Structure of a precursor to human pancreatic polypeptide

We have isolated mRNA from a human pancreatic islet cell tumor and have identified among the cell-free translation products a precursor of pancreatic polypeptide with an approximate Mr = 11,000. Recombinant DNA molecules encoding this precursor were selected from a cDNA library prepared from the islet tumor mRNA. From the nucleotide sequences of cDNAs encoding the precursor, we have deduced the complete amino acid sequence of pre-propancreatic polypeptide. These sequences encode a protein consisting of 95 amino acid residues with a Mr = 10,432. The sequence of human pancreatic polypeptide occurs in the middle of the precursor and is flanked at its carboxyl terminus by a 27-amino acid sequence which is similar to a peptide previously isolated from canine pancreatic islets. At the amino terminus of the precursor is a probable leader sequence which is rich in hydrophobic residues. A smaller pancreatic polypeptide-related protein was generated in cell-free translations of mRNA supplemented with microsomal membranes. Sequential Edman degradations of this smaller peptide indicate that the sequence of pancreatic polypeptide is located at the amino terminus of the prohormone.     

Purification and characterization of epidermal growth factor (beta-urogastrone) and epidermal growth factor fragments from large volumes of human urine

We purified human epidermal growth factor (hEGF) and hEGF fragments from a benzoic acid precipitate of the materials not adsorbed to silica gel in 330 liters of human urine by a relatively brief, simple, and efficient method employing sequential batch absorption to and stepwise elution from CM-cellulose and DEAE-cellulose, Bio-Gel P-10 chromatography in 50 mM HCl, and three reverse-phase HPLC steps for final resolution and purification of hEGF components. Recovery of hEGF was 29%. Eight apparently homogeneous hEGF components were recovered, each of which had similar activities in a homologous hEGF radioimmunoassay and an EGF radioreceptor assay using human placental membranes. Amino acid composition analysis indicated that there were four pairs of components that represented intact 53-amino acid hEGF, hEGF-(1-52), hEGF-(1-51), and hEGF-(1-50); intact hEGF accounted for one-third of the total materials recovered. Automated Edman degradation of each component for at least 10 cycles revealed a single amino acid sequence identical to that proposed for human beta-urogastrone. Similar immunoreactive hEGF components were observed in similar proportions in freshly voided urine, indicating that they were not artifacts of the purification process. Thus, multiple forms of fully biologically active hEGF (i.e., beta-urogastrone) can be relatively easily and efficiently purified from large volumes of human urine.     

Isolation and characterization of the rat proenkephalin gene

The rat proenkephalin gene has been isolated by molecular cloning and characterized by DNA-sequence analysis. The gene exhibits a structural organization similar to that of the human gene. The nucleotide sequence encoding the biologically active opioid peptides which are generated from the proenkephalin precursor as well as the 3' untranslated region of the mRNA are found on a large exon at the 3' end of the gene (Exon III). The nucleotide sequence encoding the N terminus of the mature protein and its signal peptide are located on Exon II while Exon I encodes the 5' untranslated region of the mRNA. The nucleotide sequence of these exons and their flanking regions has been determined and compared to the human proenkephalin gene. Analysis of the nucleotide sequence homology between the human and rat proenkephalin gene reveals the presence of highly conserved regions within both the coding and noncoding portions of the genes. Enkephalin-coding sequences as well as 5' flanking sequences appear to be the most highly conserved. The importance and possible function of these sequences are discussed.     

Isolation and characterization of a cDNA coding for human myeloperoxidase

A cDNA encoding the carboxyl-terminal fragment of the human myeloperoxidase heavy chain was isolated and characterized. It was then used to determine the locations of the myeloperoxidase light and heavy chains in the polypeptide precursor. A cDNA library from poly(A)+ RNA from human leukemia HL-60 cells was constructed in pBR322 and screened by differential hybridization with enriched and depleted cDNA probes and then by hybridization with an oligonucleotide probe. A cDNA clone containing 1278 bp with an open reading frame of 474 bp and a 3' noncoding region of 804 bp was isolated. The amino acid sequence deduced from the nucleotide sequence consisted of 158 residues including a sequence of 14 amino acids known to be present in the heavy chain of the molecule. The cDNA also included a stop codon of TAG followed by a noncoding sequence that included a potential recognition site for polyadenylylation and a poly(A) tail. RNA transfer blot analysis with the cDNA probe indicated that myeloperoxidase mRNA was approximately 3.3 kb in length. In vitro translation of the mRNA selected by cDNA hybridization revealed preferential synthesis of a 74,000-Da polypeptide precursor that could be precipitated with anti-myeloperoxidase IgG. Antibodies specific for the heavy and light chains of myeloperoxidase were isolated from antiserum by affinity chromatography employing Sepharose columns covalently bound to the heavy or light chains. Antibodies specific for the light chain or the heavy chain readily precipitated the 74,000-Da precursor polypeptide. These results indicated that myeloperoxidase is synthesized as a single chain which undergoes processing into a light and heavy chain. Furthermore, the heavy chain of myeloperoxidase originates from the carboxyl terminus of the precursor polypeptide.     

Eight small subunits of Euglena ribulose 1-5 bisphosphate carboxylase/oxygenase are translated from a large mRNA as a polyprotein.

The small subunit (SSU) of ribulose 1-5 bisphosphate carboxylase/oxygenase is a 15 kd protein in Euglena gracilis. The protein is synthesized as a 130 kd precursor as shown by immunoprecipitation of in vitro translation products and confirmed by immunoprecipitation of in vivo pulse-labeled Euglena proteins. From the published SSU amino acid sequence, an oligonucleotide was synthesized that specifically hybridizes to a large mRNA whose length (approximately 4.3 kb) is consistent with the precursor size. The complete nucleotide sequence of the SSU mRNA was obtained by sequencing a cDNA clone from a lambda gt11 library and completed by direct mRNA sequencing. We report for the first time the complete sequence of a large mRNA and show that it encodes eight consecutive SSU mature molecules. The deduced precursor amino acid sequence shows that the amino terminus of the first SSU molecule is preceded by a 134 amino acid peptide which is cleaved during the maturation process. This long transit peptide exhibits features characteristic of signal peptides involved in the secretion of proteins through the endoplasmic reticulum. This is in agreement with the idea that the third (outer) membrane of the Euglena chloroplast envelope is of endoplasmic reticulum origin.     

Synthesis and secretion of an epidermal growth factor (EGF) by human fibroblast cells in culture

Human fibroblast (WS-1) cells in culture synthesized and secreted an epidermal growth factor which cross-reacted with human epidermal growth factor (hEGF) purified from human urine. hEGF secreted by the cells (designated as WS-1 EGF or fibroblast EGF) and hEGF isolated from urine (designated as urine EGF) were immunologically indistinguishable. The molecular weight of fibroblast EGF estimated by gel filtration was identical with that of hEGF from urine. On chromatofocusing chromatography, fibroblast EGF was eluted mainly at pH 4.26 as a sharp symmetric peak with a minor peak at pH 4.62, like urine EGF. These results suggested that EGF synthesized and secreted by human fibroblast cells is an identical molecule to that of hEGF in human urine.     

The 120 kilodalton outer membrane protein (rOmp B) of Rickettsia rickettsii is encoded by an unusually long open reading frame: evidence for protein processing from a large precursor

A Rickettsia rickettsii outer surface membrane protein (rOmp B), of an apparent molecular mass of 120 kilodaltons, is a major surface antigen of the Rickettsiae that displays genus, species, and sub-species specific antigenic determinants. The 5' portion of this gene was found to be unstable in plasmids, but was stably cloned in a lambda vector. The nucleotide sequence of the 5' terminus has been determined, thus completing the DNA sequence of the entire gene. Genetic analysis revealed an unusually large open reading frame with the capacity to encode a product much larger than the mature protein. A 32 kilodalton peptide from purified rickettsiae was isolated and the amino terminus was sequenced, which revealed that the peptide is encoded by the 3' portion of this large open reading frame. This suggests a role for post-translational processing of rOmp B from a large precursor molecule.     

Monoclonal antibodies specific for high molecular weight form of human epidermal growth factor

Hybridomas that secrete monoclonal antibodies specific for the high molecular weight (HMW) form of human epidermal growth factor (hEGF) were established by fusing spleen cells obtained from mice immunized with purified urinary HMW-hEGF with myeloma P3 x 63Ag8.653. The resulting monoclonal antibodies were characterized basically into two groups. One group recognized both EGF and HMW-hEGF, while the other recognized HMW-hEGF specifically on radio immunoprecipitation. Surprisingly, the majority of the isolates was positive by western blotting. Utilizing these monoclonal antibodies for affinity chromatography, we purified HMW-hEGF successfully from urine. These antibodies may be an extraordinarily powerful tool for histological study related to both forms of EGF.     

Cell-free translation of purified virion-associated high-molecular-weight RNA synthesized in vitro by vaccinia virus.

Virion-associated high-molecular-weight (HMW) RNA synthesized in vitro by purified vaccinia virus particles has been translated in a wheat germ cell-free protein synthesizing system. Purified HMW RNA directs the synthesis of translation products which are identical to the translation products made in response to in vitro-synthesized, virion-released 8 to 12S mRNA. The translation of HMW RNA proceeds exclusively through a 5'-terminal cap-mediated initiation step. Furthermore, only one coding sequence is translated per HMW RNA molecule, and that sequence is probably located near the 5' end of the molecule. These conclusions are based on the following results. (i) Sodium dodecyl sulfate--polyacrylamide gel electrophoresis patterns of translation products synthesized in response to HMW RNA and in response to 8 to 12S mRNA were qualitatively identical. (ii) On an equal weight basis, HMW RNA was 25 to 30% as active as 8 to 12S mRNA in stimulating in vitro protein synthesis. (iii) Unmethylated HMW RNA was translated at 10% the efficiency of the methylated form of this RNA. (iv) m7pG inhibited the translation of fully methylated HMW RNA by 90%. (v) After the initiation step of translation was blocked by aurintricarboxylic acid, the rate with which amino acids were incorporated into individual polypeptides decreased in a similar manner for the translation of both HMW RNA and 8 to 12S mRNA. Virion-released 8 to 12S mRNA derived from virion-associated HMW RNA during a chase in the presence of ATP, GTP, and S-adenosylmethionine was also translated. At low RNA concentrations, the derived RNA appeared to stimulate amino acid incorporation more efficiently than the HMW RNA precursor. However, at higher concentrations of this RNA, protein synthesis was severely inhibited.     

Assessment by a two-site enzyme immunoassay of human epidermal growth factor (urogastrone) in the urine of patients with various gastrointestinal diseases including malignant tumors

By using our two-site enzyme immunoassay (EIA) system, the levels of human epidermal growth factor (hEGF) in the urine of patients with various gastrointestinal diseases including malignant tumors were measured. Urinary excretion of hEGF in patients having undergone gastric resection, expressed as a function of creatinine, was found to be somewhat decreased. While the levels of hEGF in patients with gastric cancer were significantly increased. Then, the molecular features of hEGF in the urine of patients with gastric cancer were examined by gel filtration. The elution profile demonstrated that high molecular weight components, which immunologically cross-reacted with hEGF, were considerably increased. On the other hand, the level of normal EGF with a molecular weight of 6500 was decreased to some extent. These results suggest that processing of the EGF precursor into an active EGF molecule is partially suppressed in patients with gastric cancer.     

Molecular nature of human epidermal growth factor (hEGF)-like immunoreactivity in human plasma

To investigate the molecular features of hEGF in the circulatory system, we analyzed hEGF-like immunoreactivity (hEGF-LI) in human serum and plasma by our two-site enzyme immunoassay (EIA), using an aliquot of each fraction obtained by gel filtration of human serum or plasma on Sephadex G-100. The results demonstrated that the majority of the hEGF-LI in the plasma (HMW X hEGF-LI) emerged in the void volume, while a small amount of hEGF-LI (LMW X hEGF-LI) eluted at a position similar with that of standard hEGF. After reduction of HMW X hEGF-LI with 2-mercaptoethanol, hEGF-LI emerged at the same elution position as that of standard hEGF, suggesting that the predominant form of hEGF may circulate as a complex with some macromolecule(s) in human blood. The biochemical properties of the plasma component(s) with high molecular weight which form the complex with hEGF and the physiological significance of this circulating complex still remain to be clarified.     

Mouse protamine 2 is synthesized as a precursor whereas mouse protamine 1 is not.

The nuclei of mouse spermatozoa contain two protamine variants, mouse protamine 1 (mP1) and mouse protamine 2 (mP2). The amino acid sequence predicted from mP1 cDNAs demonstrates that mP1 is a 50-amino-acid protein with strong homology to other mammalian P1 protamines. Nucleotide sequence analysis of independently isolated, overlapping cDNA clones indicated that mP2 is initially synthesized as a precursor protein which is subsequently processed into the spermatozoan form of mP2. The existence of the mP2 precursor was confirmed by amino acid composition and sequence analysis of the largest of a set of four basic proteins isolated from late-step spermatids whose synthesis is coincident with that of mP1. The sequence of the first 10 amino acids of this protein, mP2 precursor 1, exactly matches that predicted from the nucleotide sequence of cDNA and genomic mP2 clones. The amino acid composition of isolated mP2 precursor 1 very closely matches that predicted from the mP2 cDNA nucleotide sequence. Sequence analysis of the amino terminus of isolated mature mP2 identified the final processing point within the mP2 precursor. These studies demonstrated that mP2 is synthesized as a precursor containing 106 amino acids which is processed into the mature, 63-amino-acid form found in spermatozoa.     

Construction and characterization of cDNA encoding the alpha 2 chain of chicken type IX collagen

We have isolated and characterized a cDNA encoding the carboxy-terminal half of one of the polypeptide subunits of a novel disulfide-bonded collagen found in hyaline cartilage. This collagen has been given the type assignment type IX, and it has several unusual characteristics. First, the polypeptide subunits are shorter than alpha-chains of the fibrillar collagens types I, II, and III. Second, type IX molecules are heterotrimers of three genetically distinct polypeptide subunits. Third, type IX molecules contain three triple-helical collagenous domains interspersed with noncollagenous domains. When chicken cartilage collagens are extracted with pepsin, type IX collagen is cleaved and gives rise to the triple-helical fragments HMW and LMW. The identification of the cDNA reported here is based on a comparison of the amino acid composition of tryptic peptides derived from LMW with the composition of tryptic peptides predicted from the nucleotide sequence of the cDNA. We also show that the amino-terminal sequence of one of the subunits of LMW is identical with the sequence predicted from the nucleotide sequence of the cDNA. Finally, we demonstrate that the amino-terminal amino acid sequence of a tryptic peptide isolated from one of the subunits of HMW is identical with a sequence predicted from the cDNA. We have given the polypeptide chain encoded by the cDNA reported here the name alpha 2(IX), and we show that it is homologous to the alpha 1(IX) chain previously characterized by us.     

Characterization of a gene which encodes a mannosyltransferase homolog of Paracoccidioides brasiliensis

We screened an expression library of the yeast form of Paracoccidioides brasiliensis with a pool of human sera that was pre-adsorbed with mycelium, from patients with paracoccidioidomycosis (PCM). A sequence (PbYmnt) was obtained and characterized. A genomic clone was obtained by PCR of P. brasiliensis total DNA. The sequence contained a single open reading frame (ORF) encoding a protein of 357 amino acid residues, with a molecular mass of 39.78 kDa. The deduced amino acid sequence exhibited identity to mannosyl- and glycosyltransferases from several sources. A DXD motif was present in the translated gene and this sequence is characteristic of the glycosyltransferases. Hydropathy analysis revealed a single transmembrane region near the amino terminus of the molecule that suggested a type II membrane protein. The PbYmnt was expressed preferentially in the yeast parasitic phase. The accession number of the nucleotide sequence of PbYmnt and its flanking regions is AF374353. A recombinant protein was generated in Escherichia coli. Our data suggest that PbYmnt encodes one member of a glycosyltransferase family of proteins and that our strategy was useful in the isolation of differentially expressed genes.     

Nucleotide sequence of human rotavirus genome segment 10, an RNA encoding a glycosylated virus protein.

The complete nucleotide sequence of human rotavirus (Wa strain) genome segment 10 was determined by using a cloned DNA copy. The sequence data indicated that segment 10 is A + T rich (65%) and consists of 750 base pairs. The positive strand of segment 10 contains a single open reading frame that extends 175 codons from the first AUG triplet (residues 42 through 44). The amino acid sequence of the segment 10 product was deduced from the nucleotide sequence. There are two distinct glycosylation sites at the N-terminal hydrophobic region, consistent with previous findings that this protein exists in a glycosylated form. The apparent molecular weight (20,000) of the unglycosylated, precursor polypeptide is in good agreement with the one calculated from the predicted amino acid sequence. Structural analysis of the positive strand (mRNA from segment 10) showed that it could form, like mRNA from segment 11, a stable panhandle structure involving the 5' and 3'-terminal regions. The nucleotide sequence of segment 10 from simian rotavirus, recently determined by Both et al. (J. Virol. 48:335-339, 1983) was found to be highly homologous to, and to share several important features with, segment 10 of human rotavirus.     

Rat pre-prosomatostatin. Structure and processing by microsomal membranes

The tetradecapeptide hormone somatostatin arises from proteolytic processing of a large precursor, pre-prosomatostatin. Studies of other hormone precursors predict that the NH2 terminus of pre-prosomatostatin comprises a leader, or signal, region which is cleaved during its translation. Such co-translational cleavage would generate prosomatostatin. In these studies, we present the complete sequence of rat pre-prosomatostatin, deduced from the nucleotide sequence of cDNAs derived from a somatostatin-rich medullary thyroid carcinoma. These findings indicate that rat pre-prosomatostatin contains 116 amino acids (12,737 daltons). Cell-free translations of medullary thyroid carcinoma mRNA with dog pancreas microsomal membranes were performed to identify the cleavage point of the leader region from prosomatostatin. Partial microsequencing data indicates that the cleavage occurs between the glycine and alanine at positions 24 and 25 of pre-prosomatostatin. Thus, rat prosomatostatin contains 92 amino acids (10,388 daltons). Comparison of the amino acid sequences of the rat and human pre-prosomatostatins reveals only four amino acid substitutions. In view of the high degree of homology between rat and human pre-prosomatostatin, we expect a similar cleavage site and NH2-terminal structure for human prosomatostatin. The high level of conservation between rodents and humans of the entire pre-prosomatostatin molecule further suggests the possibility of biologic functions of the NH2-terminal portions of prosomatostatin.     

Partial purification and characterization of epidermal growth factor in human breast milk

Human epidermal growth factor (hEGF), a potent growth stimulator of many tissues in culture, has been isolated from human urine and subsequently identified in many human biological fluids including breast milk. In this study, partial purification and characterization of hEGF-like substance(s) in human milk were performed using homologous hEGF radioimmunoassay (RIA) and radioreceptor assay (RRA). hEGF-like material(s) was extracted from pooled human milk by ethanol precipitation, followed by adsorption to cation- and anion-exchange resin. DEAE-Sephadex G-25 ion-exchange chromatography of human milk extracts revealed three major components with hEGF activity (peak I, II, III) eluted with a linear gradient by ammonium acetate. The competitive binding curves for these components were parallel to those for standard hEGF in both RIA and RRA. The apparent molecular weight of peak I was approximately 6,500 and that of peak II and III was approximately 7,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The pI value for peak I was approximately 4.5 and that for peaks II and III was approximately 5.0 by isoelectric focusing. These data are comparable to the size and charge heterogeneity of hEGF in human urine extracts. In conclusion, the major components of hEGF in human milk appear to be physicochemically, immunologically and biologically (receptor binding activity) indistinguishable from hEGF of urinary origin.     

Cloning and nucleotide sequence of cDNA encoding human liver serine-pyruvate aminotransferase

Cloned cDNAs for human liver serine-pyruvate aminotransferase (Ser-PyrAT) were obtained by screening of a human liver cDNA library with a fragment of cDNA for rat mitochondrial Ser-PyrAT as a probe. Two clones were isolated from 50,000 transformants. Both clones contained approximately 1.5 kb cDNA inserts and were shown to almost completely overlap each other on restriction enzyme mapping and DNA sequencing. The nucleotide sequence of the mRNA coding for human liver Ser-PyrAT was determined from those of the cDNA clones. The mRNA comprises at least 1487 nucleotides, and encodes a polypeptide consisting of 392 amino acid residues with a molecular mass of 43,039 Da. The amino acid composition determined on acid hydrolysis of the purified enzyme showed good agreement with that deduced from the nucleotide sequence of the cDNA. In vitro translation of the mRNA derived from one of the isolated clones, pHspt12, as well as that of mRNA extracted from human liver, yielded a product of 43 kDa which reacted with rabbit anti-(rat mitochondrial Ser-PyrAT) serum. Comparison of the deduced amino acid sequences of human Ser-PyrAT and the mature form of rat mitochondrial Ser-PyrAT revealed 79.3% identity. Although human Ser-PyrAT appears to be synthesized as the mature size, the 5'-noncoding region of human Ser-PyrAT mRNA contains a nucleotide sequence which would encode, if translated, an amino acid sequence similar to that of the N-terminal extension peptide of the precursor for rat mitochondrial Ser-PyrAT.     

Structural organization of the human kininogen gene and a model for its evolution

The entire human kininogen gene has been isolated as a set of overlapping genomic DNA fragments, and the 11 exons encompassing approximately 27 kilobase pairs have been mapped by restriction enzyme analysis and nucleotide sequence determination. The nine 5'-terminal exons encode the 5'-untranslated region and the protein-coding region for the signal peptide and the heavy chain, which are common for high molecular weight (HMW) and low molecular weight (LMW) prekininogen mRNAs. Exon 10 consists of the common sequence for bradykinin and the immediately following unique sequence for HMW prekininogen mRNA. Exon 11 is then located following a 90-nucleotide sequence downstream from exon 10 and precisely specifies the sequence unique to LMW prekininogen mRNA. This, together with the hybridization analysis of total human cellular DNA, leads us to conclude that human HMW and LMW prekininogen mRNAs are produced from a single gene as a consequence of alternative RNA processing events. The structural analysis of the kininogen gene also shows that each of the nine 5'-terminal exons discretely specifies the nine protein domains observed in the amino-terminal portion of the kininogens. Furthermore, these nine genetic domains can be characterized by a thrice repeated pattern of three genetic segments, and two sets of these three domains, encompassing exons 3-5 and exons 6-8, are most closely related to each other. Therefore, we have proposed two successive duplication mechanisms as a model for the generation of the structure of the kininogen gene.     

N-terminal amino acid sequence of human urinary prokallikrein

The N-terminal amino acid sequences of human urinary prokallikrein and kallikrein have been determined. Their amino acid sequences are as follows. (Formula; see text) The results showed that prokallikrein comprises an additional seven amino acids at the amino terminus of the kallikrein, of which the sequence is (H2N)Ala-Pro-Pro-Ile-Gln-Ser-Arg(COOH). Comparison of the structure of this peptide with those of other proteins revealed extensive sequence identity with the propeptide portions of rat and mouse tissue kallikreins, that were predicted from the preproenzyme-encoded nucleotide sequences. The amino acid sequence of the peptide was also highly homologous to that of the propeptide portion of EGF-binding protein, that was predicted from the nucleotide sequence, and that of the alpha-subunit of NGF. The N-terminal amino acid sequence of kallikrein was completely identical to the reported one (Lottspeich, F., et al. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1947-1950) and shows considerable amino acid sequence homology with the porcine and rat pancreatic kallikreins. As far as the present results are concerned, it is strongly indicated that the inactive kallikrein in human urine is a tissue type prokallikrein which is activated on the release of the N-terminal peptide consisting of seven amino acids.