Diabetes and Pancreatic Exocrine Dysfunction Due to Mutations in the Carboxyl Ester Lipase Gene-Maturity Onset Diabetes of the Young (CEL-MODY)

Abstract

CEL-maturity onset diabetes of the young (MODY), diabetes with pancreatic lipomatosis and exocrine dysfunction, is due to dominant frameshift mutations in the acinar cell carboxyl ester lipase gene (CEL). As Cel knock-out mice do not express the phenotype and the mutant protein has an altered and intrinsically disordered tandem repeat domain, we hypothesized that the disease mechanism might involve a negative effect of the mutant protein. In silico analysis showed that the pI of the tandem repeat was markedly increased from pH 3.3 in wild-type (WT) to 11.8 in mutant (MUT) human CEL. By stably overexpressing CEL-WT and CEL-MUT in HEK293 cells, we found similar glycosylation, ubiquitination, constitutive secretion, and quality control of the two proteins. The CEL-MUT protein demonstrated, however, a high propensity to form aggregates found intracellularly and extracellularly. Different physicochemical properties of the intrinsically disordered tandem repeat domains of WT and MUT proteins may contribute to different short and long range interactions with the globular core domain and other macromolecules, including cell membranes. Thus, we propose that CEL-MODY is a protein misfolding disease caused by a negative gain-of-function effect of the mutant proteins in pancreatic tissues.Most monogenic forms of diabetes are due to mutations in genes expressed in the pancreatic beta cell. Previously, Raeder et al.(1) reported a novel monogenic syndrome caused by mutations in the carboxyl ester lipase gene (CEL) (OMIM 609812; CEL-MODY 2 or MODY8), characterized by dominantly inherited childhood-onset pancreatic exocrine dysfunction and diabetes mellitus from adulthood. Notably, this gene is not transcribed in beta cells but is mainly expressed in pancreatic acinar tissue (2, 3) and lactating mammary glands (4, 5). The CEL enzyme (EC 3.1.1.13), also known as bile salt-stimulated/dependent lipase, is secreted into the intestine and activated by bile salts, playing a role in the hydrolysis and absorption of cholesterol-and lipid-soluble vitamins (6).The human CEL gene is ϳ10 kb in size and consists of 11 exons. In the last exon, there is a variable number of tandem repeats (VNTR) where the 33-bp nearly identical segments are repeated usually between 7 and 23 times (1, 7-9). The VNTR of the most common CEL allele has 16 repeats, thereby encoding a protein consisting of 745 amino acids with a predicted molecular mass of ϳ79 kDa. The rat Cel is secreted from the acinar cells and is thought to follow the classical pathway of secretory proteins (for review see Ref.3). In the endoplasmic reticulum (ER), the protein is co-translationally N-glycosylated at a conserved Asn residue (Asn-210), serving as a client protein for the molecular chaperone GRP94 and being associated with ER membranes (10, 11). The VNTR-encoded protein C terminus is heavily O-glycosylated in the mature protein, probably on Thr residues before or after PVPP motifs in the first 10 repeats (12). The O-glycosylation sites are present in a region enriched in the amino ...

Endocytosis of Secreted Carboxyl Ester Lipase in a Syndrome of Diabetes and Pancreatic Exocrine Dysfunction

“…Pathway-We previously reported that secretion of both CEL-WT and CEL-MUT from HEK293 cells occurs at similar rates (26). However, the microscopic data described above indicated that the two proteins display clearly distinct intracellular distributions (Fig.…”

“…Moreover, in vivo excreted CEL-MUT can be detected in pancreatic juice from the patients. Our findings led to the suggestion that CEL-MODY is a protein misfolding disease in which the CEL-MUT protein forms aggregates leading to the stimulation of the unfolded protein response (26).…”

“…Because the CEL gene is not expressed in pancreatic beta cells, the negative effect of CEL-MUT on insulin secretion is likely to be secondary to a primary pathological event affecting the acinar cells. We previously reported that the changed C-terminal VNTR of CEL-MUT undergoes O-glycosylation and that the mutant protein is secreted to the cell exterior at a rate similar to what is seen for the WT when expressed in stably transfected HEK293 cells (26). Moreover, in vivo excreted CEL-MUT can be detected in pancreatic juice from the patients.…”

“…This made it possible to detect the recombinant proteins by commercially available epitope-tag antibodies as well as with CEL-specific antibodies. The cloning procedure is described in detail in Johansson et al (26). The CEL-WT construct has 16 repeated VNTR segments as in the most common allele found in Europeans (20,22,23,24).…”

“…We previously found that CEL-MUT has an increased tendency to aggregate (26), and we therefore wanted to investigate if autophagy was involved in the delivery of the protein to the lysosomes. HEK293 cells stably expressing CEL-MUT were transiently transfected with a plasmid encoding LC3-GFP.…”