Alternative splicing encodes functional intracellular CD59 isoforms that mediate insulin secretion and are down-regulated in diabetic islets

Abstract

Human pancreatic islets highly express CD59, which is a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein and is required for insulin secretion. How cell-surface CD59 could interact with intracellular exocytotic machinery has so far not been described. We now demonstrate the existence of CD59 splice variants in human pancreatic islets, which have unique C-terminal domains replacing the GPI-anchoring signal sequence. These isoforms are found in the cytosol of β-cells, interact with SNARE proteins VAMP2 and SNAP25, colocalize with insulin granules, and rescue insulin secretion in CD59-knockout (KO) cells. We therefore named these isoforms IRIS-1 and IRIS-2 (Isoforms Rescuing Insulin Secretion 1 and 2). Antibodies raised against each isoform revealed that expression of both IRIS-1 and IRIS-2 is significantly lower in islets isolated from human type 2 diabetes (T2D) patients, as compared to healthy controls. Further, glucotoxicity induced in primary, healthy human islets led to a significant decrease of IRIS-1 expression, suggesting that hyperglycemia (raised glucose levels) and subsequent decreased IRIS-1 expression may contribute to relative insulin deficiency in T2D patients. Similar isoforms were also identified in the mouse CD59B gene, and targeted CRISPR/Cas9-mediated knockout showed that these intracellular isoforms, but not canonical CD59B, are involved in insulin secretion from mouse β-cells. Mouse IRIS-2 is also down-regulated in diabetic db/db mouse islets. These findings establish the endogenous existence of previously undescribed non–GPI-anchored intracellular isoforms of human CD59 and mouse CD59B, which are required for normal insulin secretion.

Keywords: CD59; SNAREs; insulin secretion; intracellular complement; type 2 diabetes.

Fig. 1.

(A) Scheme representing the two alternative splice forms of human CD59, which lack the GPI-anchoring domain and have C-terminal domains. (B) The 3D structure of human CD59 is shown with the secondary structure elements labeled and the five-disulfide bonds displayed (yellow). The N-glycan at Asn18 is marked in red. (C) Predicted structure of human IRIS-1 (cyan) superimposed onto canonical CD59. The C-terminal region of IRIS-1 (the last 28 residues) is missing in the experimental template and predicted to have different conformations: a helical structure (in cyan) packing against the remaining part of the protein, a more open conformation (in purple), or the C-terminal sequence of IRIS-1 is disordered (in dark blue) but could adopt a helical conformation when in contact with other macromolecules. (D) Predicted structure of human IRIS-2 (blue) superimposed onto canonical CD59. The predicted longer loop of IRIS-2 is flexible (blue). One disulfide bond, equivalent to C64–C69 of canonical human CD59 is missing due to C-terminal amino acid changes in IRIS-2. (E) Expression of CD59 isoforms: IRIS-1, IRIS-2, and canonical CD59 in human pancreatic islets and liver, RNA level. RT-PCR, n = 3, biological repeats. (F) Expression of IRIS-1 and IRIS-2 (relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) in various human tissues was measured by RT-PCR, n = 3 technical repeats. (G) Staining with specific antibodies against IRIS-1 or IRIS-2 (red) in dispersed, primary human pancreatic islets, and colocalization with insulin granules . Nuclei stained with DAPI (blue). Results are representative for donor #317 and were repeated with human pancreatic islets from three different healthy donors.

Fig. 2.

(A and B) Transmission electron microscopy with immunogold-labeled IRIS-1 (A), and IRIS-2 (B) antibodies (20-nm particles) and insulin antibodies (10-nm particles) were used to visualize the binding between IRIS-1/2 and insulin granules in INS-1 CD59-knockout (KO) cells overexpressing human IRIS-1 or IRIS-2, after low or high glucose incubation. Colocalization between IRIS proteins and insulin granules in low and high glucose conditions was quantified in C. (D) Isolated healthy human pancreatic islets were divided into two and treated with either low (1 mM), or high (16.7 mM) glucose for 72 h, followed by blotting using specific IRIS-1 antibodies. Quantification of five biological repeats (five different donors) is shown on the Right. (E) IRIS-1 protein levels were assessed by blotting of human pancreatic islets from T2D and healthy donors. Quantification of three technical repeats from nine healthy and 10 T2D donors is shown in F. Lysates of INS-1 cells overexpressing CD59 isoforms, treated with deglycosylating enzymes, indicates lack of glycosylation of IRIS-1 in human pancreatic islets and specificity of the IRIS-1 antibody. (G) IRIS-2 protein levels were assessed by blotting of human pancreatic islets from T2D and healthy donors. Quantification of three technical repeats from nine healhy and 10 T2D donors is shown in H. Statistics (in C): two-way ANOVA with Bonferroni’s posttest; (in D): two-tailed, paired t test; (in F and H): nonparametric Mann–Whitney test. Error bars indicate SD. *P < 0.05, **P < 0.01, and ****P < 0.0001.

Fig. 3.

(A) GSIS was carried out in INS-1 CD59-KO cells transfected (transient transfection) with WT CD59, IRIS-1, or IRIS-2; n = 5. Each data point represents the average of three independent repeats for five different CD59-KO clones (untransfected [UT] or transfected with human CD59-WT, or IRIS-1, or IRIS-2). (B) GSIS (16.7 mM glucose in the presence of 200 μM diazoxide) carried out in mock control and INS-1 CD59-KO cells transiently expressing IRIS-1 and IRIS-2; n = 3 biological repeats. (C) Exocytosis by TIRF-M: Total K⁺ depolarization induced exocytosis during 40 s in control INS-1 cells (black, n = 18 cells, three preparations), CD59-KO cells (red, n = 33 cells, three preparations), and CD59-KO cells stably overexpressing IRIS-1 (blue, n = 27 cells, three preparations) or IRIS-2 (orange, n = 27 cells, three preparations). (D) Exocytosis by patch clamp: Average cell capacitance time course (ΔCm, ±SEM) during a train of 14 × 200-ms depolarizations from −70 mV to 0 mV (n = 15, 30 cells assessed per clone). (E) Average changes in membrane capacitance from H, during the first depolarization, and total increase during the train of 14 depolarizations. (F) GSIS was carried out in EndoC-βH1 cells UT or transfected with siRNA against total human CD59 (siCD59) or negative control (siCtrl). n = 3 biological repeats. (G) GSIS inducing first (12 min) and second (1 h) phase of insulin exocytosis carried out in INS-1 CD59-KO cells stably overexpressing IRIS-1 and IRIS-2; n = 3, biological repeats. (H and I) Current–voltage relationship of inward currents in control INS-1 cells (black, n = 30 cells, four preparations), CD59-KO cells (red, n = 29 cells, four preparations), and CD59-KO cells stably overexpressing IRIS-1 (blue, n = 19 cells, three preparations) or IRIS-2 (orange, n = 15 cells, three preparations). (H) Average Ca²⁺ current during 5 to 45 ms of the depolarization and (I) peak Na⁺ current during the first 5 ms of the depolarization (p = 0.0001 for IRIS-1 and 0.001 for IRIS-2 vs. CD59-KO). Statistics (in A, B, and F): two-way ANOVA with Bonferroni’s posttest, (in C): one-way ANOVA with Fisher’s posttest, (in D, E, H, and I): two-tailed, unpaired t test. (in G): two-way ANOVA with Dunnett’s posttest. Error bars indicate SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 4.

(A) Western blot representing subcellular fractionation of INS-1 CD59-KO cells overexpressing FLAG-tagged human IRIS-1 and IRIS-2. Control proteins were found in expected compartments; n = 5 biological repeats. (B) Coimmunoprecipitation of IRIS-1, IRIS-2, and WT CD59 with VAMP2 from INS-1 CD59-KO cells stably expressing FLAG-tagged CD59 isoforms; n = 4 biological repeats. ELISA was used to verify the interaction between IRIS-1, IRIS-2, and VAMP2 (C) or SNAP25 (D) in lysates from INS-1 CD59-KO cells stably overexpressing IRIS-1 or IRIS-2. Prior to ELISA, cells were incubated with either high (16.7 mM) or low (2.8 mM) glucose concentrations; n = 3 biological repeats. Background absorbance values obtained for negative control (lysates of INS-1 CD59-KO cells) were subtracted from the presented samples. (E) Proximity ligation assay was used to assess colocalization (red dots) of CD59 isoforms with VAMP2 or SNAP25, under low or high glucose conditions, and in the presence of the calcium chelator BAPTA-AM. (F) Quantification of interactions shown in E; n = 3 biologial repeats. Statistics (in C and D): two-way ANOVA with Sidak’s posttest; (F): two-way ANOVA with Bonferroni’s posttest. Error bars indicate SD. *P < 0.05, **P < 0.01,***P < 0.001, and ****P < 0.0001.

Fig. 5.

(A) Scheme of alternative splice variants of mouse CD59B, lacking the GPI-anchoring domain with C-terminal domains (of 1 amino acid [aa] and 34 aa for IRIS-1 and -2, respectively). These isoforms were named mouse IRIS-1 and IRIS-2 (Mouse Isoforms Rescuing Insulin Secretion 1 and 2). The 3D structure of human CD59 (green) superimposed onto mouse IRIS-1 (orange) (B), and IRIS-2 (gray) (C). The N-glycan at N18 is marked in red. The orientation of mouse IRIS-1 and IRIS-2 is slightly shifted as compared to Fig. 1 B–D to visualize the C-terminal regions. In the short mouse IRIS-1 model, helices alpha1 and alpha1’c and strands E and C are missing as compared to the human CD59 experimental structure. In the predicted mouse IRIS-2 structure, helices alpha1, alpha1’c, and strand E are missing as compared to human CD59. (D) CD59A expression (Left) and CD59B expression (Right) in WT and diabetic Akita mice liver and pancreatic islets, measured by qPCR; n = 3 technical repeats. (E) Expression of CD59A and CD59B in the mouse pancreatic β-cell line (MIN6) and mouse liver; n = 3 biological repeats. (F) Mouse CD59B isoforms in MIN6 cells detected by RT-PCR; n = 3 biological repeats. (G) RT-PCR results showing expression of mouse IRIS-1 and IRIS-2 in liver, spleen, and islets isolated from WT and diabetic db/db mice; n = 3 technical repeats. (H) Quantification of WT results shown in panel G. (I) Quantification of IRIS-1 and IRIS-2 in diabetic db/db mouse and WT islets shown in panel G. (J) K⁺ stimulated insulin secretion performed on MIN6 cells, MIN6 with total CD59B knockout, or MIN6 with targeted knockout of canonical CD59B (cCD59B) only, leaving IRIS-1 and IRIS-2 still expressed. Heterozygous knockouts: ^+/−; homozygous knockouts: ^−/−; n = 5 biological repeats. (K) Reexpression of mouse IRIS-1 or IRIS-2 in MIN6 cells with total CD59B KO restores K⁺-stimulated insulin secretion; n = 4 biological repeats. Statistics (in D, E, and H–K): two-way ANOVA with Bonferroni’s posttest. Error bars indicate SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.