), insulin and proinsulin in human panreas samples as desried previously [24] and in the ESM Methods. Co-immunopreipitation of PICK1 with CAR This was performed in EndoC-βH1 ells and human islets as desried in ESM Methods. Statistial analysis Immunofluoresene images of islets were seleted randomly from stained panreas setions. The Pearson orrelation oeffiient was used to estimate the oloalisation etween proteins using the JACoP plugin from Image J. Values for the Pearson orrelation oeffiient ranged from 0 for no orrelation, to 1 for a positive orrelation. GraphPad Prism 5.04 (La Jolla, CA, USA) was employed for all statistial analysis, and data are expressed as means ±SEM. Statistial signifiane was alulated using one-way ANOVA, and the Bonferroni multiple omparison test was used for multiple omparisons. A p value <0.05 was onsidered statistially signifiant. Results Differential CAR immunostaining in human panreas The availaility of antisera direted against different regions of the CAR protein (Fig. 1) enaled the expression profiles of the two isoforms earing a transmemrane domain to e studied. The antiserum designated CAR-CT was raised against a peptide ontaining the C-terminal 35 amino aids of the CAR-SIV protein and required the presene of the triplet SIV sequene at the immediate C-terminus for immunoreativity. As suh, it did not lael any other isoform of CAR. This speifiity was onfirmed y transfetion of 1.1B4 ells with onstruts enoding either the full-length CAR-SIV or a variant in whih the final three amino aids (SIV) had een removed y targeted mutagenesis (ESM Methods). Western lotting and immunofluoresene staining onfirmed immunoreognition of the full-length CAR- SIV isoform, ut not the trunated form, y the CAR-CT antiserum (ESM Fig. 1). Two further CAR antiodies, CAR-ECD (Aam, Camridge, UK) and RmB (Merk Millipore, Watford, UK), oth of whih reognise the ECD of human CAR, were also used. By deploying a panel of approahes (western lotting, immunofluoresene staining of fixed ells, flow ytometry and immunohistohemistry in FFPE setions), it was found that the CAR-ECD antiserum worked effetively in oth western lotting and immunohistohemistry, whereas the RmB lone was most suitale for immunofluoresene staining of fixed ells or flow ytometry. As noted aove, oth the CAR-ECD and RmB antiodies reognise multiple isoforms of CAR inluding CAR-SIV, CAR-TVV and CAR4/7 (ESM Fig. 1,). Use of these differentially speifi antisera revealed that various isoforms of CAR are expressed among the exorine and endorine ompartments of the human panreas (Fig. 1,d). Importantly, the CAR-CT antiserum laelled only islet ells (Fig. 1) whereas use of the CAR-ECD antiserum resulted in laelling of oth exorine and endorine tissue (Fig. 1d). An idential staining pattern was onfirmed in a total of eight panreases from non-diaeti donors (within the Exeter and npod Bioanks), ranging in age from 4 weeks to 59 years (ESM Tale 1). Confirmation of SIV isoform expression y qrt-pcr in human islets Isoform-speifi primers were used to analyse CXADR isoform expression (ESM Tale 3) y qrt-pcr in RNA extrated from isolated human islets (ESM Tale 4). This revealed that transripts enoding the CAR-SIV and CAR- TVV isoforms were expressed at the highest levels, while the solule CAR4/7 isoform was less aundant (Fig. 2a). The two shorter isoforms CAR3/7 and CAR2/7 were arely detetale. These results were onfirmed in a seond set of independent samples in whih RNA was isolated from LCM islets (Fig. 2; ESM Tale 5). This was further onfirmed y RNAseq analysis of isolated human islets (Fig. 2). Importantly, CAR-SIV was enrihed threefold ompared with CAR-TVV in the human panreati eta ell line EndoC-βH1 (ESM Fig. 2a; ESM Methods)[25], and semi-quantitative RT- PCR analysis of RNA extrated from two highly purified preparations of human islets revealed that the expression of CAR-SIV was greater than that of CAR-TVV (ESM Fig. 2). Finally, the presene of the SIV isoform was verified y western lotting in isolated human islets and EndoC-βH1 ell using the CAR-CT and CAR-ECD antisera (Fig. 2d). The SIV isoform is expressed preferentially in human eta ells To determine whether the SIV isoform of CAR is preferentially loalised to a speifi endorine ell suset, omined immunofluoresene staining using anti-car-ct, insulin and gluagon was performed. This revealed that expression of the CAR-SIV isoform was restrited solely to eta ells in human panreas setions (Fig. 3a) and isolated human islets (Fig. 3). Somewhat surprisingly, rather than eing loalised to the ell surfae, the CAR-SIV isoform was distriuted mainly within

6 Diaetologia (2018) 61: a Relative expression (2 -ΔC t) Relative expression (2 -ΔC t) CAR-SIV CAR-TVV CAR4/7 CAR3/7 CAR2/7 CAR-SIV CAR-TVV CAR4/7 CAR3/7 CAR2/7 15 d 40 kda CAR-CT RPKM CAR-SIV CAR-TVV CAR4/7 40 kda 36 kda Fig. 2 Confirmation of CAR-SIV isoform expression in human islets. qrt-pcr analysis of CXADR isoform expression in (a) isolated human islets (n = 5 individuals) and () LCM human islets (n = 2 individuals) demonstrates that the SIV and TVV isoforms are highly expressed, CAR4/7 is present at low levels, and CAR3/7 and CAR2/7 are arely detetale. Data were normalised to the relative expression of three housekeeping genes, β-atin, GAPDH and B2M. Relative expression is H. islet EndoC-βH1 CAR-ECD GAPDH presented as the mean ± SEM. () RNAseqdatashowingCARisoform expression in islets from five normoglyaemi individuals (mean ± SEM) [23]. RPKM, reads per kiloase of exon model per million mapped reads. (d) Confirmation of CAR-SIV protein expression in isolated human islets and EndoC-βH1 ells as assessed y western lotting using CAR-CT and CAR-ECD antisera and loading ontrol glyeraldehyde 3-phosphate dehydrogenase (GAPDH). H., human a Fig. 3 The SIV isoform of CAR is expressed in panreati eta ells. (a) Representative immunofluoresene staining of the CAR-SIV isoform (CAR-CT antiody; green), insulin (light lue), gluagon (red) and DAPI (dark lue) in an islet from a non-diaeti human panreas. Sale ars, 10 μm. () CAR-SIV isoform staining in FFPE isolated human islets: CAR-SIV (CAR-CT; green) and insulin (red) and DAPI (dark lue). The enlarged region (from the area outlined y the dashed white ox) demonstrates o-loalisation of CAR-CT and insulin staining (yellow). Sale ars, 10 μm. () Granular distriution of CAR-SIV (green) and oloalisation with insulin (red) and DAPI (dark lue) in the islet of a nondiaeti panreas. Sale ars, 5 μm. These results are representative of findings in the panreases of 15 non-diaeti individuals (ESM Tale 1)

7 2350 Diaetologia (2018) 61: the ytoplasm of eta ells. Confoal mirosopy revealed that CAR-SIV displayed a puntate distriution and that it speifially o-loalised with insulin, suggesting a possile assoiation with seretory granules (Fig. 3). A similar pattern of puntate immuno-o-loalisation with insulin was oserved when using an alternative CAR antiserum (CAR-ECD; data not shown). To explore these relationships further, the expression of CAR-SIV was investigated in the panreases of a series of people with type 1 diaetes (ten individuals; 3 42 years of age; ESM Tale 1, ESM Fig. 3) and islet autoantiodypositive non-diaeti individuals (two partiipants, aged 18 and 37 years, respetively). The samples from individuals with type 1 diaetes ontained oth insulin-defiient islets and insulin-ontaining islets. CAR-SIVexpression was asent from the insulin-defiient islets, ut was learly visile in residual insulin-ontaining islets. Neither the suellular distriution nor the staining intensity differed etween the type 1 diaeti, autoantiody-positive non-diaeti and non-diaeti islets (ESM Fig. 3 and data not shown). Finally, y applying the CAR-CT antiody to a human tissue miroarray (gift from Alan Foulis; University of Glasgow, UK), it was onfirmed that the SIV isoform ould e variously found at the surfae memrane (testes and ladder small ell arinoma) and/or in ytoplasmi regions (stomah and islets) within normal and anerous tissues (ESM Fig. 4). RNAseq data supported this expression profile (ESM Fig. 4). CAR-SIV o-loalises with seretory granule proteins in eta ells In order to verify the loalisation of CAR-SIV in eta ells, onfoal o-immunofluoresene studies were performed to loalise ertain other proteins (ZnT8, Fig. 4a; prohormone onvertase 1/3 [PC1/3], Fig. 4; and proinsulin, Fig. 4) known to e present in seretory granules. Calulation of the Pearson orrelation oeffiient onfirmed that insulin strongly assoiated with ZnT8 (0.94 ± 0.01; Fig. 4d). Importantly, CAR-SIV also orrelated strongly with insulin (0.95 ± 0.02; Fig. 4d) and PC1/3 (0.81 ± 0.02; Fig. 4d). In ontrast, CAR-SIV did not assoiate with gluagon (0.05 ± 0.02; Fig. 4d,e) and was more weakly assoiated with proinsulin (0.55 ± 0.04; Fig. 4d). High-resolution onfoal mirosopy onfirmed the o-loalisation of CAR-SIV with ZnT8 (Fig. 4f) and PC1/3 (Fig. 4g), and showed that CAR-SIV and proinsulin were less frequently o-loalised (Fig. 4h). Using a more sophistiated reiproal analysis, it was onfirmed that the majority of CAR-SIV o-loalised with insulin (Manders a d Pearson orrelation oeffiient CAR-SIV & gluagon ZnT8 & insulin CAR-SIV & insulin CAR-SIV & ZnT8 e f CAR-SIV & PC1/3 CAR-SIV & proinsulin g h Fig. 4 CAR-SIV o-loalisation with multiple insulin granule proteins within the eta ell. Representative immunofluoresene staining of the CAR-SIV isoform (CAR-CT antiody; green) and insulin (lue) in relation to insulin seretory granule proteins (red): (a) ZnT8, () PC1/3 and () proinsulin. (d) Pearson orrelation oeffiient demonstrating the assoiation etween CAR-SIV and gluagon, insulin, ZnT8, PC1/3 and proinsulin, and etween ZnT8 and insulin. Eah data point represents a single islet, and two islets were assessed per ase from eah of three independent samples. (e h) Representative higher magnifiation images of the CAR- SIV isoform (CAR-CT antiody; green) with (e) gluagon, (f) ZnT8, (g) PC1/3 and (h) proinsulin in red. DAPI is shown in dark lue. No assoiation was oserved etween the CAR-SIV isoform (CAR-CT antiody; green) and gluagon (red) (e). Sale, ars 10 μm

8 Diaetologia (2018) 61: o-loalisation oeffiient [MCC] ± 0.016) and that this was also true in reverse (i.e. the proportion of insulin oloalising with CAR-SIV was high (MCC ± 0.028; ESM Fig. 5). By ontrast, although a large proportion of total proinsulin o-loalised with CAR-SIV (MCC ± 0.082), this did not hold in reverse (CAR-SIV:proinsulin, MCC ± 0.042) eause little proinsulin esaped into mature seretory granules. Together, these findings suggest that CAR-SIV is present in oth immature and mature insulin seretory granules in human eta ells. a Cryo-immune eletron mirosopy onfirms that CAR-SIV is loalised to insulin seretory granules To onfirm more diretly that the CAR-SIV isoform loalises to insulin seretory granules, immunogold laelling was performed on thin frozen setions using the post-emedding Tokuyasu method [24] with antisera against CAR-SIV (10 nm gold partiles) and ZnT8 (5 nm gold partiles). This revealed that oth antisera loalise to seretory granules in human panreas setions (Fig. 5); the harateristi eletron-dense appearane of the granule ores implies that they ontain insulin. This was verified y immunostaining with anti-insulin (ESM Fig. 6). By ontrast, gold partiles were not onentrated in the seretory granules of exorine ells. Immunogold laelling of insulin seretory granules with the CAR-CT antiody demonstrated that loalisation was least aundant in the entre of the granule ores and preferentially displayed at their periphery (Fig. 5,d). Laelling of ZnT8 showed a similar distriution. Quantifiation of 841 CAR-SIV immunogold partiles from 21 different mirographs, aross 1291 different memrane intersetions, revealed that CAR-SIV was most aundant on mature insulin seretory granules (79%). CAR-SIV was also oserved in immature insulin granules (14.4%), ut was rarely oserved in eta ell nulei (2.5%), mitohondria (1.0%) or endoplasmi retiulum (1.1%) or on the plasma memrane (1.2%; Fig. 5,e). Of note, uranyl aetate yields a negative ontrast for organelle memranes in the staining method employed, and as a onsequene the appearane of the seretory granules differs from that seen with osmium laelling. To further onfirm the presene of CAR-SIV in the insulin granules at different stages of granule maturation, immunogold laelling of normal panreas setions to detet proinsulin (20 nm gold partiles), CAR-SIV (10 nm gold partiles) and insulin (5 nm gold partiles) was performed (ESM Fig. 6). This revealed the presene of CAR-SIV in immature granules, defined as proinsulin and CAR-SIV positive (ESM Fig. 6); maturing granules, defined as proinsulin, insulin and CAR-SIV positive (ESM Fig. 6d); and mature granules, defined as insulin and CAR-SIV positive (ESM Fig. 6e). Examination of 122 granules ontaining CAR-SIV revealed that six (4.9%) were also positive for proinsulin (immature d e CAR-SIV gold lael on memranes (%) Mature insulin granule Immature insulin granule Plasma memrane Intraellular vesile ER Mitohondrion Nuleus Fig. 5 Cryoimmune eletron mirosopy. Immunogold laelling of CAR-SIV (10 nm gold partiles) and ZnT8 (5 nm gold partiles) in thin frozen setions of human panreas tissue. (a) The low-magnifiation image demonstrates the presene of granules in ainar ells and in islet ells. (, ) The higher magnifiation images (of the areas outlined y the dashed lak oxes in a) reveal a lak of CAR-SIV laelling of ainar ell granules (), ut positive CAR-SIV laelling in eta ell granules (). (d) A higher resolution, magnified image onfirms that the laelling of CAR- SIV (10 nm gold; lak arrows) and ZnT8 (5 nm gold; lak arrowheads) surrounds the eta ell granules. Sale ars, 2 μm (a), 1 μm (, ), and 500 nm (d). (e) Relative distriution of CAR-SIV in organelles ased on quantifiation using line intersetion ounting. ER, endoplasmi retiulum

9 2352 Diaetologia (2018) 61: a Pearson orrelation oeffiient CAR-SIV & PICK1 CAR-SIV & insulin PICK1 & insulin Fig. 6 CAR-SIV o-loalisation with PICK1 in the eta ell. (a) Representative immunofluoresene staining of PICK1 (red), CAR-SIV isoform (CAR-CT antiody; green), insulin (light lue) and DAPI (dark lue) in non-diaeti human panreas. Overlay of CAR-SIV and PICK1 (yellow), and CAR-SIV, PICK1 and insulin (white). () Pearson orrelation oeffiient demonstrating the extent of o-loalisation of CAR-SIV with PICK1, CAR-SIV with insulin,andpick1withinsulin.() Hyvolution imaging demonstrates a lose assoiation of the CAR-SIV isoform (CAR-CT antiody; green) and PICK1 (red) in the insulin granule (light lue) of a non-diaeti panreas. Granules positive for insulin, PICK1 and CAR-SIV are indiated y the orange arrows. Sale ars, 5 μm granules); 30 (24.6%) were also positive for proinsulin and insulin (maturing granules), and the majority 86 (70.5%) were positive for insulin (mature granules). Taken together, these results suggest that CAR-SIV is present within the granule memrane at all stages of granule maturation. CAR-SIV o-loalises with PICK1 in insulin seretory granules PICK1 may play an important role in insulin granule traffiking and maturation [18], and the SIV isoform of CAR, ut not the TVV isoform, seletively interats with PICK1 in other ell types [13, 15]. To assess whether CAR-SIV o-loalises with PICK1 in human eta ells, further onfoal oimmunofluoresene studies (Fig. 6) and immunopreipitation (ESM Fig. 7) studies were performed. PICK1 was readily deteted in islet endorine ells (in oth eta and non-eta ells; Fig. 6a) and, importantly, it o-loalised with CAR- SIV in eta ells (Fig. 6a) and was o-immunopreipitated with CAR from EndoC-βH1 ells and human islets (ESM Fig. 7). Pearson s orrelation analysis of the immunofluoresene signals (Fig. 6) onfirmed a strong assoiation etween PICK1 and oth CAR-SIV (0.73 ± 0.02) and insulin (0.84 ± 0.04). To examine the o-loalisation of PICK1, CAR-SIV and insulin in more detail, onfoal mirosopy oupled with Hyvolution software was employed to provide improved resolution. This onfirmed the intimate assoiation etween PICK1 and the SIV isoform of CAR within insulin seretory granules (Fig. 6). PICK1 was also oserved in assoiation with the seretory granules of other islet non-eta endorine ells, ut CAR-SIV was not deteted in those ells (Fig. 6). Disussion The present study reveals that the SIV isoform of CAR is expressed seletively y eta ells in the human panreas. This finding was onfirmed at oth the RNA and protein levels. We also disovered that the suellular loalisation of CAR-SIV was atypial: the protein was found mainly within the ytoplasmi domain of eta ells rather than at the ell surfae. We used oth immunologial and moleular iologial approahes to investigate CAR expression in human islet ells, and the results were onordant. Thus, analysis of RNA extrated either from isolated human islets or laser apture mirodisseted islets revealed that two major isoforms of CAR, CAR-SIV and CAR-TVV, were present. Both of these ontain a single transmemrane domain, implying that they ould eah e loalised within defined, memrane-limited, ompartments in the islet ells, as in other ell types [13]. The results

10 Diaetologia (2018) 61: KEY: CAR-SIV PICK1 CVB Proinsulin Insulin Mature granule CAR-ECD CAR-CT Fig. 7 CAR-SIV in eta ells. Our data demonstrate that CAR-SIV is present at high onentrations on the insulin granule and is losely assoiated with the ytoplasmi protein PICK1. CAR-SIV has previously een shown to interat with PICK1, whih is proposed to have a role in the udding and maturation of vesiles from the trans-golgi network. We predit that the C-terminus of CAR-SIV faes the extragranular/ytoplasmi environment sine its PDZ-inding domain would e availale to interat with ytoplasmi PICK1 only in this orientation. We propose that CAR-SIV, through its interation with PICK1, ould therefore play a hitherto unsuspeted role in the maturation and traffiking of the insulin granule. Importantly, when onsidering the orientation of CAR-SIV in this model, the putative ECD of CAR-SIV, whih is required for the inding of enteroviruses, faes the interior of the seretory granule during its maturation. This suggests that, as the insulin granule fuses with the plasma memrane during insulin exoytosis, the ECD of CAR-SIV eomes displayed on the external fae of the plasma memrane and is then ale to ind to enteroviruses that use this reeptor, for example CVBs. During the susequent endoytosis of the granule, for reyling, the virus would e transported inside the ell, where it ould initiate a produtive infetion favoured a preponderane of the CAR-SIV isoform in islets, and immunohistohemial analysis onfirmed aundant prodution of CAR-SIV protein in human islet ells, ontrasting with its asene from the exorine panreas. Taken as a whole, the results suggest that the major isoform of CAR present in human islets is CAR-SIV, although CAR-TVV is also present. CAR-SIV is expressed preferentially in eta ells, sine we were unale to find evidene for its expression in non-eta islet ells. By ontrast, use of less seletive antisera suggested that additional isoforms of CAR (inluding CAR-TVV) may e present among the other non-eta endorine ells. This would e onsistent with the analysis of RNA expression. The primary suellular loalisation of CAR in eta ells has not een extensively addressed in previous work [3, 20] ut, despite this, in other ell types a onsensus has emerged that this protein is often loalised within tight juntional omplexes at the plasma memrane [13, 14, 26]. As suh, CAR would e expeted to e present at the ell surfae, where it ould fulfil a seondary (presumaly suverted) role as a vehile for viral entry. Thus, the present demonstration that CAR-SIV is present in human eta ells is onsistent with the known sensitivity of these ells to infetion y various enterovirus serotypes. However, the intriguing disovery that this isoform was loalised primarily at an intraellular site in eta ells suggests a more omplex senario (and a different physiologial role) ompared with other ells in whih CAR resides mainly on the plasma memrane. High-resolution onfoal mirosopi analysis was used to examine in more detail the unexpeted suellular loalisation of CAR-SIV in eta ells. This revealed a puntate, ytoplasmi immunolaelling pattern for CAR-SIV, onsistent with its distriution in a distint intraellular organelle ompartment. Additional studies demonstrated that this immunolaelling profile orrelated with that of insulin, as well as with two additional seretory granule proteins, ZnT8 and PC1/3, therey implying a loalisation within eta ell insulin seretory granules. Diret onfirmation of this was provided y ryoimmune eletron mirosopy studies in whih immunogold methods allowed the visualisation of CAR-SIV, prinipally in assoiation with the dense-ore granules harateristially found in eta ells. By ontrast, no laelling was seen in the equivalent seretory granules found in adjaent exorine ells, implying that CAR-SIV is not asolutely required for seretory granule iogenesis in all ell types. Importantly, and onsistent with results otained in other ells [18], we also noted that CAR-SIV o-loalised with, and ould e immunopreipitated with, the PDZ domain protein PICK1 in eta ells. PICK1 plays a speifi role in insulin seretory granule maturation [18, 27], and it is oneivale that CAR-SIV serves as a seletive inding partner for PICK1 in eta ells, therey onentrating the two proteins within maturing seretory granules. Consistent with this hypothesis, we found y immunofluoresene analysis that the SIV isoform of CAR is muh more strongly assoiated with mature insulin than with proinsulin, suggesting that CAR (and PICK1) may eome onentrated in seretory granules as these mature eyond their emergene from the trans-golgi network.

11 2354 Diaetologia (2018) 61: If this model is orret, it has additional important onsequenes. In partiular, the model predits that the C-terminus of CAR-SIV must fae the extragranular environment, sine its PDZ-inding domain is loated in this region and would only e availale to interat with ytoplasmi PICK1 (or other ytoplasmi PDZ-inding proteins) in this orientation. As suh, the putative ECD of CAR-SIV would then fae the lumen of the granule, with the single transmemrane region serving to anhor the protein in this orientation in the limiting memrane surrounding the granule. Thus, the region of SIV required for the inding of enteroviruses would fae the interior of the seretory granule during maturation. It follows from this that the ECD of CAR-SIV would eome displayed on the external fae of the plasma memrane following the fusion of the seretory granule and plasma memranes during exoytosis. These onsiderations are summarised in Fig. 7 and suggest that enteroviral entry into eta ells may e failitated under onditions in whih the rate of seretory granule exoytosis is high (and granule memrane reyling rates are orrespondingly elevated). Thus, CAR-SIV appears to e onfigured in eta ells suh that it an interat with PDZ-inding proteins during seretory granule maturation, and its virus-inding domain eomes exposed to the extraellular environment during exoytosis. Of note, most of the autoantigens in type 1 diaetes are expressed in the insulin granule [28]and the preferential loalisation of viral reeptors in the granule indiates a potential mehanism y whih CVB infetion may modify the proessing of granule proteins, to promote the generation of autoantigens. In support of our onlusions, Ylipaasto et al [3] demonstrated that the pre-treatment of human islets with a loking antiody direted solely against externally oriented CAR attenuated infetion with Coxsakie viruses. When oupled with our finding that the majority of CAR is loated intraellularly in islet ells, this suggests that externalisation of the protein ours to mediate viral entry. Moreover,wealsonoteHodiketal s [29] demonstration that enterovirus repliation omplexes and viral partile latties are present on or near to insulin granules in CVB autely infeted eta ells. Thus, this weight of evidene is strongly supportive of our hypothesis and deserves further study. In summary, we show that human eta ells express the SIV isoform of CAR in the insulin granules. We propose that the iohemial properties of CAR-SIV that onfer the physiologial importane of this protein within eta ells may also represent an Ahilles heel y whih the entry and repliation of enteroviruses is failitated. Aknowledgements This researh was performed with the support of the Network for Panreati Organ donors with Diaetes (npod; RRID:SCR_ ), a ollaorative type 1 diaetes researh projet sponsored y the JDRF (npod: 5-SRA Q-R) and The Leona M. & Harry B. Helmsley Charitale Trust (Grant no. 2018PG-T1D053). Organ Prourement Organizations (OPO) partnering with npod to provide researh resoures are listed at The Bioenter Oulu eletron mirosopy ore faility and T. Kantoluoto are aknowledged for utting thin, frozen setions and assisting with staining. We would also like to aknowledge J. L. E. Hill (Institute of Biomedial & Clinial Siene, University of Exeter Medial Shool, Exeter, UK) for assistane with the immunopreipitations and J.- V. Turatsinze (ULB Center for Diaetes Researh, Brussels) for ioinformatis support. Some of the data in this manusript were presented at the Diaetes UK Annual Professional Conferene in 2017 (P14). Data availaility The datasets generated during and/or analysed during the urrent study are availale from the orresponding author on reasonale request. Funding We are pleased to aknowledge finanial support from the European Union s Seventh Framework Programme PEVNET (FP7/ ) under grant agreement numer The partiipants of the PEVNET onsortium are desried at puliations.html. Additional support was from a JDRF Career Development Award (5-CDA A-N) to SJR, a JDRF researh grant awarded to the network of Panreati Organ Donors Virus (npod-v) onsortium (JDRF ), an MRC Projet Grant MR/P010695/1 awarded to SJR and NGM, a Foundation main grant to EI from the Funds for Women Graduates (178123), and FRFS-Welio grant CR-2015A-06, Belgium, awarded to DLE. Duality of interest The authors delare that there is no duality of interest assoiated with this manusript. Contriution statement SJR and NGM designed the study, performed data analysis and interpretation, and wrote the manusript. EI, MAR, SD, PL, GS, LN, DLE and VM performed data olletion and analysis, and edited the manusript. FD provided ritial analysis of the results and edited the manusript. All authors approved the manusript s final version. SJR and NGM are the guarantors of this work and, as suh, had full aess to all the data in the study and take responsiility for the integrity of the data and the auray of the data analysis. Open Aess This artile is distriuted under the terms of the Creative Commons Attriution 4.0 International Liense ( reativeommons.org/lienses/y/4.0/), whih permits unrestrited use, distriution, and reprodution in any medium, provided you give appropriate redit to the original author(s) and the soure, provide a link to the Creative Commons liense, and indiate if hanges were made. Referenes 1. Yeung W-CG, Rawlinson WD, Craig ME (2011) Enterovirus infetion and type 1 diaetes mellitus: systemati review and metaanalysis of oservational moleular studies. BMJ 342:d35 2. 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