GP-2 also known as ZAP75 is a phosphatidylinositol linked glycoprotein (31) present on the inner surface of the pancreatic zymogen granule (ZG). It was first recognized by MacDonald and Ronzio (26) as the major protein species in ZG membranes (ZGM) and was also noted to stain with PAS reagent. It had a molecular mass about 80 kDa although in various species it varies from 75 to 90 kDa. Its name comes from a subsequent study where pancreatic tissue slices were labeled with 3H-glucosamine and ZGM isolated and electrophoresed. Three major glycoprotein peaks were noted and named in order of increasing mobility GP-1 (120 kDa), GP-2 (74 kDa) and GP3 (52 kDa) (36). Other topological studies showed that GP-2 was present on the inner surface of the membrane (4, 24). GP-2 is the most abundant ZGM protein accounting for 25-40 percent of total protein in different species. Immunolocalizatiom showed GP-2 present on the apical plasma membrane as well as the ZG (2) (Figure 1). Lebel and Beattie and Fukuoka et al showed that GP-2 is linked to the membrane through a glycophophatidyl inositol (GPI) bond. This was based in part on its release from ZGM treated with a PI-specific PLC (12, 22). GP-2 was also shown to be homologous to Tamm-Horsfall protein, a GPI linked protein produced in kidney and excreted in urine (37).
GP-2 is present in pancreatic juice and makes up 5-8% of unstimulated juice protein in the rat and pig (38). Much of the released protein forms fibrils or aggregates and is sedimentable (3). However, it has lost its hydrophobicity as determined by Triton X-114 partitioning and contains a inositol 1,2-(cyclic)monophosphate residue (30, 32). Thus, the GP2 form present in pancreatic juice appears to have lost its GP1 modifiation throught the action of a PLC. However, since there is no PI-specific PLC present in ZGM, it may have to enter the apical acinar cell membrane to undergo processing. This could then explain the presence of significant amounts of GP-2 in basal pancreatic juice. Some GP-2 is also present in ZG content (16) and smaller cleaved forms are also present in pancreatic juice, implying proteolytic cleavage as well. However, where such processing takes place is not clear. Stimulation with secretin has no effect on GP-2 output in pancreatic juice while stimulation with caerulein or carbachol increases secretion, but with a slower time course than the secretion of digestive enzymes (21). GP-2 is also present in ductal proteinaceous plugs found in chronic pancreatitis (10).
The complete nucleotide and amino acid sequence of GP-2 was determined initially by molecular cloning technique from peptide sequence for rat, dog and humans (12, 13, 18, 43). The cDNA encodes a protein of 530 aa in humans with 67% identity to rat and 72% to dog GP-2
The primary sequence encodes 10 (human) or 8 (dog) Asn-linked glycosylation sites. This is consistent with data for in vitro translation by reticulocyte lysate that yields a protein of 55 kDa and treatment with N-glycanase which reduces the molecular mass of canine GP-2 from 75 kDa to 52 kDa (12, 18). Rat GP-2 has been shown to possess 5 or 6 N-linked high mannose carbohydrate chains (16). The primary protein sequence is also post-translationally glycosylated on its carboxyl terminal to a GPI moiety in the membrane during passage through the Golgi to reach the ZG (11, 13, 17). This GPI moiety contains a phosphoethanolamine linker attached to the protein, a 4-5 sugar core and a phosphatidylinositol whose hydrocarbon chains insert into the membrane.
2. Function in the Pancreas
The function of GP-2 remains unknown with attention to a role in the pancreas or downstream in the pancreatic juice or small intestinal lumen. The first suggested role was in the packaging of ZG. In general, secretory granule formation involves an acidic pH-dependent aggregation of secretory proteins within the trans golgi network (TGN) followed by binding of the aggregate to the membrane of the TGN and the pinching off of immature secretory granules (41). In the acinar cell, digestive enzyme proteins can aggregate along with GP-2. It has been hypothesized that GP-2 in conjunction with proteoglycan forms a matrix on the luminal side of the ZGM that can bind the aggregated protein (19, 23, 37). Isolated ZGs contain such a submembranous matrix which contains syncollin and ZGp16 as well as GP-2 (39). In support of a role for this matrix, inhibition of GPI anchor biosynthesis led to impaired granule formation (40). However, in embryonic pancreas and AR42J cells, when granule formation is increased, there is no change in GP-2 (7). Most directly, in GP-2 knockout mice there was no phenotype and granules were formed and digestive enzymes secreted normally (46). However, in the mouse, GP-2 is less abundant than other species and a different protein, muclin, a sulfated glycoprotein,may have become the primary sorting receptor (5, 6).
A second postulated role for GP-2 is in regulating endocytosis of exocytosed ZG membrane. In this model developed by Freedman, Kern and Scheele, the high bicarbonate and alkaline pH of the duct lumen activates a PI-PLC to cleave GP-2 and other PI linked proteins and their release activates apical membrane endocytosis (8, 9). Evidence for this model is that endocytosis is pH dependent and inhibited at pH 6.0 while addition of PI-PLC activates endocytosis even at pH 6.0. Latter it was observed that GP-2 is present in the acinar cell apical membrane and associated with Src family member Tyr kinases (29). However, since GP-2 expression in AR42J cells did not affect the secretory process or endocytosis (44) a role for GP-2 in vesicular trafficking remains doubtful.
A possible connection of GP-2 to pancreatic disease has also been explored. GP-2 is elevated in the plasma of rats with experimental pancreatic and humans with acute pancreatitis and appears to stay elevated longer than amylase or lipase (14, 25). In one study chronic ethanol feeding decreased GP-2 protein in the pancreas (1). Another study evaluated GP-2 for mutations in chronic pancreatitis but the frequency of mutations was similar to the normal population (42).
3. Role in Intestinal Disease
Recent studies have raised the question of whether GP-2 plays a role after pancreatic juice enters the intestine. One line of evidence began with the showing that GP-2 binds E. Coli that express Type 1 fimbria (45). Recently GP-2 was found to be expressed on M cells, a specialized antigen-transporting cell present in the gut epithelium overlying lymphoid follicles (15, 28). In this location it serves as a bacterial uptake mechanism for gram negative bacteria. However, the relation of this function to pancreatic GP-2 is unclear.
A second area is the role of autoantibodies to GP-2 found in GI disease. About 30% of patients with Crohn’s disease and 8% with ulcerative colitis have serum pancreatic autoantibodies and GP-2 is the major pancreatic antigen (33, 34). These antibodies are both IgG and IgA. IgA antibodies against GP-2 have also been reported from patients with coeliac disease (20, 35).The antibodies disappeared following a gluten free diet similar to other autoantibodies in coeliac disease. How these antibodies relate to the disease, however, is not clear.
4. Tools for Study
a. Antibodies. Our studies used a monoclonal Ab to GP-2 developed by Anson Lowe and described in (24). Currently a number of Ab are available commercially including a Atlas antipeptide antibody available from Sigma specific for human GP-2, multiple goat antibodies available from Santa Cruz, and other AB from Novus, GeneTex and OriGene. All are raised against human sequence and would need testing in rats or mice.
b. Elisa Kits. Kits stated to react with dog, human, mouse and rat GP-2 are available from Antibodies on line but have not been tested by us.
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