Louis-Jeantet Foundation

The persistence of serum IgG antibodies elicited in human infants is much shorter than when such responses are elicited later in life. The reasons for this rapid waning of antigen-specific antibodies elicited in infancy are yet unknown. We have recently shown that adoptively transferred tetanus toxoid (TT)-specific plasmablasts (PBs) efficiently reach the bone marrow (BM) of infant mice. However, TT-specific PBs fail to persist in the early-life BM, suggesting that they fail to receive the molecular signals that support their survival/differentiation. Using a proliferation-inducing ligand (APRIL)- and B-cell activating factor (BAFF) B-lymphocyte stimulator (BLyS)-deficient mice, we demonstrate here that APRIL is a critical factor for the establishment of the adult BM reservoir of anti-TT IgG-secreting cells. Through in vitro analyses of PB/plasma cell (PC) survival/differentiation, we show that APRIL induces the expression of Bcl-X(L) by a preferential binding to heparan sulfate proteoglycans at the surface of CD138(+) cells. Last, we identify BM-resident macrophages as the main cells that provide survival signals to PBs and show that this function is slowly acquired in early life, in parallel to a progressive acquisition of APRIL expression. Altogether, this identifies APRIL as a critical signal for PB survival that is poorly expressed in the early-life BM compartment.

Green fluorescent protein (GFP) is used extensively as a reporter protein to monitor cellular processes, including intracellular protein trafficking and secretion. In general, this approach depends on GFP acting as a passive reporter protein. However, it was recently noted that GFP oligomerizes in the secretory pathway of endocrine cells. To characterize this oligomerization and its potential role in GFP transport, cytosolic and secretory forms of enhanced GFP (EGFP) were expressed in GH4C1 and AtT-20 endocrine cells. Biochemical analysis showed that cytosolic EGFP existed as a 27 kDa monomer, whereas secretory forms of EGFP formed disulphide-linked oligomers. EGFP contains two cysteine residues (Cys(49) and Cys(71)), which could play a role in this oligomerization. Site-directed mutagenesis of Cys(49) and Cys(71) showed that both cysteine residues were involved in disulphide interactions. Substitution of either cysteine residue resulted in a reduction or loss of oligomers, although dimers of the secretory form of EGFP remained. Mutation of these residues did not adversely affect the fluorescence of EGFP. EGFP oligomers were stored in secretory granules and secreted by the regulated secretory pathway in endocrine AtT-20 cells. Similarly, the dimeric mutant forms of EGFP were still secreted via the regulated secretory pathway, indicating that the higher-order oligomers were not necessary for sorting in AtT-20 cells. These results suggest that the oligomerization of EGFP must be considered when the protein is used as a reporter molecule in the secretory pathway.

The tSNARE SNAP-25 is expressed in pancreatic (beta)-cells and is involved in the regulated release of insulin. It has been shown previously that SNAP-25 associates with the plasma membrane consequent to palmitoylation of one or more cysteines in the central region of the molecule. The importance of palmitolyation in the biological function of SNAP-25 in exocytosis was not addressed. Furthermore, studies on both SNAP-25 and its non-palmitoylated homologues SNAP-29 and sec9, have suggested an alternative or complementary mechanism for membrane association involving interaction with syntaxin. To address these issues, we have now studied the behavior and biological activity of cysteine mutant SNAP-25 in insulin-secreting (HIT) cells. While 91% of native SNAP-25 was associated with the membrane, this value decreased to 56% for the single cysteine mutant C85/A and to 10% for the double (C85,88/A) and quadruple (C85,88,90,92/A) mutants. The mutant SNAP-25 forms were all found to bind syntaxin 1A with equal efficacy. Over-expression of syntaxin 1A in HIT cells allowed for partial relocalization of both the double and quadruple SNAP-25 cys mutants to the membrane. By introducing a further mutation to the SNAP-25 molecules to render them resistant to botulinum neurotoxin E, it was possible to study their ability to reconstitute regulated insulin secretion in toxin-treated HIT cells. Native SNAP-25 was able to fully reconstitute secretory activity in such cells. Despite the fact that the single cysteine mutant was significantly displaced to the cytosol, it still displayed 82% activity in the secretion reconstitution assay, and a similar discrepancy was seen for the double mutant. Even the quadruple mutant with no remaining cysteines was able to support a minimal level of secretion. It is concluded that both palmitoylation and binding to syntaxin are implicated in membrane association of SNAP-25. This as well as the discrepancy between membrane localization and biological activity of the cysteine mutants, suggests a complex, multi-component process for association of SNAP-25 with the membrane and its recruitment to a biologically productive state.

Conditional expression of the glucokinase regulatory protein in insulinoma cells, under control of the reverse tetracycline-dependent transactivator, was used to investigate whether expression of this protein de novo would alter the intracellular distribution of glucokinase. The regulatory protein, which was undetectable in the basal state, could be induced by doxycycline to levels comparable to those of liver and was detected mostly in the nucleus. Concomitantly, glucokinase accumulated in the nucleus. Human embryonic kidney cells were transiently transfected to express glucokinase and the regulatory protein, either separately or together. Each protein localized predominantly to the cytoplasm when expressed alone. On co-expression, however, both proteins localized virtually entirely to the nucleus. The enzymic activity of glucokinase was not required for promoting nuclear import of the two proteins, as shown with a glucose-phosphorylation-deficient mutant. Finally, in embryonic kidney cells expressing the regulatory protein alone, treatment with leptomycin B resulted in a partial redistribution of the protein from the cytoplasm to the nucleus, suggesting that this protein can shuttle between the two compartments.

Pancreatic islets of Langerhans are composed of four major endocrine cell types with a smaller number of nonendocrine cells. To study the molecular constituents and function of just one subpopulation of islet cells, it is necessary to sort them from the other cell types. While rat beta-cells can be sorted by autofluorescence-activated flow cytometry, this has not proved possible on a routine and reproducible basis for human beta-cells. In the present study, we have selectively labeled human beta-cells with green fluorescent protein (GFP), allowing for their sorting by flow cytometry. Human islet cells were infected with replication-defective (attenuated) recombinant adenovirus expressing GFP driven by the rat insulin I promoter (Ad-RIP-GFP) for targeted expression in beta-cells, or beta-galactosidase driven by the promiscuous cytomegalovirus (CMV) promoter (Ad-CMV-beta-gal) as control. Whereas the majority of islet cells can be infected by adenovirus, as shown by control infection with Ad-CMV-beta-gal, increased fluorescence after infection with Ad-RIP-GFP was limited to insulin-containing beta-cells. Infection of islet cells with Ad-RIP-GFP resulted reproducibly in the appearance of a population of intensely fluorescent cells, when analyzed by flow cytometry. These cells were sorted using a fluorescence-activated cell sorter (FACS) and shown by immunofluorescence to consist of >95% beta-cells. The targeted expression of GFP thus allows for preparation of human beta-cells purified close to homogeneity. This method should be readily applicable in any laboratory with FACS capability.

The production of insulin from proinsulin involves cleavage of intact proinsulin into proinsulin conversion intermediates by the processing of enzymes PC2 and PC3 before fully processed insulin is produced. Intact proinsulin and these conversion intermediates are measured in many immunoreactive insulin (IRI) assays, and therefore contribute to the absolute IRI measurement. The proportion of basal IRI made up of proinsulin (PI)-like molecules (PI/IRI) is increased in NIDDM. Whether stimulated IRI levels are similarly made up of disproportionately increased PI/IRI or whether the relative proportions of proinsulin and its conversion intermediates are altered has not been evaluated. An index of the efficiency of proinsulin processing within the pancreatic beta-cell can be achieved by measuring PI/IRI immediately following acute stimulation of beta-cell secretion, and then determining the proportion of intact proinsulin and proinsulin conversion intermediates contributing to circulating proinsulin-like molecules. In this study, we determined the PI/IRI levels under basal and arginine-stimulated conditions in 17 healthy and 16 NIDDM subjects; high-performance liquid chromatography (HPLC) was also performed in a subset of these subjects to measure the relative contribution of intact proinsulin and its conversion intermediates to total proinsulin-like molecules. In NIDDM subjects, levels of both basal (44.6 +/- 9.6 vs. 9.3 +/- 1.5 pmol/l; P = 0.0007) and stimulated (64.0 +/- 12.7 vs. 19.8 +/- 2.8 pmol/l; P = 0.001) proinsulin-like molecules were higher than in healthy subjects. Although IRI was higher in NIDDM than in control subjects under basal conditions (106 +/- 19 vs. 65.1 +/- 8.1 pmol/l; P = 0.05), it was lower in NIDDM than in control subjects following stimulation (increment: 257 +/- 46 vs. 416 +/- 51 pmol/l; P = 0.03). PI/IRI ratios were increased in NIDDM subjects under both basal (43.3 +/- 5.0 vs. 14.0 +/- 1.3%; P < 0.0001) and stimulated (increment: 10.1 +/- 2.1 vs. 2.5 +/- 0.2%; P = 0.0006) conditions, compatible with the release of a disproportionately increased amount of proinsulin-like products. HPLC analysis revealed that, in the stimulated state, intact proinsulin made up 40.1 +/- 6.7% of proinsulin-like molecules in NIDDM individuals (n = 9) and 30.1 +/- 5.6% in healthy subjects (n = 7; NS). The remainder of the proinsulin-like molecules comprised the des-31,32-split proinsulin conversion intermediate. The increase in PI/IRI in NIDDM under basal and especially under stimulated conditions suggests that proinsulin conversion is indeed perturbed in this disorder. Because the relative proportions of intact and des-31,32-split proinsulin are similar in both healthy and NIDDM subjects, the orderly cleavage of proinsulin at its two junctions appears preserved. However, at the time of exocytosis, the secretory granule in the islet of NIDDM subjects contains an increased proportion of incompletely processed proinsulin, presumably reflecting a slower rate of conversion or granules' reduced time of residence in beta-cells.

Insulin and connecting peptide (C-peptide) are produced in equimolar amounts during proinsulin conversion in the pancreatic beta cell secretory granule. To determine whether insulin and C-peptide are equally stable in beta cell granules (and thus secreted in equimolar amounts), neonatal and adult rat beta cells were pulse-chased, and radiolabeled insulin and C-peptide analyzed by high performance liquid chromatography. A novel truncated C-peptide was identified and shown by mass spectrometry to be des-(27-31)C-peptide (loss of 5 C-terminal amino acids). Des-(27-31)C-peptide is a major beta cell secretory product, accounting for 37.4 +/- 1.6% (neonatal) and 8.5 +/- 0.6% (adult) of total labeled C-peptide in secretory granules after 10 h of chase. Des-(27-31)C-peptide is also secreted in a glucose-sensitive manner from the perfused adult rat pancreas, accounting for approximately 10% of total C-peptide immunoreactivity secreted. Human C-peptide is also a substrate for truncation in granules. Thus, when human proinsulin was expressed (infection with recombinant adenovirus) in transformed (INS) rat beta cells, human des-(27-31)C-peptide was secreted along with the intact human peptide and both intact and truncated rat C-peptide. In addition to truncation, 33.1 +/- 1.2% of C-peptide in neonatal but not adult rat beta cell granules was further degraded. Such degradation was completely inhibited by ammonium chloride (known to neutralize intra-granular pH), whereas truncation was only partially inhibited by approximately 50%. In conclusion, a novel beta cell secretory product, des-(27-31)C-peptide, has been identified and should be considered as a potential bioactive peptide. Both truncation and degradation of C-peptide are responsible for non-equimolar secretion of insulin and C-peptide in rat beta cells.

PC1 (PC3) and PC2, members of the mammalian family of proprotein convertases homologous to the yeast Kex2 gene product, are both expressed in pancreatic islets of Langerhans. Recent studies have suggested that PC1 and PC2 are responsible for the conversion of proinsulin to insulin and connecting peptide (C-peptide) in the islet beta cells. However, the insulin-secreting beta cells are not the only cells present in these complex micro-organs, prompting us to evaluate the expression of PC1 and PC2 in islet beta and non-beta cells. Rat islet cells were sorted by autofluorescence-activated flow cytometry to separate beta cells from non-beta cells, and conversion endoprotease levels were analysed by Western blotting. The immunolabel ratio of PC1/PC2 in beta cells was 2.6. Non-beta cells displayed much lower levels of PC1 than beta cells, but twice as much PC2 (PC1/PC2 = 0.05). Post-translational modification of the convertases themselves was found to differ between the cell types. In particular, a 75 kDa precursor form of PC2 (pro-PC2) was found to accumulate in beta cells, whereas only the fully processed 67 kDa form was detected in the non-beta cells. Finally, the quantification of PC1 and PC2 and their precursor forms in transformed cells (insulin-producing beta-TC and glucagon-producing alpha-TC) showed that transformation appeared to be accompanied by unusually high levels of the precursors.

Proinsulin is usually targetted to the regulated secretory pathway of beta cells, and converted to insulin in beta granules. Under certain pathological situations, a significant amount of proinsulin becomes diverted to the constitutive pathway. To study the kinetics of proinsulin conversion in the constitutive pathway, FAO (hepatoma) cells, which secrete proteins uniquely via this pathway and not the regulated pathway, were stably transfected with cDNA encoding human, rat I or rat II proinsulin. Products released to the medium of transfected cells were analysed by reversed phase HPLC and radioimmunoassay. For human proinsulin, des 31,32 split proinsulin (the conversion intermediate resulting from cleavage only at the B-chain/C-peptide junction followed by trimming of C-terminal basic residues by carboxypeptidase) was the only detectable conversion intermediate; for rat proinsulin II it was des 64,65 split proinsulin (cleaved and trimmed only at the C-peptide/A-chain junction); for rat proinsulin I, both intermediates were seen. Complete processing to insulin occurred for all three, but was most extensive for rat proinsulin I. When considered with the corresponding proinsulin sequences, these data show that a -4 basic residue (i.e. 4 residues N-terminal to the site of cleavage) facilitates proinsulin conversion in the constitutive pathway, and that arginine is preferred over lysine.