Coordinate regulation of secretory stress proteins (PSP/reg, PAP I, PAP II, and PAP III) in the rat exocrine pancreas during experimental acute pancreatitis.

BACKGROUND: Pancreatic stone protein (PSP/reg) is a constitutively secreted protein in pancreatic juice. Pancreatitis-associated protein (PAP) belongs to the same family of proteins. PAP is highly increased during acute pancreatitis, while no exact data exist regarding PSP/reg protein synthesis and secretion. Recently, an attempt to determine PSP/reg and PAP levels in sera of rats with acute pancreatitis showed a significant increase in PAP but failed to demonstrate changes in PSP/reg. Others reported that surgical manipulation of the pancreas, including sham controls, affected mRNA levels of PSP/reg. Neither report determined protein levels of PSP/reg. METHODS: Rats were treated intraperitoneally with a supramaximal dose of caerulein to induce pancreatitis, a physiological dose of caerulein, or a saline injection. Pancreata were analyzed for PAP and PSP/reg using ELISAs. RNA was extracted for Northern blot analysis of PAP I, II, and III and PSP/reg mRNA. RESULTS: Experimental induction of acute pancreatitis caused a coordinate increase in both PSP/reg and PAP. PAP showed an acute response and returned to low levels within 48 h while PSP/reg exhibited a more sustained response. Intraperitoneal application of a physiological dose of caerulein and even a saline injection caused an increase in PSP/reg. CONCLUSION: PSP/reg and PAP levels are increased through similar mechanisms by physiological and supramaximal doses of caerulein. However, PSP/reg regulation appears to sustain high levels while PAP levels are more transient. Since the regulation of this protein family is affected even under mild stress, we define them as secretory stress proteins.

Pancreatic acinar cell dysfunction in CFTR(-/-) mice is associated with impairments in luminal pH and endocytosis.

BACKGROUND & AIMS: We have previously shown that endocytosis at the apical plasma membrane in pancreatic acinar cells is coupled to ductal bicarbonate secretion into the lumen. We hypothesized that decreased bicarbonate secretion in cystic fibrosis (CF) inhibits apical endocytosis. The aim of this study was to determine in cftr(-/-) mice (1) if the pH of the pancreatic juice is acidic compared with wild-type (WT) controls, (2) if there is a selective block in endocytosis, and (3) if alkalinization of the luminal fluid reverses this defect. METHODS: Fluid secretion and pH of pancreatic juice were measured. Exocytosis, endocytosis, and morphology were compared in pancreatic lobules from cftr(-/-) and WT mice. RESULTS: Pancreatic juice pH was 8.12 +/- 0.06 in WT mice compared with 6.60 +/- 0.04 in cftr(-/-) mice. Although cholecystokinin-stimulated amylase secretion was not significantly different, endocytosis was markedly inhibited in cftr(-/-) compared with WT mice. Cleavage of GP2, a GPI-anchored protein tightly associated with activation of endocytosis, was also decreased. Incubation of lobules from cftr(-/-) mice at pH 8.3 reversed the luminal dilatation. CONCLUSIONS: These data indicate that apical endocytosis is selectively impaired in cftr(-/-) mice, which explains, in part, the luminal dilatation observed at the apical plasma membrane. In vitro alkalinization of luminal fluid led to reversal of defects in membrane dynamics, restored coupled exocytosis and endocytosis, and abolished the luminal dilatation in this animal model of CF. Acidic pH changes in luminal secretions may play a role in the pancreatic membrane dysfunction observed in CF.

A family of 16-kDa pancreatic secretory stress proteins form highly organized fibrillar structures upon tryptic activation.

A group of 16-kDa proteins, synthesized and secreted by rat pancreatic acinar cells and composed of pancreatic stone protein (PSP/reg) and isoforms of pancreatitis-associated protein (PAP), show structural homologies, including conserved amino acid sequences, cysteine residues, and highly sensitive N-terminal trypsin cleavage sites, as well as conserved functional responses in conditions of pancreatic stress. Trypsin activation of recombinant stress proteins or counterparts contained in rat pancreatic juice (PSP/reg, PAP I and PAP III) resulted in conversion of 16-kDa soluble proteins into 14-kDa soluble isoforms (pancreatic thread protein and pancreatitis-associated thread protein, respectively) that rapidly polymerize into insoluble sedimenting structures. Activated thread proteins show long lived resistance to a wide spectrum of proteases contained in pancreatic juice, including serine proteases and metalloproteinases. In contrast, PAP II, following activation with trypsin or pancreatic juice, does not form insoluble structures and is rapidly digested by pancreatic proteases. Scanning and transmission electron microscopy indicate that activated thread proteins polymerize into highly organized fibrillar structures with helical configurations. Through bundling, branching, and extension processes, these fibrillar structures form dense matrices that span large topological surfaces. These findings suggest that PSP/reg and PAP I and III isoforms consist of a family of highly regulated soluble secretory stress proteins, which, upon trypsin activation, convert into a family of insoluble helical thread proteins. Dense extracellular matrices, composed of helical thread proteins organized into higher ordered matrix structures, may serve physiological functions within luminal compartments in the exocrine pancreas.

Regulation of PSP/reg in rat pancreas: immediate and steady-state adaptation to different diets.

Pancreatic stone protein/reg protein (PSP/reg) is a secretory pancreatic protein of hitherto unknown function. It is precursor to a spontaneously precipitating peptide called pancreatic thread protein, which is found in protein plugs within the pancreatic ductal system. Increasing PSP/reg concentrations in pancreatic juice might augment the risk of intraductal plug formation and therefore be a condition predisposing to chronic pancreatitis. Malnutrition is associated with a high incidence of chronic pancreatitis in tropical countries. In a diet study with rats, we tested the hypothesis that protein malnutrition leads to increased PSP/reg concentrations in pancreatic juice. A highly sensitive and reliable enzyme-linked immunosorbent assay (ELISA) for rat PSP/reg was newly established. Male Sprague-Dawley rats were allocated to three nearly isocaloric experimental diets, which contained 0, 45, or 82% casein, respectively, or to a control diet (22% casein). We evaluated PSP/reg expression under these four dietary conditions on the RNA and on the protein level, performing a time-course study over a period of 28 days. Our results demonstrate that PSP/reg expression is not increased because of a protein-deficient diet if investigated under steady-state conditions. After a temporary increase in PSP/reg levels due to a carbohydrate-deficient high-protein diet, we could not find signs of a diet-dependent regulation of this protein. The regulation of PSP/reg thus differs from that of most other pancreatic secretory proteins. Our findings contradict earlier reports that had drawn conclusions based solely on messenger RNA levels.

Telenzepine-sensitive muscarinic receptors on rat pancreatic acinar cells.

To identify the muscarinic subtype present on the rat pancreatic acinar cell, we examined the effects of different muscarinic receptor antagonists on amylase secretion and proteolytic zymogen processing in isolated rat pancreatic acini. Maximal zymogen processing required a concentration of carbachol 10- to 100-fold greater (10(-3) M) than that required for maximal amylase secretion (10(-5) M). Although both secretion and conversion were inhibited by the M3 antagonist 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP) (50% inhibition approximately 6 x 10(-7) M and 1 x 10(-8) M, respectively), the most potent inhibitor was the M1 antagonist telenzepine (50% inhibition approximately 5 x 10(-10) M and 1 x 10(-11) M, respectively). Pirenzepine, another M1 antagonist, and the M2 antagonist methoctramine did not reduce amylase secretion or zymogen processing in concentrations up to 1 x 10(-5) M. Analysis of acinar cell muscarinic receptor by PCR revealed expression of both m1 and m3 subtypes. The pancreatic acinar cell has a distinct pattern of muscarinic antagonist sensitivity (telenzepine >> 4-DAMP > pirenzepine) with respect to both amylase secretion and zymogen conversion.

Acinar lumen pH regulates endocytosis, but not exocytosis, at the apical plasma membrane of pancreatic acinar cells.

A two-step exocytosis/endocytosis protocol was used in rat pancreatic acini to study membrane trafficking events at the apical plasma membrane (APM) as a function of extracellular pH. Exocytosis, as measured by cholecystokinin (CCK)-8-induced release of amylase into the incubation medium, was relatively insensitive to changes in extracellular pH from 5.5 to 9.0. In contrast, endocytosis, as measured by temperature-dependent uptake of horseradish peroxidase (HRP), was robust at pH values between 6.5 and 8.3 but abolished at acidic pH values of 5.5 to 6.0. Energy metabolism and cell viability were maintained during pH 6-induced cessation of HRP uptake, and the vesicular block could be reversed upon raising the luminal pH to 7.4. Histochemical and morphometric studies of HRP uptake examined by electron microscopy indicated that extracellular pH regulates endocytosis at the apical plasma membrane. At pH 6.0 in prestimulated cells, HRP uptake at the APM was abolished, and acinar lumen membranes remained markedly dilated with decreased density of microvilli and "arrested" exocytic images. At pH 7.4, HRP was taken up into endolysosomal structures within the Golgi complex, and acinar lumen membranes were contracted. Cleavage of GP2, a glycosyl phosphatidylinositol-anchored protein, was associated with the pH-dependent activation of HRP uptake. These studies demonstrate that acinar lumen pH regulates endocytic but not exocytic activity at the APM and suggest that alkalinization of the acinar lumen by duct cells is required for retrieval of exocytic membranes into the acinar cell via vesicular uptake mechanisms. The role of acid-base interactions within the acinar lumen provides a novel basis for understanding the cellular and luminal defects observed within the exocrine pancreas in cystic fibrosis.

Cleavage of GPI-anchored proteins from the plasma membrane activates apical endocytosis in pancreatic acinar cells.

Using rat pancreatic acini, we have recently shown that apical endocytosis is inhibited at pH 6.0 and progressively activated as the pH is increased to 8.3. Endocytotic activity correlated with the release of GP2, a GPI-linked protein, from the apical plasma membrane. To determine whether the cleavage of GPI-anchored proteins from the membrane of rat acinar cells was responsible for activation of endocytosis, cells at pH 6.0 were incubated with PI-specific phospholipase C (PI-PLC). PI-PLC treatment reversed the inhibition of endocytosis observed at pH 6.0. Reactivation of endocytosis correlated with PI-PLC-induced release of GP2 but not cleavage of phospholipids in cellular membranes. Furthermore, administration of diacylglycerol or phorbol esters had no effect on reactivation of endocytosis. PI-PLC did not alter intracellular pH or calcium levels. Two proteins were identified as GPI-linked proteins on the cell surface. One was GP2, whose release from the apical plasma membrane correlated with apical endocytosis of horseradish peroxidase (HRP). The other protein, identified by Western blotting using an antibody directed against a cryptic determinant exposed on GPI-linked proteins after cleavage with PI-PLC, has a molecular weight of 98000 in nonreducing SDS gels and 54000 in reducing SDS gels. By nondenaturing gel electrophoresis and staining with naphthylphosphate, this protein was found to be alkaline phosphatase. In contrast to GP2, alkaline phosphatase was not endogenously released at pH values of 7.4 or 8.3, conditions that activate endocytosis of HRP under physiological conditions. By electron microscopic evaluation, incubation of cells at pH 6.0 with PI-PLC led to induction of HRP uptake into vesicles at the apical pole of the cell, a reduction in apical plasma membranes, and a concomitant contraction of the acinar lumen space. Internalized HRP accumulated in the Golgi region of the cell. These results suggest that the cleavage of GPI-anchored proteins from the apical plasma membrane activates apical endocytosis.

Pancreatic stone protein (lithostathine), a physiologically relevant pancreatic calcium carbonate crystal inhibitor?

Apart from digestive enzymes, pancreatic juice contains several proteins that are not directly involved in digestion. One of these, lithostathine, has been reported to exhibit calcite crystal inhibitor activity in vitro. As pancreatic juice is supersaturated with respect to calcium carbonate, it was hypothesized that lithostathine stabilizes pancreatic juice. Lithostathine is cleaved by trace amounts of trypsin, resulting in a C-terminal polypeptide and an N-terminal undecapeptide, which has been identified as the active site of lithostathine regarding crystal inhibition. We produced rat lithostathine in a baculovirus expression system. In order to test its functional activity, the protein was purified using a nondenaturing multi-step procedure. In the low micromolar range, recombinant rat lithostathine in vitro exhibited calcite crystal inhibitor activity, confirming earlier reports. Limited tryptic proteolysis of recombinant lithostathine was performed, and the two cleavage products were separated; the C-terminal polypeptide was precipitated by centrifugation, and the N-terminal undecapeptide was purified by high performance liquid chromatography. Only the C-terminal peptide displayed measurable calcite crystal inhibitory activity. Furthermore, synthetic undecapeptides with identical sequence to the N-terminal undecapeptides of rat or human lithostathine were inactive. However, when tested in the same in vitro assays, other pancreatic or extra-pancreatic proteins show inhibitory activity in the same concentration range as lithostathine, and inorganic phosphate is active as well. Based on these findings it seems unlikely that lithostathine is a physiologically relevant calcite crystal inhibitor. The name "lithostathine" is therefore inappropriate, and the protein's key function remains to be elucidated.

Pancreatic dysfunction in cystic fibrosis occurs as a result of impairments in luminal pH, apical trafficking of zymogen granule membranes, and solubilization of secretory enzymes.

Recent progress in understanding the luminal biochemistry of regulated pancreatic exocrine secretion, including acid-base interactions between acinar and duct cells and pH-dependent processes that regulate membrane trafficking (endocytosis) at the apical plasma membrane, have led to the development of in vitro models of cystic fibrosis in the rat exocrine pancreas. Based on investigations in these model systems, a unifying hypothesis is presented that proposes that pancreatic dysfunction in cystic fibrosis occurs as a result of progressive acidification of the acinar and duct lumen, which leads to secondary defects in (i) apical trafficking of zymogen granule membranes and (ii) solubilization of secretory (pro)enzymes. By directly acidifying the pH of the acinar lumen in cholescystokinin-stimulated acini, the early cytological findings observed in cystic fibrosis, including (i) massive dilatation of the acinar lumen, (ii) decreased appearance of zymogen granules, (iii) loss of the apical pole of the acinar cell, and (iv) persistent aggregation of secretory (pro)enzymes released into the luminal space, have been reproduced in primary cultures of pancreatic tissue.

High-level secretion of native rat pancreatic lithostathine in a baculovirus expression system.

We have constructed a recombinant baculovirus expression vector containing rat pancreatic lithostatin cDNA. Baculovirus infection of Spodoptera frugiperda (sf9) insect cells resulted in the de novo synthesis and secretion of a recombinant protein demonstrating an apparent molecular weight of about 16.5 kDa. Under optimal conditions [multiplicity of infection of 5 plaque-forming units (pfu)/cell and culture times of 48-56 h postinfection] recombinant protein was secreted into the culture medium at 5-10 mg/L. The secretory form of the recombinant protein was judged to be rat pancreatic lithostatin by the following criteria: (a) Trypsin cleavage resulted in limited proteolysis of the secreted product giving rise to a trypsin-resistant 15.5-kDa peptide, consistent with the size of the "pancreatic stone/thread protein"; (b) polyclonal antibodies raised against the recombinant protein identified 16.5-kDa secretory proteins in both rat pancreatic juice and sf9 culture medium; and (c) immunohistochemistry indicated that the native antigen resides within zymogen granules in pancreatic acinar cells.

Apical membrane trafficking during regulated pancreatic exocrine secretion--role of alkaline pH in the acinar lumen and enzymatic cleavage of GP2, a GPI-linked protein.

The GP2/THP family of glycosyl phosphatidylinositol (GPI)-anchored proteins is targeted to apical secretory compartments in polarized epithelial cells. We demonstrate in the rat exocrine pancreas that enzyme-mediated release of GP2 from acinar cell membranes represents a pH-dependent process regulated by bicarbonate secreted from ductular cells. Release of GP2 from secretin-stimulated pancreatic lobules, which retain intralobular ducts, was inhibited by (i) bicarbonate substitution, (ii) chloride substitution, and (iii) DIDS, a potent inhibitor of chloride-bicarbonate exchange. These inhibitory effects were not observed in preparations of pancreatic acini devoid of ductal elements. Enzymatic cleavage of GP2 and amylase release from pancreatic acini varied directly as a function of pH of the acinar human. Alkali-induced GP2 release could be correlated with ultrastructural and biochemical evidence for stimulated retrieval (endocytosis) of exocytic membranes at the acinar lumen. Our study defines functional roles for ductal bicarbonate in acinar cell and lumen physiology and provides a potential explanation for the biological significance of enzyme-mediated cleavage of GP2 from the apical plasma membrane.

Nonparallel secretion of GP-2 from exocrine pancreas implies luminal coupling between acinar and duct cells.

The in vivo and in vitro secretion of glycoprotein-2 (GP-2), a glycosyl phosphatidylinositol (GPI)-anchored protein from the rat exocrine pancreas, was characterized. GP-2 was secreted in a nonparallel manner compared with amylase, a marker of secretory enzymes. Attenuated GP-2 secretion correlated with hormones that stimulated exocytosis in acinar cells. Augmented GP-2 secretion correlated with hormones that stimulated fluid and bicarbonate secretion from ductal elements. Immunofluorescence studies identified an enriched pool of GP-2 tightly bound to the apical membranes of acinar cells in addition to zymogen granules. This non-zymogen granule pool appears to represent the source of GP-2 released from acinar cells in a nonparallel manner. With the use of dispersed pancreatic acini largely devoid of ductal elements, GP-2 release was found to be augmented by alkaline pH. Thus GP-2 secretion appears to be modulated by two discrete cellular processes: 1) delivery of prereleased GP-2 within zymogen granules to the ductal lumen by exocytic mechanisms and 2) enzymatic release of GPI-anchored GP-2 from the luminal membranes, a kinetic process that appears to be regulated by secretin- or carbachol-induced secretion of bicarbonate.

Acid-base interactions during exocrine pancreatic secretion. Primary role for ductal bicarbonate in acinar lumen function.

The role of acid-base interactions during coordinated acinar and duct cell secretion in the exocrine pancreas is described. The sequence of acid-base events may be summarized as follows: (1) Sorting of secretory proteins and membrane components into the regulated secretory pathway of pancreatic acinar cells is triggered by acid- and calcium-induced aggregation and association mechanisms located in the trans-Golgi network. (2) Cholecystokinin-stimulated exocytosis in acinar cells releases the acidic contents of secretory granules into the acinar lumen. (3) Secretin-stimulated bicarbonate secretion from duct and duct-like cells neutralizes the acidic pH of exocytic contents, which leads to dissociation of protein aggregates and solubilization of (pro)enzymes within the acinar lumen. (4) Stimulated fluid secretion transports solubilized enzymes through the ductal system. (5) Further alkalinization of acinar lumen pH accelerates the enzymatic cleavage of the glycosyl phosphatidyl-inositol anchor associated with GP2 and thus releases the GP2/proteoglycan matrix from lumenal membranes, a process that appears to be required for vesicular retrieval of granule membranes from the apical plasma membrane and their reuse in the secretory process. We conclude that the central function of bicarbonate secretion by centroacinar and duct cells in the pancreas is to neutralize and then alkalinize the pH of the acinar lumen, sequential process that are required for (a) solubilization of secreted proteins and (b) cellular retrieval of granule membranes, respectively.

Role of the GP2/THP family of GPI-anchored proteins in membrane trafficking during regulated exocrine secretion.

Identification and characterization of the GP2/THP family of GPI-anchored membrane proteins associated with apical secretory membranes suggest that this new class of GPI-linked proteins plays a critical role in regulated protein secretion and ion transport in polarized epithelial cells in pancreas, liver, lung, kidney, and gastrointestinal tract. Based on recent information obtained from the world literature and from our own investigations we present the following two hypotheses capable of unifying previously diverse observations. Hypothesis 1 is that formation of GP2 tetramers in the acidic milieu of the trans-Golgi network (TGN) organizes a GP2/proteoglycan (PG) matrix tightly associated with the luminal surface of zymogen granule (ZG) membranes, and proposes that this matrix functions in (a) membrane sorting during granule assembly in the TGN, (b) inactivation of ZG membranes during the storage phase of secretion, and (c) regulated trafficking of ZG membranes from the apical plasma membrane (APM) after exocytosis. Hypothesis 2 is that the acinar lumen constitutes a distinct physiologic compartment for coupled biochemical reactions between acinar and duct cells. Because the acidic pH of the TGN plays a critical role in condensation of secretory proteins, alkalinization of the acinar lumen is required for (a) neutralization of the acidic pH of exocytic contents and (b) solubilization of aggregated (pro)enzymes. Further alkalinization appears to be required for pH-dependent release of the GP2/PG matrix from the APM, a process that may regulate internalization of ZG membranes for reuse during secretion. Taken together, the two hypotheses suggest that luminal factors including acid-base interactions and matrix assembly and disassembly processes perform critical functions during regulated storage and release of pancreatic (pro)enzymes. The requirement that coupling reactions be coordinated through the actions of separate hormones [cholecystokinin (CCK) and secretin] on divergent epithelial cells (acinar and duct cells, respectively) provides a new appreciation for the importance of combined CCK and secretin stimulation during pancreatic secretion in response to food intake.

Regulated secretory proteins in the exocrine pancreas aggregate under conditions that mimic the trans-Golgi network.

Fifteen pancreatic secretory proteins, including seven serine-endoproteinases (isoenzyme forms of trypsinogen, chymotrypsinogen and proelastase), four metallo-exoproteinases (isoenzymic forms of procarboxypeptidase A and procarboxypeptidase B), amylase, lipase, and two forms of carboxyl ester lipase were observed to aggregate under conditions of acidic pH (5.5) and calcium that mimic the trans-Golgi network. Subsequent neutralization of the pH resulted in disruption of protein aggregates and solubilization of pancreatic (pro)enzymes. In the absence of secretory granule membranes, granule contents display an "intrinsic" property for reversible, pH-dependent aggregation under conditions of mild acidification.

Reversible pH-induced homophilic binding of GP2, a glycosyl-phosphatidylinositol-anchored protein in pancreatic zymogen granule membranes.

GP2, the major zymogen granule membrane (ZGM) protein in the pancreas, is linked to the lumenal leaflet of the lipid bilayer via a glycosyl-phosphatidylinositol (GPI) moiety. We demonstrate that the peptide domain of GP2 (pGP2, approximately 75 kDa), purified from pancreatic ZGMs after phospholipase C cleavage, shows pH- and calcium-dependent self-association into sedimenting complexes. This homophilic binding process is progressive as pH is reduced from 7.0 to 5.5 and calcium is increased from 0 to 10-20 mM. This self-association reaction is temperature-dependent, optimal between 20 and 37 degrees C, progressively reduced below 20 degrees C, and eliminated at 10 degrees C. The reaction is reversible as a function of pH and abolished in the presence of nonionic detergents. Specificity in the homophilic reaction is demonstrated by the exclusion of heterologous proteins (globin, serum albumin, and IgG) from sedimenting complexes. At pH 5.5 in the presence of 20 mM calcium, oligomeric structures (approximately 300 kDa) consistent with tetrameric complexes were observed by gel filtration chromatography and elliptical structures (14-18 nm), frequently arranged in variegated clusters, were observed in the electron microscope by negative staining techniques. The pH- and calcium-dependent self-association observed for GP2 may represent an important mechanism by which GPI-anchored membrane proteins engage in homotypic binding reactions to establish highly functional membrane (micro)- domains targeted to regulated secretory compartments in polarized epithelial cells.

Structure of the canine pancreatic colipase gene includes two protein-binding sites in the promoter region.

Characterization of a lambda phage genomic clone, CL5A, which encodes the canine pancreatic colipase gene, revealed the primary structure of 987 nucleotides (nt) of 5'-flanking sequence, 2066 nt defining the primary transcriptional unit, which is organized into three exon sequences, and 130 nt of 3'-flanking sequence. Exon 1 encodes the amino-terminal signal peptide, the propeptide (Val1-Pro-Asp-Pro-Arg), and the hydrophobic lipid-binding region (Gly6-Ile-Ile-Ile) at the amino terminus of the mature coenzyme. Exon 2 encodes carboxylate residues (Glu12 and Glu15) likely to be involved in binding of pancreatic lipase to colipase at the aqueous-lipid interface. Exon 3 encodes the hydrophobic sequence (Leu54-Tyr-Gly-Tyr-Tyr) that is essential for binding the central tightly structured disulfide-bonded region of the coenzyme to lipid. Southern blot analysis was consistent with the presence of a single-copy colipase gene and a potential colipase gene homologue. Among 16 tissues examined by Northern blot analysis, colipase expression was detected only in pancreas. Proteins contained in nuclear extracts prepared from dog pancreas conferred two regions of DNase I protection coincident for both coding and noncoding strands (positions -62 to -44 (CL-I site) and -128 to -106 (CL-II site) in the coding strand). Competition gel mobility shift experiments indicated that protein-DNA interactions that occur at colipase sites I and II are sequence- and protein-specific and unrelated to the PAN-binding sequence described in the 5'-enhancer region of the rat chymotrypsin B gene (Nelson, C., Shen, L.-P., Meister, A., Fodor, E., and Rutter, W. J. (1990) Genes & Dev. 4, 1035-1043). Nuclear extracts from pancreas and brain, but not liver, contain similar CL-I- and CL-II-binding proteins. CL-I and CL-II represent protein-binding elements that may participate as additional promoter regions in regulated expression of the colipase gene. CL-I contains a central homopolymeric d(G) sequence. CL-II shows a GC-rich region on the noncoding strand (5' GGGGGCGTGT 3') that is similar (8/9 match) to the Sp1-binding sequence.

GP-2/THP gene family encodes self-binding glycosylphosphatidylinositol-anchored proteins in apical secretory compartments of pancreas and kidney.

A family of homologous genes is shown to encode GP-2, the major glycosylphosphatidylinositol (GPI)-linked glycoprotein of pancreatic zymogen granule membranes, and Tamm-Horsfall protein (THP), a GPI-linked glycoprotein associated with apical vesicles in kidney thick ascending limb of Henle (TALH) cells. The C-terminal regions of GP-2 (Asp54-Phe530) and THP (Asp175-His644) from rat show 53% identity, 86% similarity, and 26 conserved cysteine residues including one epidermal growth factor motif. The unique N-terminal domain of rat THP (unique-THP, Pro29-Gln174) shows four conserved epidermal growth factor motifs, three in tandem and one in reverse orientation. GP-2 homologues are observed in a wide variety of epithelial cells, several of which contain highly regulated secretory processes. GP-2 released from zymogen granule membranes with phosphatidylinositol phospholipase C reacts with anti-cross-reactive determinant antibody (anti-CRD), confirming the GPI nature of the pancreatic homologue. In contrast, GP-2 and THP, released endogenously from pancreas and kidney, respectively, do not react with anti-cross-reactive determinant antibody, suggesting alternative enzymatic mechanisms for their physiological release. Globular domains of GP-2 and THP, but not albumin, show pH- and ion-dependent self-association in vitro. The GP-2/THP family appears to represent a newly discovered class of GPI-anchored proteins, which may utilize pH- and ion-dependent self-association mechanisms for establishing membrane (micro)domains targeted to intracellular secretory compartments.

A single gene encodes membrane-bound and free forms of GP-2, the major glycoprotein in pancreatic secretory (zymogen) granule membranes.

GP-2, a 75-kDa glycoprotein, was isolated from dog pancreatic zymogen granule membranes (ZGMs). In a carbohydrate-shift strategy, N-terminal and internal peptide sequences were obtained on glycosylated and deglycosylated forms of GP-2, respectively, by gas-phase sequencing. Sets of mixed oligonucleotides and the polymerase chain reaction were used to obtain a double-stranded cDNA probe, which was used to isolate overlapping cDNA clones from a dog pancreatic cDNA library. The sequence of these clones revealed an open reading frame that encodes a protein of 509 amino acids, eight N-linked oligosaccharide attachment sites, and an N-terminal signal sequence absent from the mature form of GP-2 associated with ZGMs. The C terminus shows a 20-residue hydrophobic transmembrane domain preceded by a decapeptide containing potential phosphatidylinositol-glycan attachment sites. GP-2 completely released from ZGMs by exogenous phospholipase C showed similar immunochemical properties and electrophoretic mobilities compared to the form associated with ZGMs. A similar form of GP-2 was released from zymogen granules permeabilized with saponin and incubated in the absence of added phospholipase C. Kinetic analysis of GP-2 release at 0 degrees C and 37 degrees C suggested the presence of a granule enzyme responsible for endogenous release of GP-2 to granule contents and into the apical medium. The data indicate that GP-2 is a phosphatidylinositol-glycan-linked membrane protein released from the membrane of mature zymogen granules by an enzymatic mechanism. The cDNA structure presented here thus encodes both membrane-bound and free forms of GP-2.