Biosynthesis of rat insulin-like growth factor II. I. Immunochemical demonstration of a approximately 20-kilodalton biosynthetic precursor of rat insulin-like growth factor II in metabolically labeled BRL-3A rat liver cells

BRL-3A rat liver cells synthesize mature 7484-dalton rat insulin-like growth factor II (rIGF-II) as a approximately 22-kDa precursor, presumably prepro-rIGF-II. In the present study, we have biosynthetically labeled intact BRL-3A cells with [35S]cysteine and immunoprecipitated cell lysates and media with antisera to rIGF-II. A approximately 20-kDa protein was identified in immunoprecipitates of cell lysates having properties consistent with pro-rIGF-II. The approximately 20-kDa protein is precipitated by immune sera but not by nonimmune serum. Its immunoprecipitation is specifically inhibited by unlabeled rIGF-II but not by insulin. It is not precipitated from labeled lysates of a subclone of BRL-3A cells (BRL-3A2) that does not synthesize rIGF-II. The approximately 20-kDa protein is rapidly labeled intracellularly (10 min) but is not detected in BRL-3A media. In pulse-chase experiments, radioactivity in the approximately 20-kDa protein disappears during the chase and appears, at later times, in specifically immunoprecipitated approximately 19-, approximately 10-, approximately 8-, and approximately 7-kDa proteins in media and, to a limited extent, intracellularly. A protein with electrophoretic mobility identical to that of the approximately 20-kDa protein observed in cell lysates is immunoprecipitated from 35S-proteins whose synthesis is directed by BRL-3A RNA in a reticulocyte lysate cell-free translation system supplemented with microsomal membranes, and presumably arises by cotranslational removal of the signal peptide from approximately 22-kDa prepro-rIGF-II. Processing of the approximately 20-kDa protein in intact BRL-3A cells to intermediate and mature rIGF-II species appears to occur at the time of secretion and/or shortly thereafter, with the different forms appearing at approximately the same time.     

Identification and molecular cloning of two new 30-kDa insulin-like growth factor binding proteins isolated from adult human serum.

Three insulin-like growth factor binding proteins (IGFBP) with apparent molecular masses of 24, 28-30, and 30 kDa, nonreduced, have been isolated from human serum. The 15 NH2-terminal amino acids of the 24-kDa binding protein are identical with those of the 30-kDa BP. The apparent molecular mass of the latter is reduced to 24 kDa by N-glycanase, suggesting that the 30-kDa BP is the glycosylated form of the isolated 24-kDa BP. The complete amino acid sequences derived from the cloned cDNAs represent two new IGFBPs. They are tentatively termed IGFBP-4 and -5. The prepeptide sequences of BP-4 and -5 contain 27 and 21, the mature proteins 213 and 237 amino acids, respectively (Mr = 22,610 and 25,980). The NH2- and COOH-terminal thirds of BP-4 and -5 display pronounced homology to the other three human BPs. 16 of the 16-20 cysteines and 37 of the 213-289 amino acids (12.8-17.1%) are conserved in all five mature BPs. 10 amino acid positions located in the NH2-terminal region and shared by BP-1, -2, -3, and -5 are different in BP-4. These differences may account for the preferential affinity of BP-4 for IGF II. A most intriguing homology exists between the COOH-terminal quarter of the five IGFBPs, 10 repetitive domains of human thyroglobulin, a gastrointestinal tumor-associated antigen, and the invariant chain of the class II histocompatibility antigen. The cDNAs of five human IGFBPs are now available. They will allow their expression and production in sufficient quantities for in vivo studies to unravel their role in growth and metabolism.     

Isolation from adult human serum of four insulin-like growth factor (IGF) binding proteins and molecular cloning of one of them that is increased by IGF I administration and in extrapancreatic tumor hypoglycemia.

We have isolated four insulin-like growth factor binding proteins (IGFBPs) from adult human serum by insulin-like growth factor (IGF) I affinity chromatography and high performance liquid chromatography. A 36-kDa binding protein (BP), not digestible with N-glycanase, is increased in patients with extrapancreatic tumor hypoglycemia and during IGF I administration in healthy adults. Its 38 NH2-terminal amino acids are identical to those of an IGFBP sequence derived from a human cDNA that cross-hybridizes with the rat IGFBP-2 cDNA. With probes encoding a NH2-terminal, COOH-terminal, and a middle region of this protein we have obtained three cDNA clones from a Hep G2 cDNA library; one encodes human IGFBP-2, and the other two presumably represent unspliced heteronuclear and alternatively spliced mRNA, respectively. A 28-30-kDa IGFBP represents a novel BP species in human serum. Its 30 NH2-terminal amino acids are not homologous to IGFBP-1, -2, or -3. It is not digestible with N-glycanase and does not bind 125I-IGF I. The NH2-terminal sequences of a 42/45- and a 31-kDa IGFBP are identical to that of human IGFBP-3. The 42/45-kDa proteins are two glycosylation variants of BP-3. The 31-kDa protein presumably is a degradation product of BP-3 that lacks the COOH terminus. It is likely that the different IGFBPs modulate auto-/paracrine and endocrine effects of IGFs on growth and metabolism in a different and specific manner.     

Amino-terminal sequence and structure of monoclonal antibody immunopurified cytochromes P-450

Six hepatic cytochromes P-450 were isolated from 3-methylcholanthrene-treated animals by immunopurification with monoclonal antibodies. The purified cytochromes P-450 include 57- and 56-kDa polypeptides from Sprague-Dawley rats, 57- and 56-kDa polypeptides from C57BL/6 mice, a 56-kDa polypeptide from DBA/2 mice, and a 53-kDa polypeptide from guinea pigs. These isozymes were structurally compared by peptide mapping using both sodium dodecyl sulfate--polyacrylamide gel electrophoresis and high-pressure liquid chromatography and by amino acid and NH2-terminal sequence analyses. The 57-kDa polypeptides from rats and mice have similar but nonidentical peptide maps and amino acid compositions and are about 80% homologous in their NH2-terminal amino acid sequence. The 56-kDa polypeptides from rats and both mice strains have very similar peptide maps and amino acid compositions and identical NH2-terminal sequences. The NH2-terminal sequence of the mice 56-kDa polypeptides corresponds to that reported for the mouse P1-450 isozyme except that we identified two additional residues, proline and serine, at the NH2 terminus in the 57-kDa polypeptide from C57BL/6 mice that were not deduced from the cDNA sequence of the mouse P1-450 isozyme. The guinea pig 53-kDa polypeptide has a distinct peptide map relative to the other polypeptides studied and an NH2-terminal sequence with only partial homology to the 56- and 57-kDa polypeptides from rats and mice. This report shows the varying degree of structural relatedness among the isozymes examined and demonstrates the suitability and advantage of immunopurified cytochromes P-450 for sequencing and structural studies.     

Nucleotide sequence and expression of a cDNA clone encoding a fetal rat binding protein for insulin-like growth factors

The insulin-like growth factors (IGFs), IGF-I and IGF-II, occur in plasma and tissue fluids complexed to specific binding proteins. Although the role of the binding proteins is not completely defined, they are capable of modulating the biological activity of the IGFs. In order to better understand the function of these proteins, we have isolated a clone from the BRL-3A rat liver cell line that encodes a protein corresponding to the IGF binding protein in fetal rat serum. The cDNA clone encodes a precursor protein of 304 amino acids (32,886 daltons), comprised of a 34-residue hydrophobic prepeptide and a 270-residue mature protein (29,564 daltons). The deduced amino acid sequence agrees with the sequence of 173 amino acid residues determined by Edman degradation. The mature protein contains 18 cysteines and no N-glycosylation sites. It contains an Arg-Gly-Asp (RGD) sequence near the carboxyl terminus. A similar sequence is present on many extracellular matrix proteins and contributes to their recognition by cellular adhesion receptors. The cloned cDNA has been transcribed in vitro and the resulting RNA expressed in Xenopus oocytes. Injected oocytes secrete a 33-kDa protein that is immunoprecipitated by polyclonal antibodies to the BRL-3A binding protein and binds IGF-I and IGF-II with the same affinity and specificity as does purified BRL-3A binding protein. The binding protein cDNA probe hybridizes to an approximately 2-kilobase mRNA in BRL-3A cells and in multiple fetal rat tissues including liver, kidney, intestine, and lung. Levels of this mRNA are greatly reduced in the corresponding adult tissues. The rat IGF binding protein is closely related to the partial amino acid sequences reported for a bovine IGF binding protein and more distantly related to a human IGF binding protein that recently has been cloned. No significant homologies were identified to other proteins. Thus, the rat IGF binding protein that we have cloned appears to be a distinct member of a family of related IGF binding proteins. We postulate that the structurally distinct IGF binding proteins may have different biological functions.     

NH2-terminal processing of Drosophila melanogaster actin. Sequential removal of two amino acids

Class II actins, such as Drosophila and mammalian skeletal muscle actins, have genes that code for a Met-X-Asp NH2 terminus where X is usually cysteine. These actins have an Ac-Asp NH2 terminus so two amino acids must be removed. To determine the nature of this processing, we labeled Drosophila Schneider L-2 cells with [35S]methionine or cysteine, isolated the actin, and analyzed the NH2-terminal actin tryptic peptides and their thermolysin digestion products. After a 4-h labeling period, we detected completed actin polypeptide chains with either an unblocked Asp or an Ac-Asp NH2 terminus. No intermediate with an NH2-terminal Cys or Met could be demonstrated. If, however, Drosophila mRNA was translated in a mRNA-dependent rabbit reticulocyte lysate system, an additional 43-kDa actin intermediate was observed. On the basis of thermolysin digestion studies and experiments using mild acid hydrolysis of a labeled actin NH2-terminal tryptic peptide fragment, we identified this intermediate as having an Ac-Cys-Asp NH2 terminus. In a time-dependent fashion, Ac-Cys was removed generating actin with an exposed NH2-terminal Asp which was subsequently acetylated to produce the mature form of actin. The removal of Met and the acetylation of Cys may occur early in translation while the nascent polypeptide chain is still attached to the ribosome. Subsequent processing occurs following completion of the synthesis of the actin polypeptide. The removal of Ac-Cys from Drosophila actin is thus similar to removal of Ac-Met from the NH2 terminus of class I actins although in the case of the class II actins, it is the second amino acid that is removed as an acetylated species.     

Processing and amino acid sequence analysis of the mouse mammary tumor virus env gene product.

The envelope proteins of mouse mammary tumor virus (MMTV) are synthesized from a subgenomic 24S mRNA as a 75,000-dalton glycosylated precursor polyprotein which is eventually processed to the mature glycoproteins gp52 and gp36. In vivo synthesis of this env precursor in the presence of the core glycosylation inhibitor tunicamycin yielded a precursor of approximately 61,000 daltons (P61env). However, a 67,000-dalton protein (P67env) was obtained from cell-free translation with the MMTV 24S mRNA as the template. To determine whether the portion of the protein cleaved from P67env to give P61env was removed from the NH2-terminal end of P67env and as such would represent a leader sequence, the NH2-terminal amino acid sequence of the terminal peptide gp52 was determined. Glutamic acid, and not methionine, was found to be the amino-terminal residue of gp52, indicating that the cleaved portion was derived from the NH2-terminal end of P67env. The NH2-terminal amino acid sequences of gp52's from endogenous and exogenous C3H MMTVs were determined though 46 residues and found to be identical. However, amino acid composition and type-specific gp52 radioimmunoassays from MMTVs grown in heterologous cells indicated primary structure differences between gp52's of the two viruses. The nucleic acid sequence of cloned MMTV DNA fragments (J. Majors and H. E. Varmus, personal communication) in conjunction with the NH2-terminal sequence of gp52 allowed localization of the env gene in the MMTV genome. Nucleotides coding for the NH2 terminus of gp52 begin approximately 0.8 kilobase to the 3' side of the single EcoRI cleavage site. Localization of the env gene at that point agrees with the proposed gene order -gag-pol-env- and also allows sufficient coding potential for the glycoprotein precursor without extending into the long terminal repeat.     

Pre-ornithine transcarbamylase. Properties of the cytoplasmic precursor of a mitochondrial matrix enzyme

Biogenesis of the mitochondrial matrix enzyme, ornithine transcarbamylase, has been shown to begin with synthesis on cytoplasmic ribosomes of a precursor, designated pre-ornithine transcarbamylase, which is approximately 4000 daltons larger than its corresponding mitochondrial subunit, followed by post-translational uptake and proteolytic processing of the precursor to its mature counterpart by mitochondria. We now report initial studies on the structure and properties of preornithine transcarbamylase. When this precursor is labeled at the NH2 terminus with N-formyl[35S]methionine and processed by mitochondria, no label is recovered with the mature subunit. This demonstrates that the amino acid extension which is characteristic of the precursor and which is removed during mitochondrial processing is NH2-terminal. This NH2-terminal extension is found intact in two peptides produced by limited proteolysis of the labeled precursor. Moreover, this amino acid extension modifies the behavior of the precursor during immunoprecipitation in the presence of ionic detergents and plays a critical role in facilitating uptake of the precursor by mitochondria.     

Primary structure of rat liver 5'-nucleotidase deduced from the cDNA. Presence of the COOH-terminal hydrophobic domain for possible post-translational modification by glycophospholipid

Rat liver 5'-nucleotidase was purified from a crude microsomal fraction, and its molecular mass was estimated to be 73 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein was subjected to cleavage with CNBr or lysyl endopeptidase, and the resulting 21 peptides as well as the NH2 terminus of the native protein were sequenced by Edman degradation. For further information on the molecular structure, we constructed a lambda gt11 liver cDNA library and isolated two cDNA clones for 5'-nucleotidase, lambda cNTP6 and lambda cNT34. The 3.2-kilobase cDNA insert of lambda cNTP6 contains an open reading frame that encodes a 576-residue polypeptide with a calculated size of 63,965 Da, which is in reasonable agreement with that of 5'-nucleotidase (62 kDa) immunoprecipitated from cell-free translation products. The NH2-terminal 28 residues comprise a signal peptide, which is followed by the NH2-terminal sequence of the purified protein. The predicted structure contains all the other peptide sequences determined by Edman degradation. Five potential N-linked glycosylation sites are found in the molecule, accounting for the difference in mass between the precursor and mature forms. Another characteristic feature is that the primary structure contains a highly hydrophobic amino acid sequence at the COOH terminus, a possible signal for the post-translational modification by glycophospholipid. In fact, labeling experiments of rat hepatocytes demonstrated that 3H-labeled compounds such as ethanolamine, myo-inositol, and palmitic acid, components of the glycolipid anchor, were incorporated into 5'-nucleotidase. Phosphatidylinositol-specific phospholipase C released 5'-nucleotidase from the cell surface, and the released protein no longer contained the radioactivity of [3H]palmitic acid incorporated.     

Unique precursor structure of an extracellular protease, aqualysin I, with NH2- and COOH-terminal pro-sequences and its processing in Escherichia coli

Aqualysin I is a subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extremely thermophilic Gram-negative bacterium. The nucleotide sequence of the entire gene for aqualysin I was determined, and the deduced amino acid sequence suggests that aqualysin I is produced as a large precursor, consisting of at least three portions, an NH2-terminal pre-pro-sequence (127 amino acid residues), the protease (281 residues), and a COOH-terminal pro-sequence (105 residues). When the cloned gene was expressed in Escherichia coli cells, aqualysin I was not secreted. However, a precursor of aqualysin I lacking the NH2-terminal pre-pro-sequence (38-kDa protein) accumulated in the membrane fraction. On treatment of the membrane fraction at 65 degrees C, enzymatically active aqualysin I (28-kDa protein) was produced in the soluble fraction. When the active site Ser residue was replaced with Ala, cells expressing the mutant gene accumulated a 48-kDa protein in the outer membrane fraction. The 48-kDa protein lacked the NH2-terminal 14 amino acid residues of the precursor, and heat treatment did not cause any subsequent processing of this precursor. These results indicate that the NH2-terminal signal sequence is cleaved off by a signal peptidase of E. coli, and that the NH2- and COOH-terminal pro-sequences are removed through the proteolytic activity of aqualysin I itself, in that order. These findings indicate a unique four-domain structure for the aqualysin I precursor; the signal sequence, the NH2-terminal pro-sequence, mature aqualysin I, and the COOH-terminal pro-sequence, from the NH2 to the COOH terminus.     

The 43-kilodalton protein of Torpedo nicotinic postsynaptic membranes: purification and determination of primary structure

The primary structure of the 43-kilodalton peripheral membrane protein (43-kDa protein) of Torpedo nicotinic postsynaptic membrane has been determined. The 43-kDa protein, which was isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, has an amino terminus resistant to Edman degradation, while the sequence at the carboxyl terminus is Tyr-Val. An amino acid sequence of 405 residues was obtained by NH2-terminal sequence analysis of complementary peptides generated by digestion with trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and endoproteinase Lys-C, as well as by chemical cleavage at methionine. This sequence of molecular mass 45,618 daltons lacks the amino terminus but extends to the carboxyl terminus of the 43-kDa protein. Unusual structural features of the 43-kDa protein include two regions of approximately 80 residues, each containing 10% cysteine, as well as stretches predicted to exist as amphipathic alpha-helices. Other than the group blocking the amino terminus, no evidence was found for posttranslational modification of amino acids. The 43-kDa protein may represent a novel protein family because a computer search of this sequence with the National Biomedical Research Foundation data base (Release 12.0) did not reveal any significant homology to known protein sequences.     

Yolk vitronectin. Purification and differences from its blood homologue in molecular size, heparin binding, collagen binding, and bound carbohydrate.

This is the first report on a unique vitronectin molecule, yolk vitronectin, which is similar to its blood homologue in cell spreading activity but different in molecular size, bound carbohydrate, and heparin and collagen binding activity. Yolk vitronectin was purified 2,500-fold from chick egg yolk by a combination of hydroxylapatite, DEAE-cellulose, and anti-vitronectin-Sepharose column chromatographies. In SDS-polyacrylamide gel electrophoresis under reducing conditions, yolk vitronectin was separated into 54- and 45-kDa bands, which are 16 and 25 kDa smaller, respectively, than the 70-kDa major band of chick blood vitronectin. The 54-kDa band shares the same NH2-terminal sequence as chick blood vitronectin. In contrast, the NH2-terminal sequence of the 45-kDa band is somewhat homologous with the internal sequences of mammalian vitronectins beginning at the 50th amino acid from the NH2 terminus. The bound carbohydrate of the 54- and 45-kDa species of yolk vitronectin is similar to, but distinct from, that of blood vitronectin. Unlike blood vitronectin, yolk vitronectin cannot bind to either heparin or collagen.     

Characterization of the inactive fragment resulting from limited proteolysis of human alpha1-proteinase inhibitor by Crotalus adamanteus proteinase II.

The inactive 50,000-dalton fragment of human plasma alpha1-proteinase inhibitor resulting from limited proteolysis of the inhibitor by Crotalus adamanteus proteinase II has been isolated and partially characterized. The amino acid composition of the inactivated inhibitor indicates the loss of a peptide fragment from the intact inhibitor. Both intact and inactivated inhibitor contain COOH-terminal lysine. However, the NH2 terminus of the intact inhibitor is Glx, whereas that of inactivated inhibitor is methionine. NH2-terminal analysis of the inactive inhibitor fragment revealed the following sequence: -Met-Phe-Leu-Glu-Ala-Ile-Pro-Met-Ser-Ile-Pro-Pro-Gln-Val-Lys-Phe-Asn. The data show that the venom proteinase has inactivated alpha1- proteinase inhibitor by cleavage of a single bond which differs from that reported for trypsin or papain.     

Correct NH2-terminal processing of cardiac muscle alpha-isoactin (class II) in a nonmuscle mouse cell

Both mammalian nonmuscle and muscle actins possess an AcAsp(Glu)NH2 terminus. The nonmuscle actin genes code for a polypeptide with a Met-Asp NH2 terminus (class I) whereas the muscle actin genes code for a polypeptide with a Met-Cys-Asp NH2 terminus (class II). Two amino acids must be removed for mature muscle actin synthesis, whereas only the Met must be removed for nonmuscle actin synthesis. We wished to know whether a nonmuscle cell which normally does not synthesize a class I actin can correctly process a muscle actin with its extra NH2-terminal amino acid in vivo. To answer this question we have used L/LK165 cells, a mouse L-cell transfected with a human cardiac muscle actin gene. When these cells were labeled overnight with [35S]Cys, an actin with an NH2-terminal tryptic peptide corresponding to that of mature cardiac muscle actin was detected. When the cells were pulse-labeled for 20 min, a new actin intermediate containing an AcCys-Asp amino terminus was observed which then disappeared with time. Furthermore, the muscle actin was processed as fast if not faster than the nonmuscle actin in these cells. This actin intermediate was also seen in chick myotube cultures. Our results show that the ability to correctly process muscle specific actins is not tissue specific. Furthermore, these results confirm a processing pathway for class II actins proposed by us earlier on the basis of experiments with a cell-free translation system.     

Cell-free synthesis of rat parotid preamylase.

Poly(A)-containing RNA from rat parotid gland directs the cell-free synthesis of several products in the reticulocyte lysate translation system including a very prominent 58,000-dalton polypeptide which is immunoreactive with anti-alpha-amylase. Purified alpha-amylase has a molecular weight estimated as 56,000 daltons. The 58,000-dalton, cell-free product and alpha-amylase share common peptides as determined by analysis of their limited proteolysis digests. The cross-reactivity and peptide homology suggest that the cell-free product may be a precursor of mature alpha-amylase. While the NH2 terminus of alpha-amylase is blocked, that of the 58,000-dalton product evidently is not, and automated sequence analysis has yielded its partial sequence as: Met-X-Phe-Phe-Leu-Leu-Leu-X-Leu-Ile-X-Leu-X-X-X-X-X-X-X-X-X-Phe-X-X-X-X-X-Ile-X-X-Leu-Phe. The highly hydrophobic nature of the NH2 terminus of the 58,000-dalton, cell-free product suggests that, like other secreted polypeptides, the extra piece may play a role in the transport and secretion of the mature alpha-amylase.     

Characterization of beta 1----4 galactosyltransferase purified from rat liver microsomes.

beta 1----4 Galactosyltransferase was purified from rat liver microsomes. Catalytic properties of the enzyme resembled those of previously purified soluble and membrane-bound beta 1----4 galactosyltransferases. The enzyme purified in the present study showed a major band around a molecular weight of 53,000 on SDS-PAGE. The NH2-terminal sequence of the enzyme was determined up to the 20th residue. The sequence was identical to the amino acid sequence from Ala-13 to Lys-32 deduced from mouse beta 1----4 galactosyltransferase cDNA. These results suggest that most of the mature enzyme in rat liver microsomes is produced by removal of the NH2-terminal 12 amino acids from a precursor polypeptide.