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

Aims/hypothesis The Coxsackie and adenovirus receptor (CAR) is a transmembrane cell-adhesion protein that serves as an entry receptor for enteroviruses and may be essential for their ability to infect cells. Since enteroviral infection of beta cells has been implicated as a factor that could contribute to the development of type 1 diabetes, it is often assumed that CAR is displayed on the surface of human beta cells. However, CAR exists as multiple isoforms and it is not known whether all isoforms subserve similar physiological functions. In the present study, we have determined the profile of CAR isoforms present in human beta cells and monitored the subcellular localisation of the principal isoform within the cells. Methods Formalin-fixed, paraffin-embedded pancreatic sections from non-diabetic individuals and those with type 1 diabetes were studied. Immunohistochemistry, confocal immunofluorescence, electron microscopy and western blotting with isoform-specific antisera were employed to examine the expression and cellular localisation of the five known CAR isoforms. Isoform-specific qRT-PCR and RNA sequencing (RNAseq) were performed on RNA extracted from isolated human islets. Results An isoform of CAR with a terminal SIV motif and a unique PDZ-binding domain was expressed at high levels in human beta cells at the protein level. A second isoform, CAR-TVV, was also present. Both forms were readily detected by qRT-PCR and RNAseq analysis in isolated human islets. Immunocytochemical studies indicated that CAR-SIV was the principal isoform in islets and was localised mainly within the cytoplasm of beta cells, rather than at the plasma membrane. Within the cells it displayed a punctate pattern of immunolabelling, consistent with its retention within a specific membrane-bound compartment. Co-immunofluorescence analysis revealed significant co-localisation of CAR-SIV with zinc transporter protein 8 (ZnT8), prohormone convertase 1/3 (PC1/3) and insulin, but not proinsulin. This suggests that CAR-SIV may be resident mainly in the membranes of insulin secretory granules. Immunogold labelling and electron microscopic analysis confirmed that CAR-SIV was localised to dense-core (insulin) secretory granules in human islets, whereas no immunolabelling of the protein was detected on the secretory granules of adjacent exocrine cells. Importantly, CAR-SIV was also found to co-localise with protein interacting with C-kinase 1 (PICK1), a protein recently demonstrated to play a role in insulin granule maturation and trafficking. Conclusions/interpretation The SIV isoform of CAR is abundant in human beta cells and is localised mainly to insulin secretory granules, implying that it may be involved in granule trafficking and maturation. We propose that this subcellular localisation of CAR-SIV contributes to the unique sensitivity of human beta cells to enteroviral infection. Electronic supplementary material The online version of this article (10.1007/s00125-018-4704-1) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

reaching 80% confluence and both cell lines were maintained at 37 0 C, 100% humidity and 5% CO2. All cells were mycoplasma negative.

Western blotting
Cells were collected and lysed in buffer (20mmol/l Tris, 150mmol/l Nacl, 1mM EDTA and 1% Triton-X including protease and phosphatase inhibitors (Sigma, Poole, UK)) on ice for 10mins.
Protein lysates was separated from insoluble fraction by centrifugation at 5000rpm for 10mins at 4 0 C. Total protein was estimated with the Pierce TM (BCA) Protein Assay (Thermofisher Scientific, UK) and absorbance was measured at 562nm with a PHERAstar (BMG Labtech).

Real Time RTqPCR
Total RNA from isolated human islets was extracted using miRNeasy mini kit (Qiagen, Hilden, Germany). Briefly, human isolated islets were lysed in Qiazol solution and chloroform was added to separate DNA, RNA and proteins. The aqueous phase was then loaded on to RNA binding columns and eluted in 30µl of nuclease-free water. RNA quality were evaluated using a 2100 Bioanalyzer RNA 6000 Pico kit (Agilent Technologies, Santa Clara, CA, USA) and only samples with RNA Integrity Number (RIN) >5.0 were used for further analyses.
Differential expression of alternatively spliced isoforms of the human CXADR gene were evaluated using custom designed TaqMan primers and probes (Thermofisher) (ESM Table 3).
For human isolated islets (ESM Table 4), 250ng of total RNA was retro-transcribed using Superscript III Reverse Transcriptase kit (Thermofisher) and 10ng of the resultant cDNA were loaded into 2x TaqMan Universal Master Mix, 20x TaqMan gene expression assay buffer and nuclease-free water in a final volume of 20µl. Data were normalised using the expression of β-Actin, GAPDH and β2-microglobulin. Reactions were performed on a Verity Thermal Cycler and ViiA7 Real Time PCR instruments (Thermofisher). Data were analysed and exported using Expression Suite software 1.1 (Thermofisher) and finally elaborated using the 2 -ΔCt method.
For LCM captured islets (ESM Table 5), 1ng of total RNA was retro-transcribed using Superscript III Reverse Transcriptase kit (Thermofisher). The resultant cDNA was preamplified using 0.2x Tris-EDTA-diluted TaqMan assays pool (designed to amplify CXADR isoforms plus 3 housekeeping genes (β-Actin, GAPDH, β2-microglobulin)) and 2x Preamp Master Mix in a final volume of 50µl. The pre-amplification reaction was diluted in Tris-EDTA and 5µl of each pre-amplified cDNAs were used in a Real-Time PCR reaction in a final volume of 20µl.

Semi-quantitative RT-PCR
Total RNA was isolated from human islets (ESM Table 4

Quantification of CAR-SIV in Cryo-immune EM
Quantification of CAR-CT immuno-gold labelling was performed by conventional line

Co-immunoprecipitation of PICK1 with CAR
Whole cell extracts were prepared by lysing 5 x 10 5 EndoC-βH1 cells or human islets with buffer containing 50mmol/l Tris (pH 7.5); 137mmol/l NaCl; 5mmol/l EDTA; 1mmol/l EGTA; 10µg/ml protease inhibitor (Sigma) and 10µg/ml phosphatase inhibitor cocktail 2 & 3 (Sigma, UK). The lysates were incubated with 3µg of anti-CAR clone RmcB or negative control mouse IgG (Dako; X0931) overnight at 4⁰C. Protein G Sepharose beads were then added for 4 hours at 4⁰C followed by three washes [first -lysis buffer; second -10% lysis buffer in TBS (50mmol/l Tris (pH 7.5) and 137mmol/l NaCl); Third -TBS). Proteins were eluted with 4x LDS and 10% β-mercaptoethanol at 70⁰C for 10min. Western blot analysis was then performed as described above.       Table 4), EndoC-βH1 (Mean±SEM). CAR-SIV is expressed at levels 3-fold higher than CAR-TVV. CAR4/7 and CAR3/7 are present at low levels and CAR2/7 was undetectable. (b) Semi-quantitative RT-PCR (30 cycles) analysis using specific CAR-SIV and CAR-TVV primers in RNA extracted from purified human islets, suggests in two independent islet preparations that CAR-SIV is present at a higher abundance than CAR-TVV.  Table 1). CAR-SIV is expressed in T1D donor islets but only in beta cells (upper panel). Once the beta cells have been destroyed CAR-SIV expression is lost (lower panel). CAR-SIV expression in beta cells from autoantibody positive nondiabetic donors did not differ from that seen in autoantibody negative donors without diabetes (data not shown).  This analysis allows us to assess how much of the proinsulin or insulin immunostaining is coincident with CAR-SIV and vice versa. This more detailed analysis has shown that the vast majority of CAR-SIV co-localises with insulin (MCC: 0.914±0.016) and that the same is also true for insulin co-localising with CAR-SIV (MCC: 0.912±0.028). This is consistent with them both being present in mature insulin secretory granules. In contrast,