A Family of 16-kDa Pancreatic Secretory Stress Proteins Form Highly Organized Fibrillar Structures upon Tryptic Activation
2001; Elsevier BV; Volume: 276; Issue: 24 Linguagem: Inglês
10.1074/jbc.m010717200
ISSN1083-351X
AutoresRolf Graf, Marc Schiesser, George A. Scheele, Klaus Marquardt, Thomas W. Frick, R Ammann, Daniel Bimmler,
Tópico(s)Galectins and Cancer Biology
ResumoA 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. 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. pancreatic stone protein, regenerating protein C-type lectin domain (Ca2+-dependent carbohydrate-recognition domain) pancreatitis-associated protein pancreatitis-associated thread protein pancreatic thread protein scanning electron microscope scanning/transmission electron microscope polymerase chain reaction 4-morpholineethanesulfonic acid polyacrylamide gel electrophoresis Pancreatic juice in vertebrates contains a group of 16-kDa proteins without known enzyme, proenzyme, or inhibitor function in the digestive process. This group, without defined function, is composed of the following protein species. Pancreatic stone protein (PSP/reg)1 is a 16-kDa acidic protein with an isoelectric point in the range of pH 5.5–6. A truncated form of this protein was originally isolated from calcium carbonate stones surgically removed from the main pancreatic duct of humans with chronic pancreatitis (1De Caro A. Lohse J. Sarles H. Biochem. Biophys. Res. Commun. 1979; 87: 1176-1182Crossref PubMed Scopus (137) Google Scholar). For several years it was believed that PSP/reg served as an inhibitor of calcium carbonate precipitation in pancreatic juice, and it was proposed that its name should be changed to "lithostathine" (2Sarles H. Dagorn J.C. Giorgi D. Bernard J.P. Gastroenterology. 1990; 99: 900-901Abstract Full Text PDF PubMed Scopus (0) Google Scholar). However, it was later shown that PSP/reg has no more crystal inhibitory activity than several of the pancreatic digestive enzymes (3Bimmler D. Graf R. Scheele G.A. Frick T.W. J. Biol. Chem. 1997; 272: 3073-3082Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 4De Reggi M. Gharib B. Patard L. Stoven V. J. Biol. Chem. 1998; 273: 4967-4971Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Other studies have demonstrated that the expression of PSP/regprotein is increased during the regeneration of islets after nicotinamide treatment and partial pancreatectomy (5Terazono K. Uchiyama Y. Ide M. Watanabe T. Yonekura H. Yamamoto H. Okamoto H. Diabetologia. 1990; 33: 250-252Crossref PubMed Scopus (112) Google Scholar, 6Unno M. Itoh T. Watanabe T. Miyashita H. Moriizumi S. Teraoka H. Yonekura H. Okamoto H. Adv. Exp. Med. Biol. 1992; 321: 61-69Crossref PubMed Scopus (28) Google Scholar). These observations led to the conclusion that PSP/reg may be a protein involved in regeneration (7Watanabe T. Yonemura Y. Yonekura H. Suzuki Y. Miyashita H. Sugiyama K. Moriizumi S. Unno M. Tanaka O. Kondo H. Bone A.J. Takasawa S. Okamoto H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3589-3592Crossref PubMed Scopus (236) Google Scholar) and furthermore may act as a growth mediator stimulating the proliferation of β-cells. Tissue culture studies implied a mitogenic activity of PSP/reg on the growth of various cell types (8Zenilman M.E. Magnuson T.H. Swinson K. Egan J. Perfetti R. Shuldiner A.R. Gastroenterology. 1996; 110: 1208-1214Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 9Fukui H. Kinoshita Y. Maekawa T. Okada A. Waki S. Hassan S. Okamoto H. Chiba T. Gastroenterology. 1998; 115: 1483-1493Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), and application of PSP/reg was observed to partially ameliorate diabetes in NOD mice (10Gross D.J. Weiss L. Reibstein I. van den Brand J. Okamoto H. Clark A. Slavin S. Endocrinology. 1998; 139: 2369-2374Crossref PubMed Google Scholar). Recently, a receptor was cloned from regenerating islets that binds PSP/reg and causes an increase in proliferation of cells transfected with a vector containing the receptor cDNA (11Kobayashi S. Akiyama T. Nata K. Abe M. Tajima M. Shervani N.J. Unno M. Matsuno S. Sasaki H. Takasawa S. Okamoto H. J. Biol. Chem. 2000; 275: 10723-10736Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Reg II (PAP I) appears to be involved in regeneration of motor neurons by acting as a Schwann cell mitogen (12Livesey F.J. O'Brien J.A. Li M. Smith A.G. Murphy L.J. Hunt S.P. Nature. 1997; 390: 614-618Crossref PubMed Scopus (170) Google Scholar). Still other investigations sought to implicate PSP/reg in the digestive process. However, recent studies did not show regulated PSP/reg synthesis and secretion in response to changes in nutritional substrates in the diet (13Bimmler D. Angst E. Valeri V. Bain M. Scheele G.A. Frick T.W. Graf R. Pancreas. 1999; 19: 255-267Crossref PubMed Google Scholar). Pancreatitis-associated protein (PAP) is a 16-kDa basic protein with an isoelectric point in the range of pH 6.5–7.6. Although most species contain a single PAP form, rat contains three isoforms, PAP I, PAP II, and PAP III, transcribed from three separate genes (14Iovanna J. Orelle B. Keim V. Dagorn J.-C. J. Biol. Chem. 1991; 266: 24664-24669Abstract Full Text PDF PubMed Google Scholar, 15Frigerio J.M. Dusetti N.J. Keim V. Dagorn J.C. Iovanna J.L. Biochemistry. 1993; 32: 9236-9241Crossref PubMed Scopus (46) Google Scholar, 16Frigerio J.M. Dusetti N.J. Garrido P. Dagorn J.C. Iovanna J.L. Biochim. Biophys. Acta. 1993; 1216: 329-331Crossref PubMed Scopus (49) Google Scholar). PAP levels increase in pancreatic juice during experimental (17Keim V. Rohr G. Stockert H.G. Haberich F.J. Digestion. 1984; 29: 242-249Crossref PubMed Scopus (72) Google Scholar) and clinical (18Keim V. Iovanna J.L. Orelle B. Verdier J.M. Busing M. Hopt U. Dagorn J.C. Gastroenterology. 1992; 103: 248-254Abstract Full Text PDF PubMed Scopus (49) Google Scholar) pancreatitis. Although showing an acute phase response under conditions of pancreatic disease, the function of PAP remains unknown. PSP/reg and PAP forms, cloned in the rat, mouse, cow, and man, show similarities in amino acid sequence. At the C terminus there is a C-type lectin binding sequence, and it has been proposed that this site might confer bacterial resistance on PAP (14Iovanna J. Orelle B. Keim V. Dagorn J.-C. J. Biol. Chem. 1991; 266: 24664-24669Abstract Full Text PDF PubMed Google Scholar, 19Iovanna J. Frigerio J.M. Dusetti N. Ramare F. Raibaud P. Dagorn J.C. Pancreas. 1993; 8: 597-601Crossref PubMed Scopus (37) Google Scholar). Recent studies have demonstrated that PSP/reg and PAP both act as acute phase reactants in pancreatic juice under a variety of conditions including acute pancreatitis (20Keim V. Iovanna J.L. Dagorn J.C. Digestion. 1994; 55: 65-72Crossref PubMed Scopus (59) Google Scholar), chronic pancreatitis in male WBN/Kob rats, and during the post-weaning period (21Bimmler D. Schiesser M. Angst E. Bain M. Frick T.W. Graf R. Pancreas. 1999; 19: 416Crossref Scopus (20) Google Scholar). Trypsin cleavage of PSP/reg and PAP has resulted in the appearance of precipitated proteins believed to represent insoluble thread structures in humans (22Gross J. Carlson R.I. Brauer A.W. Margolies M.N. Warshaw A.L. Wands J.R. J. Clin. Invest. 1985; 76: 2115-2126Crossref PubMed Scopus (108) Google Scholar) and cows (23Gross J. Brauer A.W. Bringhurst R.F. Corbett C. Margolies M.N. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5627-5631Crossref PubMed Scopus (37) Google Scholar). However, it remains difficult to understand how precipitation properties could serve useful functions in pancreatic physiology, and it is not clearly known whether these precipitated protein forms demonstrate specific or nonspecific structures. By taking a different approach in this study, we have attempted to define the functional as well as structural similarities shared between these molecules as a means to generate clues related to their function. To this end we have cloned, expressed, and purified recombinant forms of PSP/reg, PAP I, PAP II, and PAP III in the rat (24Bimmler D. Frick T.W. Scheele G.A. Pancreas. 1995; 11: 63-76Crossref PubMed Scopus (13) Google Scholar, 25Schiesser M. Bimmler D. Frick T.W. Graf R. Pancreas. 2001; 22: 186-192Crossref PubMed Scopus (39) Google Scholar). These purified reagents have allowed us to investigate the structural and functional properties of these proteins before and after trypsin cleavage to search for a unifying hypothesis that might explain the function of these proteins in pancreatic physiology and pathology. In this paper we have studied the structural/functional consequences of trypsin activation on these proteins with respect to (i) resistance of the processed forms to trypsin as well as to the heterogeneous mixture of proteases in pancreatic juice, (ii) the kinetics of conversion from soluble to insoluble protein forms, (iii) the kinetics of assembly of protein subunits into polymerized thread structures, and (iv) the morphology of polymerized thread structures by scanning, transmission scanning, and transmission electron microscopy. Recombinant PSP/reg was produced in the baculovirus system and purified as described previously (3Bimmler D. Graf R. Scheele G.A. Frick T.W. J. Biol. Chem. 1997; 272: 3073-3082Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 24Bimmler D. Frick T.W. Scheele G.A. Pancreas. 1995; 11: 63-76Crossref PubMed Scopus (13) Google Scholar). Monospecific antibodies directed against rat PSP/reg were generated in rabbits as described earlier (24Bimmler D. Frick T.W. Scheele G.A. Pancreas. 1995; 11: 63-76Crossref PubMed Scopus (13) Google Scholar). PAP I cDNA and PAP II cDNA were amplified using the polymerase chain reaction (PCR) from a rat pancreas cDNA library using PAP I- and II-specific primers and confirmed by DNA sequencing. PAP III cDNA was isolated by reverse transcriptase-PCR using rat ileum mRNA as a template. First, total RNA was extracted from rat ileum as described (13Bimmler D. Angst E. Valeri V. Bain M. Scheele G.A. Frick T.W. Graf R. Pancreas. 1999; 19: 255-267Crossref PubMed Google Scholar), and then mRNA was prepared with an Oligotex mRNA minikit (Qiagen). Reverse transcription of 5 μg of ileal mRNA was performed in a volume of 25 μl at 37 °C for 30 min using 19 units of Moloney murine leukemia virus-reverse transcriptase (Amersham Pharmacia Biotech) and a PAP III-specific primer. All three PAP isoforms are secretory proteins; therefore, we attempted to direct the accumulation of recombinant proteins in the media rather than intracellularly. To ensure that these proteins are secreted byP. pastoris, the endogenous PAP signal peptides were substituted by the signal peptide of the α-mating factor. The latter contains a Kex2 signal cleavage site consisting of a Lys-Arg followed by a Glu. Since all PAP isoforms start with a Glu, the PCR and subcloning strategy was designed to produce a cDNA with the Lys-Arg site followed by the first amino acid of the PAP isoforms (Glu). At the 5′-end of the cDNAs an XhoI site was included that facilitates ligation of the cDNA with the pBlueScript and recreates the correct reading frame of the α-mating factor signal peptide in pPIC9 (viz. manual of the Pichia Expression Kit, Invitrogen). The cDNAs were processed using a two-step PCR amplification procedure. In a first PCR, an antisense primer was combined with the matching sense primer (all primer sequences are described in Ref. 25Schiesser M. Bimmler D. Frick T.W. Graf R. Pancreas. 2001; 22: 186-192Crossref PubMed Scopus (39) Google Scholar). Second, the purified PCR products were combined in another PCR with the same antisense primers and a set of sense primers causing an elongation of the cDNA. PCR was performed on a PerkinElmer Life Sciences Gene Amp PCR system 9600 using Taq DNA polymerase (Amersham Pharmacia Biotech). 30 cycles were run (1 min at 94 °C, 2 min at 55 °C, and 3 min at 72 °C). After each amplification step the cDNAs were purified by excising them from agarose gels (26Heery D.M. Gannon F. Powell R. Trends Genet. 1990; 6: 173-175Abstract Full Text PDF PubMed Scopus (178) Google Scholar). The recovered cDNAs were purified by phenol extraction and sodium acetate/ethanol precipitation. The cDNAs were subsequently digested with XhoI andEcoRI restriction endonucleases (Amersham Pharmacia Biotech). The digests were purified and subcloned into pBlueScript (Stratagene) using T4 ligase (Amersham Pharmacia Biotech). TOP F10′Escherichia coli cells were transformed with the ligated plasmids by electroporation using a Bio-Rad Pulser II. Purified plasmids from individual colonies were digested with restriction enzymes to check for inserts, and some were subsequently sequenced to confirm the correct orientation of the coding sequence. Plasmids carrying the correct sequence were then digested with XhoI and EcoRI restriction enzymes, and the inserts were subcloned into the Pichia shuttle vector pPIC9 (Invitrogen). One recombinant vector for each isoform was sequenced again to confirm the correct insertion and sequence. ThePichia strains GS115 and KM71 (Invitrogen) were transformed with the linearized recombinant pPIC9 vector (SalI restriction endonuclease) by electroporation according to the supplier's recommendations. The transformants were plated on histidine-deficient minimal dextrose (MD) agar plates (1.34% yeast nitrogen base, 0.00004% biotin and 1% dextrose), and colonies were analyzed for insert integration by PCR, using the α-factor primer and the 3′ AOX1 (alcohol oxidase) primer. Positive clones were selected and tested with respect to their expression levels. By using a shaking incubator at 280 rpm, colonies were grown at 29 °C in baffeled flasks, each containing 80 ml of buffered minimal glycerol (BMG) medium (100 mm potassium phosphate, pH 6.0, 1.34% yeast nitrogen base, 0.00004% biotin, and 1% glycerol). Culture growth was monitored by measurement of the absorbance (600 nm); when anA 600 between 2 and 6 was reached, the cells were harvested by centrifugation. To induce expression of the recombinant protein, the cells were resuspended in 12 ml of BMM medium (BMG medium, containing 0.5% methanol instead of glycerol) and cultured for 6 days. The protein secretion was monitored in 24-h intervals by electrophoretic analysis of the supernatant (Coomassie-stained SDS-15% polyacrylamide gels). The most productive clones were selected and cultured in increased culture volumes. The highest yields were found in 5-day-old cultures. High yield media were collected by centrifugation at 1500 ×g for 5 min at 4 °C. The supernatants were centrifuged again at 5000 × g for 15 min. The supernatants were diluted 1:3 with chilled MilliQ filtered water (Millipore) and adjusted to pH 3.6 with HCl. Diluted protein solution (1200–1600 ml) was applied at a rate of 5 ml/min to a cation exchange column (SP-Sepharose, Amersham Pharmacia Biotech, 26 × 80 mm) with a bed volume of 40 ml. The column was washed with 2 volumes of starting buffer (50 mm MES, 10 mm LiCl, pH 5.3). The proteins were eluted with a linear LiCl gradient (0–35% elution buffer: 50 mm MES, 2 m LiCl, pH 6.3), generated by an AKTA purifier system (Amersham Pharmacia Biotech). To verify the identity of each of the isoforms, both mass spectrometry (electrospray mass analysis, PerkinElmer Life Sciences) and N-terminal amino acid sequencing was performed. The secreted, purified forms of PAP I, II, and III were desalted by ultrafiltration. To identify the new N terminus of the trypsin-resistant 14-kDa protein, each isoform was digested with trypsin as described below. The solutions were centrifuged to pellet the fibrils and remove the cleaved undecapeptide. For PAP II, the solution was filtered after digestion with an ultrafiltration device that retained the C-terminal peptide (10-kDa molecular weight cut-off, Centricon, Millipore). For mass analysis, aliquots were adsorbed to C18 ZIP tips (Millipore), eluted with 78% methanol, 1% formic acid, and injected into the analyzer. One hundred micrograms of recombinant PAP II were injected in Freund's complete adjuvant into several subcutaneous deposits in the back of a New Zealand White rabbit and a guinea pig. After 1 month the animals were boosted with 50 μg of PAP II in Freund's incomplete adjuvant followed by a similar boost a month later. Antibody titers were monitored in serum after venopuncture of the ear vein. Terminal bleeding was performed under anesthesia (Ketamine/Xylazine) by heart puncture. The antibody directed against PAP II reacts with PAP II (100%), PAP III (35%), PAP I (<10%), PATP II (80%), PATP III (<10%), and PATP I (<10%) but does not react with PSP/reg. Twenty microliters of rat pancreatic juice were diluted in 400 μl of Tris-Calcium buffer (10 mm, pH 8.0, 1 mm CaCl2) and equilibrated at 37 °C. An aliquot was withdrawn, and enteropeptidase (Worthington) was added at a final concentration of 0.1 unit/ml. The reaction was continued at 37 °C for 16 h during which several aliquots were withdrawn as a function of time as indicated in Fig. 1. The aliquots were transferred into a tube containing 50 μg of FOY 305, a potent trypsin inhibitor, and snap-frozen in liquid nitrogen until all aliquots had been collected. The samples were then rapidly thawed and centrifuged at 10,000 × g for 10 min in a cooled Beckman centrifuge (TL-100). The supernatant fraction was transferred to a new tube, and the pellet fraction was washed with 50 μl of Tris-Calcium buffer and centrifuged as described above. The pellets were dissolved in Tris-Calcium buffer in the original volume of the sample. They were prepared for electrophoresis by adding a 0.5 volume of 3-fold concentrated SDS-sample buffer (150 mmTris-HCl, pH 6.8, 3% SDS, 0.015% bromphenol blue, 15% glycerol v/v) followed by heating for 5 min at 90 °C. To maximize immunoreactivity of the activated protein forms, β-mercaptoethanol was omitted from the buffer solutions. Activation of the recombinant proteins was performed in a volume of 100 or 200 μl in Tris-Calcium buffer. Proteins (10 or 20 μg) were activated with 0.5–1 μg of trypsin (Worthington) for 30 min at 37 °C. Standard 15% polyacrylamide gels were prepared in SDS. Protein samples were heat-denatured at 90 °C in SDS-sample buffer (50 mm Tris-HCl, pH 6.8, 1% SDS, 0.005% bromphenol blue, and 5% glycerol) in the presence or absence of β-mercaptoethanol as indicated above. Proteins resolved in gels were stained with Coomassie Brilliant Blue (0.1% in 30% methanol, 10% acetic acid, Bio-Rad). For Western blot analysis, the proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) (24Bimmler D. Frick T.W. Scheele G.A. Pancreas. 1995; 11: 63-76Crossref PubMed Scopus (13) Google Scholar) on a semidry blotting apparatus (Amersham Pharmacia Biotech). The membranes were blocked with 1% bovine serum albumin in Tris-buffered saline (20 mm Tris, pH 7.5, 150 mm NaCl). Guinea pig anti-PAP II, diluted 1:3000 (25Schiesser M. Bimmler D. Frick T.W. Graf R. Pancreas. 2001; 22: 186-192Crossref PubMed Scopus (39) Google Scholar), and phosphatase-coupled anti-guinea pig IgG, diluted 1:10,000 (Sigma), were used to detect PAP and PATP. For the detection of PSP/regand PTP, chemiluminescence (ECLplus, Amersham Pharmacia Biotech) was employed. The primary rabbit anti-PSP/reg antibody was diluted 1:20,000 in Tris-buffered saline containing 1% bovine serum albumin. The membranes were washed in Tris-buffered saline containing 0.05% Tween 20. The secondary antibody, peroxidase-coupled goat anti-rabbit IgG (Sigma) was diluted 1:25,000 in the same buffer. Development and detection followed the manufacturer's recommendations. Relative quantities of individual protein bands were estimated by densitometry. Coomassie Blue-stained gels or Western blots were scanned with a Scanjet 6300C (Hewlett-Packard). The files were imported into Adobe Photoshop and quantified using ImageQuant (Molecular Dynamics) software. Intensities were expressed in percent of the maximal value as indicated in the figure legends. Recombinant thread proteins were diluted to 10 μg/50 μl in Tris-Calcium buffer and centrifuged in Microfuge tubes (Beckman) using a table top ultracentrifuge (Beckman TL 100). Following centrifugation of the sample at 1000 × g (4 °C, 30 min), the supernatant fractions were transferred to new tubes and centrifuged at 10,000 × g for 30 min at 4 °C. The resulting supernatant fractions were centrifuged at 100,000 × g for 30 min at 4 °C. The pellets were washed with Tris-Calcium buffer (described above) and redissolved in the original volume. Pellet and supernatant fractions were denatured in the presence of SDS-sample buffer, including 1% β-mercaptoethanol, for 5 min at 90 °C and submitted to SDS-PAGE. For scanning electron microscopy, pouches of nylon mesh, ∼1 cm2, were produced by folding a small piece of nylon mesh. The edges were heat-sealed, except for a small hole through which the samples could be introduced. The activation mixtures were transferred into the pouch, closed with a clamp, and submerged in 50 mm sodium cacodylate, pH 7.5, for 30 min at 4 °C. The pouch was then transferred to 2% glutaraldehyde in 100 mmsodium cacodylate, pH 7.5. After fixation at 4 °C overnight, the pouches were opened and processed for SEM analysis using standard procedures. The samples were viewed on a JEOL (JSM-25S II) scanning electron microscope. Photographs were taken via an attached computer using the software DISS (Digital Image Scanning System, Prophysics, Switzerland). For scanning transmission electron microscopy (STEM, Philips CM 120) analysis, a small amount of fibrillar material was removed from the surface of the pouch and plated with gold. To exclude gold-derived artifacts, the following pilot study was performed. After glutaraldehyde fixation, the pouch (see above) was immersed in 2% OsO4, 0.1 m sodium cacodylate buffer for 3 h. The pouch was rinsed and processed for SEM and STEM. In the absence of gold plating, the quality of resolution of the protein matrix was inferior compared with the gold-plated sample. However, it was concluded that the structure of the unplated sample was comparable to the structure of the gold-plated sample. To prepare thin sections of fibrils for examination in the electron microscope, the activation mixtures were initially embedded in Epon. However, this approach required extensive centrifugation after each medium change and a polymerization step at 60 °C. The resulting preparations appeared amorphous due to the loss of the fibrillar architecture. To circumvent this problem, we omitted centrifugation and used an alternative procedure that did not require polymerization at 60 °C. The digests were mixed with 0.1 volume of 25% glutaraldehyde and left at 4 °C overnight. Then OsO4 was added to a final concentration of 2 mm, and fixation was continued at room temperature for another hour. The fibrils demonstrated a black appearance and settled to the bottom of the vessel. Unicryl (British Biocell Int., Cardiff, UK), an embedding medium that polymerizes at low temperatures under UV light, was used according to the manufacturer's recommendation. Thin sections were cut with a diamond knife and examined in an electron microscope (Philips 400). As a negative control for this procedure, buffer was substituted for the activation mixture and processed as described above. Although some amorphous material was generated during OsO4 fixation, the fibrillar structures were not observed in the control. For negative contrast staining of fibrils, a small drop of activated sample was placed on the surface of a grid, dried, exposed to phosphotungstic acid (27Brenner S. Horne W.R. Biochim. Biophys. Acta. 1959; 34: 103-110Crossref PubMed Scopus (574) Google Scholar), and examined in the electron microscope. The multiple sequence alignment was created by "PileUp," an algorithm for progressive, pairwise sequence alignments by Feng and Doolittle (28Feng D.F. Doolittle R.F. J. Mol. Evol. 1987; 25: 351-360Crossref PubMed Scopus (1535) Google Scholar). The same program produces a dendrogram that depicts the clustering relationships. The determination of sequence similarity and identity was performed by pairwise analysis using the program "Gap." Gap and PileUp are part of the Wisconsin package version 9, supplied by Genetics Computer Group (Madison, WI). GCG is run on a UNIX system (Silicon Graphics) maintained by the Rechenzentrum der Universität Zürich. Fig. 1shows the sequence alignment of the N-terminal and C-terminal regions of three isoforms of PAP with PSP/reg in the rat. The mature secretory proteins are defined by two peptide domains separated by a highly conserved trypsin cleavage site. In three of the four secretory stress proteins (PSP/reg, PAP I, and PAP III), the Arg11–Ile12 bond represents the most sensitive cleavage site for trypsin. Upon trypsin cleavage the N-terminal undecapeptide is separated from the C-terminal peptide, which varies from 138 residues in PAP to 133 residues in PSP/reg. In addition to similarities in size, these proteins show similarities in sequence and protein domains. PSP/reg shows 43% identity and 54% similarity with PAP I, 45% identity and 52% similarity with PAP II, and 50% identity and 57% similarity with PAP III. PSP/reg is five amino acids shorter than the three PAP isoforms. The conservation of six cysteine residues suggests that three disulfide bonds are conserved. At the C terminus there is a conserved sequence indicating a C-type lectin domain (Fig. 1). Although the function of this signature sequence has not been elucidated, CTLs have been found in a variety of proteins that demonstrate diverse functions in different cellular and extracellular compartments (29Drickamer K. Curr. Opin. Struct. Biol. 1993; 3: 393-400Crossref Scopus (211) Google Scholar). However, the conserved N-terminal trypsin cleavage site is not observed in other proteins bearing CTLs. The secreted and proteolytic processed forms of recombinant PSP/reg and recombinant PAP shown in Fig. 1 were analyzed by mass spectrometry and N-terminal sequencing. The secreted forms of PAP I, II, and III all conformed with the expected amino acid sequence (first 10 amino acids determined), starting with a glutamine residue in each case. The processed forms of PAP also conformed with the expected sequence (first 5–7 amino acids determined) starting with an isoleucine (PATP I, III) or a threonine (PATP II) residue. Mass analysis yielded the following measurements: PAP I, 16623.6 (theoretical 16623.6); PATP I, 15414.0 (15414.2); PAP II, 16404.2 (16403.2); PATP II, 15203.9 (15203.9); PAP III, 16247.8 (16248.0); and PATP III, 15021.5 (1
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