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

Abstract

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.

Keywords: Beta cells; Coxsackie and adenovirus receptor; Coxsackievirus B; Enterovirus; Insulin granule; Pancreas; Protein interacting with C-kinase 1 (PICK1).

Fig. 1

A description of the CAR isoforms and the selective expression of CAR-SIV in human islets. (a) CAR protein structure. The signal peptide (red) is cleaved to yield a mature protein with an ECD comprising two immunoglobulin (Ig)-like domains, type 1 (blue) and type 2 (green). The transmembrane domain (yellow) bridges the extracellular and cytoplasmic regions (pink), which terminates with a PDZ-binding domain (red). (b) CXADR exon maps of the five differentially spliced isoforms. The type 1 Ig domain is encoded by exons 2 and 3, while type 2 Ig-like domain is encoded by exons 4 and 5. Isoforms 1 and 2 contain a transmembrane domain and are named CAR-SIV (or hCAR1, CAR^Ex7) and CAR-TVV (or hCAR2, CAR^Ex8), respectively (denoted by the three terminal amino acids on their C-termini). The soluble isoforms 3, 4 and 5 are named CAR4/7, CAR3/7 and CAR2/7, respectively, reflecting exon inclusion or exclusion and lack of the transmembrane domain. The binding regions of the different CAR antibodies are also shown. The CAR-CT antiserum recognises amino acids 335–365 located at the C-terminus of the CAR-SIV isoform but does not recognise the other CAR isoforms. Both the CAR-ECD and CAR-RmcB antisera recognise epitopes within the ECD, and are predicted to recognise the majority of the longer CAR isoforms (CAR-SIV, CAR-TVV, CAR4/7 and potentially CAR3/7). (c, d) Representative immunocytochemical analysis of the expression of different CAR isoforms in normal control pancreas tissue stained with (c) CAR-CT and (d) CAR-ECD antisera. Scale bars, 20 μm. The insets below represent higher magnification images (of the areas outlined by the black boxes) demonstrating the differential staining profile of the CAR-CT antisera in the endocrine (+++) and exocrine pancreas (−), and the CAR-ECD antisera in the endocrine (+++) and exocrine pancreas (+); +++, strong; +, weak; −, negative. These results are representative of findings in pancreas from eight non-diabetic individuals; aa, amino acids, Ab., antibody

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 (b) 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 barely detectable. Data were normalised to the relative expression of three housekeeping genes, β-actin, GAPDH and B2M. Relative expression is presented as the mean ± SEM. (c) RNAseq data showing CAR isoform expression in islets from five normoglycaemic individuals (mean ± SEM) [23]. RPKM, reads per kilobase of exon model per million mapped reads. (d) Confirmation of CAR-SIV protein expression in isolated human islets and EndoC-βH1 cells as assessed by western blotting using CAR-CT and CAR-ECD antisera and loading control glyceraldehyde 3-phosphate dehydrogenase (GAPDH). H., human

Fig. 3

The SIV isoform of CAR is expressed in pancreatic beta cells. (a) Representative immunofluorescence staining of the CAR-SIV isoform (CAR-CT antibody; green), insulin (light blue), glucagon (red) and DAPI (dark blue) in an islet from a non-diabetic human pancreas. Scale bars, 10 μm. (b) CAR-SIV isoform staining in FFPE isolated human islets: CAR-SIV (CAR-CT; green) and insulin (red) and DAPI (dark blue). The enlarged region (from the area outlined by the dashed white box) demonstrates co-localisation of CAR-CT and insulin staining (yellow). Scale bars, 10 μm. (c) Granular distribution of CAR-SIV (green) and co-localisation with insulin (red) and DAPI (dark blue) in the islet of a non-diabetic pancreas. Scale bars, 5 μm. These results are representative of findings in the pancreases of 15 non-diabetic individuals (ESM Table 1)

Fig. 4

CAR-SIV co-localisation with multiple insulin granule proteins within the beta cell. Representative immunofluorescence staining of the CAR-SIV isoform (CAR-CT antibody; green) and insulin (blue) in relation to insulin secretory granule proteins (red): (a) ZnT8, (b) PC1/3 and (c) proinsulin. (d) Pearson correlation coefficient demonstrating the association between CAR-SIV and glucagon, insulin, ZnT8, PC1/3 and proinsulin, and between ZnT8 and insulin. Each data point represents a single islet, and two islets were assessed per case from each of three independent samples. (e–h) Representative higher magnification images of the CAR-SIV isoform (CAR-CT antibody; green) with (e) glucagon, (f) ZnT8, (g) PC1/3 and (h) proinsulin in red. DAPI is shown in dark blue. No association was observed between the CAR-SIV isoform (CAR-CT antibody; green) and glucagon (red) (e). Scale, bars 10 μm

Fig. 5

Cryoimmune electron microscopy. Immunogold labelling of CAR-SIV (10 nm gold particles) and ZnT8 (5 nm gold particles) in thin frozen sections of human pancreas tissue. (a) The low-magnification image demonstrates the presence of granules in acinar cells and in islet cells. (b, c) The higher magnification images (of the areas outlined by the dashed black boxes in a) reveal a lack of CAR-SIV labelling of acinar cell granules (b), but positive CAR-SIV labelling in beta cell granules (c). (d) A higher resolution, magnified image confirms that the labelling of CAR-SIV (10 nm gold; black arrows) and ZnT8 (5 nm gold; black arrowheads) surrounds the beta cell granules. Scale bars, 2 μm (a), 1 μm (b, c), and 500 nm (d). (e) Relative distribution of CAR-SIV in organelles based on quantification using line intersection counting. ER, endoplasmic reticulum

Fig. 6

CAR-SIV co-localisation with PICK1 in the beta cell. (a) Representative immunofluorescence staining of PICK1 (red), CAR-SIV isoform (CAR-CT antibody; green), insulin (light blue) and DAPI (dark blue) in non-diabetic human pancreas. Overlay of CAR-SIV and PICK1 (yellow), and CAR-SIV, PICK1 and insulin (white). (b) Pearson correlation coefficient demonstrating the extent of co-localisation of CAR-SIV with PICK1, CAR-SIV with insulin, and PICK1 with insulin. (c) Hyvolution imaging demonstrates a close association of the CAR-SIV isoform (CAR-CT antibody; green) and PICK1 (red) in the insulin granule (light blue) of a non-diabetic pancreas. Granules positive for insulin, PICK1 and CAR-SIV are indicated by the orange arrows. Scale bars, 5 μm

Fig. 7

CAR-SIV in beta cells. Our data demonstrate that CAR-SIV is present at high concentrations on the insulin granule and is closely associated with the cytoplasmic protein PICK1. CAR-SIV has previously been shown to interact with PICK1, which is proposed to have a role in the budding and maturation of vesicles from the trans-Golgi network. We predict that the C-terminus of CAR-SIV faces the extragranular/cytoplasmic environment since its PDZ-binding domain would be available to interact with cytoplasmic PICK1 only in this orientation. We propose that CAR-SIV, through its interaction with PICK1, could therefore play a hitherto unsuspected role in the maturation and trafficking of the insulin granule. Importantly, when considering the orientation of CAR-SIV in this model, the putative ECD of CAR-SIV, which is required for the binding of enteroviruses, faces the interior of the secretory granule during its maturation. This suggests that, as the insulin granule fuses with the plasma membrane during insulin exocytosis, the ECD of CAR-SIV becomes displayed on the external face of the plasma membrane and is then able to bind to enteroviruses that use this receptor, for example CVBs. During the subsequent endocytosis of the granule, for recycling, the virus would be transported inside the cell, where it could initiate a productive infection