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

Abstract

Aims/hypothesisThe 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.MethodsFormalin-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.ResultsAn 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/interpretationThe 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 materialThe online version of this article (10.1007/s00125-018-4704-1) contains peer-reviewed but unedited supplementary material, which is avai...

Enterovirus and Encephalitis

“…There is evidence that immature neurons express relatively high levels of CAR compare to their fully differentiated counterparts (Ahn et al, 2008), suggesting that the level of virus receptor is one of the critical determinants in preferential virus replication in undifferentiated neural progenitor cells. Besides, CAR exists as multiple isoforms and a specific isoform of CAR that is expressed at high levels in human pancreatic beta cells has been suggested to be prone to coxsackievirus infection (Ifie et al, 2018). Whether this specific isoform of CAR plays a role in coxsackievirus infection in the CNS is not known.…”

Islet–immune interactions in type 1 diabetes: the nexus of beta cell destruction

“…result in the generation and presentation of neoantigens [67]. Indeed, T cells reactive to post-translationally modified proteins such as a citrullinated form of GRP78 292-305 and islet amyloid polypeptide (IAPP) [65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80][81][82][83][84] , as well as defective ribosomal insulin gene products (DRiPs) and peptides formed from the fusion of insulin C-peptide fragments with chromogranin A or IAPP (termed hybrid insulin peptides, HIPs) have been isolated from the islets of T1D donors [57,68]. Thus, β-cells may play a role in their own destruction through stress-induced protein processing defects, which may serve to induce a critical break in tolerance or to exacerbate existing pathology by expanding the β-cell antigenic repertoire.…”

“…Extensive efforts to identify precise environmental triggers that might initiate or potentiate the autoimmune process have proven challenging thus far, but examination of donor pancreata have revealed viral response signatures which could offer clues into early T1D etiology. Biopsies of pancreas from living donors with recentonset T1D, aged 24-35 years, through DiViD contained some indications of viral capsid protein VP1 immunostaining, HLA Class I hyperexpression, and enterovirus RNA within the islets [69], possibly the result of a β-cell tropism mediated by expression of a specific isoform of the Coxsackie and Adenovirus Receptor [70]. Furthermore, islets infected with coxsackievirus B (CVB) in-vitro demonstrated reduced insulin mRNA expression and insulin secretion [71], suggesting viral infection could impair β-cell function.…”

From immunohistological to anatomical alterations of human pancreas in type 1 diabetes: New concepts on the stage

“…Moreover, capsid protein VP1 was detected in islets (but not in exocrine tissue) of pancreatic autopsy specimens from patients with T1D, as well as in β‐cells (but not in α‐cells) of islets from pancreatic biopsies of recent‐onset T1D patients recruited in the DiViD study . We can hypothesize a scenario in which enteroviruses (in particular CVBs) could infect pancreatic β‐cells by binding to specific receptors (such as human Coxsackie‐ and adenovirus receptor, hCAR) . After internalization into β‐cells, viruses could be recognized by innate immune pattern recognition receptors (PRRs), triggering inflammatory signalling cascades, and leading to the production of proinflammatory chemokines and cytokines, with subsequent expression of interferon‐stimulated genes (ISGs), whose products could limit infection.…”