Secretory apparatus assessed by analysis of pancreatic secretory stress protein expression in a rat model of chronic pancreatitis

Secretory stress proteins (SSP) are a family of proteins including isoforms of pancreatitis-associated protein (PAP) and pancreatic stone protein (PSP/reg). In vitro exposure to trypsin results in the formation of insoluble fibrillar structures. SSP are constitutively secreted into pancreatic juice at low levels. The WBN/Kob rat is a model for chronic pancreatitis, displaying focal inflammation, destruction of the parenchyma and changes in the architecture of the acinar cell; the synthesis and secretion of SSP are also increased. We have investigated the secretory apparatus by SSP immunohistochemistry at the light- and electron-microscopical (EM) levels. Immunocytochemistry of PSP/reg in Wistar control rats reveals low levels, with individual acinar cells exhibiting high immunoreactivity in zymogen granules. PAP is not detectable. In the WBN/Kob rat, PSP/reg and PAP immunoreactivity is markedly increased. Double immunofluorescence for PSP/reg and PAP I or II demonstrates that these proteins colocalize to the same cell. Acinar cells change their secretory architecture by fusion of zymogen granules and elongation of the fused organelles. The immunogold technique has demonstrated an increase of SSP in zymogen granules in WBN/Kob rats. PSP/reg-positive zymogen granules fuse to form elongated structures with fibrillar contents. An extensive PSP/reg-positive fibrillar network is established in the cytosol. Extracellular fibrils have been observed in several ductules. Thus, SSP-derived fibrils form concomitantly with acinar damage in the WBN/Kob rat. Based on the known tryptic cleavage site of SSP, the in vivo generation of fibrils is presumably the result of premature trypsin activation.     

Comprehensive proteomic analysis of human pancreatic juice

Proteomic technologies provide an excellent means for analysis of body fluids for cataloging protein constituents and identifying biomarkers for early detection of cancers. The biomarkers currently available for pancreatic cancer, such as CA19-9, lack adequate sensitivity and specificity contributing to late diagnosis of this deadly disease. In this study, we carried out a comprehensive characterization of the "pancreatic juice proteome" in patients with pancreatic adenocarcinoma. Pancreatic juice was first fractionated by 1-dimensional gel electrophoresis and subsequently analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). A total of 170 unique proteins were identified including known pancreatic cancer tumor markers (e.g., CEA, MUC1) and proteins overexpressed in pancreatic cancers (e.g., hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) and lipocalin 2). In addition, we identified a number of proteins that have not been previously described in pancreatic juice (e.g., tumor rejection antigen (pg96) and azurocidin). Interestingly, a novel protein that is 85% identical to HIP/PAP was identified, which we have designated as PAP-2. The proteins identified in this study could be directly assessed for their potential as biomarkers for pancreatic cancer by quantitative proteomics methods or immunoassays.     

Messenger RNA sequence and expression of rat pancreatitis-associated protein, a lectin-related protein overexpressed during acute experimental pancreatitis.

Rat pancreatitis-associated protein (PAP) is an additional protein appearing in pancreatic juice after induction of prancreatic inflammation. Its messenger RNA was cloned and sequenced from pancreas. The deduced amino acid sequence revealed that PAP was synthetized as a preprotein with, in its mature form, a predicted molecular weight of 16,630. A search in protein data bases revealed a marked homology with the carbohydrate binding region of animal lectins; no hemagglutination activity could be shown for PAP, but the protein induced extensive bacterial aggregation. In healthy rats, the very low level of PAP expression in pancreas could be increased up to 4-fold by physiological stimuli such as chronic hormonal or cholinergic stimulation of pancreatic secretion and adaptation of rats to a carbohydrate-rich diet. By contrast, induction of acute experimental pancreatitis by retrograde injection of sodium taurocholate resulted in dramatic overexpression. Pancreatic concentration of PAP mRNA increased more than 300 x within 12 h whereas concentrations of mRNAs encoding major secretory proteins such as amylase decreased. PAP overexpression persisted during the 2 days of the acute phase and then returned to the control level during pancreatic recovery. PAP mRNA could not be evidenced in liver, stomach, salivary glands, brain, kidney, or testis. Its pattern of expression during severe pancreatic aggression suggests that it might be a stress protein involved in the control of bacterial proliferation.     

Characterization in rat pancreatic juice of a protein homologous to the human pancreatic stone protein

1. The pancreatic stone protein (PSP, Mr 15,000) which has been discovered in human calculi derives from the native glycosylated forms of the protein (Mrs 17,500-22,000) which are present in human pancreatic juice through tryptic cleavage of the Arg 11-Ile 12 bond. 2. In the present study, a homologous native form of the protein (Mr 17,000) was purified from rat pancreatic juice. 3. Its N-terminal amino acid sequence was found to display a high degree of homology with that of the human native protein forms, apart from the fact that it was not glycosylated. 4. In rat as in human, tryptic cleavage of the Arg 11-Ile 12 bond transforms a soluble protein into one which is practically insoluble at neutral pH.     

The multifunctional family of secreted proteins containing a C-type lectin-like domain linked to a short N-terminal peptide

PSP/Lithostathine/PTP/regI, PAP/p23/HIP, reg1L, regIV and "similar to PAP" are the members of a multifunctional family of secreted proteins containing a C-type lectin-like domain linked to a short N-terminal peptide. The expression of this group of proteins is controlled by complex mechanisms, some members being constitutively expressed in certain tissues while, in others, they require activation by several factors. These members have several apparently unrelated biological effects, depending on the member studied and the target cell. These proteins may act as mitogenic, antiapoptotic or anti-inflammatory factors, can regulate cellular adhesion, promote bacterial aggregation, inhibit CaCO3 crystal growth or increase resistance to antitumoral agents. The presence of specific receptors for these proteins is suggested because biological effects were observed after the addition of purified protein to culture media or after systemic administration to animals, whereas other biological effects could be explained by their biochemical capacity to form homo or heteromers or to form insoluble fibrils at physiological pH.     

Ligand-induced association of surface immunoglobulin with the detergent insoluble cytoskeleton may involve alpha-actinin

B cell surface immunoglobulin (SIg) plays an important role in antigen recognition and cellular activation. Cross-linking of SIg by bivalent antibody converts it into a detergent insoluble state. The resultant SIg may be partially solubilized by incubating the detergent insoluble cytoskeleton in buffers that convert F actin to G actin. Immunoprecipitation of SIg from the detergent soluble fraction of [35S]methionine-labeled B cells results in the co-isolation of 112 kDa, 42 kDa, (actin), and three additional proteins in the 70- to 73-kDa molecular mass range, isoelectric point 4.8 to 5.6. Analysis of anti-Ig immunoprecipitates made after preclearing with anti-alpha-actinin showed complete depletion of the 112-kDa protein, suggesting that the 112-kDa protein is immunologically related to alpha-actinin. These immunoprecipitates also showed partial depletion of 70- to 73-kDa proteins and mu and delta heavy chains. After treatment of a rat B cells with anti-Ig, much of the Ig-associated 112-kDa protein and 70- to 73-kDa proteins became detergent insoluble, concomitant with most of the SIg. The migration of the SIg-associated 112-kDa and 70- to 73-kDa proteins from the detergent soluble fraction to the detergent insoluble fraction after ligand treatment, suggests that these proteins might be involved in linking SIg to the underlying cytoskeleton and could be involved in the transmission of mitogenic signals.     

The human pancreatic stone protein

Chronic calcifying pancreatitis (CCP) is characterized by the presence of stones in pancreatic ducts. Calcium carbonate (CaCO3) is the main constituent of stones, to which is associated an organic matrix consisting primarily of one protein of Mr 14,000, the pancreatic stone protein or PSP. PSP is not present as such in pancreatic juice, but in polymorphic forms with higher molecular weights. These secretory forms (PSP S2-5, Mr 16-19,000) are synthesized in the acinar cells of the pancreas and secreted along the same secretory pathway as the exocrine enzymes. The heterogeneity of the forms of higher Mr (PSP S2-5) is probably due to different glycosylation patterns. PSP and PSP S1 are generated by the cleavage of an Arg-Ile bond in the N-terminal part of PSP S2-5. The N-terminal sequence of PSP (40 amino acids) is identical to that of PSP S1, whose complete sequence (133 amino acids) has been determined. Yet, the two proteins differ by their pI. Pancreatic juice is normally supersaturated in CaCO3, suggesting the presence of a stabilizer preventing CaCO3 precipitation. The PSP S could play that role, since an activity inhibiting the nucleation and growth in vitro of CaCO3 crystals was found in pancreatic juice, associated with these proteins. Moreover, PSP S concentration was significantly lower in the pancreatic juice of patients with CCP than in control patients. Proteins homologous to PSP S were also found in the dog, rat, swine, monkey and ox. They constitute a new family of pancreatic secretory proteins, whose biological role would be to maintain pancreatic juice in a stable state towards CaCO3.     

胰腺应激蛋白PSP/reg抑制胰腺星状细胞TIMPs:MMPs及RECK表达

目的 观察胰腺应激蛋白PSP/reg对胰腺星状细胞(PSC)合成和分泌基质金属蛋白酶(MMPs)及其组织抑制剂(TIMPs)以及RECK表达的影响.方法 分离纯化慢性胰腺炎患者纤维化区的PSC,基因重组胰腺应激蛋白PSP/reg,以终浓度为10和100 ng/mL对PSC进行干预,实时荧光定量PCR检测MMP1/2、TIMP1/2及RECK基因表达,Western blot测定MMP1/2、TIMP1/2及RECK蛋白,细胞免疫荧光观察细胞膜表面RECK分布.结果 PSP/reg对MMP1/2、TIMP1/2及RECK表达无明显影响;PSP/reg轻度抑制PSC培养上清中MMP2水平(P＜0.05),而显著抑制TIMP1/2水平(P ＜0.01);PSC细胞膜表面发现有RECK蛋白,PSP/reg减少PSC的RECK含量(P＜0.01).结论 胰腺应激蛋白PSP/reg能够降低TIMPs:MMPs比率、减少RECK蛋白水平表达,从而解除对MMPs的部分抑制,使MMPs活性相对增高,有利于纤维化的分解消散,促进胰腺损伤后的再生修复.     

N-terminal sequence extension in the glycosylated forms of human pancreatic stone protein. The 5-oxoproline N-terminal chain is O-glycosylated on the 5th amino acid residue

The pancreatic stone protein isolated from human calculi (PSP) derives from the immunoreactive protein forms detected in human pancreatic juice (PSP S2-5) through the tryptic cleavage of the Arg-11-Ile-12 bond. Among the eleven amino acids of the PSP S2-5 N-terminal extension Z-E-A-Q-T-E-L-P-Q-A-R, the first residue is an oxoproline and the fifth, a threonine, bears the single carbohydrate chain of the protein molecules. Variations in the glycan chain composition account for the differences in the Mr of PSP S2-5. The PSP S2-5 forms are very soluble in aqueous solutions between the pH values 5.0-9.0, whereas the proteolysated form is scarcely soluble.     

Hyperphosphorylation of the rotavirus NSP5 protein is independent of serine 67, [corrected] NSP2, or [corrected] the intrinsic insolubility of NSP5 is regulated by cellular phosphatases

The NSP5 protein is required for viroplasm formation during rotavirus infection and is hyperphosphorylated into 32- to 35-kDa isoforms. Earlier studies reported that NSP5 is not hyperphosphorylated without NSP2 coexpression or deleting the NSP5 N terminus and that serine 67 is essential for NSP5 hyperphosphorylation. In this report, we show that full-length NSP5 is hyperphosphorylated in the absence of NSP2 or serine 67 and demonstrate that hyperphosphorylated NSP5 is predominantly present in previously unrecognized cellular fractions that are insoluble in 0.2% sodium dodecyl sulfate. The last 68 residues of NSP5 are sufficient to direct green fluorescent protein into insoluble fractions and cause green fluorescent protein localization into viroplasm-like structures; however, NSP5 insolubility was intrinsic and did not require NSP5 hyperphosphorylation. When we mutated serine 67 to alanine we found that the NSP5 mutant was both hyperphosphorylated and insoluble, identical to unmodified NSP5, and as a result serine 67 is not required for NSP5 phosphorylation. Interestingly, treating cells with the phosphatase inhibitor calyculin A permitted the accumulation of soluble hyperphosphorylated NSP5 isoforms. This suggests that soluble NSP5 is constitutively dephosphorylated by cellular phosphatases and demonstrates that hyperphosphorylation does not direct NSP5 insolubility. Collectively these findings indicate that NSP5 hyperphosphorylation and insolubility are completely independent parameters and that analyzing insoluble NSP5 is essential for studies assessing NSP5 phosphorylation. Our results also demonstrate the involvement of cellular phosphatases in regulating NSP5 phosphorylation and indicate that in the absence of other rotavirus proteins, domains on soluble and insoluble NSP5 recruit cellular kinases and phosphatases that coordinate NSP5 hyperphosphorylation.     

Immunocytochemical demonstration of a pancreatic secretory protein of unknown function in human duodenum

We have previously isolated from human pancreatic juice a secretory glycoprotein of 19 KD (P19), devoid of known enzymatic activity. P19 gave by proteolysis a protein of 14 KD (P14), at first named protein X and also called pancreatic thread protein or pancreatic stone protein. Specific rabbit immunosera prepared against P19 and P14 were applied to localize these proteins in human small intestine. By comparison, antibodies directed against some human pancreatic enzymes (amylase, lipase, chymotrypsin, trypsinogen 1, trypsinogen 2, and trypsin 1) were also tested. Positive immunoreactivity was observed on Paneth cells with antisera directed against trypsinogens, trypsin 1, and P19-related proteins. In addition, antisera directed against P19-related proteins stained the columnar cells located in the crypts of Lieberkühn. These original findings are a further indication of the resemblance between Paneth and pancreatic acinar cells but show that their functional analogy is only partial. On the other hand, the presence of P19-related proteins on non-mature columnar cells suggests that this differential distribution is a consequence of differentiation.     

Degradation of proteins microinjected into HeLa cells. The role of substrate flexibility

Increasing the flexibility of a protein enhances its susceptibility to defined proteases in vitro. To ascertain whether flexibility also affects protein stability in vivo, radioiodinated proteins with similar structures, but dissimilar flexibilities, were introduced into HeLa cells using red cell-mediated microinjection. Intracellular proteolysis was then measured as the rate of release of 125I-tyrosine into the medium. Ribonuclease A was considerably more resistant to degradation by purified proteases or in reticulocyte lysate than its flexible derivatives ribonuclease S and S-protein. In contrast, all three proteins were equally stable within HeLa cells. Like the results obtained for RNases, the rates of degradation of trypsin inhibitors, trypsin analogs, and their complexes correlated with flexibility in reticulocyte lysate. However, the intracellular half-lives of anhydrotrypsin and various proteinaceous trypsin inhibitors were not affected upon formation of enzyme-inhibitor complexes. Furthermore, trypsinogen was degraded more slowly than the structurally similar anhydrotrypsin in HeLa cells, although trypsinogen has additional segmental flexibility in its activation domain. Electrophoretic analyses revealed that trypsin-inhibitor complexes remained intact following injection into HeLa cells, and that neither free inhibitors nor anhydrotrypsin formed Triton-stable complexes with soluble cytoplasmic proteins. The observation that the components of the trypsin-inhibitor complexes were degraded simultaneously indicates that neither constituent unfolded prior to the onset of proteolysis. These studies provide evidence that RNases, trypsin, and trypsin inhibitors are degraded by an intracellular proteolytic pathway(s) which recognizes surface features of the folded proteins.     

Presence of cathepsin B in the human pancreatic secretory pathway and its role in trypsinogen activation during hereditary pancreatitis

The lysosomal cysteine protease cathepsin B is thought to play a central role in intrapancreatic trypsinogen activation and the onset of experimental pancreatitis. Recent in vitro studies have suggested that this mechanism might be of pathophysiological relevance in hereditary pancreatitis, a human inborn disorder associated with mutations in the cationic trypsinogen gene. In the present study evidence is presented that cathepsin B is abundantly present in the secretory compartment of the human exocrine pancreas, as judged by immunogold electron microscopy. Moreover, pro-cathepsin B and mature cathepsin B are both secreted together with trypsinogen and active trypsin into the pancreatic juice of patients with sporadic pancreatitis or hereditary pancreatitis. Finally, cathepsin B- catalyzed activation of recombinant human cationic trypsinogen with hereditary pancreatitis-associated mutations N29I, N29T, or R122H were characterized. In contrast to a previous report, cathepsin B-mediated activation of wild type and all three mutant trypsinogen forms was essentially identical under a wide range of experimental conditions. These observations confirm the presence of active cathepsin B in the human pancreatic secretory pathway and are consistent with the notion that cathepsin B-mediated trypsinogen activation might play a pathogenic role in human pancreatitis. On the other hand, the results clearly demonstrate that hereditary pancreatitis-associated mutations do not lead to increased or decreased trypsinogen activation by cathepsin B. Therefore, mutation-dependent alterations in cathepsin B-induced trypsinogen activation are not the cause of hereditary pancreatitis.     

Two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis of protein mixtures containing active or potentially active proteases: Analysis of human exocrine pancreatic proteins

Analysis of human pancreatic juice in two dimensions using isoelectric focusing followed by sodium dodecyl sulfate gel electrophoresis indicated that human pancreatic trypsinogen (IEP_n = 6.4) rapidly autoactivated in the absence of the secretory trypsin inhibitor. The addition of 4 to 6 m urea to the protein sample and 8 m urea to the isoelectric focusing gel inhibited this autoactivation process and allowed the analysis of human exocrine pancreatic proteins. Thirteen discrete proteins were separated by the two-dimensional gel procedure including two forms each for trypsinogen, proelastase, and procarboxypeptidase A, and single forms each for amylase, lipase, procarboxypeptidase B, and chymotrypsinogen. The kinetics of inhibition of human trypsin by 8 m urea in the presence of ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid indicated that samples containing active proteases could also be analyzed by this procedure.     

Protein X, a proteolysis product of human pancreatic juice. Immunological relationship with trypsinogen 1

Protein X (PX) previously isolated from human pancreatic juice is an inactive protein of 14 kDa which has been shown to be a degradation product liberated by proteolysis of 19 kDa precursors. Polyclonal antibodies against P19 and PX were prepared in rabbits by injection of the two proteins purified by SDS polyacrylamide gel electrophoresis. These antibodies reacted with a form of trypsin 1 (DFP-trypsin 1) which was shown to be partly proteolysed. Immunological studies were performed with pancreatic juice proteins and partially purified trypsinogen 1 using antibodies directed against PX, P19 and trypsin 1. The results of immunoprecipitation and immunoadsorbent chromatography show that these different antisera recognized a protein of 25 kDa. Immunoblotting has permitted to characterize this protein as a trypsinogen 1-like molecule which would be a form of inert protein generated by uncontrolled trypsinogen activation.     

Serine proteases mediate inflammatory pain in acute pancreatitis

Acute pancreatitis is a life-threatening inflammatory disease characterized by abdominal pain of unknown etiology. Trypsin, a key mediator of pancreatitis, causes inflammation and pain by activating protease-activated receptor 2 (PAR(2)), but the isoforms of trypsin that cause pancreatitis and pancreatic pain are unknown. We hypothesized that human trypsin IV and rat P23, which activate PAR(2) and are resistant to pancreatic trypsin inhibitors, contribute to pancreatic inflammation and pain. Injections of a subinflammatory dose of exogenous trypsin increased c-Fos immunoreactivity, indicative of spinal nociceptive activation, but did not cause inflammation, as assessed by measuring serum amylase and myeloperoxidase activity and by histology. The same dose of trypsin IV and P23 increased some inflammatory end points and caused a more robust effect on nociception, which was blocked by melagatran, a trypsin inhibitor that also inhibits polypeptide-resistant trypsin isoforms. To determine the contribution of endogenous activation of trypsin and its minor isoforms, recombinant enterokinase (ENK), which activates trypsins in the duodenum, was administered into the pancreas. Intraductal ENK caused nociception and inflammation that were diminished by polypeptide inhibitors, including soybean trypsin inhibitor and a specific trypsin inhibitor (type I-P), and by melagatran. Finally, the secretagogue cerulein induced pancreatic nociceptive activation and nocifensive behavior that were reversed by melagatran. Thus trypsin and its minor isoforms mediate pancreatic pain and inflammation. In particular, the inhibitor-resistant isoforms trypsin IV and P23 may be important in mediating prolonged pancreatic inflammatory pain in pancreatitis. Our results suggest that inhibitors of these isoforms could be novel therapies for pancreatitis pain.     

Ultrastructural localization of GP2 in acinar cells of pancreas: presence of GP2 in endocytic and exocytic compartments

GP2 is a glycoprotein found in pancreatic acinar cells. Its subcellular distribution suggests that it may be involved both in exocytosis and endocytosis. Immunocytochemical studies have demonstrated GP2 to be present on the membrane and in the matrix of zymogen granules, on Golgi saccules, on the apical and basolateral surfaces of the plasma membrane, and in the lumina of acini. In addition, this protein was observed in small vacuoles and tubular structures previously identified as "basal lysosomes," "snake-like tubules," and in lysosomes. Because the latter group of structures are involved in endocytosis, it is possible that GP2 may be involved in this phenomenon. GP2 was readily detectable in pancreatic juice and was totally sedimentable by ultracentrifugation, as assessed by Western blot analysis. Induced lysis of isolated zymogen granules also caused release of GP2 in a sedimentable form which, by electron microscopy, appeared as a fibrillar structure. Immunocytochemical localization of amylase was studied in parallel with GP2 and was found in the secretory product to be associated with thread-like structures, presumably the pancreatic thread protein. The physiological significance of these observations is discussed.     

A tale of two polymers: new insights into helical filaments

Many proteins function as helical polymers within the cell. Two intensively studied examples are eukaryotic actin and bacterial RecA, which belong to two different protein superfamilies. However, most other members of these superfamilies do not polymerize into helical filaments. General features of polymorphism, cooperativity and allostery that emerge from studies of eukaryotic actin and bacterial RecA raise more general issues about how conserved these filamentous structures have been during evolution.     

Difference of acrosomal serine protease system between mouse and other rodent sperm

A fraction of acrosomal proteins dispersed during calcium ionophore A23187‐induced acrosome reaction was prepared from cauda epididymal sperm of wild‐type and acrosin‐deficient mice, rat, and hamster. The acrosome‐reacted sperm were further extracted by Nonidet P‐40 to obtain the detergent‐soluble protein fraction. Activities of serine proteases in the two protein fractions were examined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the presence of gelatin. A mixture of 42‐ and 41‐kDa gelatin‐hydrolyzing proteases was found in both fractions of the wild‐type mouse sperm, whereas the acrosin‐deficient mouse sperm contained the active 42‐kDa protease and apparently lacked the activity of the 41‐kDa protease. However, exogenous bovine pancreatic trypsin compensated for the absence of acrosin in the protein fractions of the mutant mouse sperm; the gelatin‐hydrolyzing activity of the 41‐kDa protease appeared when the sperm proteins of the mutant mice were treated with pancreatic trypsin. Two‐dimensional polyacrylamide gel electrophoresis revealed that the 42‐ and 41‐kDa proteases were distinguished from acrosin by the isoelectric point and immunoreactivity with affinity‐purified antibody against an oligopeptide corresponding to the N‐terminal amino acid sequence of mouse proacrosin. Moreover, the gelatin‐hydrolyzing proteins corresponding to these two proteases were not detected in rat and hamster sperm, in spite of the treatment of the sperm extracts with pancreatic trypsin, and the total amount of gelatin‐hydrolyzing activities in mouse was much smaller than those in rat and hamster. These results may reflect the difference of the serine protease system for the sperm penetration through the egg zona pellucida between mouse and other rodent animals, possibly explaining why the acrosin‐deficient mouse sperm are capable of penetrating the zona pellucida. Dev. Genet. 25:115–122, 1999. © 1999 Wiley‐Liss, Inc.