Proprotein Processing Research Articles

Quantitative Characterization of Furin Specificity: ENERGETICS OF SUBSTRATE DISCRIMINATION USING AN INTERNALLY CONSISTENT SET OF HEXAPEPTIDYL METHYLCOUMARINAMIDES

Furin, an essential mammalian proprotein processing enzyme of the kexin/furin family of subtilisin-related eukaryotic processing proteases, is implicated in maturation of substrates involved in development, signaling, coagulation, and pathogenesis. We examined the energetics of furin specificity using a series of peptidyl methylcoumarinamide substrates. In contrast to previous reports, we found that furin can cleave such substrates with kinetics comparable to those observed with extended peptides and physiological substrates. With the best of these hexapeptidyl methylcoumarinamides, furin displayed k(cat)/K(m) values greater than 10(6) M(-1) s(-1). Furin exhibited striking substrate inhibition with hexapeptide but not tetrapeptide substrates, an observation of significance to the evaluation of peptide-based furin inhibitors. Quantitative comparison of furin and Kex2 recognition at P(1), P(2), and P(4) demonstrates that whereas interactions at P(1) make comparable contributions to catalysis by the two enzymes, furin exhibited a approximately 10-fold lesser dependence on P(2) recognition but a 10-100-fold greater dependence on P(4) recognition. Furin has recently been shown to exhibit P(6) recognition and we found that this interaction contributes approximately 1.4 kcal/mol toward catalysis independent of the nature of the P(4) residue. We have also shown that favorable residues at P(2) and P(6) will compensate for less than optimal residues at either P(1) or P(4). The quantitative analysis of furin and Kex2 specificity sharply distinguish the nature of substrate recognition by the processing and degradative members of subtilisin-related proteases.

Various functions of selenols and thiols in anaerobic gram-positive, amino acids-utilizing bacteria.

Electron transfer reactions for the reduction of glycine in Eubacterium acidaminophilum involve many selenocysteine (U)- and thiol-containing proteins, as shown by biochemical and molecular analysis. These include an unusual thioredoxin system (-CXXC-), protein A (-CXXU-) and the substrate-specific protein B of glycine reductase (-UXXCXXC-). Most probably a selenoether is formed at protein B by splitting the C-N-bond after binding of the substrate. The carboxymethyl group is then transferred to the selenocysteine of protein A containing a conserved motif. The latter protein acts as a carbon and electron donor by giving rise to a protein C-bound acetyl-thioester and a mixed selenide-sulfide bond at protein A that will be reduced by the thioredoxin system. The dithiothreitol-dependent D-proline reductase of Clostridium sticklandii exhibits many similarities to protein B of glycine reductase including the motif containing selenocysteine. In both cases proprotein processing at a cysteine residue gives rise to a blocked N-terminus, most probably a pyruvoyl group. Formate dehydrogenase and some other proteins from E. acidaminophilum contain selenocysteine, e.g., a 22 kDa protein showing an extensive homology to peroxiredoxins involved in the detoxification of peroxides.

Role of endoproteolytic dibasic proprotein processing in maturation of secretory proteins in Trichoderma reesei.

Cell extracts of Trichoderma reesei exhibited dibasic endopeptidase activity toward the carboxylic side of KR, RR, and PR sequences. This activity was stimulated by the presence of Ca2+ ions and localized in vesicles of low bouyant density; it therefore exhibited some similarity to yeast Kex2. Analytical chromatofocusing revealed a single peak of activity. The dibasic endopeptidase activity was strongly and irreversibly inhibited in vitro as well as in vivo by 1 mM p-amidinophenylmethylsulfonyl fluoride (pAPMSF) but not by PMSF at concentrations up to 5 mM. We therefore used pAPMSF to study the role of the dibasic endopeptidase in the secretion of protein by T. reesei. Secretion of xylanase I (proprotein processing sequence -R-R- downward arrow-R- downward arrow-A-) and xylanase II (-K-R- downward arrow-Q-) was strongly inhibited by 1 mM pAPMSF, and a larger, unprocessed enzyme form was detected intracellularly under these conditions. Secretion of cellobiohydrolase II (CBH II; -E-R- downward arrow-Q-) was only slightly inhibited by pAPMSF, and no accumulation of unprocessed precursors was detected. In contrast, secretion of CBH I (-R-A- downward arrow-Q-) was stimulated by pAPMSF addition, and a simultaneous decrease in the concentration of intracellular CBH I was detected. Similar experiments were also carried out with a single heterologous protein, ShBLE, the phleomycin-binding protein from Streptoalloteichus hindustanus, fused to a series of model proprotein-processing sequences downstream of the expression signals of the Aspergillus nidulans gpdA promoter. Consistent with the results obtained with homologous proteins, pAPMSF inhibited the secretion of ShBLE with fusions containing dibasic (RK and KR) target sequences, but it even stimulated secretion in fusions to LR, NHA, and EHA target sequences. Addition of 5 mM PMSF, a nonspecific inhibitor of serine protease, nonspecifically inhibited the secretion of heterologous proteins from fusions bearing the NHA and LR targets. These data point to the existence of different endoproteolytic proprotein processing enzymes in T. reesei and demonstrate that dibasic processing is obligatory for the secretion of the proproteins containing this target.

A Leucine-based Motif Mediates the Endocytosis of Vesicular Monoamine and Acetylcholine Transporters

Specific transport proteins mediate the packaging of neurotransmitters into secretory vesicles and consequently require targeting to the appropriate intracellular compartment. To identify residues in the neuron-specific vesicular monoamine transporter (VMAT2) responsible for endocytosis, we examined the effect of amino (NH2-) and carboxyl (COOH-)-terminal mutations on steady state distribution and internalization. Deletion of a critical COOH-terminal domain sequence (AKEEKMAIL) results in accumulation of VMAT2 at the plasma membrane and a 50% reduction in endocytosis. Site-directed mutagenesis shows that replacement of the isoleucine-leucine pair within this sequence by alanine-alanine alone reduces endocytosis by 50% relative to wild type VMAT2. Furthermore, the KEEKMAIL sequence functions as an internalization signal when transferred to the plasma membrane protein Tac, and the mutation of the isoleucine-leucine pair also abolishes internalization of this protein. The closely related vesicular acetylcholine transporter (VAChT) contains a similar di-leucine sequence within the cytoplasmic COOH-terminal domain that when mutated results in accumulation of VAChT at the plasma membrane. The VAChT di-leucine sequence also confers internalization when appended to two other proteins and in one of these chimeras, conversion of the di-leucine sequence to di-alanine reduces the internalization rate by 50%. Both VMAT2 and VAChT thus use leucine-based signals for efficient endocytosis and as such are the first synaptic vesicle proteins known to use this motif for trafficking.

Structural and functional properties of Bos taurus tryptase: a search for a possible propeptide processing role

Some structural features of bovine tryptase were discussed based on spectroscopic analysis. The far UV-CD spectrum of the enzymatically active bovine tryptase is consistent with a structure containing very little, if any α-helix, as found for other serine proteases. The analysis of near UV-CD and UV absorption spectra reveals the presence of a high number of Trp residues arranged probably in strong structural motifs. At variance with other tryptases, the bovine enzyme shows an electrophoretic behaviour in native and denaturating conditions compatible with an association state larger than a tetramer (probably a dodecamer). From a biochemical point of view, the bovine tryptase shares with the human counterpart, the preference for cleaving substrates bearing dibasic cleavage sites. Thus, it is hyphotesized that tryptase may be involved in some proprotein processing mechanism(s).

Structural and functional analysis of the protein products derived from mutant fur alleles in an endoprotease-deficient Chinese hamster ovary cell strain.

The fur gene encodes the endoprotease, furin. We recently demonstrated mutations in both fur alleles in the mutant Chinese hamster ovary (CHO)-K1 strain, RPE.40, and hypothesized that these mutations were responsible for the endoprotease-deficient phenotype of these cells. We now present the structural and functional properties of three protein products derived from the mutant fur alleles. None of these protein products were able to process the precursor to von Willebrand factor, which is processed by wild-type furin. Pro-protein processing activity initially attributed to one of the mutant proteins was due to wild-type furin produced inadvertently from one of the expression constructs used in these experiments. None of the mutant proteins exhibited evidence of autocatalysis, consistent with the lack of activity versus the test substrate, and glycosylation patterns suggested at least two of them remained in the endoplasmic reticulum. These results confirm that RPE.40 cells are furin null mutants, as earlier evidence had suggested.

ChemInform Abstract: Peptides as Tools for Studying Propeptide and Proprotein Processing

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Interplay between S1 and S4 subsites in Kex2 protease: Kex2 exhibits dual specificity for the P4 side chain.

The yeast Kex2 protease is the prototype of a family of eukaryotic proprotein processing proteases that includes PC1, PC2, and furin. The catalytic domains of these enzymes are homologous to the degradative serine proteases of the subtilisin family. Kex2 exhibits optimal activity toward substrates with Lys or Arg at P2 and Arg at P1 (Lys-Arg or Arg-Arg cleavage sites). However, mammalian proprotein processing proteases such as furin exhibit more stringent requirements for basic residues at P4 than at P2. Here we demonstrate that Kex2 protease also recognizes P4, with dual specificity for aliphatic and basic residues. Recognition of P4 is even more readily apparent in substrates having a poor P1 residue (Lys). Kinetic analysis of a series of otherwise identical fluorogenic substrates with Lys at P1 and different residues at P4 indicates that large, aliphatic P4 residues increase kcat/KM by 100-fold. However, smaller residues or acidic residues at P4 do not. P4 Arg also confers efficient cleavage on such a substrate, but the uncharged isostere of Arg, citrulline, does not. Kex2 may thus possess distinct subsites that recognize aliphatic or basic P4 side chains. Although a favorable P4 residue can partially compensate for the defects in kcat and kcat/KM seen with Lys in place of Arg at P1, this substitution resulted in a change in rate-determining step for all substrates examined. As previously seen in the case of subtilisin, effects of substitutions at the P1 and P4 positions were not independent, suggesting that interplay between these two positions is a common feature of substrate specificity for both processing proteases and degradative enzymes of the subtilisin superfamily.

The processing of yeast pheromones

Yeast provides an attractive system in which to study proprotein processing. Many of their processing events are remarkably similar to those in higher eukaryotes and their amenability to experimental manipulation permits approaches that are not always feasible in multicellular organisms. Analysing the biosynthesis of the yeast mating pheromones has been particularly rewarding and has provided insights into both the proteolytic aspects of processing and other post-translational events such as prenylation and carboxymethylation.

Molecular diversity in neurosecretion: reflections on the hypothalamo-neurohypophysial system.

1. The diversity of molecules involved in various aspects of neurosecretion, such as proprotein processing, axonal transport of large dense core vesicles (LDCVs), and regulated secretion, is discussed in the context of the hypothalamo-neurohypophysial system (HNS). 2. Recent studies have uncovered a family of at least seven processing enzymes known as proprotein convertases (PCs) which are involved in proteolytically cleaving protein precursors at paired basic amino acid motifs to yield biologically active peptides. Three of these, PC1(3), 2, and 5, are found in neurons and are involved in producing regulated secretory peptide products. 3. The axonal transport of LDCVs occurs on microtubule tracks by still unknown mechanisms. There are over 11 distinct kinesin-related molecules that have now been identified as possible microtubule motor candidates. 4. Calcium channels in the nervous system are known to be derived from at least five alpha-subunit and four beta-subunit genes with multiple alternatively spliced isoforms in each case. These could account, in part, for the varied calcium currents found in the HNS. 5. The large number of proteins and isoforms now demonstrated to be involved in regulated secretion are discussed, with a focus on LDCV compositions and the synaptotagmin gene family.

Proteolytic processing of chromogranin B and secretogranin II by prohormone convertases.

Two experimental approaches were used to study the processing of chromogranin B and secretogranin II by prohormone convertases. In GH3 cells various prohormone convertases were overexpressed together with the substrate chromogranin B by use of a vaccinia virus infection system. PC1 appeared to be by far the most active enzyme and converted chromogranin B to several smaller molecules, including the peptide PE-11. In brain this peptide is cleaved physiologically from chromogranin B. Some processing of chromogranin B and formation of free PE-11 were also observed with PC2 and PACE4. Furin produced larger fragments, whereas PC5-A and PC5-B had negligible effects. As a second model, PC12 cells were stably transfected with PC1 or PC2 to investigate the processing of endogenous chromogranins. Both enzymes effectively cleaved chromogranin B and secretogranin II, liberating the peptides PE-11 and secretoneurin, respectively. However, in transfection experiments the ability to generate the free peptides was more pronounced with PC2 than with PC1. The extent of proprotein processing achieved by prohormone convertases apparently differed depending on the experimental system applied. This suggests that in vivo mechanisms to support and fine-tune the activity of the processing enzymes exist, which might be overlooked by using only one methodological approach.

Cytoskeletal protein ABP-280 directs the intracellular trafficking of furin and modulates proprotein processing in the endocytic pathway.

Furin catalyzes the proteolytic maturation of many proproteins within the trans-Golgi network (TGN)/endosomal system. Furin's cytosolic domain (cd) directs both the compartmentalization to and transit between its manifold processing compartments (i.e., TGN/biosynthetic pathway, cell surface, and endosomes). Here we report the identification of the first furin cd sorting protein, ABP-280 (nonmuscle filamin), an actin gelation protein. The furin cd was used as bait in a yeast two-hybrid screen to identify ABP-280 as a furin-binding protein. Binding analyses in vitro and coimmunoprecipitation studies in vivo showed that furin and ABP-280 interact directly and that ABP-280 tethers furin molecules to the cell surface. Quantitative analysis of both ABP-280-deficient and genetically replete cells showed that ABP-280 modulates the rate of internalization of furin but not of the transferrin receptor, a cycling receptor. However, although ABP-280 directs the rate of furin internalization, the efficiency of sorting of the endoprotease from the cell surface to early endosomes is independent of expression of ABP-280. By contrast, efficient sorting of furin from early endosomes to the TGN requires expression of ABP-280. In addition, ABP-280 is also required for the correct localization of late endosomes (dextran bead uptake) and lysosomes (LAMP-1 staining), demonstrating a pleiotropic role for this actin binding protein in the organization of cellular compartments and directing protein traffic. Finally, and consistent with the trafficking studies on furin, we showed that ABP-280 modulates the processing of furin substrates in the endocytic but not the biosynthetic pathways. The novel roles of ABP-280 and the cytoskeleton in the sorting of furin in the TGN/ endosomal system and the formation of proprotein processing compartments are discussed.

Peptides as tools for studying propeptide and proprotein processing

In this overview, we discuss on the use of synthetic peptides reproducing tand/or mimicking propeptides and proproteins to study various aspects of the post-translational maturation of these biosynthetic precursors. Examples chosen in the series of pro-ocytocin, prosomatostatin illustrate the use of these peptides to purify putative converting enzymes and to study structure-function relationships with particular emphasis on the importance of privileged secondary structures and of given amino acid residues around dibasic cleavage sites in substrate recognition by the processing endoproteases.

Biosynthesis, Distinct Post-translational Modifications, and Functional Characterization of Lymphoma Proprotein Convertase

Proprotein convertases are responsible for the endoproteolytic processing of prohormones, neuropeptide precursors, and other proproteins within the constitutive and regulated secretory pathways. Cleavage occurs carboxyl-terminally of basic amino acid motifs, such as RX(K/R)R, RXXR, and (R/K)R. As already available for the other known mammalian members of this enzyme family, we here define structural and functional features of human lymphoma proprotein convertase (LPC). Analysis of expression of recombinant LPC in stably transfected Chinese hamster ovary cells reveals biosynthesis of a 92-kDa nonglycosylated precursor (proLPC) and a 102-kDa endoglycosidase H-sensitive glycosylated form of proLPC. Only the latter is further processed and after propeptide removal converted into a complexly N-glycosylated mature form of LPC of about 92 kDa. Co-expression experiments of truncated LPC with an active site mutant of LPC (LPCS265A) indicate that prodomain removal of LPC occurs via an autoproteolytic, intramolecular mechanism, as was demonstrated before for some of the other members of this enzyme family. Prodomain removal is shown to be required for LPC to exit the endoplasmic reticulum. As far as subcellular localization is concerned, immunocytochemical, ultrastructural, and biochemical analyses show that LPC is concentrated in the trans-Golgi network, associated with membranes, and not secreted. Carboxyl-terminal domains are critically involved in this cellular retention, because removal of both the hydrophobic region and the cytoplasmic tail of LPC results in secretion. Of interest are the observations that LPC is not phosphorylated like furin but is palmitoylated in its cytoplasmic tail. Finally, substrate specificity of LPC is similar to that of furin but not identical. Whereas for furin a basic substrate residue at position P-2 is dispensable, it is essential for LPC. For optimal LPC substrate processing activity, an arginine at position P-6 is preferred over an arginine at P-4.

Inhibition of intracellular proteolytic processing of soluble proproteins by an engineered alpha 2-macroglobulin containing a furin recognition sequence in the bait region.

The bait region of the general protease inhibitor alpha 2-macroglobulin (alpha 2M) was mutated by introducing a recognition sequence of furin. This did not interfere with folding, S-ester formation or tetramerization of the mutant recombinant alpha 2M (r alpha 2M). Mutant r alpha 2M inhibited furin in vitro, by a similar mechanism to that used by plasma alpha 2M to inhibit high-molecular-mass proteases. The mutant alpha 2M was intracellularly active in COS-1 cells in inhibiting the endogenous processing of the soluble substrates for furin (von Willebrand factor, transforming growth factor beta1 and a soluble form of the envelope glycoprotein gp160 from HIV-1) but not the membrane-bound form of gp160. The intracellular activity of mutant alpha 2M strongly indicated that alpha 2M attains its native conformation, and thus that the unusual internal S-ester is formed, before alpha 2M passes through the cleavage compartment(s). Our results show for the first time that modulation of the bait region of alpha 2M allows the creation of an inhibitor against membrane-bound proteases. It can be expected that the use of alpha 2M-bait mutants will become important as a technique for the study of various proteolytic processes and for the identification of the proteases involved.

Two Activation States of the Prohormone Convertase PC1 in the Secretory Pathway

PC1, a neuroendocrine member of the prohormone convertase family of serine proteinases, is implicated in the processing of proproteins in the secretory pathway. PC1 is synthesized as a zymogen and cleaves not only its own profragment in the endoplasmic reticulum, but a subset of protein substrates in the Golgi apparatus and in the Golgi-distal compartments of the regulated secretory pathway. Likewise, mouse PC1 (mPC1) has previously been shown to cleave human prorenin in GH4 cells (that contain secretory granules) while being unable to cleave prorenin in cells, such as Chinese hamster ovary (CHO) or BSC-40, which are devoid of secretory granules. In the current study, we show that removal of a C-terminal tail of mPC1 allows the efficient cleavage of prorenin in the constitutive secretory pathway of CHO cells. The C-terminal tail thus appears to act as an inhibitor of PC1 activity against certain substrates in the endoplasmic reticulum and Golgi apparatus, and its removal, which occurs naturally in secretory granules, may explain the observed granule-specific processing of certain proproteins. These results also demonstrate that PC1 is present in a partially active state prior to the secretory granules where it is processed to a maximally active state.

High-Level Expression of the Prohormones Proenkephalin, Pro-Neuropeptide Y, Proopiomelanocortin, and β-Protachykinin forin VitroProhormone Processing

Prohormone substrates are required for investigation of the proteolytic processing of prohormones and proproteins into active peptide hormones and neurotransmitters. However, the lack of prohormone proteins has been a limiting factor in elucidating proteolytic mechanisms for conversion of prohormones into active peptides. Therefore, in this study, cloned cDNAs encoding the prohormones proenkephalin (PE), pro-neuropeptide Y (pro-NPY), proopiomelanocortin (POMC), and β-protachykinin (β-PT) were utilized to express recombinant prohormones inEscherichia coli.High-level expression of milligrams of prohormones was achieved with the pET3c expression vector utilizing the T7 promoter for production of PE, pro-NPY, and POMC, as demonstrated by SDS–PAGE gel electrophoresis, Western blots, and35S-methionine labeling. In addition, β-PT was expressed at high levels as fusion proteins with the maltose-binding protein and glutathione S-transferase by the pMAL-c and pGEX-2T expression vectors, respectively. Relative rates of processing by the established processing proteases “prohormone thiol protease” (PTP), 70-kDa aspartyl protease, and PC1/3 and PC2 (PC, prohormone convertase) were examined with purified PE, pro-NPY, and POMC. Distinct preferences of processing enzymes for different prohormones was demonstrated. PTP prefered PE and pro-NPY substrates, whereas little processing of POMC was detected. In contrast, the 70-kDa aspartyl protease cleaved POMC more readily than pro-NPY or PE. However, PC1/3 and PC2 prefer POMC as substrate. Demonstration of selectivity of processing enzymes for prohormone substrates illustrates the importance of expressing recombinant prohormones forin vitroprocessing studies.

Endoplasmic reticulum Ca2+ is important for the proteolytic processing and intracellular transport of proinsulin in the pancreatic beta-cell.

The role of intracellular Ca2+ in the proteolytic processing and intracellular transport of secretory granule proproteins was investigated by pulse-chase radiolabelling of isolated rat islets of Langerhans. The conversion of proinsulin was inhibited by depletion of medium Ca2+ with EGTA and by blocking the transport of Ca2+ into cells with the Ca2+-channel antagonists verapamil, nifedipine and NiCl2. Proinsulin conversion was also reduced by the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin, indicating that the process requires transport of Ca2+ into the endoplasmic reticulum. This was supported by the finding that proinsulin processing was inhibited when Ca2+ was depleted before or during pulse-labelling, but not after transport of the protein to post-endoplasmic-reticulum compartments. Similarly, the inhibition of proinsulin processing was reversed by re-introduction of medium Ca2+ around the time of radiolabelling, but not after 15 min of chase incubation. Ca2+ depletion also decreased proteolytic maturation of the prohormone convertases PC1, PC2 and carboxypeptidase H. Secretion experiments suggested that the rate and extent of proinsulin transport into secretory granules were inhibited marginally by Ca2+ depletion, whereas those of the convertases were markedly impeded. Inhibition of proinsulin conversion by Ca2+ depletion was thus not simply related to the Ca2+-dependencies of mature PC1 and PC2, but also to a requirement for endoplasmic reticulum Ca2+ in proteolytic maturation of the convertases and in their transfer to secretory granules. The results also suggest that the Ca2+ required for prohormone processing in the granules enters the secretory pathway via the endoplasmic reticulum.

Maturation of mammalian 20 S proteasome: purification and characterization of 13 S and 16 S proteasome precursor complexes

The maturation of the eukaryotic 20 S proteasome complex occurs via 13 S and 16 S precursor complexes in a multistep assembly pathway. These precursor complexes contain α-subunits as well as unprocessed β-subunit proproteins. We have purified and characterized the different proteasome assembly intermediates and analysed their ability to support β-subunit proprotein processing in vitro. Our data show that 13 S and 16 S proteasome precursor complexes differ not only in size but also in their protein content and behaviour during hydrophobic chromatography. By establishing conditions which allowed us to analyse β-prosubunit maturation in vitro we demonstrate that the processing of the homologous proproteins of the β-subunits LMP2 and δ essentially takes place in 16 S precursor complexes. No proprotein processing activity was observed in 13 S precursor complexes. Furthermore, proprotein processing in vitro can be inhibited with a proteasome specific inhibitor, but with different efficiency for LMP2 and δ. A peptide, which represents the sequence of the proprotein processing site HGTT, exhibited no inhibitory effect on the processing of either subunit. These data provide further evidence that proprotein processing occurs via an autocatalytic mechanism. Our experiments also demonstrate that the chaperone protein hsc73 is associated with 16 S but not with 13 S precursor complexes. In support of the specificity of this interaction incubation with ATP leads to the dissociation of hsc73 from 16 S complexes and to the formation of high molecular weight aggregates. Prosubunit processing in isolated 16 S complexes does not, however, result in the formation of proteolytically active 20 S proteasomes which may me due to the fact that not all β-subunits can be efficiently processed in vitro. In contrast to previous assumptions subunit processing and formation of proteolytic activity do not coincide and final 20 S complex assembly seems to represent in part a separate event which requires additional factors or proteins which are not present or active in the purified 16 S precursor complexes.

Ontogeny of prohormone convertases in rat prenatal development.

It has been well established that peptide precursors usually undergo limited proteolysis at pairs or single basic amino acids during their biosynthetic process. This posttranslational modification paradigm is common for numerous membrane-spanning and secreted proteins, neuropeptides, and peptide hormones of physiological significance, in which endoproteolytic cleavage is invariably essential for the accurate biosynthesis and full activity of the mature products. Establishment of an effective peptide profile is dependent on not only the presence of peptide precursor, but also the presence and the enzymatic specificities of cleavage enzymes. We have, therefore, characterized the spatial and temporal patterns of six subtilisin-like serine endoproteases known to be involved in proprotein processing, including furin, PC1, PC2, PC4, PC5, and PACE4, in rat prenatal development and related the results to the expression patterns of several peptide precursors. We have observed largely distinct and sometimes complementary expression patterns of individual PCs in various embryonic structures, suggesting PCs may be functionally distinct in processing different sets of proprotein substrates in development. From these studies, numerous tentative enzyme-substrate relationships in various embryonic structures have been proposed and should encourage more studies to test the in vitro cleavage potentialities of individual PCs toward these precursors. In the future, knowledge gained from these studies, when combined with insights gained from in vivo perturbation and genetic ablation studies, should lead to final comprehensive understanding of specific precursors cleaved by specific enzymes at specific cleavage sites in known spatial and temporal expression patterns during development.