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.     

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.     

Partial amino acid sequence of human pancreatic stone protein, a novel pancreatic secretory protein.

Pancreatic stone protein (PSP) is the major organic component of human pancreatic stones. With the use of monoclonal antibody immunoadsorbents, five immunoreactive forms (PSP-S) with close Mr values (14,000-19,000) were isolated from normal pancreatic juice. By CM-Trisacryl M chromatography the lowest-Mr form (PSP-S1) was separated from the others and some of its molecular characteristics were investigated. The Mr of the PSP-S1 polypeptide chain calculated from the amino acid composition was about 16,100. The N-terminal sequences (40 residues) of PSP and PSP-S1 are identical, which suggests that the peptide backbone is the same for both of these polypeptides. The PSP-S1 sequence was determined up to residue 65 and was found to be different from all other known protein sequences.     

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.     

人PSP94全长cDNA的获得及PSP94-TNF~Δ融合蛋白的构建

利用ＲＴ－ＰＣＲ从人肥大前列腺组织钓取９４个氨基酸的人前列腺分泌蛋白（ＰＳＰ９４）全长ｃＤＮＡ,序列分析结果与文献报道的完全一致．将ＰＳＰ９４成熟肽与人ＴＮＦα衍生物（ＴＮＦΔ）通过Ｌｉｎｋｅｒ－ＳＡＰＧＴＰ在基因水平上融合成５′ＰＳＰ９４－ＴＮＦΔ,融合基因ＤＮＡ序列分析结果与设计的相符合．５′ＰＳＰ９４－ＴＮＦΔ在大肠杆菌中表达产物分子量约为３１ｋＤ,表达量约占菌体总蛋白量的３５％．以Ｌ９２９细胞和人前列腺癌细胞株ＰＣ－３为靶细胞进行细胞毒分析结果表明,５′ＰＳＰ９４－ＴＮＦΔ融合蛋白既具有ＴＮＦ的细胞毒活性,又具有对前列腺癌细胞ＰＣ－３的杀伤作用     

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.     

Recombinant perchloric acid-soluble protein suppresses the immunoglobulin production of human-human hybridoma HB4C5 cells

Because perchloric acid-soluble protein (PSP) has been conserved evolutionally in various species from Escherichia coli to humans, it may reflect an involvement in basic cellular regulation. However, the precise function of PSP is currently unknown. In this study, we examined the direct effect of PSP on the production of immunoglobulin (Ig) using human B, HB4C5, NAT-30, and U266 cells because it has been reported that subcutaneous administration of PSP affects rodent immune systems. Suppression of Ig productivity and decrement of the cell viability was recognized only in HB4C5 cells by the addition of PSP into the medium. On the other hand, PSP had no effect on Ig productivity and cell viability in NAT-30 and U266 cells. In addition, PSP was clearly incorporated by HB4C5 but not by the other cells. These results suggest that the Ig production suppressed by PSP, which has been previously reported to inhibit protein synthesis, contributed to the incorporation of PSP into the HB4C5 cells.     

Dual host-defence functions of SPLUNC2/PSP and synthetic peptides derived from the protein

PSP (parotid secretory protein)/SPLUNC2 (short palate, lung and nasal epithelium clone 2) is expressed in human salivary glands and saliva. The protein exists as an N-glycosylated and non-glycosylated form and both appear to induce agglutination of bacteria, a major antibacterial function for salivary proteins. Both forms of PSP/SPLUNC2 bind LPS (lipopolysaccharide), suggesting that the protein may also play an anti-inflammatory role. Based on the predicted structure of PSP/SPLUNC2 and the location of known antibacterial and anti-inflammatory peptides in BPI (bactericidal/permeability-increasing protein) and LBP (LPS-binding protein), we designed GL13NH2 and GL13K, synthetic peptides that capture these proposed functions of PSP/SPLUNC2. GL13NH3 agglutinates bacteria, leading to increased clearance by macrophages and reduced spread of infection in a plant model. GL13K kills bacteria with a minimal inhibitory concentration of 5-10 μg/ml, kills bacteria in biofilm and retains activity in 150?mM NaCl and 50% saliva. Both peptides block endotoxin action, but only GL13K appears to bind endotoxin. The peptides do not cause haemolysis, haemagglutination in serum, inhibit mammalian cell proliferation or induce an inflammatory response in macrophages. These results suggest that the GL13NH2 and the modified peptide GL13K capture the biological activity of PSP/SPLUNC2 and can serve as lead compounds for the development of novel antimicrobial and anti-inflammatory peptides.     

hSP, the domain-duplicated homolog of pS2 protein, is co-expressed with pS2 in stomach but not in breast carcinoma.

Approximately 50% of human breast tumors secrete a small cysteine-rich protein, pS2, of unknown function. pS2 protein was recently found to be homologous to a porcine protein with hormonogastric activity, pancreatic spasmolytic polypeptide (PSP), in which the 5-cysteine domain present in pS2 is tandemly duplicated. We have characterized cDNA species encoding PSP and its human and mouse counterparts, hSP and mSP. We show that hSP and pS2 are separately encoded in the genome, and that the two proteins are co-expressed in normal stomach epithelium. However, expression of hSP was not detected in breast tumors. Computer analysis revealed that the pattern of conserved cysteine residues in hSP and pS2, the P domain, is present at the N termini of two other mammalian proteins, intestinal sucrase-isomaltase and lysosomal alpha-glucosidase.     

Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5

Ribosomal protein (rp) S5 belongs to a family of ribosomal proteins that includes bacterial rpS7. rpS5 forms part of the exit (E) site on the 40S ribosomal subunit and is essential for yeast viability. Human rpS5 is 67% identical and 79% similar to Saccharomyces cerevisiae rpS5 but lacks a negatively charged (pI approximately 3.27) 21 amino acid long N-terminal extension that is present in fungi. Here we report that replacement of yeast rpS5 with its human homolog yielded a viable yeast strain with a 20%-25% decrease in growth rate. This replacement also resulted in a moderate increase in the heavy polyribosomal components in the mutant strain, suggesting either translation elongation or termination defects, and in a reduction in the polyribosomal association of the elongation factors eEF3 and eEF1A. In addition, the mutant strain was characterized by moderate increases in +1 and -1 programmed frameshifting and hyperaccurate recognition of the UAA stop codon. The activities of the cricket paralysis virus (CrPV) IRES and two mammalian cellular IRESs (CAT-1 and SNAT-2) were also increased in the mutant strain. Consistently, the rpS5 replacement led to enhanced direct interaction between the CrPV IRES and the mutant yeast ribosomes. Taken together, these data indicate that rpS5 plays an important role in maintaining the accuracy of translation in eukaryotes and suggest that the negatively charged N-terminal extension of yeast rpS5 might affect the ribosomal recruitment of specific mRNAs.     

Identification of cell-binding sites on the Laminin alpha 5 N-terminal domain by site-directed mutagenesis

The newly discovered laminin alpha(5) chain is a multidomain, extracellular matrix protein implicated in various biological functions such as the development of blood vessels and nerves. The N-terminal globular domain of the laminin alpha chains has an important role for biological activities through interactions with cell surface receptors. In this study, we identified residues that are critical for cell binding within the laminin alpha(5) N-terminal globular domain VI (approximately 270 residues) using site-directed mutagenesis and synthetic peptides. A recombinant protein of domain VI and the first four epidermal growth factor-like repeats of domain V, generated in a mammalian expression system, was highly active for HT-1080 cell binding, while a recombinant protein consisting of only the epidermal growth factor-like repeats showed no cell binding. By competition analysis with synthetic peptides for cell binding, we identified two sequences: S2, (123)GQVFHVAYVLIKF(135) and S6, (225)RDFTKATNIRLRFLR(239), within domain VI that inhibited cell binding to domain VI. Alanine substitution mutagenesis indicated that four residues (Tyr(130), Arg(225), Lys(229), and Arg(239)) within these two sequences are crucial for cell binding. Real-time heparin-binding kinetics of the domain VI mutants analyzed by surface plasmon resonance indicated that Arg(239) of S6 was critical for both heparin and cell binding. In addition, cell binding to domain VI was inhibited by heparin/heparan sulfate, which suggests an overlap of cell and heparin-binding sites. Furthermore, inhibition studies using integrin subunit monoclonal antibodies showed that integrin alpha(3)beta(1) was a major receptor for domain VI binding. Our results provide evidence that two sites spaced about 90 residues apart within the laminin alpha(5) chain N-terminal globular domain VI are critical for cell surface receptor binding.     

N-terminal domain, residues 1-91, of ribosomal protein TL5 from Thermus thermophilus binds specifically and strongly to the region of 5S rRNA containing loop E.

In this work we show for the first time that the overproduced N-terminal fragment (residues 1-91) of ribosomal protein TL5 binds specifically to 5S rRNA and that the region of this fragment containing residues 80-91 is a necessity for its RNA-binding activity. The fragment of Escherichia coli 5S rRNA protected by TL5 against RNase A hydrolysis was isolated and sequenced. This 39 nucleotides fragment contains loop E and helices IV and V of 5S rRNA. The isolated RNA fragment forms stable complexes with TL5 and its N-terminal domain. Crystals of TL5 in complex with the RNA fragment diffracting to 2.75 A resolution were obtained.     

Differences in C-terminal amino acid sequences between erythrocyte and liver cytochrome b5 isolated from pig and human. Evidence for two tissue-specific forms of cytochrome b5

Two forms of cytochrome b5, a soluble erythrocyte form and a membrane-bound liver form, were purified from pig and human, and structural differences between them were analyzed. Porcine and human erythrocyte cytochrome b5 consisted of 97 amino acid residues and contained the same catalytic domain structure (residues 1-96) as that of the corresponding liver cytochrome b5, but had one amino acid replacement at the C-terminus (residue 97). These results suggest that erythrocyte cytochrome b5 is not derived from the liver protein by proteolysis but a translational product from another distinct mRNA of cytochrome b5.     

N-terminal residues of plasmatocyte-spreading peptide possess specific determinants required for biological activity.

Plasmatocyte-spreading peptide (PSP) is a 23-amino acid cytokine that activates a class of insect immune cells called plasmatocytes. The tertiary structure of PSP consists of an unstructured N terminus (residues 1-6) and a well structured core (residues 7-23). A prior study indicated that deletion of the N terminus from PSP eliminated all biological activity. Alanine substitution of the first three residues (Glu(1)-Asn(2)-Phe(3)) further indicated that only replacement of Phe(3) resulted in a loss of activity equal to the N-terminal deletion mutant. Here, we characterized structural determinants of the N terminus. Adding a hydroxyl group to the aromatic ring of Phe(3) (making a Tyr) greatly reduced activity, whereas the addition of a fluorine (p-fluoro) did not. Substitutions that changed the chirality or replaced the aromatic ring of Phe(3) with a branched aliphatic chain (making a Val) also greatly decreased activity. The addition of a methylene group to Val (making a Leu) partially restored activity, whereas the removal of a methylene group from Phe (phenyl-Gly) eliminated all activity. These results indicated that a branched carbon chain with a methylene spacer at the third residue is the minimal structural motif required for activity. The deletion of Glu(1) also eliminated activity. Additional experiments identified the charged N-terminal amine and backbone of Glu(1) as key determinants for activity.     

1Hepatitis C virus NS5A protein modulates c-Jun N-terminal kinase through interaction with tumor necrosis factor receptor-associated factor 2

The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) is a phosphoprotein possessing various functions. We have previously reported that the HCV NS5A protein interacts with tumor necrosis factor (TNF) receptor-associated factor (TRAF) domain of TRAF2 (Park, K.-J., Choi, S.-H., Lee, S. Y., Hwang, S. B., and Lai, M. M. C. (2002) J. Biol. Chem. 277, 13122-13128). Both TNF-alpha- and TRAF2-mediated nuclear factor-kappaB (NF-kappaB) activations were inhibited by NS5A-TRAF2 interaction. Because TRAF2 is required for the activation of both NF-kappaB and c-Jun N-terminal kinase (JNK), we investigated HCV NS5A protein for its potential capacity to modulate TRAF2-mediated JNK activity. Using in vitro kinase assay, we have found that NS5A protein synergistically activated both TNF-alpha- and TRAF2-mediated JNK in human embryonic kidney 293T cells. Furthermore, synergism of NS5A-mediated JNK activation was inhibited by dominant-negative form of MEK kinase 1. Our in vivo binding data show that NS5A does not inhibit interaction between TNF receptor-associated death domain and TRAF2 protein, indicating that NS5A and TRAF2 may form a ternary complex with TNF receptor-associated death domain. These results indicate that HCV NS5A protein modulates TNF signaling of the host cells and may play a role in HCV pathogenesis.     

Ribonuclease inhibitor from human placenta. Purification and properties

A soluble ribonuclease inhibitor from the human placenta has been purified 4000-fold by a combination of ion exchange and affinity chromatography. The inhibitor has been isolated in 45% yield (about 2 mg/placenta) as a protein that is homogeneous by sodium dodecyl sulfate-gel electrophoresis. In common with the inhibitors of pancreatic ribonuclease from other tissues that have been studied earlier, the placental inhibitor is an acidic protein of molecular weight near 50,000; it forms a 1:1 complex with bovine pancreatic RNase A and is a noncompetitive inhibitor of the pancreatic enzyme, with a Ki of 3 X 10(-10) M. The amino acid composition of the protein has been determined. The protein contains 30 half-cystine plus cysteine residues determined as cysteic acid after performic acid oxidation. At pH 8.6 the nondenatured protein alkylated with iodoacetic acid in the presence of free thiol has 8 free sulfhydryl groups. The inhibitor is irreversibly inactivated by sulfhydryl reagents and also by removal of free thiol from solutions of the protein. Inactivation by sulfhydryl reagents causes the dissociation of the RNase - inhibitor complex into active RNase and inactive inhibitor.     

A novel fibroblast growth factor receptor-5 preferentially expressed in the pancreas(1)

Using the polymerase chain reaction on human embryonic cDNAs, we isolated a cDNA encoding a novel 504 amino acid protein, termed fibroblast growth factor receptor (FGFR)-5, which is highly homologous to known FGFRs. The NH(2)-terminal portion of FGFR5 contains a putative secretory signal sequence, three typical immunoglobulin-like domains, six cysteines, and an acidic box, but no HAV motif. The COOH-terminal portion of FGFR5 contains one transmembrane domain but no intracellular kinase domain. Recombinant FGFR5 expressed in COS-7 cells is not secreted, but recombinant truncated FGFR5 lacking the predicted transmembrane domain is secreted. Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) do not bind to FGFR5. Among 23 adult human tissues, FGFR5 mRNA is preferentially expressed in the pancreas. These results suggest that FGFR5 may provide a binding site for some other fibroblast growth factors and may regulate some pancreatic function.     

Growth stimulatory effect of pancreatic spasmolytic polypeptide on cultured colon and breast tumor cells

The effects of a novel polypeptide, pancreatic spasmolytic polypeptide (PSP) on a colon carcinoma cell line (HCT 116) were examined. PSP stimulated the incorporation of [3H]thymidine into HCT 116 cells as well as cell proliferation in a dose-dependent manner. Maximal increase in [3H]thymidine incorporation of 50-60% occurred at 3-300 microM PSP. The VIP-mediated-increase in cAMP levels was reduced by PSP at greater than 1 microM concentrations. PSP is highly homologous to the estrogen-induced pS2 protein in MCF-7 breast cancer cells. We find that PSP also enhanced [3H]thymidine incorporation in MCF-7 cells. These findings indicate for the first time that PSP has growth stimulatory properties.     

Secretin N-terminal hexapeptide potentiates insulin release in mouse islets

Peptides representing the N-terminal part of secretin were synthesized and their effects tested on column-perifused isolated mouse pancreatic islets. Insulin release induced by D-glucose was potentiated by the two peptides His-Ser-Asp-Gly-Thr-Phe-OMe (S1-6) and Ser-Asp-Gly-Thr-Phe-OMe (S2-6). The consecutive smaller N-terminal peptides Asp-Gly-Thr-Phe-OMe (S3-6) and Gly-Thr-Phe-OMe (S4-6) had no effects while the dipeptide ester Thr-Phe-OMe (S5-6) also potentiated the release of insulin. The results suggest that the N-terminal part of secretin may be involved in the marked in vitro glucose-dependent insulin release induced by secretin.