Biosynthesis of calcitonin by a rat medullary thyroid carcinoma cell line

The 32-amino acid form of the peptide hormone calcitonin is the product of a series of post-translational processing steps of a 13,400-dalton precursor, procalcitonin. We have now identified the steps involved in proteolytic paring of the precursor to the mature secretory form. Cultures of the CA-77 cell line were radiolabeled and the various forms of calcitonin were isolated by specific immunoprecipitation followed by fractionation on gel filtration and reversed-phase high performance liquid chromatography. Pulse-chase kinetics showed that procalcitonin was cleaved to a 6,500-dalton biosynthetic intermediate which was subsequently processed to the size of mature calcitonin (3,400 daltons). Partial microsequencing of the [35S] methionine-labeled intermediate indicated that the sequence consisted of the COOH-terminal 52 residues of procalcitonin. Partial microsequencing of the [35S]methionine- or [3H]proline-labeled 3,400-dalton species revealed that it was indistinguishable from naturally occurring, amidated calcitonin. These data define the major pathway for calcitonin biosynthesis in this neoplastic cell line and presumably in normal cells.     

Biosynthesis of an asparagine-linked oligosaccharide-containing calcitonin by a rat medullary thyroid carcinoma cell line

Calcitonin contains an amino acid sequence that provides a potential site for glycosylation of the peptide at the asparagine at position 3. Preliminary evidence has suggested that there are glycosylated forms of calcitonin and its precursor, procalcitonin. The CA-77 rat medullary thyroid carcinoma cell line, recently developed to study calcitonin biosynthesis, was used to demonstrate the synthesis of glycosylated forms of this hormone by intact cells. Cultures were incubated in medium containing either [3H]mannose or [35S]methionine. Two species incorporating both labels were specifically immunoprecipitated when cell extracts were treated with calcitonin antibodies. Gel filtration chromatography in 6 M guanidine hydrochloride indicated that one peptide had a molecular weight of 5500, approximately 2000 daltons larger than calcitonin, while the second peptide had a molecular weight of 14 400, the approximate size of procalcitonin. Treatment of the [3H]mannose-labeled cell extract with endo-beta-N-acetylglucosaminidase H before immunoprecipitation removed the labeled sugar from the calcitonin species. Microsequence analysis of the radiolabeled immunoreactive 5500-dalton calcitonin species showed methionine at cycle 8 and mannose at cycle 3, suggesting that this peptide is calcitonin containing an N-linked oligosaccharide at Asn-3. These results suggest that in this cell line a minor but significant biosynthetic pathway exists for the production of glycosylated calcitonin from glycosylated procalcitonin.     

Molecular cloning, sequencing, deletion, and overexpression of a methionine aminopeptidase gene from Saccharomyces cerevisiae.

A yeast gene for a methionine aminopeptidase, one of the central enzymes in protein synthesis, was cloned and sequenced. The DNA sequence encodes a precursor protein containing 387 amino acid residues. The mature protein, whose NH2-terminal sequence was confirmed by Edman degradation, consists of 377 amino acids. The function of the 10-residue sequence at the NH2 terminus, containing 1 serine and 6 threonine residues, remains to be established. In contrast to the structure of the prokaryotic enzyme, the yeast methionine aminopeptidase consists of two functional domains: a unique NH2-terminal domain containing two motifs resembling zinc fingers, which may allow the protein to interact with ribosomes, and a catalytic COOH-terminal domain resembling other prokaryotic methionine aminopeptidases. Furthermore, unlike the case for the prokaryotic gene, the deletion of the yeast MAP1 gene is not lethal, suggesting for the first time that alternative NH2-terminal processing pathway(s) exist for cleaving methionine from nascent polypeptide chains in eukaryotic cells.     

Calcitonin COOH-terminal cleavage peptide as a model for identification of novel neuropeptides predicted by recombinant DNA analysis

A strategy is presented for identification of putative neurohormones that were predicted by sequence analysis of recombinant cDNa molecules. The nucleotide sequence of a cloned calcitonin cDNA insert predicts that the proteolytic excision of calcitonin from a 136-amino acid precursor should generate two additional peptides derived from regions flanking the calcitonin sequence on the NH2 and carboxyl termini. The predicted 16-amino acid COOH-terminal cleavage peptide (NH2-Asp-Met-Ala-Lys-Asp-Leu-Glu-Thr-Asn-His-His-Pro-Thr-Phe-Gly-Asn-COOH) was chemically synthesized using solid phase procedures. Antibodies against this synthetic hexadecapeptide detect immunologically cross-reactive material in tissue extracts from normal thyroid glands and calcitonin-producing medullary thyroid carcinomas. Each of the four calcitonin mRNA-directed cell-free translation products contain the immunological determinants for both calcitonin and the COOH-terminal cleavage peptide.     

Sequence of the precursor to rat bone gamma-carboxyglutamic acid protein that accumulates in warfarin-treated osteosarcoma cells

A biosynthetic precursor to rat bone gamma-carboxyglutamic acid protein (BGP) was isolated from warfarin-treated ROS 17/2 osteosarcoma cells by antibody affinity chromatography followed by reverse phase high performance liquid chromatography. Thirty-two residues of its NH2-terminal sequence were determined by gas-phase protein sequence analysis. Comparison of this sequence with the known structure of rat BGP established that the intracellular precursor is a 76-residue molecule of Mr = 9120 that differs from 6000-Da bone BGP in having an NH2-terminal extension of 26 residues. This precursor appears to be generated from the primary translation product by cleavage of a hydrophobic signal peptide and is the probable substrate for gamma-carboxylation by virtue of its accumulation in the presence of warfarin. The putative targeting region for gamma-carboxylation previously identified in the leader sequences of vitamin K-dependent proteins is found in the propeptide portion of the precursor. Since the immunoreactive component secreted by warfarin-treated cells is identical in sequence to the 6000-Da BGP from bone, propeptide cleavage from the precursor is independent of gamma-carboxylation and precedes secretion of BGP from the cell.     

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.     

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.     

Cellular processing of pre-proparathyroid hormone involves rapid hydrolysis of the leader sequence.

The cellular synthesis of parathyroid hormone (PTH) involves two consecutive cleavages of NH2-terminal peptide sequences from a larger precursor, pre-proparathyroid hormone (Pre-ProPTH). The initial cleavage consists of the removal of an NH2-terminal leader sequence either during or shortly after biosynthesis of the polypeptide chain is complete. To determine the fate of the cleaved leader sequence, we prepared, by chemical synthesis, a peptide based on the known structure of the leader sequence of pre-proparathyroid hormone and used this peptide labeled with 125iodine as a marker to monitor the recovery of the putative cellular leader peptide during extraction and electrophoresis of [35S]methionine-labeled proteins from pulse-labeled parathyroid gland slices. Under conditions in which the recovery of the synthetic leader peptide was 50 to 70%, we found no detectable 35S-labeled product in the region of sodium dodecyl sulfate gels where the synthetic peptide migrates. In view of the known methionine content of pre-proparathyroid hormone and proparathyroid hormone (ProPTH), it would have been possible to detect endogenously labeled leader peptide if present in amounts equal to 0.05% of the amount of labeled ProPTH present in the tissues. These observations indicate that the cellular conversion of Pre-ProPTH to ProPTH involves a rapid hydrolysis of the leader peptide either during or immediately after its removal from the precursor.     

Interchain disulfide bonding in the regulatory subunit of cAMP-dependent protein kinase I

The two protomers of the purified regulatory subunit from porcine cAMP-dependent protein kinase I have been shown to be covalently cross-linked by interchain disulfide bonding. Limited proteolysis which cleaves the polypeptide chain into two fragments demonstrated that the disulfide bonding was associated exclusively with the fragment that corresponded to the NH2-terminal region of the polypeptide chain. This NH2-terminal fragment accounted for approximately 15 to 20% of the molecule. The disulfide bonding was further characterized by alkylating the cysteines in the native regulatory subunit. Following oxidation with performic acid, each regulatory subunit contained 7 cysteic acid residues; however, under denaturing conditions, but without prior reduction, only 5 cysteine residues could be alkylated with iodoacetic acid. Following limited proteolysis, all five of these cysteines were associated with the larger COOH-terminal, cAMP binding domain. In contrast, if the denatured subunit was first reduced prior to alkylation, all 7 cysteine residues were alkylated. The 2 cysteines that were only accessible to alkylation after prior reduction were both associated with the NH2-terminal end of the polypeptide chain ultimately with a 5,400 peptide. Alkylation of the isolated, denatured NH2-terminal domain with iodoacetic acid resulted in no covalent modification unless the fragment was first reduced with dithiothreitol. The NH2-terminal and COOH-terminal domains were shown to be linked by a region of the polypeptide chain that is rich in both proline and arginine. It is the arginine-rich site that is readily prone to proteolytic cleavage.     

Assembly of the sarcoplasmic reticulum. Cell-free synthesis of te Ca2+ + Mg2+-adenosine triphosphatase and calsequestrin

Polyadenylated RNA prepared from neonatal rat muscle was translated in a rabbit reticulocyte cell-free system. Two sarcoplasmic reticulum proteins, the Ca2+ + Mg2+-dependent adenosine triphosphatase (ATPase) and calsequestrin, were isolated from the translation mixture by immunoprecipitation, followed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The [35S]methionine-labeled translation products were characterized by molecular weight, peptide mapping, and NH2-terminal sequence analysis. The ATPase synthesized in the cell-free system was found to have the same molecular weight (Mr = 100,000) and [35S]-methionine-labeled peptide map as the mature ATPase. The methionine residue present at the NH2 terminus of the mature ATPase was donated by initiator methionyl-tRNArMet and it became acetylated during translation. These results suggest that the ATPase was synthesized without an NH2-terminal signal sequence. Calsequestrin (Mr - 63,000) was synthesized as a higher molecular weight precursor (Mr = 66,000) that contained an additional [35S]methionine-labeled peptide when compared to mature calsequestrin. The NH2-terminal sequence of the precursor was different from the mature protein. The precursor was processed to a polypeptide with a molecular weight identical with mature calsequestrin when microsomal membranes prepared from canine pancreas were included during translation. These results show that calsequestrin is synthesized with an NH2-terminal signal sequence that is removed during translation. These data add to the evidence that the ATPase and calsequestrin follow distinctly different biosynthetic pathways, even though, ultimately, they are both located in the same membrane.     

Study of the primary structures of the peptide core of bovine estrus cervical mucin. Possible existence of small similar subunits

Two populations of tryptic peptides were isolated from bovine estrus cervical mucin (BCM). One contained all the carbohydrate, and was rich in threonine and serine. These glycopeptides had, like the whole mucin, alanine as their NH2-terminal residues. Their COOH-terminal residues were arginine. The second population of peptides was rich in carboxylic amino acids, contained two cysteinyl residues, and had, like the whole mucin, leucine as COOH-terminal residues. Their NH2-terminal residues were aspartic acid. The sum of the residues of one glycopeptide plus one cysteinyl-containing peptide corresponded to the number of residues constituting a putative subunit of BCM. The amino acid sequence of the major cysteinyl peptide was determined. A cluster of hydrophobic residues was found in the COOH-terminal region. The amino acid sequences of two of the glycopeptides were found identical up to the 22nd residue. The small number of tryptic peptides, as well as the large amount of NH2- and COOH-terminal amino acids found in BCM indicate that this glycoprotein is made up of similar subunits with a molecular weight of about 22,000, one of the glycopeptides representing the NH2-terminal part, and one of the cysteinyl peptides, the COOH-terminal part. However, the existence of these subunits was not confirmed by ultracentrifugation of BCM in dithiothreitol and sodium dodecyl sulfate. BCM was polydisperse and had a mean molecular weight of 507,000.     

Antibodies against synthetic peptides reveal that the unidentified reading frame A6L, overlapping the ATPase 6 gene, is expressed in human mitochondria

Antibodies prepared against chemically synthesized peptides predicted from the DNA sequence have been used to detect human mitochondrial gene products. In particular, antibodies directed against either the NH2-terminal decapeptide or the COOH-terminal undecapeptide of cytochrome c oxidase subunit II (COII) were both very effective in immunoprecipitating the previously identified COII polypeptide from an SDS lysate of mitochondria from HeLa cells. Similarly, antibodies directed against the COOH-terminal nonapeptide of the putative polypeptide encoded in the unidentified reading frame A6L, which overlaps the ATPase 6 gene, immunoprecipitated specifically a component (#25) of the HeLa cell mitochondrial translation products; antibodies directed against the NH2-terminal octapeptide also precipitated protein 25, although less efficiently. The size of protein 25, as estimated from its electrophoretic mobility, is compatible with its being the unidentified reading frame A6L product. Furthermore, a fingerprinting analysis of this protein after trypsin digestion has given results consistent with this identification.     

Unusual COOH-terminal structure of staphylococcal protease

The extracellular enzyme, staphylococcal protease, carries a COOH-terminal tryptic peptide of 43 amino acid residues most of which are aspartic acid, asparagine, and proline. This peptide might have a function equivalent to that of a similar segment previously observed at the NH2-terminal end of the membrane-bound penicillinase precursor of Bacillus licheniformis (Yamamoto, S., and Lampen, J. O. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 1457-1461). These observations would suggest that bacterial exoproteins which are secreted in the form of precursors differ from extracellular proteins by the presence of an extra segment at their NH2- and/or COOH-terminal ends.     

Comparative studies on the primary structure of human cystatin as from epidermis, liver, spleen, and leukocytes

We have studied the primary structure of human cystatin As from epidermis, liver, spleen, and leukocytes. These molecules were indistinguishable on PAGE in the presence and absence of SDS, by fast protein liquid chromatography (FPLC) chromatofocusing on a Mono P column, and in amino acid composition. The NH2- and COOH-terminal amino acid sequences of human cystatin As from epidermis, liver, and spleen were identical with those of human leukocyte cystatin A previously reported except for the lack of the NH2-terminal methionine residue in human epidermal cystatin A. The peptides obtained upon digestion of four human cystatin As with Achromobacter protease I (AP) showed identical peptide maps on HPLC except for different retention times of the NH2-terminal peptides. Furthermore, the amino acid compositions of corresponding separated peptide quartets were identical. We also determined the complete amino acid sequence of human epidermal cystatin A by sequencing peptides obtained from AP digestion and cyanogen bromide (CNBr) cleavage. It consisted of 97 amino acid residues, and was identical with those of human cystatin As from liver, spleen, and leukocytes except for the lack of the NH2-terminal methionine residue.     

Primary structure of rat liver mannan-binding protein deduced from its cDNA sequence

cDNA clones encoding rat liver mannan-binding protein (MBP), a lectin specific for mannose and N-acetylglucosamine, were isolated from a rat liver cDNA library carried in lambda gt 11, by screening with affinity purified polyclonal rabbit anti-rat liver MBP antibodies. The nucleotide sequence of the cDNA determined by the dideoxy method revealed the complete amino acid sequence of the MBP (226 residues). The NH2-terminal residue of the MBP, glutamic acid, was preceded by a predominantly hydrophobic stretch of 18 amino acids, which was assumed to be a signal peptide. Near the NH2-terminal, there was a collagen-like domain, which consisted of 19 repeats of the sequence Gly-X-Y. Here, X and Y were frequently proline and lysine. Three proline and lysine residues were hydroxylated, and one of the latter appeared to link to galactose. Computer analysis of several lectins for sequence homology suggested that the COOH-terminal quarter of the MBP is associated with the calcium binding as well as carbohydrate recognition.     

Purification and characterization of a novel thiol protease involved in processing the enkephalin precursor

Proteolytic processing enzymes are required to convert the enkephalin precursor to active opioid peptides. In this study, a novel 33-kDa thiol protease that cleaves complete precursor in the form of [35S]methionine preproenkephalin was purified from bovine adrenal medullary chromaffin granules. Chromatography on concanavalin A-Sepharose and Sephacryl S-200, chromatofocusing, and chromatography on thiopropyl-Sepharose resulted in an 88,000-fold purification with a recovery of 35% of enzyme activity. The thiol protease is a glycoprotein with a pI of 6.0. It cleaves [35S]methionine preproenkephalin with a pH optimum of 5.5, indicating that it is functional at the intragranular pH of 5.5-6.0. Interestingly, production of trichloroacetic acid-soluble products was optimal at pH 4.0, suggesting that processing of initial precursor and intermediates may require slightly different pH conditions. The protease requires dithiothreitol for activity and is inhibited by the thiol protease inhibitors iodoacetate, p-hydroxymercuribenzoate, mercuric chloride, and cystatin. These properties distinguish it from other thiol proteases (cathepsins B, H, L, N, and S), indicating that a unique thiol protease has been identified. The enzyme converted [35S]cysteine preproenkephalin (possessing [35S]cysteine residues specifically within the precursor's NH2-terminal segment) to 22.1-, 21.6-, 17.7-, 17.3-, and 15.0-kDa intermediates that contain the precursor's NH2-terminal segment; proenkephalin in vivo is converted to similar intermediates. The enzyme cleaves peptide F at Lys-Arg and Lys-Lys dibasic amino acid sites to generate methionine enkephalin and intermediates. The appropriate vesicular localization, pH optimum, proteolytic products, and cleavage site specificity suggest that this thiol protease may be involved in enkephalin precursor processing. Most interestingly, [35S]methionine beta-preprotachykinin, a precursor of substance P, is minimally cleaved, suggesting that the thiol protease may possess some selectivity for the enkephalin precursor.     

Isolation and partial characterization of two different subunits from the molybdenum-iron protein of Azotobacter vinelandii nitrogenase

The molybdenum-iron protein of Azotobacter vinelandii nitrogenase was separated into two subunits of equal concentration by ion exchange chromatography on sulfopropyl (SP) Sephadex at pH 5.4 in 7 M urea. Better than 90% yield of each subunit was obtained on a preparative scale if the reduced carboxymethylated molybdenum-iron protein was incubated at 45 degrees C for 45 min prior to chromatography. Without the heating step low yields of the subunits were obtained. Although the amino acid compositions of the two subunits were very similar, the NH2-terminal sequences were completely different as determined by automated sequential Edman degradation. The sequence for the alpha subunit was NH2-Ser-Gln-Gln-Val-Asp-Lys-Ile-Lys-Ala-Ser-Tyr-Pro-Leu-Phe-Leu-Asp-Gln-Asp-Tyr- and for the beta subunit the sequence was NH2-Thr-Gly-Met-Ser-Arg-Glu-Glu-Val-Glu-Ser-Leu-Ile-Gln-Glu-Val-Leu-Glu-Val-Tyr-. Likewise the COOH-terminal sequences for the two subunits, as determined with carboxypeptidase Y, were tota-ly different. The sequence for the alpha subunit was -Leu-Arg-Val-COOH and that for the beta subunit was -Ile-(Phe, Glu)-Ala-Phe-COOH. Radioautographs of tryptic peptide maps were prepared for the molybdenum-iron protein and the two subunits which had been labeled at the cysteinyl residues with iodo[2-14C]acetic acid. These maps indicated that the two subunits had no cysteinyl peptides in common and that the cysteinyl residues were clustered in both subunits.     

Human adenine phosphoribosyltransferase. Complete amino acid sequence of the erythrocyte enzyme

We defined the amino acid sequence of adenine phosphoribosyltransferase isolated from human erythrocytes. Peptide fragments formed by cleavage at arginine, lysine, glutamic acid, and methionine were purified by high pressure liquid chromatography and sequenced by manual Edman degradation. The complete primary structure of human adenine phosphoribosyltransferase was established by sequence analysis of 19 peptide fragments. Presumed homology between the human and rodent enzymes was used to order fragments that had inadequate overlapping sequences. The enzyme has 179 residues with a calculated subunit molecular weight of 19,481. Mass spectrometry indicated that the NH2-terminal residue is acetylated. Human adenine phosphoribosyltransferase has sequence homology with xanthine-guanine phosphoribosyltransferase from Escherichia coli in 110-amino acid region encompassing the NH2-terminal section of the enzyme.