Paired Basic Residues Research Articles

A putative opioid-peptid processing activity in enriched golgi fraction from rat brain

A Golgi enriched fraction from rat brain was prepared. The preparation has no carboxypeptidase activity and is not contaminated with cytosol, mitochondria and lysosomes as judged by marker enzyme activities for these constituents. Associated with the Golgi membranes a putative opioid peptide processing activity was demonstrated, which acts on Dynorphin 1–13, α- and β-Neoendorphin. The enzyme cleaves the bond between the paired basic residues, releasing Leucine-enkephalin-Arg 6. The activity has a pH-optimum around 9 and is inhibited by serine-protease inhibitors. Intracellular location and substrate specificity suggest that this endopeptidase activity may be involved in proenkephalin processing.

Secretion of somatostatin by Saccharomyces cerevisiae. Correct processing of an alpha-factor-somatostatin hybrid.

Somatostatin is a 14-amino acid peptide hormone that is proteolytically processed from its precursor, prosomatostatin, by a paired-basic-specific protease localized in the Golgi apparatus and secretory vesicles. Yeast (Saccharomyces cerevisiae MAT alpha) synthesize an analogous peptide hormone precursor, pro-alpha-factor, that contains tandem repeats of alpha factor (13 amino acids) flanked by spacers that include paired basic residues. To investigate the role of these two pro regions in mediating intracellular transport and processing, cloned genes specific for preprosomatostatin and prepro-alpha-factor were used to generate recombinants encoding hybrids between the alpha-factor pro region (amino-terminal) and somatostatin (carboxyl-terminal). These recombinants were inserted into yeast expression vectors under control of either the native alpha-factor promoter or the inducible yeast PHO5 (acid phosphatase) promoter. Yeast transformed with these plasmids expressed the hybrid messenger RNAs constitutively (alpha-factor promoter) or when induced in phosphate-deficient medium (PHO5 promoter). Radioimmunoassay of culture media revealed the secretion of up to 200 ng of immunoreactive somatostatin/10(7) cells. Metabolic labeling with [35S]cysteine, followed by immunoprecipitation with anti-somatostatin antibodies revealed two forms of hybrid precursor intracellularly, one of Mr 25,000, containing core carbohydrates, and a second of Mr 11,000, which was unglycosylated. Translation of mRNA extracted from these transformants in the wheat germ cell-free system revealed that the Mr 11,000 form was the primary translation product, whereas the Mr 25,000 species could be generated in vitro by inclusion of mammalian rough microsomes. The secreted immunoreactive material was shown to be authentic somatostatin by high pressure liquid chromatography analysis and protein sequencing. These results demonstrate that the yeast processing enzymes recognize these chimeric precursors, resulting in the secretion of the mature peptide hormone.

An endopeptidase associated with bovine neurohypophysis secretory granules cleaves pro-ocytocin/neurophysin peptide at paired basic residues

The octacosapeptide sequence [Tyr 18] pro-ocytocin/neurophysin (1–18)NH 2 [ pro-OT Np(1–18)NH 2 ] was synthesized and used as substrate to detect endoprotease(s) possibly involved in the processing of this precursor in bovine hypothalamo-neurohypophyseal tract. An endopeptidase (58 Kda) was detected in lysates made from highly purified neurosecretory granules. This protease which cleaves the peptide bond on the carboxyl side of the Lys-Arg doublet, and no single basic residue, generates both OT-Gly 10-Lys 11-Arg 12+Ala 13-Val-Leu-Asp-Leu-Tyr 18(NH 2) from the octacosapeptide substrate. In addition, a carboxypeptidase B-like activity converting OT-Gly 10-Lys 11-Arg 12 into OT-Gly 10 was detected in the same granule lysates. It is hypothesized that a combination of these endoprotease and carboxypeptidase B-like activities together with the amidating enzyme of secretory granules might participate in the cleavage and processing of pro-OT Np in vivo .

Identification and primary structural analysis of peptide II, an end-product of precursor processing in cells R 3-R 14 of Aplysia

Peptide II, which is encoded on a gene for a precursor protein in abdominal ganglion neurons R 3-R 14, was purified from extracts of abdominal ganglia of Aplysia californica. Native peptide II comigrates with synthetic standards on HPLC under isocratic conditions. Amino acid sequence and composition analyses indicate that the sequence of peptide II is Glu-Ala-Glu-Glu-Pro-Ser-Phe-Met-Thr-Arg-Leu, as predicted from the precursor. The molluscan cardioexcitatory peptide Phe-Met-Arg-Phe-amide was also identified in abdominal ganglion extracts by similar means. The large amount of peptide II recovered (100 ng/ganglion), and its location on the precursor between two pairs of basic residues, strongly suggest that the precursor is processed into peptide II and at least two other peptides. Although cells R 3-R 14 have been postulated to play a role in cardiovascular control, peptide II was without effect at ≤10 −4 M concentrations on identified abdominal ganglion neurons, the gastroesophageal artery or the heart. The physiological role of peptide II therefore remains to be elucidated.

Extracellular processing of proapolipoprotein A-II in Hep G2 cell cultures is mediated by a 54-kDa protease immunologically related to cathepsin B.

Apolipoprotein A-II is the second most abundant polypeptide found in human plasma high density lipoprotein particles. The primary translation product of human apo-A-II mRNA is a prepropolypeptide. We have previously reported (Gordon, J. I., Sims, H. F., Edelstein, C., Scanu, A. M., and Strauss, A. W. (1984) J. Biol. Chem. 259, 15556-15563) that the prosegment of apo-A-II was removed following export from a human hepatoma cell line (Hep G2). This represented a novel processing compartment for prosegments terminating with paired basic residues and differed from the processing of proalbumin which occurred with high efficiency prior to export from these cells. We have now characterized the enzyme responsible for this extracellular cleavage. The proapo-A-II converting activity is blocked by the thiol protease inhibitors antipain, E-64, leupeptin, and Ala-Lys-Arg chloromethyl ketone. Incubation of 125I-iodotyrosylated Ala-Lys-Arg chloromethyl ketone with serum-free media harvested from cell cultures over a 12-h period revealed a time-dependent accumulation of a 54-kDa protease. Although small quantities of the 54-kDa protease were detected in cell lysates, the major intracellular sequences labeled by the affinity probe had masses of 31.5 and 6 kDa. The 54-kDa extracellular, as well as 31.5- and 6-kDa intracellular, species were all immunoprecipitated by monospecific anti-human liver cathepsin B IgG. Addition of this antibody to media inhibited extracellular conversion of proapo-A-II to the mature protein. Based on these observations, we conclude that a "pro" cathepsin B-like protease exported by Hep G2 cells is responsible for proapo-A-II prosegment removal. It appears that cathepsin B-like proteases exhibit a complex pattern of segregation within the secretory pathway and that larger molecular weight forms of cathepsin B-like proteases are capable of accurately processing propolypeptides.

Isolation and characterization of an endogenous C-terminal fragment of the alpha-neo-endorphin/dynorphin precursor from bovine caudate nucleus

Antibodies have been raised to a synthetic peptide corresponding to the C-terminal 15-amino acid residues of prodynorphin, the common precursor to the neo-endorphins and dynorphins. The amino acid sequence of the antigen was based on the sequence deduced from mRNA isolated and cloned from porcine hypothalamus (Kakidani, H., Y. Furutani, H. Takahashi, M. Noda, Y. Morimoto, T. Hirose, M. Asai, S. Inayama, S. Nakanishi, and S. Numa (1982) Nature 298: 245-248). Using a radioimmunoassay developed from these antibodies we have isolated an endogenous prodynorphin C-fragment from bovine caudate nucleus. The isolated peptide displayed characteristics on gel filtration similar to those of synthetic prodynorphin C-fragment predicted from the porcine mRNA sequence but had low cross-reactivity in the radioimmunoassay. Sequencing and amino acid analysis showed a substitution of serine for asparagine at position 6 in the porcine sequence. Dynorphin B (rimorphin), which is adjacent to prodynorphin C-fragment in the precursor, was isolated from the same extract. Amino acid analysis and elution position on a gel filtration column confirmed its structure as that previously characterized from bovine pituitary extracts. The release of prodynorphin C-fragment and the C-terminus of dynorphin B from the porcine precursor would require cleavage at a single arginine residue. However, a terminal arginine was not present on either of these prodynorphin peptides isolated from bovine caudate. The data would suggest that processing at a single arginine residue results in elimination of the arginine, a feature in common with processing at paired basic residues.

Purification and characterization of a paired basic residue-specific pro-opiomelanocortin converting enzyme from bovine pituitary intermediate lobe secretory vesicles.

Pro-opiomelanocortin (adrenocorticotropin/endorphin prohormone) is processed to yield active hormones by cleavages at paired basic amino acid residues. In this study, an enzyme that specifically cleaves at the paired basic residues of this prohormone has been purified from bovine pituitary intermediate lobe secretory vesicles, the intracellular processing site of proopiomelanocortin. This enzyme, named pro-opiomelanocortin converting enzyme, has been characterized as a glycoprotein of Mr approximately 70,000. It has an apparent isoelectric point between 3.5 and 4.0. The pH optimum of the pro-opiomelanocortin converting enzyme is between 4 and 5, but the enzyme is highly active at the intravesicular pH of 5.1-5.6. The enzyme specifically cleaved the Lys-Arg pairs of pro-opiomelanocortin to yield Mr = to 21,000-23,000 ACTH, beta-lipotropin, Mr 13,000 and 4,500 ACTH, beta-endorphin, and a Mr = 16,000 NH2-terminal glycopeptide, the products synthesized by the pituitary intermediate lobe in situ. NH2- and COOH-terminal analysis of the products indicated that the pro-opiomelanocortin converting enzyme cleaves the peptide bond either between the Lys and Arg or on the carboxyl side of the Arg at Lys-Arg pairs of pro-opiomelanocortin. The intracellular localization, pH optimum, and cleavage specificity of the enzyme suggest that it may function as a pro-opiomelanocortin processing enzyme in the pituitary intermediate lobe in vivo.

Cloning and sequence analysis of a cDNA for rat transforming growth factor-alpha.

Transforming growth factors (TGFs) are mitogenic polypeptides produced most conspicuously by transformed cells and conferring on normal cells several phenotypic alterations associated with transformation. TGFs comprise two distinct sets of molecules: TGF-alpha s are structurally similar to epidermal growth factor (EGF), binding to and inducing the tyrosine phosphorylation of the EGF receptor in a manner indistinguishable from that of EGF. In addition, the 50-amino acid rat TGF-alpha has 33 and 44% homologies with mouse and human EGFs, respectively, and shares with EGFs a conserved pattern of three disulphide bridges. Thus, it has been proposed that TGF-alpha s belong to a family of EGF-like polypeptides. TGF-beta s, on the other hand, display no measurable binding to EGF receptors, but potentiate the growth-stimulating activities of TGF-alpha. Here we report the isolation of a complementary DNA clone encoding rat TGF-alpha. This cDNA hybridizes to a 4.5-kilobase (kb) messenger RNA that is 30 times larger than necessary to code for a 50-amino acid polypeptide and is present not only in retrovirus-transformed rat cells but also at lower levels in normal rat tissues. The nucleotide sequence of the cDNA predicts that TGF-alpha is synthesized as a larger product and that the larger form may exist as a transmembrane protein. However, unlike many polypeptide hormones (including EGF), cleavage of the 50-amino acid TGF-alpha from the larger form does not occur at paired basic residues, but rather between alanine and valine residues, suggesting the role of a novel protease.

Proenkephalin processing enzyme with specificity toward paired basic residues purified from bovine adrenal chromaffin granules

A novel protease exhibiting substrate specificity toward paired basic residues has been partially purified from the soluble fraction of bovine adrenal chromaffin granules by utilizing an affinity chromatography on STI-Sepharose. The enzyme, with optimal pH around 7.5–9.5, is classified into a serine-protease by its inhibitor spectrum. The enzyme specifically cleaved the Lys-Arg bonds of two synthetic peptides containing the subsequence of proenkephalin A, but endogenous opioid peptides containing a single basic residue in the molecule ((Met)enk-Arg-Phe, (Met)enk-Arg-Gly-Leu) were not affected by the enzyme. The unique substrate specificity of the enzyme, which is well in accord with the processing pattern of proenkephalin A in adrenal medulla, indicates that the enzyme may be physiologically involved in proenkephalin processing.

Isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla.

Biologically active peptide hormones and neurotransmitters have been shown to be enzymatically liberated from larger, inactive precursor molecules by tissue-specific post-translational processing, particularly at the typical cleavage signals of paired basic residues. Subsequent N-terminal or C-terminal modifications may be of importance in regulating the biological activities of these peptides. C-terminal alpha-amidation is considered to be essential for the biological function of several non-opioid peptides. Here we present the isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla. Amidorphin and the recently isolated octapeptide metorphamide (adrenorphin) are the only endogenous opioid peptides in mammals known to possess a C-terminal amide group. The amino acid sequence of amidorphin corresponds to the sequence 104-129 of bovine proenkephalin A. Very high concentrations of amidorphin were detected in bovine adrenal medulla and in a further endocrinological system, the hypothalamic-neurohypophyseal axis. Amidorphin may therefore be considered to be a major gene product of the opioid peptide precursor proenkephalin A in these endocrine tissues.

Human proapolipoprotein A-II is cleaved following secretion from Hep G2 cells by a thiol protease.

The two principal high-density lipoprotein apolipoproteins A-I and A-II are both initially synthesized as preproproteins. The prosegment of apo-A-I is unusual: it ends with paired glutamine residues and is removed extracellularly. The apo-A-II prosegment resembles the propeptides of prohormones and proalbumin: it ends with paired basic amino acids. We have studied the processing of proapo-A-II in a human hepatoma cell line (Hep G2) which is known to accurately and efficiently remove the prosegment from proalbumin prior to secretion. Pulse-chase experiments were performed in order to determine if the apo-A-II prosegment is removed prior to or after secretion. Apo-A-II was purified from cell lysates and media at various times during the chase and subjected to automated sequential Edman degradation. The results indicate that proteolytic processing of proapo-A-II is largely an extracellular event. These cells secrete the protease responsible for prosegment removal. The converting activity present in media is not blocked by serine protease inhibitors (phenylmethanesulfonyl fluoride, aprotinin, and furoyl saccharin) or by a metalloprotease inhibitor (o-phenanthroline). It is inhibited by the thiol protease reagents p-chloromercuribenezene-sulfonic acid and leupeptin. Prosegment removal changes the pI of the dominant apo-A-II isoform from 6.61 to 4.95. The presence of the propeptide does not prevent specific in vitro recombination of apo-A-II with high-density lipoprotein3 particles present in normolipemic serum. Extracellular processing after a single basic amino acid has been described for a variety of precursor proteins. Extracellular cleavage of the apo-A-II propeptide after paired COOH-terminal basic residues represents a novel processing pathway.

A novel protease from yeast with specificity towards paired basic residues

Paired basic residues have been observed as sites of proteolytic processing of prohormones in a wide range of eukaryotic species. This strongly suggests that proteases exhibiting specificity towards paired basic residues may be involved in prohormone processing, but candidate enzymes have not so far been identified. Yeast Saccharomyces cerevisiae alpha-cells synthesize and secrete alpha-mating factor, a peptide of 13 amino acids, the processing of which from a larger precursor involves cleavage at paired basic residues (-Lys-Arg-). We have therefore used them as a simple model system for the study of prohormone processing and report here the identification, in cell lysates, of a novel protease which specifically recognizes and cleaves the peptide bonds between consecutive basic residues. The purified enzyme, which we have called propheromone -convertase Y, has a molecular weight (MW) of around 43,000. It cleaves various peptide substrates at paired basic residues, but not at single basic residues, implying it is distinct from trypsin-like proteases. Its unique substrate specificity suggests the enzyme may be involved in propheromone processing in vivo.

Golgi/granule processing of peptide hormone and neuropeptide precursors: a minireview.

Proteolytic processing of precursor proteins is a phylogenetically ancient and widely used mechanism for producing biologically active peptides. Proteolytic cleavage of proproteins begins only after transport to the Golgi apparatus has been completed and in most systems may continue for many hours within newly formed secretory vesicles as these are stored in the cytosol or transported along axons to more peripheral sites of release. Paired basic residues are required for efficient proteolysis in most precursors, suggesting that a small number of specialized tryptic proteases exist that have great site selectivity but can process many sites within the same precursor or in different precursors within the same cell, or in different cells or tissues. Cleavage-site choice may be strongly influenced by other factors, such as secondary and tertiary structure, but definitive structural information on precursor proteins is lacking. Modifications such as glycosylation, phosphorylation, and sulfation also are Golgi associated but are not known to influence proteolytic processing patterns. Golgi/granule processing also rarely occurs at sites other than pairs of basic amino acids, including single basic residues ( trypsinlike ), Leu-Ala, Leu-Ser, or Tyr-Ala bonds ( chymotrysinlike ) as well as other specialized nontryptic cleavages, suggesting that mixtures of proteases coexist in the Golgi/granule system. Cathepsin B-like thiol proteases, or their precursors, have been implicated as the major processing endopeptidases in several systems. Carboxypeptidase B-like enzymes also have been identified in secretion granules in several tissues and appear to be metalloenzymes similar in mechanism to the pancreatic carboxypeptidases, but with a lower pH optimum. The role of the Golgi apparatus in sorting newly formed secreted products from lysosomal hydrolases may have permitted the development in evolution of an intimate relationship between certain of the lysosomal degradative enzymes, such as cathepsin B or its precursors, and the Golgi/granule processing systems. The sequestration of the proteolytic products of precursors within secretion granules leads to the coordinate discharge of highly complex mixtures of peptides having related or overlapping biological activities. The cosecretion of nonfunctional peptide " leftovers ," such as the proinsulin C-peptide, can serve as useful markers of secretion or cellular localization, as well as of evolutionary relation ships. Errors in cleavage due to point mutations in precursors have been identified in several systems, leading to the accumulation of incorrectly processed materials in the circulation. These and/or defects (ABSTRACT TRUNCATED AT 400 WORDS)

Degradation of neuropeptides by calcium-activated neutral protease.

The substrate specificity of calcium-activated neutral protease (CANP) from monkey cardiac muscle was examined with various neuropeptides as substrates. The enzyme required mM order calcium ions for activation and had an enkephalinase activity, hydrolyzing Leu-enkephalin at the 1Tyr-2Gly and 3Gly-4Phe bonds. Furthermore, it showed the tendency to cleave especially the bonds around the paired basic amino acid residues in alpha- and beta-neoendorphins and dynorphin(1-13), while it could not hydrolyze substance P.

Biosynthesis of human preproapolipoprotein A-II.

The primary translation product of human apolipoprotein A-II was purified from wheat germ and ascites cell-free lysates programmed with RNA isolated from either a hepatocellular carcinoma cell line (HepG2) or intestinal epithelium. A-II mRNA represents 0.2% of the translatable RNA in these hepatocytes and in jejunal epithelium. Plasma high density lipoprotein-associated A-II is a 77-amino acid polypeptide. The primary translation product is 100 amino acids long and contains a 23-amino acid NH2-terminal extension. Cotranslational cleavage of the cell-free product indicated that this NH2-terminal sequence consists of an 18-amino acid long signal peptide, Met-Lys-Leu-Leu-Ala-Ala-X-Val-Leu-Leu-Leu-X-X-Cys-X-Leu-X-X-, and a 5-amino acid long propeptide, Ala-Leu-Val-Arg-Arg. This functional division was confirmed by sequencing the stable intracellular form of apolipoprotein A-II isolated from HepG2 cells. Approximately 45% of the proapo-A-II is cleaved to the mature form during export from HepG2 cells. The COOH-terminal dipeptide conforms to the rule that prosegments are cleaved after paired basic residues. We have previously shown (Gordon, J. I., Sims, H. F., Lentz, S. R., Edelstein, C., Scanu, A. M., and Strauss, A. W. (1983) J. Biol. Chem. 258, 4037-4044) that proapolipoprotein A-I is not cleaved during export from these cells and contains a prosegment with a COOH-terminal Gln-Gln dipeptide. Therefore, proteolytic processing of the two principal high density lipoprotein-associated apolipoproteins proceeds along different pathways.

Processing of prodynorphin at single and paired basic residues in porcine neurointermediate lobe

The [Leu] enkephalin-containing peptides derived from prodynorphin have been quantitated in porcine neurointermediate lobe. Dynorphin-17, β-neo-endorphin, [Leu] enkephalin, rimorphin and dynorphin-24 were found in highest concentrations (200–700 pmol/gm). No detectable amounts of the carboxyl terminal-extended rimorphin sequence (rimorphin 1–29) were observed, indicating that in addition to processing at paired basic residues, cleavage at the single Arg residue following rimorphin must be a major step in the processing of prodynorphin. Processing at Arg-Arg and/or Arg-Lys sequences to yield free [Leu] enkephalin also occurs to an appreciable extent in this tissue.

Pituitary endopeptidases.

This review summarizes our knowledge of pituitary endopeptidases. Emphasis has been placed on well-characterized enzymes and their potential roles in proteolytic processes of the pituitary. Because of space limitations, degradation of biologically active peptide by crude preparations has generally not been discussed. Only a few proteolytic enzymes are at present adequately characterized, and knowledge of their physiological function in vivo is insufficient. Among the many functions of proteolytic enzymes, those that are specific for the pituitary as an endocrine gland are of primary interest. Such functions include inactivation of neuropeptides and factors that control the secretory function of the pituitary, processing of precursors destined for secretion, selective cleavage of prohormones into active fragments, and degradation of inactive fragments. While some of the enzymes described here, such as cathepsin D, could be expected to have primarily a degradative function, others could potentially be involved in hormonal metabolism, since they exhibit trypsin-like, chymotrypsin-like, and dipeptidyl carboxypeptidase-like activities, all potentially useful in hormonal conversions. Data suggestive of the presence in the pituitary of enzymes involved in removal of the 'signal sequence', and enzymes involved in hormone processing by cleavage of bonds after a pair of basic residues and in the subsequent removal of these residues by a carboxypeptidase B-like activity have been published. None of these enzymes, however, has been isolated or purified to a degree that would allow determination of its specificity, mechanisms of action, physicochemical properties, and susceptibility to specific inhibitors. Questions that remain unresolved ask whether differences in the processing pathways in various anatomical parts of the pituitary are due to the presence of proteases with different specificities, or to different disposition of these enzymes, and factors, such as conformation of the substrate and its secondary modification, for example by glycosylation or phosphorylation. Proof of a functional involvement of a protease in hormonal processing should include demonstration that inhibition of activity results in inhibition of processing in the intact cell. Specific inhibitors of processing enzymes could potentially be used to modulate pituitary function, and thus have pharmacological interest. Although there are few answers to the above problems at present, the questions are well defined, and it can be expected that the rapidly expanding research on pituitary proteases will soon provide some of the answers.

Pro-opiocortin processing in the pituitary: A model for neuropeptide biosynthesis

The biosynthesis of α-MSH, β-endorphin and ACTH in the pituitary is reviewed. These neuropeptides are synthesized from a common pro-protein, pro-opiomelanocortin. The pro-protein is cleaved intragranularly, at pairs of basic residues in the molecule to yield the respective peptide products. An unique, thiol protease (pro-opiocortin converting enzyme) and a carboxypeptidase B-like enzyme, both localized within pituitary secretory granules and having a pH optimum of 5–6, appear to be involved in the proteolytic processing of pro-opiomelanocortin.

Cloning and sequence analysis of cDNA for bovine adrenal preproenkephalin.

The nucleotide sequence of cloned cDNA for preproenkephalin from bovine adrenal medulla indicates that the precursor protein contains four copies of Met-enkephalin and one copy each of Leu-enkephalin, Met-enkephalin-Arg6-Phe7 and Met-enkephalin-Arg6-Gly7-Leu8, a previously undetected opioid peptide. The enkephalin and extended enkephalin sequences are each bounded by paired basic amino acid residues. Preproenkephalin may represent a multi-hormone precursor, like the corticotropin-beta-lipotropin precursor.

The primary translation product of rat intestinal apolipoprotein A-I mRNA is an unusual preproprotein.

We have characterized the NH2-terminal sequence of the primary translation product of intestinal apolipoprotein A-I mRNA. Co-translational cleavage of this in vitro product and NH2-terminal sequence analysis of plasma high density lipoprotein-associated apolipoprotein A-I showed that it is initially synthesized as a preproprotein. The 24-amino-acid NH2-terminal extension consists of an 18-amino-acid presegment; Met-Lys-Ala-Ala-Val-Leu-Ala-Val-Ala-Leu-Val-Phe-Leu-Thr-Gly-Cys-Gln-Ala, and a hexapeptide prosegment; -X-Glu-Phe-X-Gln-Gln, followed by the NH2 terminus of the "mature" plasma protein: Asp-Glu-Pro-Gln-X-Gln-Trp-Asp-Arg-Val-Lys-Asp-Phe-Ala-X-Val-Tyr-X-Asp-Ala-Val. Although the prepeptide resembles other signal peptides, the prosegment differs from other propeptides in that it does not terminate with paired basic amino acid residues. These results suggest that translocation of nascent preproapolipoprotein A-I is similar to that of other secretory proteins. However, post-translational proteolytic processing of proapolipoprotein A-I must be unique, and could play a role in the formation of nascent high density lipoprotein particles.