Pro Segment Research Articles

Structural insights into latency of the metallopeptidase ulilysin (lysargiNase) and its unexpected inhibition by a sulfonyl-fluoride inhibitor of serine peptidases.

Peptidases are regulated by latency and inhibitors, as well as compatibilization and cofactors. Ulilysin from Methanosarcina acetivorans, also called lysargiNase, is an archaeal metallopeptidase (MP) that is biosynthesized as a zymogen with a 60-residue N-terminal prosegment (PS). In the presence of calcium, it self-activates to yield the mature enzyme, which specifically cleaves before basic residues and thus complements trypsin in proteomics workflows. Here, we obtained a low-resolution crystal structure of proulilysin, in which 28 protomers arranged as 14 dimers form a continuous double helix of 544 Å pitch that parallels cell axis b of the crystal. The PS includes two α-helices and obstructs the active-site cleft of the catalytic domain (CD) by traversing it in the opposite orientation of a substrate, and a cysteine blocks the catalytic zinc according to a "cysteine-switch mechanism". Moreover, the PS interacts through its first helix with an "S-loop" of the CD, which acts as an "activation segment" that lacks one of two essential calcium cations. Upon PS removal during maturation, the S-loop adopts its competent conformation and binds the second calcium ion. Next, we found that in addition to general MP inhibitors, ulilysin was competitively and reversibly inhibited by 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF; Ki = 4 μM). This is a compound that normally forms an irreversible covalent complex with serine peptidases but does not inhibit MPs. A high-resolution crystal structure of the complex revealed that the inhibitor penetrates the specificity pocket of ulilysin. A primary amine of the inhibitor salt-bridges an aspartate at the pocket bottom, thus mimicking the basic side chain of substrates. In contrast, the sulfonyl fluoride warhead is not involved and the catalytic zinc ion is freely accessible. Thus, the usage of inhibitor cocktails of peptidases, which typically contain AEBSF at ∼25-fold higher concentrations than the determined Ki, should be avoided when working with ulilysin. Finally, the structure of the complex, which occurred as a crystallographic dimer recurring in previous mature ulilysin structures, unveiled an N-terminal product fragment that delineated the non-primed side of the cleft. These results complement prior structures of ulilysin with primed-side product fragments and inhibitors.

Foldase and inhibitor functionalities of the pepsinogen prosegment are encoded within discrete segments of the 44 residue domain

Pepsin is initially produced as the zymogen pepsinogen, containing a 44 residue prosegment (PS) domain. When folded without the PS, pepsin forms a thermodynamically stable denatured state (refolded pepsin, Rp). To guide native folding, the PS binds to Rp, stabilizes the folding transition state, and binds tightly to native pepsin (Np), thereby driving the folding equilibrium to favor the native state. It is unknown whether these functionalities of the PS are encoded within the entire sequence or within discrete segments. PS residues 1p–29p correspond to a highly conserved region in pepsin-like aspartic proteases and we hypothesized that this segment is critical to PS-catalyzed folding. This notion was tested in the present study by characterizing the ability of various truncated PS peptides to bind Rp, catalyze folding from Rp to Np, and to inhibit Np. Four PS truncations were examined, corresponding to PS residues 1p–16p (PS1–16), 1p–29p (PS1–29), 17p–44p (PS17–44) and 30p–44p (PS30–44). The three PS functionalities could be ascribed primarily to discrete regions within the highly conserved motif: 1p–16p dictated Rp binding, 17p–29p dictated Np binding/inhibition, while the entire 1p–29p dictated transition state binding/catalyzing folding. Conversely, PS30–44 played no obvious role in PS-catalyzed folding; it is hypothesized that this more variable region may serve as a linker between PS1–29 and the mature domain. The high sequence conservation of PS1–29 and its role in catalyzing pepsin folding strongly suggest that there is a conserved PS-catalyzed folding mechanism shared by pepsin-like aspartic proteases with this motif.

Understanding the Mechanism of Prosegment-catalyzed Folding by Solution NMR Spectroscopy

Multidomain protein folding is often more complex than a two-state process, which leads to the spontaneous folding of the native state. Pepsin, a zymogen-derived enzyme, without its prosegment (PS), is irreversibly denatured and folds to a thermodynamically stable, non-native conformation, termed refolded pepsin, which is separated from native pepsin by a large activation barrier. While it is known that PS binds refolded pepsin and catalyzes its conversion to the native form, little structural details are known regarding this conversion. In this study, solution NMR was used to elucidate the PS-catalyzed folding mechanism by examining the key equilibrium states, e.g. native and refolded pepsin, both in the free and PS-bound states, and pepsinogen, the zymogen form of pepsin. Refolded pepsin was found to be partially structured and lacked the correct domain-domain structure and active-site cleft formed in the native state. Analysis of chemical shift data revealed that upon PS binding refolded pepsin folds into a state more similar to that of pepsinogen than to native pepsin. Comparison of pepsin folding by wild-type and mutant PSs, including a double mutant PS, indicated that hydrophobic interactions between residues of prosegment and refolded pepsin lower the folding activation barrier. A mechanism is proposed for the binding of PS to refolded pepsin and how the formation of the native structure is mediated.

1H, 13C, and 15N backbone resonance assignments of the porcine pepsin and porcine pepsin complexed with pepstatin

Pepsin is formed as the zymogen, pepsinogen, which includes an additional 44 residue prosegment (PS) on the N-terminus. Upon acidification (pH <3) the PS is removed, yielding active pepsin. The PS is critical to such processes as the initiation of correct folding and protein stability. In the present study, the NMR assignments of the 34.6 kDa native porcine pepsin and porcine pepsin complexed with pepstatin are reported in order to obtain structural information regarding PS-catalyzed protein folding. Such information would contribute to a better understanding of the nature of folding/unfolding energy barrier of pepsin and other aspartic proteases.

Dynamics of Thermodynamically Stable, Kinetically Trapped, and Inhibitor-Bound States of Pepsin

The pepsin folding mechanism involves a prosegment (PS) domain that catalyzes folding, which is then removed, resulting in a kinetically trapped native state. Although native pepsin (Np) is kinetically stable, it is irreversibly denatured due to a large folding barrier, and in the absence of the PS it folds to a more thermodynamically stable denatured state, termed refolded pepsin (Rp). This system serves as a model to understand the nature of kinetic barriers and folding transitions between compact states. Quasielastic neutron scattering (QENS) was used to characterize and compare the flexibility of Np, as a kinetically trapped state, with that of Rp, as a thermodynamically stable fold. Additionally, the dynamics of Np were compared with those of a partially unfolded form and a thermally stabilized, inhibitor-bound form. QENS revealed length-scale-dependent differences between Np and Rp on a picosecond timescale and indicated greater flexibility in Np, leading to the conclusion that kinetic stabilization likely does not correspond to reduced internal dynamics. Furthermore, large differences were observed upon inhibition, indicating that QENS of proteins in solution may prove useful for examining the role of conformational entropy changes in ligand binding.

The water-born protein pheromones of the polar protozoan ciliate, Euplotes nobilii: Coding genes and molecular structures

The protozoan ciliate Euplotes nobilii found in Antarctic and Arctic coastal waters relies on secretion of water-soluble cell type-specific signal proteins (pheromones) to regulate its vegetative growth and sexual mating. For three of these psychrophilic pheromones we previously determined the three-dimensional structures by nuclear magnetic resonance (NMR) spectroscopy with protein solutions purified from the natural sources, which led to evidence that their adaptation to cold is primarily achieved by increased flexibility through an extension of regions free of regular secondary structures, and by increased exposure of negative charges on the protein surface. Then we cloned the coding genes of these E. nobilii pheromones from the transcriptionally active cell somatic nucleus (macronucleus) and characterized the full-length sequences. These sequences all contain an open reading frame of 252–285 nucleotides, which is specific for a cytoplasmic pheromone precursor that requires two proteolytic cleavages to remove a signal peptide and a pro segment before release of the mature protein into the extracellular environment. The 5′ and 3′ non-coding regions are two- to three-fold longer than the coding region and appear to be tightly conserved, probably in relation to the inclusion of intron sequences destined to be alternatively removed to play key regulatory roles in the mechanism of the pheromone gene expression.

The Prosegment Catalyzes Pepsin Folding to a Kinetically Trapped Native State

Investigations of irreversible protein unfolding often assume that alterations to the unfolded state, rather than the nature of the native state itself, are the cause of the irreversibility. However, the present study describes a less common explanation for the irreversible denaturation of pepsin, a zymogen-derived aspartic peptidase. The presence of a large folding barrier combined with the thermodynamically metastable nature of the native state, the formation of which depends on a separate prosegment (PS) domain, is the source of the irreversibility. Pepsin is unable to refold to the native state upon return from denaturing conditions due to a large folding barrier (24.6 kcal/mol) and instead forms a thermodynamically stable, yet inactive, refolded state. The native state is kinetically stabilized by an unfolding activation energy of 24.5 kcal/mol, comparable to the folding barrier, indicating that native pepsin exists as a thermodynamically metastable state. However, in the presence of the PS, the native state becomes thermodynamically stable, and the PS catalyzes pepsin folding by stabilizing the folding transition state by 14.7 kcal/mol. Once folded, the PS is removed, and the native conformation exists as a kinetically trapped state. Thus, while PS-guided folding is thermodynamically driven, without the PS the pepsin energy landscape is dominated by kinetic barriers rather than by free energy differences between native and denatured states. As pepsin is the archetype of a broad class of aspartic peptidases of similar structure and function, and many require their PS for correct folding, these results suggest that the occurrence of native states optimized for kinetic rather than thermodynamic stability may be a common feature of protein design.

Recombinant prosegment peptide acts as a folding catalyst and inhibitor of native pepsin

Porcine pepsin A, a gastric aspartic peptidase, is initially produced as the zymogen pepsinogen that contains an N-terminal, 44 residue prosegment (PS) domain. In the absence of the PS, native pepsin (Np) is irreversibly denatured and when placed under refolding conditions, folds to a thermodynamically stable denatured state. This denatured, refolded pepsin (Rp) state can be converted to Np by the exogenous addition of the PS, which catalyzes the folding of Rp to Np. In order to thoroughly study the mechanism by which the PS catalyzes pepsin folding, a soluble protein expression system was developed to produce recombinant PS peptide in a highly pure form. Using this system, the wild-type and three-mutant PS forms, in which single residue substitutions were made (V4A, R8A and K36A), were expressed and purified. These PS peptides were characterized for their ability to inhibit Np enzymatic activity and to catalyze the folding of Rp to Np. The V4A, R8A and K36A mutant PS peptides were found to have nanomolar inhibition constants, K i, of 82.4, 58.3 and 95.6 nM, respectively, approximately a two-fold increase from that of the wild-type PS (36.2 nM). All three-mutant PS peptides were found to catalyze Np folding with a rate constant of 0.06 min − 1 , five-fold lower than that of the wild-type. The observation that the mutant PS peptides retained their inhibition and folding-catalyst functionality suggests a high level of resilience to mutations of the pepsin PS.

The platelet glycoprotein GPIbbeta intracellular domain participates in von Willebrand factor induced-filopodia formation independently of the Ser 166 phosphorylation site.

Circulating platelets are initially recruited at the site of vessel injury by von Willebrand factor (VWF) immobilized on collagen fibers. This process, mediated by the GPIb-V-IX complex, is accompanied by specific intracellular signaling leading to reorganization of the platelet actin cytoskeleton and extension of filopodia. To evaluate the GPIbalpha and GPIbbeta intracellular domains contribution to this signaling, we generated Chinese hamster ovary (CHO) cells expressing a GPIb-IX complex with mutant forms of the two subunits and we measured their ability to extend filopodia upon adhesion on a VWF matrix. Complete intracellular deletion or elimination of the filamin or the 14-3-3zeta binding sites in GPIbalpha did not prevent filopodia extension. In contrast, deletion of the juxtamembrane (Leu(150)-Arg(160)) or central (Ala(159)-Pro(170)) intracellular segment of GPIbbeta resulted in a 21% and 23% reduction in the number of cells extending filopodia, respectively. This occurred without decreasing adhesion efficiency or GPIb-IX association with filamin A or 14-3-3zeta. Alanine scanning mutagenesis of the Leu(150)-Pro(170) segment identified Arg(164), Leu(165), Leu(167), Thr(168) and Pro(170) as important residues for efficient filopodia formation. Surprisingly, mutation of the Ser(166) PKA phosphorylation site did not alter adhesion and shape change. A role for the GPIbbeta subunit was reinforced by the decreased capacity to extend filopodia upon adhesion on VWF of platelets from knock-in mice expressing a GPIbbeta intracellular deletion mutant. Altogether, our results strongly support participation of GPIbbeta and its intracellular region in GPIb-dependent platelet activation and shape change triggered by a VWF matrix.

Prosegment Catalyzes Pepsin Folding to a Kinetically Trapped Native State

Pepsin, an archetypical aspartic peptidase, initially contains an N-terminal, 44 residue prosegment (PS) that, upon folding, is removed to yield native pepsin (Np). Np is irreversibly denatured above pH 6, the basis for which was not understood. Our previous studies comparing Np and a denatured and subsequently refolded state (Rp) indicated that Rp was thermodynamically more stable than Np, having a higher Tm and being reversibly unfolded (Dee, D., et al., 2006, Biochemistry 45, 13982). Thus, it was suspected that Np is blocked from refolding by a large energy barrier, which is reduced by the action of the PS.

Successful production of recombinant buckwheat cysteine-rich aspartic protease in Escherichia coli

Herein, the expression of recombinant cysteine-rich atypical buckwheat (Fagopyrum esculentum) aspartic protease (FeAPL1) in five Escherichia coli strains differing in their expression capabilities is presented. It was shown that the expression success depended highly on the choice of FeAPL1 fusion partner. His6-FeAPL1 was produced in large quantities as an insoluble protein localized in inclusion bodies. On the other hand, MBP-FeAPL1 was localized in both the cytoplasm and inclusion bodies in BL21 and Rosetta-gami strains. Only purified soluble MBP-FeAPL1 from Rosetta-gami cells showed proteolytic activity at pH 3.0 with BSA as the substrate. The results also indicated that FeAPL1 contained a PRO segment that had to be removed for the enzyme activity to appear. The activity of FeAPL1 produced in the Rosetta-gami strain, which enables disulfide bond formation, indicated the importance of the twelve cysteine residues for correct folding and functionality.

Tuning the β‐turn segment in designed peptide β‐hairpins: Construction of a stable type I′ β‐turn nucleus and hairpin–helix transition promoting segments

Designed octapeptides Boc-Leu-Val-Val-Aib-(D)Xxx-Leu-Val-Val-OMe ((D)Xxx = (D)Ala, 3a;(D)Val, 3c and (D)Pro, 5a) and Boc-Leu-Phe-Val-Aib-(D)Ala-Leu-Phe-Val-OMe (3b) have been investigated to construct models of a stable type I' beta-turn nucleated hairpin and to generate systems for investigating helix-hairpin conformational transitions. Peptide 5a, which contains a central Aib-(D)Pro segment, is shown to adopt a stable type I' beta-turn nucleated hairpin structure, stabilized by four cross-strand hydrogen bonds. The stability of the structure in diverse solvents is established by the observation of all diagnostic NOEs expected in a beta-hairpin conformation. Replacement of (D)Pro5 by (D)Ala/(D)Val (3a-c) results in sequences that form beta-hairpins in hydrogen bonding solvents like CD(3)OH and DMSO-d(6). However, in CDCl(3) evidence for population of helical conformations is obtained. Peptide 6b (Boc-Leu-Phe-Val-Aib-Aib-Leu-Phe-Val-OMe), which contains a centrally positioned Aib-Aib segment, provides a clear example of a system, which exhibits a helical conformation in CDCl(3) and a significant population of both helices and hairpins in CD(3)OH and DMSO-d(6). The coexistence of multiple conformations is established by the simultaneous observation of diagnostic NOEs. Control over stereochemistry of the central beta-turn permits generation of models for robust beta-hairpins and also for the construction of systems that may be used to probe helix-hairpin conformational transitions.

Insight into autoproteolytic activation from the structure of cephalosporin acylase: a protein with two proteolytic chemistries.

Cephalosporin acylase (CA), a member of the N-terminal nucleophile hydrolase family, is activated through sequential primary and secondary autoproteolytic reactions with the release of a pro segment. We have determined crystal structures of four CA mutants. Two mutants are trapped after the primary cleavage, and the other two undergo secondary cleavage slowly. These structures provide a look at pro-segment conformation during activation in N-terminal nucleophile hydrolases. The highly strained helical pro segment of precursor is transformed into a relaxed loop in the intermediates, suggesting that the relaxation of structural constraints drives the primary cleavage reaction. The secondary autoproteolytic step has been proposed to be intermolecular. However, our analysis provides evidence that CA is processed in two sequential steps of intramolecular autoproteolysis involving two distinct residues in the active site, the first a serine and the second a glutamate.

Context-dependent conformation of diethylglycine residues in peptides.

Diethylglycine (Deg) residues incorporated into peptides can stabilize fully extended (C5) or helical conformations. The conformations of three tetrapeptides Boc-Xxx-Deg-Xxx-Deg-OMe (Xxx=Gly, GD4; Leu, LD4 and Pro, PD4) have been investigated by NMR. In the Gly and Leu peptides, NOE data suggest that the local conformations at the Deg residues are fully extended. Low temperature coefficients for the Deg(2) and Deg(4) NH groups are consistent with their inaccessibility to solvent, in a C5 conformation. NMR evidence supports a folded beta-turn conformation involving Deg(2)-Gly(3), stabilized by a 4-->1 intramolecular hydrogen bond between Pro(1) CO and Deg(4) NH in the proline containing peptide (PD4). The crystal structure of GD4 reveals a hydrated multiple turn conformation with Gly(1)-Deg(2) adopting a distorted type II/II' conformation, while the Deg(2)-Pro(3) segment adopts a type III/III' structure. A lone water molecule is inserted into the potential 4-->1 hydrogen bond of the Gly(1)-Deg(2) beta-turn.

Regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in innate host defense.

Precursors of alpha-defensin peptides require activation for bactericidal activity. In mouse small intestine, matrilysin colocalized with alpha-defensins (cryptdins) in Paneth cell granules, and in vitro it cleaved the pro segment from cryptdin precursors. Matrilysin-deficient (MAT-/-) mice lacked mature cryptdins and accumulated precursor molecules. Intestinal peptide preparations from MAT-/- mice had decreased antimicrobial activity. Orally administered bacteria survived in greater numbers and were more virulent in MAT-/- mice than in MAT+/+ mice. Thus, matrilysin functions in intestinal mucosal defense by regulating the activity of defensins, which may be a common role for this metalloproteinase in its numerous epithelial sites of expression.

Functional diversity and interactions between the repeat domains of rat intestinal lactase.

Lactase-phlorizin hydrolase (LPH), a major digestive enzyme in the small intestine of newborns, is synthesized as a high-molecular-mass precursor comprising four tandemly repeated domains. Proteolytic cleavage of the precursor liberates the pro segment (LPHalpha) corresponding to domains I and II and devoid of known enzymic function. The mature enzyme (LPHbeta) comprises domains III and IV and is anchored in the brush border membrane via a C-terminal hydrophobic segment. To analyse the roles of the different domains of LPHalpha and LPHbeta, and the interactions between them, we have engineered a series of modified derivatives of the rat LPH precursor. These were expressed in cultured cells under the control of a cytomegalovirus promoter. The results show that recombinant LPHbeta harbouring both domains III and IV produces lactase activity. Neither domain III nor IV is alone sufficient to generate active enzyme, although the corresponding proteins are transport-competent. Tandem duplication of domains III or IV did not restore lactase activity, demonstrating the separate roles of both domains within LPHbeta. Further, the development of lactase activity did not require LPHalpha; however, LPHalpha potentiated the production of active LPHbeta but the individual LPHalpha subdomains I and II were unable to do so. Lactase activity and targeting required the C-terminal transmembrane anchor of LPH; this requirement was terminal transmembrane anchor or LPH; this requirement was not satisfied by the signal/anchor region of another digestive enzyme: sucrase-isomaltase. On the basis of this study we suggest that multiple levels of intramolecular interactions occur within the LPH precursor to produce the mature enzyme, and that the repeat domains of the precursor have distinct and specific functions in protein processing, substrate recognition and catalysis. We propose a functional model of LPHbeta in which substrate is channelled from an entry point located within domain II to the active site located in domain IV.

Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe.

PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site. It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles. The C-terminal mature region is highly conserved in other serine carboxypeptidases. In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues. The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each. Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor form and via the 51-kDa single-polypeptide-chain intermediate form which has had its pro segment removed is finally converted to a heterodimer, the mature form, which is detected as a 32-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Like S. cerevisiae CPY, S. pombe Cpy1p does not require the N-linked oligosaccharide moiety for vacuolar delivery. To investigate the vacuolar sorting signal of S. pombe Cpy1p, we have constructed cpy1+-SUC2 gene fusions that direct the synthesis of hybrid proteins consisting of N-terminal segments of various lengths of S. pombe Cpy1p fused to the secreted enzyme S. cerevisiae invertase. The N-terminal 478 amino acids of Cpy1 are sufficient to direct delivery of a Cpy1-Inv hybrid protein to the vacuole. These results showed that the pro peptide of Cpy1 contains the putative vacuolar sorting signal.

Production of Human Pancreatic Ribonuclease inSaccharomyces cerevisiaeandEscherichia coli

Human pancreatic ribonuclease (HP-RNase) has considerable promise as a therapeutic agent. Structure–function analyses of HP-RNase have been impeded by the difficulty of obtaining the enzyme from its host. Here, a gene encoding HP-RNase was designed, synthesized, and inserted into two expression vectors that then direct the production of HP-RNase inSaccharomyces cerevisiae(fused to either an unmodified or a modified α-factor pre–pro segment) orEscherichia coli(fused to the pelB signal sequence). HP-RNase produced inS. cerevisiaewas secreted into the medium as an active enzyme, isolable at 0.1–0.2 mg/liter of culture. This isolate was heterogeneous due to extensive glycosylation and incomplete maturation of the pre–pro segment. HP-RNase produced inE. coliwith the pET expression system was purified from the insoluble fraction of the cell lysate. Renaturation of the reduced and denatured protein produced active, homogeneous enzyme recoverable at 1 mg/liter of culture. The N terminus of the HP-RNase produced from the bacterial expression system was processed fullyin vivo.The yeast system, combined with techniques that allow detection of picograms of ribonuclease activity, offers a sensitive probe for studies of post-translational modification and secretory targeting in eukaryotic cells. The bacterial system enables studies both to reveal new structure–function relationships in ribonucleases and to evaluate the use of HP-RNase as a cytotoxin that is tolerated by the human immune system.

Incorporation of a Potentially Helix-Breaking D-Phe-Pro Sequence into the Center of a Right-Handed 16-Residue Peptide Helix

Non protein amino acids with strong secondary structure preferences are potentially useful in peptide design. α-Aminoisobutyric acid (Aib) is a powerful ′stereochemical director′ of polypeptide chain folding, stabilizing helical conformations in diverse oligopeptide sequences. In an approach to the de novo design of α,α motifs, the 16 residue peptides Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Xxx-Pro-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe (Xxx = D-Phe 1; Xxx = L-Phe 2) have been spectroscopically studied in solution. Analysis of nuclear Overhauser effects, delineation of solvent shielded NH groups and circular dichroism spectra establish helical conformations in both the peptides. Despite the presence of a potentially helix breaking, central, (D)Phe-(L)Pro segment, peptide 1 is forced into a continuous helical fold presumably as a consequence of the overriding stereochemical dominance of the Aib residues.

Evidence for Cleavage of the PC1/PC3 Pro-Segment in the Endoplasmic Reticulum

AtT-20 cells contain two molecular weight forms (87 and 66 kDa) of the prohormone convertase PC1 (also known as PC3), thought to be involved in prohormone maturation. In this study we found that PC1 is first synthesized as a 94-kDa protein, which is then rapidly converted to a 84-kDa form. Two lines of evidence suggest that the generation of the 84-kDa protein from its 94-kDa precursor occurs in the endoplasmic reticulum (ER). The processing of the 94-kDa protein to the lower molecular weight form was extremely rapid, occurring with a half-life less than 2 min. The 84-kDa form was initially endoglycosidase H-sensitive, indicating lack of acquisition of sugars transferred in the medial Golgi. Within 40 min after the labeling period, the 84-kDa protein was converted to an endoglycosidase H-resistant form of 87 kDa, which was then processed to an endoglycosidase H-resistant 66-kDa protein. Radiosequencing of the 87- and 66-kDa proteins indicated that the biosynthesis of the 87-kDa protein involves the removal of the 83 amino acid Pro segment and that the processing of the 87-kDa to the 66-kDa form occurred by cleavage of a carboxy-terminal segment. Brefeldin A did not interrupt the cleavage of the 94-kDa to the 87-kDa protein, but completely blocked the processing of the 84- to 87-kDa proteins to the 66-kDa species. The 84-kDa protein produced in brefeldin-treated cells remained sensitive to endoglycosidase H, indicating a lack of exposure to Golgi sugar transferases. Collectively, our results indicate that while the initial cleavage of the PC1 cleavage most likely occurs in the ER, the generation of the 66-kDa proteins is performed in later cellular compartments such as the trans-Golgi network or secretory granules. These data imply that the various molecular forms of PC 1 may participate in prohormone maturation at different points along the secretory pathway.