Identification of a receptor for reg (regenerating gene) protein, a pancreatic beta-cell regeneration factor

Reg (regenerating gene) was isolated as a gene specifically expressed in regenerating islets (Terazono, K., Yamamoto, H., Takasawa, S., Shiga, K., Yonemura, Y., Tochino, Y., and Okamoto, H. (1988) J. Biol. Chem. 263, 2111-2114). Rat and human Reg gene products, Reg/REG proteins, have been demonstrated to stimulate islet beta-cell growth in vitro and in vivo and to ameliorate experimental diabetes. In the present study, we isolated a cDNA for the Reg protein receptor from a rat islet cDNA library. The cDNA encoded a cell surface 919-amino acid protein, and the cells into which the cDNA had been introduced bound Reg protein with high affinity. When the cDNA was introduced into RINm5F cells, a pancreatic beta-cell line that shows Reg-dependent growth, the transformants exhibited significant increases in the incorporation of 5'-bromo-2'-deoxyuridine as well as in the cell numbers in response to Reg protein. A homology search revealed that the cDNA is a homologue to a human multiple exostoses-like gene, the function of which has hitherto been unknown. These results strongly suggest that the receptor is encoded by the exostoses-like gene and mediates a growth signal of Reg protein for beta-cell regeneration.     

Hepatic lipase: a member of a family of structurally related lipases

Partial amino acid sequence of rat hepatic lipase was obtained by gas-phase microsequence analysis of proteolytic fragments. Sequence comparison to bovine lipoprotein lipase and porcine pancreatic lipase reveals a highly conserved region existing among these three physiologically distinct lipolytic enzymes. In a stretch of 36 amino acid residues previously reported for pancreatic lipase (De Caro, J., Boudouard, M., Bonicel, J., Guidoni, A., Desnuelle, P. and Rovery, M. (1981) Biochim. Biophys. Acta 671, 129-138), nineteen residues are identical for all three enzymes, whereas 27 of 36 are identical in rat hepatic lipase and bovine lipoprotein lipase. The fact that this primary structural conservation extends to three different animal species emphasizes the conclusion that these lipolytic enzymes comprise a protein family originating from a common ancestral gene.     

Cloning and characterization of human pancreatic lipase cDNA

Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) hydrolyzes dietary long chain triacylglycerol to free fatty acids and monoacylglycerols in the intestinal lumen. In the presence of bile acids, the activity of lipase is stimulated by colipase. As a prelude to studying the relationship of the protein structures to the functional properties of lipase and colipase, a cDNA encoding human pancreatic lipase was isolated from a lambda gt11 cDNA library screened with a rabbit polyclonal anti-human pancreatic lipase antibody. The full length cDNA clone of 1477 base pairs contained an open reading frame encoding a 465-amino acid protein, including a 16-amino acid signal peptide. The nucleotide sequence was 69% identical to the dog pancreatic lipase cDNA. The predicted NH2-terminal protein sequence agreed with the published NH2-terminal sequence of human pancreatic lipase and the predicted protein sequence was 85 and 70% identical to the protein sequences of pig and dog pancreatic lipase, respectively. A region of homology around Ser-153 is conserved in a number of lipid-binding proteins. Human hepatic lipase and lipoprotein lipase share extensive homology with pancreatic lipase, suggesting that the three proteins are members of a small gene family. In vitro translation of mRNA transcribed from the cDNA resulted in a protein of the expected molecular size that could be processed by microsomal membranes to yield a glycolated protein with proper signal peptide cleavage. RNA blot analysis demonstrated tissue specificity for pancreatic lipase. Thus, for the first time, a full length human pancreatic lipase cDNA has been isolated and characterized. The demonstrated regions of homology with other lipases will aid definition of interactions with substrate and colipase through site-specific mutagenesis.     

Expression of reg/PSP, a pancreatic exocrine gene: relationship to changes in islet beta-cell mass

A cDNA termed reg was recently isolated by differential screening of a library prepared from regenerating islets isolated from pancreatic remnants of rats subjected to 90% pancreatectomy and nicotinamide treatment. This led to speculation that this gene may be involved in expansion of beta-cell mass. In the current study we have measured reg expression after implantation and resection of a solid insulinoma tumor into rats, maneuvers known, respectively, to reduce and reexpand the volume of beta-cells in the islet. Animals with an implanted insulinoma tumor became profoundly hypoglycemic. Islet beta-cells declined from the normal 75% of total islet volume to less than 30%, in concert with a marked reduction in the reg mRNA level. Removal of the tumor resulted in a sharp increase in beta-cell replication, as measured by [3H]thymidine incorporation and a return to normal beta-cell volume within 4 days of tumor resection. This was associated with a transient induction in reg expression compared to that in tumor-bearing animals, effectively returning the amount of reg mRNA to the levels found in normal animals within 48 h; at later time points after tumor removal (3-7 days) reg expression declined, but then rose toward normal. In situ hybridization analysis localized the initial induction in reg mRNA expression to the exocrine pancreas. Continuous infusion of insulin into normal rats for 4 days, a maneuver that does not significantly reduce beta-cell mass, resulted in dramatically reduced insulin mRNA in islets, but no change in the levels of reg mRNA. We conclude that the diminution in pancreatic beta-cell mass caused by subcutaneous implantation of an insulinoma is associated with reduced reg gene expression and that the increase in beta-cell replication after resection of the tumor is preceded by return of reg gene expression toward normal.     

Preferential expression of reg I beta gene in human adult pancreas

In human pancreas two genes, reg I alpha and reg I beta, have been characterized but only the reg I alpha protein has been isolated from human pancreatic secretion. To examine their respective physiological roles in fetal and adult pancreas we have compared the patterns of gene expression using a specific RT-PCR method. No progressive evolution in the two mRNAs levels was observed during fetal development (16--41 weeks). A discoordinate expression of the two genes was found with a higher level of reg I alpha mRNA in fetus and a higher level of regI beta in adult. In addition, if reg I alpha mRNA level was correlated with the expression of genes encoding exocrine proteins in adults, reg I beta mRNA level presented no correlation with any ductular, endocrine, or exocrine gene expression. In human pancreatic cell lines we showed the only expression of reg I beta gene and protein. All these data suggest that the two reg genes and proteins could play different roles in the pancreas.     

A novel gene activated in regenerating islets

Administration of poly(ADP-ribose) synthetase inhibitors such as nicotinamide to 90% depancreatized rats induces regeneration of pancreatic islets, thereby ameliorating the surgical diabetes (Yonemura, Y., Takashima, T., Miwa, K., Miyazaki, I., Yamamoto, H., and Okamoto, H. (1984) Diabetes 33, 401-404). In screening the regenerating islet-derived cDNA library, we came across a novel gene encoding a 165-amino acid protein. The gene was expressed in regenerating islets but not in normal pancreatic islets, insulinomas, or regenerating liver. In 90% depancreatized and nicotinamide-injected rats, the expression of the gene was increased 1 month after the partial pancreatectomy and reached a peak 3 months after the operation. The increase in expression of the gene was temporally correlated with the increase in size of regenerating islets and the decrease in urinary glucose level. The gene was also found to be activated in hyperplastic islets of aurothioglucose-treated mice. Thus, the expression of the gene in both regenerating and hyperplastic islets suggests possible roles for this gene in replication, growth, and maturation of islet beta-cells. We also found that a human pancreas-derived cDNA library contained a homologue to the gene.     

Isolation of a cDNA encoding a human serum marker for acute pancreatitis. Identification of pancreas-specific protein as pancreatic procarboxypeptidase B.

A human pancreas-specific protein (PASP), previously characterized as a serum marker for acute pancreatitis and pancreatic graft rejection, has been identified as pancreatic procarboxypeptidase B (PCPB). cDNAs encoding PASP/PCPB were isolated from a human pancreas cDNA library using a combination of nucleic acid hybridization screening and immunoscreening with antisera raised against native PASP. The deduced amino acid sequence of PASP/PCPB cDNA predicts the translation of a 416-amino acid preproenzyme with a 15-amino acid signal/leader peptide and a 95-amino acid activation peptide. The proenzyme portion of this protein has 76% identity with rat PCPB and 84% identity with bovine carboxypeptidase B. DNA and RNA blot analyses indicate that human PCPB mRNA (1,400 nucleotides) is transcribed from a single locus in the human genome in a tissue-specific fashion. N-terminal sequencing of native PASP and the specific immunoreactivity of bacterially expressed PASP/PCPB with native PASP antibodies confirm the identification of PASP as human pancreatic PCPB.     

Novel organization and processing of the guinea pig pancreatic polypeptide precursor

Studies on New World hystricomorph rodents have revealed interesting structural divergences in the peptide hormones of the islets of Langerhans, particularly with respect to insulin and glucagon. Herein we report the isolation and sequencing of a cDNA encoding the precursor of pancreatic polypeptide (PP) from a guinea pig pancreas cDNA library. The 126-residue precursor sequence is predicted to include a 26-residue NH2-terminal signal peptide followed by the 36-amino acid PP hormonal sequence and a large COOH-terminal extension. The sequence identity between guinea pig and human PP is 89% (32/36 residues), and the predicted sequence is in agreement with that reported by Eng et al. (Eng, J., Huang, C.-G., Pan, Y.-C. E., Hulmes, J. D., and Yalow, R. S. (1987) Peptides 8, 165-168). In contrast, the icosapeptide domain in the guinea pig precursor exhibits only 40% (8/20) identity with the corresponding human precursor domain, and the COOH-terminal extension differs greatly in both sequence and size. The guinea pig precursor lacks the monobasic processing site (Pro-Arg) found at the COOH terminus of the icosapeptide domain in human, ovine, canine, and feline proPP. An icosapeptide is thus not likely to be liberated as such from this precursor. Of particular interest in guinea pig proPP is the substitution of serine for arginine at the dibasic amino acid processing site on the COOH-terminal side of the PP domain. Results of radioimmunoassays of gel-filtered protein fractions from a guinea pig pancreas extract indicate that efficient proteolytic cleavage takes place at this Lys-Ser site and that mature guinea pig PP is normally carboxyamidated.     

Exons of the human pancreatic polypeptide gene define functional domains of the precursor

Pancreatic polypeptide is a 36-amino acid peptide which inhibits pancreatic exocrine function. We have previously determined from the nucleotide sequence of a cDNA that pancreatic polypeptide is derived from a 95-amino acid precursor, prepropancreatic polypeptide. Pulse-chase studies have suggested that the precursor is cleaved to produce three peptides: pancreatic polypeptide, an icosapeptide, and a smaller peptide. In the present study, we have used the cloned cDNA as a hybridization probe to isolate the pancreatic polypeptide gene from a human bacteriophage genomic library. The nucleotide sequence of 2.8 kilobases of DNA representing the entire human pancreatic polypeptide gene was determined. The gene contains four exons and three introns. Exon 1 encodes the 5'-untranslated region of the mRNA, exon 2 encodes the signal sequence and the sequence of pancreatic polypeptide, exon 3 encodes the icosapeptide, and exon 4 encodes a carboxyl-terminal heptapeptide and the 3'-untranslated region of the mRNA. By Southern blot analysis, the gene detected in a pancreatic polypeptide-producing islet cell tumor was indistinguishable from that in normal human leukocytes. The structure of the human pancreatic polypeptide gene is consistent with the hypothesis that prepropancreatic polypeptide generates three distinct peptides, each encoded by a separate exon. Increased expression of pancreatic polypeptide in the islet cell tumor does not appear to be correlated with major alterations in pancreatic polypeptide gene structure.     

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.     

Structural analysis of bovine pancreatic thread protein

Pancreatic thread protein (PTP) forms double helical threads in the neutralpH range after purification, undergoing freely reversible,pH-dependent globule-fibril transformation. The purified bovine PTP consists on SDS gels of two carbohydrate-free polypeptide chains (Grosset al., 1985). Plasma desorption mass spectrometry and amino acid sequence analysis now confirm that bovine PTP contains two disulfide-bonded polypeptides, an A chain of 101 amino acid residues with a molecular weight of 11,073 and a B chain of 35 residues with a molecular weight of 3970. The intact protein exhibits a molecular weight of 15,036, agreeing >99.9% with the molecular weight calculated from the sequence. The B chain sequence was determined by gas-phase Edman degradation of the intact polypeptide. The A chain sequence was determined from overlapping peptides generated by cleavage at lysyl, tryptophanyl, and aspartyl-prolyl residues. Based upon the bovine PTP cDNA structure, the two chains of the protein result from cleavage of a single polypeptide with removal of a dipeptide between the NH₂-terminal A chain and COOH-terminal B chain. Comparison of bovine PTP with other proteins reveals significant structural relatedness with the single-chain homologues from human and rat pancreas and with the motif associated with Ca²⁺-dependent carbohydrate recognition domains. The physiological role of PTP has not yet been resolved. The protein is present in very high concentration in pancreatic secretion and it has been detected in brain lesions in Alzheimer's disease and Down syndrome and in regenerating rat pancreatic islets. The present results provide a firm protein base for ongoing molecular, physical-chemical, and structure-function studies of this unusual protein.     

Murine polypyrimidine tract binding protein. Purification, cloning, and mapping of the RNA binding domain.

A complex of nucleic acid binding proteins (100, 35, and 25 kDa) was purified to apparent homogeneity from nuclear extracts of the murine plasmacytoma J558L. Amino-terminal sequence analysis of the 25-kDa subunit enabled the isolation of a cDNA that encodes a 528-amino acid protein that is highly homologous to the human 62-kDa human polypyrimidine tract binding protein (PTB) (Garcia-Blanco, M. A., Jamison, S. F., and Sharp, P. A. (1989) Genes & Dev. 3, 1874-1886; Gil, A., Sharp, P. A., Jamison, S. F., and Garcia-Blanco, M. A. (1991) Genes & Dev. 5, 1224-1236; Patton, J. G., Mayer, S. A., Tempst, P., and Nadal-Ginard, B. (1991) Genes & Dev. 5, 1237-1251). Sequence comparison programs suggested the presence of domains related to the RNA recognition motif found in other RNA-binding proteins, and deletion analysis revealed that the carboxyl-terminal 195 amino acids of the recombinant PTB was sufficient for specific binding to pre-mRNAs. Cross-linking experiments identified a 25-kDa protein in crude nuclear extracts of J558L cells that possessed the RNA binding properties of PTB, while a approximately 60-kDa protein is detected in other murine cell lines tested. Thus, the 25-kDa protein found in J558L is likely a proteolytic product of the murine polypyrimidine tract binding protein. A probe derived from the PTB cDNA detected a ubiquitous 3.3-kb mRNA in murine cell lines and a 3.6-kb mRNA in human lines. Southern blot analysis revealed three strongly hybridizing DNA fragments and several more weakly hybridizing bands in mouse, human, and yeast DNA. The role of PTB in pre-mRNA splicing is discussed.     

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.     

Structural analysis of the sequence coding for an inducible 26-kDa protein in human fibroblasts

When human fibroblast cells were stimulated with poly(I) X poly(C) in the presence of cycloheximide for the production of interferon-beta (IFN-beta), a 26-kDa protein could be immunoprecipitated by antiserum raised against partially purified human IFN-beta [Content, J., De Wit, L., Pierard, D., Derynck, R., De Clercq, E. & Fiers, W. (1982) Proc. Natl Acad. Sci. USA 79, 2768-2772]. In our hands this 26-kDa protein showed no antiviral activity. Other investigators have, however, reported the presence in the same conditions of a second type of IFN, a so-called beta 2 species [Weissenbach, J., Chernajovsky, Y., Zeevi, M., Shulman, L., Soreq, H., Nir, U., Wallach, D., Perricaudet, M., Tiollais, P. & Revel, M. (1980) Proc. Natl Acad. Sci. USA 77, 7152-7156] of which the mRNA structure and protein characteristics strongly suggests identity with the 26-kDa product. In this paper we describe the nucleotide sequence of the 26-kDa cDNA and part of the corresponding genomic clone. The cDNA clones were isolated from a library made with mRNA from induced human fibroblasts. As, however, the information thus obtained was still incomplete, genomic clones were isolated from a total human DNA library. In this way, the entire region coding for the 26-kDa protein was established, as well as the neighbouring sequences including the inducible promoter area. From the deduced polypeptide sequence a number of characteristics of the 26-kDa protein can be explained. It turns out that the 26-kDa protein gene and the so-called 'IFN-beta 2' gene are identical. However, extensive homology searches indicate that the 26-kDa protein does not show statistically significant sequence homology with any known interferon species. Hence, the question of whether the 26-kDa product represents a novel IFN species remains open.     

Cloning of the cDNAs encoding the cellular retinaldehyde-binding protein from bovine and human retina and comparison of the protein structures

A 1173-base pair cDNA encoding bovine cellular retinaldehyde-binding protein (CRALBP) was cloned from a bovine retinal cDNA expression library using as probes both anti-CRALBP polyclonal and monoclonal antibodies. The amino acid sequence deduced from the cDNA corresponds exactly to that determined by direct analysis of NH2-terminally acetylated bovine CRALBP (Crabb, J. W., Johnson, C. M., Carr, S. A., Armes, L. G., and Saari, J. C. (1988) J. Biol. Chem. 263, 18678-18687). Nick-translated bovine CRALBP cDNA probes were then used to clone from a human retinal cDNA library a 1317-base pair cDNA encoding human CRALBP. Bovine and human CRALBP are 92% identical in amino acid sequence and not related to any other known protein sequence. Both the bovine and human proteins contain 316 residues and have calculated molecular weights of 36,378 and 36,347, respectively, exclusive of the NH2-terminal blocking groups. The CRALBP cDNA clones should prove valuable as tools for studying the physiological role of the protein in vision and visual disorders.     

Novel multigene families encoding highly repetitive peptide sequences. Sequence analyses of rat and mouse proline-rich protein cDNAs

Multigene families encode the proline-rich proteins that are so prominent in human saliva and are dramatically induced in mouse and rat salivary glands by isoproterenol treatment and by feeding tannins. A cDNA encoding an acidic proline-rich protein of rat has been sequenced (Ziemer, M. A., Swain, W. F., Rutter, W. J., Clements, S., Ann, D. K., and Carlson D. M. (1984) J. Biol. Chem. 259, 10475-10480). This study presents the nucleotide sequences of five additional proline-rich protein cDNAs complementary to both mouse and rat parotid and submandibular gland mRNAs. Amino acid compositions deduced from the nucleotide sequences are typical for proline-rich proteins: 25-45% proline, 18-22% glycine, and 18-22% glutamine and generally an absence of sulfur-containing amino acids except for the initiator methionine. These proline-rich proteins display unusual repeating peptide sequences of 14-19 amino acids. The derived amino acid sequence of the cDNA insert of plasmid pMP1 from mouse has a 19-amino acid sequence which is repeated four times. The inserts of plasmids pUMP40 and pUMP4 also from mouse encode for 12 and 11 repeats of a 14-amino acid peptide, respectively. These repetitive sequences, and others from rat and mouse cDNAs and from human genomic clones, all show very high homologies and likely evolved from duplication of internal portions of an ancestral gene. Gene conversion could account for the high degree of conservation of nucleotide sequences of the repeat regions. Protein derived from the nucleotide sequences are all characterized by four general regions: a putative signal peptide, a transition region, the repetitive region, and a carboxyl-terminal region. The 5'-flanking sequences and sequences encoding the putative signal peptides are highly conserved (greater than 94%) in all six cDNAs. This sequence conservation may be important in the regulation of the biosynthesis of these unusual proteins.     

Kirrel2, a novel immunoglobulin superfamily gene expressed primarily in beta cells of the pancreatic islets

A novel immunoglobulin superfamily (Igsf) protein gene was discovered by computational analysis of human draft genomic DNA, and multiple cDNA clones were obtained. The protein encoded by this gene contains five Ig domains, one transmembrane domain, and an intracellular domain. It has significant similarity with several known Igsf proteins, including Drosophila RST (irregular chiasm C-roughest) protein and mammalian KIRREL (kin of irregular chiasm C-roughest), NEPH1, and NPHS1 (nephrin) proteins. All these proteins have multiple Ig domains, possess properties of cell adhesion molecules, and play important roles in organ development. RT-PCR and Northern blot results indicate this gene is predominantly expressed in pancreas, and public sequence databases indicate there is also expression in the nervous system. We have named this gene Kirrel2 (kin of irregular chiasm-like 2), to reflect its similarity to irregular chiasm C-roughest and Kirrel. Four splice forms of Kirrel2 were observed, including two that we cloned from pancreas mRNA as well as two GenBank entries, one from the brain and one from a retinoblastoma cell line. A partial cDNA clone of the mouse orthologue was obtained by RT-PCR from mouse brain, and the inferred protein sequence has 85% sequence identity to the human protein. Immunohistochemical staining results indicate that the KIRREL2 protein is conserved from rodents to primates, and it is highly expressed in pancreatic islets. RT-PCR results on mouse pancreatic cell lines indicate that expression in the pancreas is restricted to beta cells. Thus, KIRREL2 protein is a beta-cell-expressed Ig domain protein and may be involved in pancreas development or beta cell function.