Total error profiling of a proinsulin time-resolved fluorescence immunoassay.

We applied total error profiling to evaluate the conversion of a known proinsulin (PI) enzyme-linked immunosorbent assay (ELISA) into a time-resolved fluorescence immunoassay (TRFIA). The formula and acceptance criteria proposed by the Ligand Binding Assay Bioanalytical Focus Group (LBABFG) of the American Association of Pharmaceutical Scientists (AAPS) were applied. We found that the expected dynamic range enlargement with TRFIA compared to ELISA ([0.5-240] versus resp. [0.7-98] pmol/L) is limited by an interference of C-peptide when present in the sample at high concentrations (>7000 pmol/L).

Streamlined procedure for the production of normal and altered versions of recombinant human proinsulin.

A method for the simplified, reproducible production of both normal and altered versions of human proinsulin has been developed. A polyhistidine/proinsulin fusion protein was expressed using a prokaryotic expression system and partially purified by affinity chromatography. Disulfide bonds within the polypeptide were formed prior to removal of the affinity tag. The proinsulin cleaved from the fusion protein was then subjected to a final purification step of semipreparative reversed-phase high-performance liquid chromatography. Integrity of both the normal and mutant proinsulins was confirmed by peptide mapping and mass spectrometry. The different versions of proinsulin will be used to map those residues of the substrate used in cleavage site recognition by members of the furin/PC family of converting enzymes.

Proinsulin: recent observations and controversies.

Proinsulin has generally been regarded as an inert precursor to insulin. However, over the past few years, proinsulin has established itself as a useful research tool for understanding how cells synthesize and secrete peptide hormones. Last year, proinsulin attracted renewed interest for its role as the precursor to C peptide, which may prove useful in the treatment of patients suffering from insulin-dependent diabetes mellitus. This mini-review focuses on three aspects of proinsulin, each of which attracted attention in 1997. These three aspects illustrate how this peptide hormone precursor may yet prove to be more important than its primary role as a prohormone, with only one bioactive product, would suggest.

Expression, purification and characterization of recombinant human proinsulin.

We have recently developed a method to produce native human proinsulin using a bacterial expression system. A proinsulin fusion protein was recovered from inclusion bodies and cleaved using cyanogen bromide. The released proinsulin polypeptide was S-sulfonated and purified by anion exchange chromatography. Following refolding, proinsulin was purified by reversed-phase high-performance liquid chromatography. Combined peptide mapping and mass spectrometric analysis indicated that the proinsulin contained the correct disulfide bridging pattern. This proinsulin will be used to study the specificity of the furin/PC family of converting enzymes by using it as a substrate in a recently developed assay.

Evidence for redundancy in propeptide/prohormone convertase activities in processing proglucagon: an antisense study.

To further examine the physiological roles of the neuroendocrine prohormone convertases (PCs) in proglucagon processing, alpha TC1-6 cells were transiently transfected with PC1/3 and PC2 expression vectors containing either antisense or sense encoding cDNAs. PC1/3- and PC2-directed RIAs were used to determine that the PC1/3 antisense transfections lowered endogenous levels of PC1/3 by 40 +/- 7.9% but did not alter the levels of PC2. The PC2 antisense transfections decreased the endogenous levels of PC2 by 91 +/- 11.7% without affecting the levels of PC1/3. To quantitate the levels of proglucagon and proglucagon-derived products, transfected cells were metabolically labeled with [3H]tryptophan, and extracts were chromatographed by reversed-phase HPLC. Recovered peptides were then subjected to peptide mapping analyses, allowing precise quantification of 3H-radioactivity incorporated into proglucagon and its cleavage products. Product-precursor ratios were determined, and percent change in the proportion of products generated in antisense-transfected vs. sense-transfected cells was calculated. The decrease in PC1/3 after antisense treatment significantly reduced the amounts of glicentin produced and partially reduced the levels of all other proglucagon cleavage products. PC2 antisense treatment significantly reduced the levels of glicentin and 9K glucagon generated but had no significant effect on the remainder of the proglucagon-derived peptides. These results suggest the existence of redundant mechanisms that ensure the production of each of the intermediate and product peptides derived from proglucagon. PC1/3 is potentially an important enzyme in the processing of most proglucagon-derived peptides, whereas PC2-processing activity appears to predominate at only two of the four potential cleavage sites.

Processing of mouse proglucagon by recombinant prohormone convertase 1 and immunopurified prohormone convertase 2 in vitro.

The mouse tumor cell line alpha TC1-6 was used as a model system to examine the post-translational processing of proglucagon. Determination of the mouse preproglucagon cDNA sequence and comparison with the published sequences of rat and human preproglucagons revealed nucleic acid homologies of 89.1 and 84%, respectively, and amino acid homologies of 94 and 89.4%, respectively. Immunohistochemical analyses with antibodies directed against PC2 and glucagon colocalized both the enzyme and substrate within the same secretory granules. PC1 was also immunolocalized in secretory granules. Cells were metabolically labeled with [3H]tryptophan, and extracts were analyzed by reverse-phase high pressure liquid chromatography. Radioactive peptides with retention times identical to those of synthetic peptide standards were recovered and subjected to peptide mapping to verify their identities. To determine the potential role of PC1 and PC2 in proglucagon processing, 3H-labeled proglucagon was incubated in vitro with recombinant PC1 and/or immunopurified PC2. Both enzymes cleaved proglucagon to yield the major proglucagon fragment, glicentin, and oxyntomodulin, whereas only PC1 released glucagon-like peptide-I from the major proglucagon fragment. Neither PC1 nor PC2 processed glucagon from proglucagon in vitro. These results suggest a potential role for PC1 and/or PC2 in cleaving several of the normal products, excluding glucagon, from the mouse proglucagon precursor.

The anglerfish somatostatin-28-generating propeptide converting enzyme is an aspartyl protease.

An enzyme that performs the conversion of anglerfish prosomatostatin-II (pro-SS-II) to anglerfish SS-28 has been identified using an improved two-dimensional electrophoresis procedure. The enzyme is a single chain 39 kDa polypeptide with its isoelectric point at pH 5.9. The converting enzyme has an acidic pH optimum, consistent with the lowered pH of the intracellular site of propeptide conversion. Secretory granule extracts were examined to determine the inhibitor sensitivity and pH optimum of the conversion of anglerfish pro-SS-II to anglerfish SS-28 in this organelle. Production of anglerfish SS-28 by secretory granules was maximal at pH 4.2 and was completely inhibited by the addition of pepstatin. Since pepstatin is a specific inhibitor of aspartyl proteases, these results indicate that the purified enzyme is a member of this enzyme family. This conclusion was supported by the data from partial amino acid sequence analysis. Because these results are consistent with the role of the purified enzyme in the in vivo production of anglerfish SS-28, the identified aspartyl protease has been termed the anglerfish SS-28-generating propeptide-converting enzyme.

Identification of a somatostatin-14-generating propeptide converting enzyme as a member of the kex2/furin/PC family.

A propeptide converting enzyme capable of producing somatostatin-14 has been identified and partially characterized from anglerfish pancreatic islets. Results from N-terminal protein sequence analysis indicate that this enzyme is a member of the kex2/furin/PC family. This observation provides strong corroborative evidence that the kex2/furin/PC protease family is involved in propeptide conversion. Comparison of the obtained protein sequence with the cDNA sequence of mammalian PC2 also suggests that the active enzyme is derived from a precursor by cleavage at a site containing four consecutive basic amino acids.

Improved method for identification of proteins using two-dimensional electrophoresis with immobilized pH gradient isoelectric focusing.

Recent advances in protein sequence analysis now permit the determination of partial N-terminal and internal primary structure from low picomole quantities of protein. The major remaining hurdles to sequence analysis of small amounts of protein are the identification, isolation, and handling of microgram and submicrogram quantities of protein. The technique of two-dimensional electrophoresis using immobilized pH gradient isoelectric focusing circumvents many of these problems. However, poor correlation between the first and second dimension have prevented use of this technique for the identification of some proteins which can only be assayed prior to the denaturing conditions used in the second dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis procedure. An improved method is presented which allows correlation of the native biological activity (first dimension) to a silver stained protein (second dimension) with a high degree of confidence.

Primary structure and tissue distribution of anglerfish carboxypeptidase H.

Most peptide hormones are synthesized as part of larger precursor proteins which must be processed after translation to generate bioactive peptides. This usually involves cleavage of the precursor by an endopeptidase at sites marked by basic amino acids, followed by removal of N- or C-terminal basic residues by the action of an aminopeptidase or carboxypeptidase. These processing events have been observed in a variety of species, from yeast to mammals. As part of an effort to characterize prohormone processing enzymes in the anglerfish, Lophius americanus, we have cloned and sequenced a cDNA for the fish prohormone processing carboxypeptidase H (CPH). Polyadenylated RNA from anglerfish (AF) islet organs was used to construct a cDNA library in phage lambda gt11. The library was screened with a probe derived from the cDNA for rat CPH. A 2400 base pair AF cDNA clone was isolated. This cDNA encodes a polypeptide which is similar in size and composition to mammalian CPH. The sequence data indicate that the AF CPH precursor is a 454 amino acid polypeptide. The derived amino acid sequence of the putative fish CPH is 81% homologous to the rat and bovine CPH enzymes. Significantly, all of the amino acid residues thought to be important for metal ion and substrate binding, glycosylation, and catalytic activity of mammalian CPH are conserved in the fish enzyme. Northern hybridization using RNA from AF tissues indicates that a 2.5 kb fish CPH mRNA is expressed in brain, pituitary and islet organs, but not in other tissues which do not secrete peptide hormones.

Purification of prosomatostatin-converting enzymes.

The enzymes responsible for performing cleavage of propeptides at basic amino acids have proven difficult to characterize. Using the processing of anglerfish islet prosomatostatin (PSS) as a model system, we are pursuing the characterization of both a single basic amino acid-specific and a dibasic amino acid-specific converting enzyme. We describe here the model system and protein isolation methods that have allowed significant progress toward complete characterization of the somatostatin-generating propeptide converting enzymes (PCEs).

Peptidyl-glycine alpha-amidating monooxygenase is present in islet secretory granules of the anglerfish, Lophius americanus.

Anglerfish islet secretory granules have been examined for the presence of an enzyme which could perform C-terminal amidation of glucagon-like peptide II and possibly anglerfish peptide Y. Using [125I]D-Tyr-Val-Gly as substrate, a peptidyl-glycine alpha-amidating monooxygenase (PAM) was detected in islet secretory granule lysates. The enzyme is active between pH 6.0 and 8.5 and exhibits maximal activity near pH 7.0. The islet PAM requires Cu2+, ascorbate, and molecular oxygen for activity. Other divalent metal ions and redox cofactors were tested and found to be inactive in the assay. Even though added Cu2+ and ascorbate are required for detecting islet PAM activity, when these factors were incubated with substrate in the absence of secretory granule lysate, no activity was observed. It was also found that the addition of higher than optimal concentrations of either Cu2+ or ascorbate inhibited amidating activity. The results demonstrate that a PAM is present in secretory granules of anglerfish islet tissue. The characteristics of the islet PAM are similar to those of PAMs identified and characterized in other tissues which produce bioactive C-terminally amidated peptides.

Direct evidence for two distinct prosomatostatin converting enzymes. Detection using a rapid, sensitive, and specific assay for propeptide converting enzymes.

Many bioactive peptides are initially synthesized via larger precursors from which they are released by proteolytic cleavage at basic amino acids. Some precursors contain more than one final product peptide, multiple copies of a single peptide, or both. Different product peptides can be produced from a common precursor in different tissues. It is not currently known whether this cell-type specific production of bioactive peptides is mediated by different, specific propeptide converting enzymes (PCEs) or by a small number of similar PCEs. To resolve this issue for the conversion of prosomatostatin, the processing of prosomatostatin-I (aPSS-I) and prosomatostatin-II (aPSS-II) to either somatostatin-14 (SS-14) or somatostatin-28 (aSS-28), respectively, was examined in anglerfish islets. Two distinct forms of PSS PCE activity were detected using a rapid, sensitive, and specific assay. Examination of the specificity of these two enzyme activities showed that one proteolytic activity performs the aPSS-I to SS-14 conversion, while the other protease liberates aSS-28 from aPSS-II. The SS-14-generating PCE also cleaves aPSS-II to produce [Tyr7,Gly10]SS-14 (a tetra-decapeptide analog of SS-14) and converts proinsulin to insulin. The aSS-28-generating PCE does not process proinsulin. These results provide direct evidence that different, specific PCEs are required for liberation of SS-14 and aSS-28 from their precursors.

Characterization of an islet carboxypeptidase B involved in prohormone processing.

An islet carboxypeptidase B-like enzyme (CP B) has been identified and characterized in secretory granules of anglerfish islets. By employing several different column chromatography methods (gel filtration, ion exchange, and hydroxylapatite), it was determined that the islet secretory granules contained only one detectable CP B. This enzyme is present in both secretory granule- and microsome-enriched subcellular fractions and is membrane associated at pH 5.2. The specific activity of the islet CP B was approximately 4-fold higher in the secretory granule- and microsome-enriched subcellular fractions than in the lysosome-enriched fraction. It is a metallo-enzyme that is stimulated by Co++, and has a pH optimum in the range of 5.2-6.2. The isoelectric point of the islet CP B is at pH 4.9. The enzyme is a glycoprotein and has an approximate molecular size of Mr 30,000 by gel filtration. The substrate analogs guanidinoethylmercaptosuccinic acid, guanidinopropylsuccinic acid, and aminopropylmercaptosuccinic acid competitively inhibited the islet CP B with inhibition constant (Ki) values of 23, 21, and 230 nM, respectively. In experiments employing purified prohormone substrates it was demonstrated that the action of a CP B-like enzyme was required for the complete processing of anglerfish proinsulin and prosomatostatin-II. These results indicate that the anglerfish islet CP B is involved in prohormone processing and has properties which are very similar to those of enkephalin convertase.

Islet secretory granules contain cytochrome b561.

A cytochrome has been detected in secretory granules prepared from anglerfish islets of Langerhans. The heme moiety was determined to be of the b type, and the dithionite-reduced cytochrome exhibited an alpha-band maximum at 561 nm with an extinction coefficient of 13.8 mM-1 X cm-1. The protein was present at a concentration of 40 +/- 4 pmol/mg of secretory granule protein. The cytochrome was found to be an integral membrane protein and to be reduced by ascorbic acid but not by NADH, NADPH, reduced glutathione (GSH), or succinate. Because of the similarity to previously characterized secretory granule cytochrome b561's from neuroendocrine tissues, this cytochrome is also referred to as cytochrome b561. Although its function has not yet been elucidated, the apparent specificity for ascorbate suggests that it may be a component of the ascorbate-dependent peptidyl-glycine alpha-amidating monooxygenase system that functions in the amidation of islet hormones.

Organ specificity of glucocorticoid-sensitive tyrosine aminotransferase. Separation from aspartate aminotransferase isoenzymes.

In order to study whether hormone-sensitive tyrosine aminotransferase exists in tissues other than liver, we have devised means to separate the liver-specific enzyme from other enzymes that transaminate tyrosine and to distinguish between the authentic enzyme and the principal "pseudotyrosine aminotransferases," which are the isoenzymes of aspartate aminotransferase. We accomplish this by suppressing proteolysis of the authentic enzyme using a buffer of pH 8.0 containing 0.1 M potassium chloride; enzyme extracted from liver in this buffer migrates as a single peak during chromatography on hydroxylapatite and represents the undegraded native form. A much smaller peak of tyrosine aminotransferase activity elutes at higher ionic strength and corresponds to a mixture of mitochondrial aspartate aminotransferase and partially degraded tyrosine aminotransferase. Cytosolic aspartate aminotransferase, in contrast, adsorbs weakly to the hydroxylapatite column and transaminates tyrosine very poorly although it readily utilizes monoiodotyrosine. The aspartate aminotransferase isoenzymes separate completely from tyrosine aminotransferase during chromatography on DEAE-Sepharose CL-6B. By combining these techniques with the use of specific antibodies, we show that brain, heart, and kidney do not contain tyrosine aminotransferase. Furthermore, we locate both isoenzymes of aspartate aminotransferase on polyacrylamide gels and show that both react histochemically as tyrosine aminotransferases when monoiodotyrosine is used as substrate. Use of these techniques, therefore, permits unambiguous identification of tyrosine aminotransferase and its separation from the background of nonspecific transamination.