The mucinous domain of pancreatic carboxyl-ester lipase (CEL) contains core 1/core 2 O-glycans that can be modified by ABO blood group determinants

Abstract

Carboxyl-ester lipase (CEL) is a pancreatic fat-digesting enzyme associated with human disease. Rare mutations in the CEL gene cause a syndrome of pancreatic exocrine and endocrine dysfunction denoted MODY8, whereas a recombined CEL allele increases the risk for chronic pancreatitis. Moreover, CEL has been linked to pancreatic ductal adenocarcinoma (PDAC) through a postulated oncofetal CEL variant termed feto-acinar pancreatic protein (FAPP). The monoclonal antibody mAb16D10 was previously reported to detect a glycotope in the highly O-glycosylated, mucin-like C terminus of CEL/FAPP. We here assessed the expression of human CEL in malignant pancreatic lesions and cell lines. CEL was not detectably expressed in neoplastic cells, implying that FAPP is unlikely to be a glycoisoform of CEL in pancreatic cancer. Testing of the mAb16D10 antibody in glycan microarrays then demonstrated that it recognized structures containing terminal GalNAc-␣1,3(Fuc-␣1,2)Gal (blood group A antigen) and also repeated protein sequences containing GalNAc residues linked to Ser/Thr (Tn antigen), findings that were supported by immunostainings of human pancreatic tissue. To examine whether the CEL glycoprotein might be modified by blood group antigens, we used high-sensitivity MALDI-TOF MS to characterize the released O-glycan pool of CEL immunoprecipitated from human pancreatic juice. We found that the O-glycome of CEL consisted mainly of core 1/core 2 structures with a composition depending on the subject's FUT2 and ABO gene polymorphisms. Thus, among digestive enzymes secreted by the pancreas, CEL is a glycoprotein with some unique characteristics, supporting the view that it could serve additional biological functions to its cholesteryl esterase activity in the duodenum.

Pathogenic Carboxyl Ester Lipase (CEL) Variants Interact with the Normal CEL Protein in Pancreatic Cells

“…Using this model, we found that CEL-HYB and CEL-MODY affected the intracellular fate of a FLAG-tagged CEL-WT protein (Figures 6 and 7), as the latter was found to be more intracellularly accumulated when coexpressed with a pathogenic CEL variant than with CEL-WT-V5. Intriguingly, the rate of CEL secretion has been suggested to positively correlate with the cells' capability to O-glycosylate the CEL VNTR region [9], partially explaining the impaired secretion of CEL-HYB and CEL-MODY, as the VNTR regions of these enzymes are shorter and potentially less O-glycosylated compared to CEL-WT [19,23,27]. However, it does not explain how the pathogenic CEL variants affect the intracellular fate of CEL-WT.…”

“…Thus, it was important to investigate CEL in acinar cells such as the murine 266-6 cell line, as these cells provide the transfected and endogenously expressed CEL proteins with a proper exo-and endocytic machinery. The human pancreatic ductal cell line PANC-1 does not express CEL [27]. However, being a cancer cell line, these cells might exhibit properties other than normal ductal cells.…”

“…Still, many of them were positive for CEL protein, and we interpret this observation as evidence for ductal uptake of CEL taking place in vivo. The tissue stemmed from patients diagnosed with pancreatic cancer, but CEL mRNA is absent also from normal human pancreatic ductal cells [27].…”

“…This is reflected in a less complex pattern of O-glycans attached to the pathogenic proteins [37], which again could be of relevance for endocytosis and pathogenicity. Moreover, modifications of the CEL VNTR region by ABO blood group antigens [27] might affect the distinct affinity and uptake kinetics of each cell type toward CEL protein variants, by altering the receptor-binding properties of CEL.…”

“…FFPE tissue sections (3-5 µm) were treated as previously described [27] and stained at 4 • C overnight in a 1:100 dilution with polyclonal anti-CEL antibody (Sigma-Aldrich). Using a custom-made CEL-specific probe covering exons 2-7, the RNAscope 2.0HD assay was employed for in situ hybridization according to supplier's instructions (Advanced Cell Diagnostics, Newark, CA, USA) and [27]. Images were acquired at various magnifications using an MC170HD camera attached to a DM2000LED microscope (Leica) and processed using LAS V4.8 software.…”