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Yapsin 1 is an aspartic protease from Saccharomyces cerevisiae and belongs to a class of aspartic proteases that demonstrate specificity for basic amino acids. It is capable of processing prohormone substrates at specific basic residue cleavage sites, similar to that of the prohormone convertases, to generate bioactive peptide hormones. An antibody raised against yapsin 1 was previously shown to immunostain endocrine cells of rat pituitary and brain as well as lysates from bovine pituitary secretory granules demonstrating the existence of yapsin 1-like aspartic proteases in mammalian endocrine tissues, potentially involved in peptide hormone production. Here, we show the specific staining of yapsin 1 immunoreactivity in the α-cells of human pancreatic islets. No staining was observed in the β- or δ-cells, indicating a specificity of the staining for glucagon-producing and not insulin- or somatostatin-producing cells. Purified yapsin 1 was also shown to process proglucagon into glucagon in vitro, demonstrating that the prototypical enzyme of this subclass of enzymes can correctly process proglucagon to glucagon. These findings suggest the existence of a yapsin 1-like enzyme exclusively in the α-cells of the islets of Langerhans in humans, which may play a role in the production of glucagon in that tissue.

The biosynthesis of most biologically active peptides involves the action of prohomone convertases, including PC3 (also known as PC1), that catalyze limited proteolysis of precursor proteins. Proteolysis of prohormones occurs mainly in the granules of the regulated secretory pathway. It has been proposed that the targeting of these processing enzymes to secretory granules involves their association with lipid rafts in granule membranes. We now provide evidence for the interaction of the 86 and 64 kDa forms of PC3 with secretory granule membranes. Furthermore, both forms of PC3 were resistant to extraction with TX-100, were floated to low-density fractions in sucrose gradients, and were partially extracted upon cholesterol depletion by methyl-beta-cyclodextrin, indicating that they were associated with lipid rafts in the membranes. Protease protection assays, immunolabeling, and biotinylation of proteins in intact secretory granules identified an approximately 115-residue cytoplasmic tail for 86 kDa PC3. Using two-dimensional gel electrophoresis and a specific antibody, a novel, raft-associated form of 64 kDa PC3 that contains a transmembrane domain consisting of residues 619-638 was identified. This form was designated as 64 kDa PC3-TM, and differs from the 64 kDa mature form of PC3. We present a model of the membrane topology of PC3, where it is anchored to lipid rafts in secretory granule membranes via the transmembrane domain. We demonstrate that the transmembrane domain of PC3 alone was sufficient to target the extracellular domain of the IL2 receptor alpha-subunit (Tac) to secretory granules.

The potent peptidic inhibitor, Y1, of the basic residue-specific yeast aspartyl protease, yapsin 1, was synthesized and characterized. The inhibitor was based on the peptide sequence of a cholecystokinin(13-33) analog that yapsin 1 cleaved with an efficiency of 5.2 x 10(5) m(-1) s(-1) (Olsen, V., Guruprasad, K., Cawley, N. X., Chen, H. C., Blundell, T. L., and Loh, Y. P. (1998) Biochemistry 37, 2768-2777). The apparent K(i) of Y1 for the inhibition of yapsin 1 was determined to be 64.5 nm, and the mechanism is competitive. Y2 was also developed as an analog of Y1 for coupling to agarose beads. The resulting inhibitor-coupled agarose beads were successfully used to purify yapsin 1 to apparent homogeneity from conditioned medium of a yeast expression system. Utilization of this new reagent greatly facilitates the purification of yapsin 1 and should also enable the identification of new yapsin-like enzymes from mammalian and nonmammalian sources. In this regard, Y1 also efficiently inhibited Sap9p, a secreted aspartyl protease from the human pathogen, Candida albicans, which has specificity for basic residues similar to yapsin 1 and might provide the basis for the prevention or control of its virulence. A single-step purification of Sap9p from conditioned medium was also accomplished with the inhibitor column. N-terminal amino acid sequence analysis yielded two sequences indicating that Sap9p is composed of two subunits, designated here as alpha and beta, similar to yapsin 1.

A C-terminally truncated form of yapsin 1 (yeast aspartic protease 3) was overexpressed in yeast and its processing through the secretory pathway was followed by pulse-labeling and immunoprecipitation studies. In the soluble cell extract, three forms of yapsin 1-87, 74, and 18 kDa-were found. Identification of these forms of yapsin 1 using different antisera suggests that the 87-kDa form is pro-yapsin 1, which is processed into two subunits, alpha (18 kDa) and beta (74 kDa), by cleavage at a loop region not found in traditional aspartic proteases. By use of a temperature-sensitive mutant strain, sec18, the generation of the two subunits was found to occur in the endoplasmic reticulum. An active site-mutated yapsin 1 was not processed into the two subunits, suggesting that this process occurs in an autocatalytic manner., (Copyright 2000 Academic Press.)

A new aspartic protease from Saccharomyces cerevisiae, with a high degree of similarity with yapsin 1 and yapsin 2 and a specificity for basic residue cleavage sites of prohormones, has been cloned. This enzyme was named yapsin 3. Expression of a C-terminally truncated non-membrane anchored yapsin 3 in yeast yielded a heterogeneous protein between 135-200 kDa which, upon treatment with endoglycosidase H, migrated as a 60 kDa form. Amino-acid analysis of the N-terminus of expressed yapsin 3 revealed two different N-terminal residues, serine-48 and phenylalanine-54, which followed a dibasic and a monobasic residue respectively. Cleavage of several prohormones by non-anchored yapsin 3 revealed a specificity distinct from that of yapsin 1.

Yapsin 1, a novel aspartic protease with unique specificity for basic residues, was shown to cleave CCK13-33 at Lys23. Molecular modeling of yapsin 1 identified the active-site cleft to have negative residues close to or within the S6, S3, S2, S1, S1', S2', and S3' pockets and is more electronegative than rhizopuspepsin or endothiapepsin. In particular, the S2' subsite has three negative charges in and close to this pocket that can provide strong electrostatic interactions with a basic residue. The model, therefore, predicts that substrates with a basic residue in the P1 position would be favored with additional basic residues binding to the other electronegative pockets. A deletion of six residues close to the S1 pocket in yapsin 1, relative to rhizopuspepsin and other aspartic proteases of known 3D structure, is likely to affect its specificity. The model was tested using CCK13-33 analogues. We report that yapsin 1 preferentially cleaves a CCK13-33 substrate with a basic residue in the P1 position since the substrates with Ala in P1 were not cleaved. Furthermore, the cleavage efficiency of yapsin 1 was enhanced for CCK13-33 analogues with arginine residues flanking the P1 position. An alanine residue, substituting for the arginine residue in the P6 position in CCK13-33, resulted in a 50% reduction in the cleavage efficiency. Substitution with arginine residues downstream of the cleavage site at the P2', P3', or P6' position increased the cleavage efficiency by 21-, 3- and 7-fold, respectively. Substitution of Lys23 in CCK13-33 with arginine resulted not only in cleavage after the substituted arginine residue, but also forced a cleavage after Met25, suggesting that an arginine residue in the S2' pocket is so favorable that it can affect the primary specificity of yapsin 1. These results are consistent with the predictions from the molecular model of yapsin 1.

A C-terminally truncated form of yapsin 1 (yeast aspartic protease 3), the first member of the novel sub-class of aspartic proteases with specificity for basic residues (designated the Yapsins), was overexpressed and purified to apparent homogeneity, yielding approximately 1 microg of yapsin 1/g of wet yeast. N-terminal amino acid analysis of the purified protein confirmed that the propeptide was absent and that the mature enzyme began at Ala68. The mature enzyme was shown to be composed of approximately equimolar amounts of two subunits, designated alpha and beta, that were associated to each other by a disulfide bond. C-terminally truncated proyapsin 1 was also expressed in the baculovirus/Sf9 insect cell expression system and secreted as a zymogen that could be activated upon incubation at an acidic pH with an optimum at approximately 4.0. When expressed without its pro-region, it was localized intracellularly and lacked activity, indicating that the pro-region was required for the correct folding of the enzyme. The activation of proyapsin 1 in vitro exhibited linear kinetics and generated an intermediate form of yapsin 1 or pseudo-yapsin 1.

The subcellular localization and functionality of transfected yeast aspartic protease 3 (YAP3p) in a mammalian cell line were investigated. The complementary DNAs encoding the prohormone-processing enzyme (YAP3p) and a prohormone, bovine POMC, were cotransfected into PC12 (rat pheochromocytoma) cells. Immunocytochemical analysis of the cells using a YAP3p antibody showed a perinuclear punctate distribution of YAP3p in the cell body as well as immunostaining in the tips of the neurites. This pattern of immunostaining indicates localization of YAP3p in secretory granules. Analysis of the processing of POMC showed that in cells transfected with the POMC complementary DNA alone, only POMC was found, indicating a lack of processing of the prohormone. However, in cells coexpressing YAP3p, the POMC was completely processed to yield ACTH-(1-39) and ACTH-(1-14), consistent with the specificity of YAP3p found in vitro. Pulse-chase studies showed that POMC was processed after 20 min of chase, suggesting that processing occurred in the late Golgi network and continued in the secretory granules. Western blot analysis determined that YAP3p was secreted from the cells in a regulated manner. This study provides the first demonstration that a yeast prohormone-processing enzyme (YAP3p) of the aspartic protease class can be sorted correctly to secretory granules and activated to process a prohormone (POMC) in a highly efficient manner in mammalian cells.

The novel aspartic proteases, yeast aspartic protease 3 and the mammalian POMC-converting enzyme (PCE), can process prohormones at specific basic residue cleavage sites. We show that an antibody against yeast aspartic protease 3 (YAP3p) cross-reacted with purified bovine PCE on Western blot, indicating structural homology between these two enzymes, but not with other aspartic proteases, such as renin or cathepsin D. A PCE-sized anti-YAP3p-immunoreactive band was detected on Western blots of bovine intermediate lobe where PCE activity has been found. YAP3p antiserum also cross-reacted with a protein of approximately 90 kDa from mouse hypothalamus and anterior pituitary, and bovine anterior pituitary secretory granules. Distribution studies showed the presence of anti-YAP3p-immunopositive cells in bovine pituitary and peptide-rich brain regions, including the mouse arcuate nucleus and hippocampus and the rat supraoptic nucleus, paraventricular nucleus, cortex, striatum, and reticular nucleus. In the bovine intermediate pituitary, a subpopulation of cells was intensely stained with the YAP3p antiserum, and in combination with in situ hybridization, these cells were shown to contain POMC messenger RNA (mRNA). Only a subpopulation of cells was immunopositive for anti-YAP3p in bovine anterior pituitary, and most of these cells were identified by double immunostaining with ACTH antiserum as corticotrophs. In situ hybridization in combination with immunocytochemistry provided evidence for the localization of arginine vasopressin mRNA in YAP3p-immunopositive neurons in the rat supraoptic nucleus, whereas cholecystokinin mRNA was detected in YAP3p-immunopositive cells in the rat cortex and hippocampus. These results support the hypothesis that YAP3p-like aspartic proteases, including PCE, play a role in prohormone processing in endocrine/neuroendocrine cells in vivo.

The yeast aspartic protease Yap3 is localised to the secretory pathway and correctly cleaves pro-alpha-mating factor at its dibasic sites. We determined the specificity of Yap3 for mono-, di-, and multi-basic cleavage sites in the context of 15 residue synthetic proalbumin peptides. Yap3 cleaved after dibasic ArgArg and LysArg sites but not after monobasic Arg sites even when there was an additional arginine at -6 and/or -4. Yap3 did not cleave a tetra-arginine site and tri-basic sites (RRR and RRK) were poor substrates. Cleavage always occurred C-terminal to the last arginine in the di- or tri-basic sequence. The optimal cleavage site sequence was RR DR and this substrate was cleaved 8-9-fold faster than the normal RR DA sequence. In contrast to Kex2, Yap3 did not remove the propeptide from normal proalbumin or a range of natural or recombinant proalbumin variants. However at pH 4.0 Yap3 slowly cleaved proalbumin and albumin between domains 2 and 3.

The specificity and relative efficiency of cleavage of mono- and paired-basic residue processing sites by YAP3p was determined in vitro for a number of prohormone substrates: human ACTH1 39, bovine proinsulin, porcine cholecystokinin 33, cholecystokinin (CCK) 13-33, dynorphin A(1-11), dynorphin B(1-13), and amidorphin. YAP3p generated ACTH1-15 from ACTH1-39. It cleaved proinsulin at the paired-basic residue sites of the B-C junction as well as the C-A junction. Leu-enkephalin-Arg and Leu-enkephalin-Arg-Arg were generated from dynorphin A and dynorphin B, respectively. YAP3p generated Met-enkephalin-Lys-Lys from amidorphin showing that cleavage by this enzyme can occur at a lone pair of Lys residues. CCK33 was cleaved at Lys23 and Arg9, each containing an upstream Arg residue at the P6 and P5 position, respectively. Km values were between 10(-4) and 10(-5) M for the various substrates, with the highest affinity exhibited for the tetrabasic site of ACTH1-39 (1.8 x 10(-5) M). The tetrabasic residue site of ACTH1-39 was cleaved with the highest relative efficiency (kcat/Km = 3.1 x 10(6) m-1 s-1), while that of the monobasic site of CCK13-33 and the paired-basic site of proinsulin B-C junction, were cleaved less efficiently at 4.2 x 10(4) m-1 s-1 and 1.6 x 10(4) m-1 s-1, respectively.

Pro-thyrotropin-releasing hormone (proTRH) is the precursor to thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH2), the hypothalamic releasing factor that stimulates synthesis and release of thyrotropin from the pituitary gland. Five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven cryptic peptides are formed following posttranslational proteolytic cleavage of the 26-kDa rat proTRH precursor. The endopeptidase(s) responsible for the physiological conversion of proTRH to the TRH progenitor form is currently unknown. We examined the in vitro processing of [3H]leucine-labeled or unlabeled proTRH by partially purified recombinant PC1. Recombinant PC1 processed the 26-kDa TRH precursor by initially cleaving the prohormone after the basic amino acid at either position 153 or 159. Based on the use of our well-established antibodies, we propose that the initial cleavage gave rise to the formation of a 15-kDa N-terminal peptide (preproTRH25-152 or pre-proTRH25-158) and a 10-kDa C-terminal peptide (pre-proTRH154-255 or preproTRH160-255). Some initial cleavage occurred after amino acid 108 to generate a 16.5-kDa C-terminal peptide. The 15-kDa N-terminal intermediate was further processed to a 6-kDa peptide (prepro-TRH25-76 or preproTRH25-82) and a 3.8-kDa peptide (preproTRH83-108), whereas the 10-kDa C-terminal intermediate was processed to a 5.4-kDa peptide (prepro-TRH206-255). The optimal pH for these cleavages was 5.5. ZnCl2, EDTA, EGTA, and the omission of Ca2+ inhibited the formation of pYE27 (preproTRH25-50), one of the proTRH N-terminal products, by 48, 82, 72, and 45%, respectively. This study provides evidence, for the first time, that recombinant PC 1 enzyme can process proTRH to its predicted peptide intermediates.

TRH is synthesized from a larger 26-kilodalton (kDa) prohormone (pro-TRH). Rat pro-TRH contains five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven other cryptic peptides. Each of the five TRH progenitor sequences is flanked by pairs of basic amino acids. We used a bovine intermediate lobe secretory vesicle membrane preparation, which contains the prohormone convertases (PCs) PC1 and PC2, to study the in vitro processing of pro-TRH. Pro-TRH was radiolabeled using [3H]Leu in AtT20 cells transfected with prepro-TRH complementary DNA, and the labeled 26-kDa pro-TRH was isolated from the cell extract by preparative sodium dodecyl sulfate-gel electrophoresis. Incubation of [3H]pro-TRH with the intermediate lobe secretory vesicle membrane preparation was followed by immunoprecipitation with antibodies specific for various regions of the pro-TRH sequence, and the immunoprecipitates were analyzed by sodium dodecyl sulfate-gel electrophoresis. Immunoprecipitation of the reaction mixture with anti-pCC10 antibody (an antibody that recognizes the intact precursor and amino-terminal intermediate products of processing) showed a time-dependent appearance of a 15-kDa and a 6-kDa peptide and, at times, a 3.8-kDa peptide with diminution of the 26-kDa substrate. Immunoprecipitation of the incubate with the C-terminal-directed antibody, pYE17 (an antibody that recognizes the intact precursor and C-terminal intermediate products of processing), showed the generation of 16.5-, 10-, and 5.4-kDa products in a time-dependent manner, with disappearance of the substrate. Western blot analysis demonstrated that the secretory vesicle membrane preparation contains PC1 and PC2. Immunodepletion studies with antiserum specific for PC1 or PC2 demonstrated that PC1 and PC2 can process pro-TRH to these intermediate products. An initial site of cleavage appeared to be either at the 152-153 or the 158-159 pair of basic residues to yield a 15-kDa N-terminal fragment that was then processed to the 6-kDa [TRH-(25-74)] and 3.8-kDa [TRH-(83-112)] forms. The 10-kDa C-terminal peptide generated by this cleavage was then processed to a 5.4-kDa peptide [TRH-(208-255)]. Alternatively, an initial cleavage at the 107-108 or the 112-113 bonds was also observed, yielding a 16.5-kDa C-terminal product that was further processed to the 5.4-kDa peptide. The pH profile for the appearance of both C- and N-terminal products showed a bimodal distribution, with optima at both 5.5 and 7.5. The cleavage of pro-TRH was enhanced by Ca2+ and partially inhibited by Zn2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Yeast aspartic protease 3 (YAP3p), a basic-residue specific proprotein processing enzyme, was shown to be a membrane-associated protease. The membrane association of YAP3p was demonstrated to be through a glycophosphatidylinositol anchor situated in the carboxy terminus of the enzyme. Carboxy-terminal truncation of YAP3p by 37 amino acids resulted in secretion of YAP3p into the growth medium. Western blot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two secreted forms of YAP3p with apparent molecular masses of approximately 180 and approximately 90 kDa. YAP3p has an isoelectric point of approximately 4.5 as determined by isoelectric focusing gel electrophoresis. Treatment of YAP3p with endoglycosidase H reduced the size of both forms of the protein to approximately 65 kDa, consistent with the presence of 10 potential N-linked glycosylation sites in the deduced amino acid sequence of this protein. Removal of the N-linked sugars did not affect the enzymatic activity of YAP3p. Analysis of the effect of temperature on the stability and the rate of enzymatic activity of YAP3p showed that the enzyme retained 100% of its activity when incubated for 1 h at 37 degrees C, while incubation at 50 degrees C for 1 h resulted in approximately 80% loss of activity. The dependence of activity on temperature demonstrated a calculated Q10 of 1.95.

Protective antigen (PA), an 83-kDa protein produced by Bacillus anthracis, requires proteolytic activation at a tetrabasic site (RKKR167) before it can combine with either edema factor or lethal factor on the cell surface. The complex is then endocytosed and the target cell intoxicated. Previous work has demonstrated that furin, a ubiquitously distributed, subtilisin-like protease, can perform this cleavage. In this study, another member of the furin family, PC1 (SPC3), was tested as a putative processing enzyme for PA. Recombinant PC1, partially purified from the medium of stably transfected L-cells, cleaved PA to a 63-kDa fragment (PA63) and a 20-kDa fragment (PA20). Amino-terminal sequence analysis of the 63 kDa product demonstrated that cleavage occurred between Arg167 and Ser168. The pH optimum for in vitro PA cleavage was 6.0 and the enzymatic activity was calcium-dependent. Medium from untransfected L-cells did not cleave PA. Site-directed mutagenesis of the tetrabasic cleavage site revealed that PC1 preferred to cleave sequences containing basic residues at positions -1 and -4 relative to the wild-type cleavage site, demonstrating that PC1 can cleave substrates at a monobasic residue site in vitro. Substrates having basic residues at the -1 and -2 positions were cleaved with approximately twofold less efficiency than wild-type PA. Mutants of PA containing basic residues in positions -1 and either -2 or -4 of the cleavage site were predicted to be substrates for PC1 and were more toxic to L-cells expressing PC1 than to untransfected L-cells. These results demonstrate that PA is cleaved by PC1 in vivo. Membranes from bovine intermediate lobe secretory vesicles which contain both prohormone convertases, PC1 and PC2, also cleaved PA to PA63 with a pH optimum of 5.5. Immunodepletion studies using antisera against PC1 and PC2 showed that these are the enzymes primarily responsible for the cleavage of PA in the membrane preparation. Thus, both recombinant PC1 and a membrane preparation containing endogenous PC1 can activate PA.

The prohormone convertases, PC1 (SPC3) and PC2, are subtilisin-like serine proteases capable of processing neuropeptide precursors. In cotransfection experiments, other investigators have found that PC1 and PC2 can process POMC to appropriate peptide products. In this study, recombinant rat PC1 was stably expressed in a mouse L-cell line and partially purified. Mouse POMC was cleaved by recombinant PC1 to generate ACTH intermediates, ACTH, ACTH linked to joining peptide, joining peptide, 16-kilodalton N-POMC, N-POMC-(1-74), and beta-lipotropin. Recombinant PC1 was also found to cleave ACTH to ACTH-(1-15) and bovine N-POMC-(1-77) to gamma 3 MSH. The pH optimum of the cleavages was 6.0. We conclude that recombinant PC1 is capable of processing POMC in vitro at all of the paired basic residues, with the exception of Lys-Arg and Lys-Lys in beta-lipotropin and beta-endorphin, respectively. This in vitro study showed a more general specificity of recombinant PC1 for paired and tetrabasic residues of POMC than was previously found in cotransfection experiments. Other cellular regulatory mechanisms probably play a role in limiting the processing of POMC in vivo in the anterior pituitary, where gamma 3 MSH and alpha MSH are not found in significant amounts.

Anglerfish somatostatin-14 (SS-14) and somatostatin-28 (aSS-28) are derived from pro-somatostatin I (aPSS-I) and pro-somatostatin II (PSS-II), respectively. Purified yeast aspartic protease 3 (YAP3), was shown to cleave aPSS-I at the Arg81-Lys82 to yield SS-14 and Lys-1SS-14. In contrast, YAP3 cleaved aPSS-II only at the monobasic residue, Arg73 to yield aSS-28. Since the paired basic and monobasic sites are present in both precursors, the results indicate that the structure and conformation of these substrates dictate where cleavage occurs. Furthermore, the data show that YAP3 has specificity for both monobasic and paired basic residues.

Yeast cells express an alternate enzyme encoded by the YAP3 gene which can process pro-alpha-mating factor when this pheromone is overexpressed in KEX2-deficient mutants. The YAP3 gene product is an aspartic protease (YAP3) that cleaves at paired basic residues (Egel-Mittani, M., Flygenring, H.P., and Hansen, M. T. (1990) Yeast 6, 127-137). In this study, the YAP3 gene was overexpressed in the BJ 3501 strain of Saccharomyces cerevisiae. YAP3 was purified to apparent homogeneity using concanavalin A and pepstatin A affinity chromatography. The enzyme was characterized as an M(r) 68,000 glycoprotein with a pH optimum of 4.0-4.5. It was inhibited by pepstatin A and activated by 5 mM Ca2+. YAP3 cleaved at paired basic residues of mouse pro-opiomelanocortin (POMC) to yield adrenocorticotropin (ACTH) and beta-lipotropin (LPH); human beta-LPH to yield beta-endorphin-(1-31), beta-endorphin-(1-29), beta-endorphin-(1-28), gamma-LPH, and beta-melanocyte-stimulating hormone; and bovine N-POMC1-77 to yield gamma 3-melanocyte-stimulating hormone. It also cleaved the tetrabasic residues of ACTH1-39 to yield primarily ACTH1-15 and Lys-Arg-corticotropin-like intermediate lobe peptide. The physical properties, pH optimum, and specificity of YAP3 indicate that it is a homologue of the mammalian POMC-converting enzyme (EC 3.4.23.17), a paired basic residue-specific aspartic protease from bovine pituitary intermediate lobe secretory granules (Loh, Y. P., Parish, D.C., and Tuteja, R. (1985) J. Biol. Chem. 260, 7194-7205).

Oligonucleotides directed towards the active site regions of aspartic proteases were used as primers for the polymerase chain reaction to identify a unique sequence (asppcr1) from the AtT-20 anterior pituitary corticotrope cell line. Asppcr1 showed the greatest similarity (85% identity) to human cathepsin E [(1989) J. Biol. Chem. 264, 16748-16753]. Northern blot analysis of AtT-20 RNA revealed a single 1.9 kB message. Nuclease protection experiments indicated that asppcr1 mRNA was present in pancreas, spleen, testis and liver at low levels and undetectable in heart and brain. This contrasted with the lysosomal aspartic protease, cathepsin D whose mRNA showed a broader tissue distribution. The restricted message distribution of asppcr1 supports a more specific role for this aspartic protease in aspect(s) of cellular physiology.