Activation and Processing of Non-anchored Yapsin 1 (Yap3p)*

Niamh X. CawleyDagger §, Vicki OlsenDagger , Chun-Fa ZhangDagger , Hao-Chia Chen, Marian TanDagger , and Y. Peng LohDagger

From the Dagger  Section on Cellular Neurobiology, Laboratory of Developmental Neurobiology and the  Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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 ~1 µg 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 ~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 in vitro exhibited linear kinetics and generated an intermediate form of yapsin 1 or pseudo-yapsin 1.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Peptide hormones are synthesized as larger precursors that require endoproteolytic cleavage at basic residues in the secretory pathway. In yeast, the Golgi resident, subtilisin-like serine protease, Kex2p (1, 2), is responsible for the processing of pro-alpha -mating factor and pro-killer toxin at specific basic residue cleavage sites (3-5). However, in Kex2p-deficient mutants, two genes encoding aspartic proteases, YAP3 and MKC7, have been cloned. Yapsin 1 (previously named Yap3p) was able to suppress the processing defect of pro-alpha -mating factor (6), and Mkc7p (now named yapsin 2) was able to suppress the cold-sensitive phenotype (7) observed in these mutant cells. Yapsin 1 and yapsin 2 are glycophosphatidylinositol (GPI)1 membrane-anchored proteins (7-9) and share 53% amino acid identity (7). Both enzymes have specificity for basic amino acid residues and appear to have a common substrate pool with that of Kex2p in vivo. Such properties allow these enzymes to be characterized as members of a sub-class of aspartic proteases involved in proprotein processing which demonstrate a unique specificity for arginine and/or lysine residue cleavage sites. This is in contrast to other aspartic proteases whose specificity is for hydrophobic residues (10, 11). This sub-class of aspartic proteases is now named the yapsin family due to its partial homology with pepsin (12, 13).

Yapsin 1 has previously been expressed in Saccharomyces cerevisiae as a carboxyl-terminally truncated soluble form and purified from the cell extract (14). This yapsin 1 enzyme was shown to process the mammalian prohormone, pro-opiomelanocortin and its fragments (14). It was characterized as a ~70-kDa glycoprotein with a pH optimum of 4.0-4.5 and observed to have a limited shelf life. In the same expression system, yapsin 1 activity was also found in the culture supernatant and was characterized as ~150-180- and ~90-kDa hyperglycosylated forms that appeared to be highly stable (8).2 Secreted yapsin 1 has been shown to process a variety of mammalian prohormones in vitro with catalytic efficiencies (kcat/Km) of 1.6 × 104 M-1 s-1 for the B-C junction of bovine proinsulin and 3.1 × 106 M-1 s-1 for human adrenocorticotropin (ACTH1-39) (15), demonstrating an enhancement of the efficiency by additional basic residues upstream and especially downstream of the cleavage site (15, 16).

Our recent work has shown that yapsin 1 is immunologically related to pro-opiomelanocortin converting enzyme, an enzyme with similar backbone size and specificity. Moreover, immunocytochemical studies showed colocalization of immunoreactive yapsin 1-related processing enzymes in neuropeptide-rich regions of mammalian brain and pituitary (13) supporting the existence of mammalian homologues of yapsin 1 involved in pro-neuropeptide processing. We also showed that yapsin 1 can function in vivo to process pro-opiomelanocortin in the mammalian PC12 cell line (17). This indicates a close structural and functional relationship of yapsin 1 with mammalian yapsins. Thus yapsin 1, which has been the most extensively studied member of the yapsin family, represents an ideal model enzyme for learning how the yapsin class of aspartic proteases are synthesized. Studies on the biosynthesis of yapsin 1 will also further define the structure of the enzyme and shed light on the best substrates that can interact with the enzyme. This will ultimately provide an insight into the physiological role that yapsin 1 plays in yeast.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Purification and Analysis of Yapsin 1 Secreted from Overexpressing Yeast

Carboxyl-terminally truncated soluble yapsin 1 enzymatic activity was found to be secreted into the culture supernatant of a previously described yeast expression system (14) and was used as the starting material for the purification of the enzyme.

Concentration-- The yeast culture supernatant containing the secreted yapsin 1 enzyme was concentrated by tangential flow filtration at room temperature using a 30-kDa molecular mass cutoff omega membrane in an ultrasette cassette (Filtron, Northborough, MA). A 4-fold diafiltration step to replace the media with 20 mM sodium phosphate buffer, pH 7.0, was also performed. The sample was further concentrated by centrifugation filtration using a Filtron 30-kDa Macrosep omega membrane filter. All subsequent procedures were carried out at 4 °C.

Anion Exchange Chromatography (MonoQ1)-- The concentrated medium was applied to a MonoQ HR 5/5 column (Pharmacia Biotech Inc.) connected to a fast protein liquid chromatography system (Pharmacia). The column was allowed to re-equilibrate in buffer A (20 mM sodium phosphate, pH 7.0) before a gradient of 0-25% buffer B (buffer A with 1 M NaCl) was applied. The flow rate was 1 ml/min, and 0.5-ml fractions were collected. The fractions were assayed by the standard ACTH1-39 assay, previously described (15). Two fractions on either side of the peak of activity were pooled and re-run on the MonoQ. The peaks of activity were combined and concentrated by centrifugation filtration through a Filtron 30-kDa Microsep omega membrane filter.

Gel Filtration Chromatography (Superdex G-200)-- The concentrated sample of activity from MonoQ1 was applied to a High Load 16/60 Superdex G-200 superfine column (Pharmacia) that was connected to the fast protein liquid chromatography system. The buffer used was 20 mM sodium phosphate, pH 7.0. The flow rate was 1 ml/min, and 1-ml fractions were collected. The column was previously calibrated with dextran blue 2000 (V0), bovine serum albumin (69 kDa), and ovalbumin (43 kDa), where V0 = 44 ml and Vt = 124 ml. Fractions containing yapsin 1 activity were pooled and used for the next step in the purification.

Anion Exchange Chromatography (MonoQ2)-- The pool of activity from the G-200 column was applied to the MonoQ2 using the same buffer system as that in MonoQ1. After re-equilibration, however, the protein was eluted in 0.5-ml fractions with a shallower gradient as follows: 0-10% buffer B in 5 min, 10-12% buffer B in 20 min, and 12-25% buffer B in 5 min. A selected portion of the yapsin 1 activity peak was pooled and designated as purified yapsin 1.

Analysis of Yapsin 1 from Yeast Culture Supernatant-- 280 ng of the purified yapsin 1 were run on a 12% Tris/glycine precast SDS-polyacrylamide gel and analyzed by silver stain (Novex, San Diego, CA). Also, an aliquot from the Superdex G-200 pool of activity (1.6 µg of protein) was deglycosylated as described before (8), run on an 8-16% Tris/glycine SDS-polyacrylamide gel in either the presence or absence of 5% beta -mercaptoethanol, and stained with Coomassie Blue. A similar gel was run in parallel but the proteins (6 µg) were analyzed by amino-terminal amino acid sequencing after they had been transferred to polyvinylidene difluoride membrane (Trans-Blot, Bio-Rad), in a Tris/glycine buffer containing 20% methanol. The proteins were visualized by staining with 0.2% Ponceau S in 1% acetic acid and destaining in water. Direct amino-terminal amino acid sequencing of the bands was carried out by Edman degradation using a Perkin-Elmer/Applied Biosystems model 494A Procise 228 Protein Sequencer. beta -Lactoglobulin was used to determine the sequencing efficiency.

Expression and Analysis of an Active Site Mutant of Yapsin 1 in Yeast

An active site mutant of the carboxyl-terminally truncated yapsin 1 was engineered by site-directed mutagenesis of Asp101 to Glu101 using an "overlap extension" mutagenesis procedure involving three PCR steps (18). Primers 3 and 4 were used as "outside" primers (see Table I), whereas primers 5 and 6, containing the Asp to Glu point mutation, were used as "inside" primers. The resulting fragment from the PCR steps was digested with BamHI and PstI and subcloned into the pEMBLyex4 vector previously described (14) and called pYAP3LC(Asp101right-arrowGlu101).

                              
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Table I
Nucleotide sequences of primers used in the construction of the various YAP3 constructs

The strain JHRY20-2C-Delta yap3::LEU2 (Mat a, his3-Delta 200, leu2-3-112, Delta yap3::LEU2) (19, 20) was transformed with either the pYAP3LC (8) or pYAP3LC(Asp101right-arrowGlu101) using standard procedures (21). Growth of the yeast and expression of the enzymes were performed as described before (8). Aliquots of the media (0.07 µg of protein) from each clone were analyzed by Western blot with two different antisera. Anti-yapsin 1 serum, MW283, previously characterized (8), and antiserum, VO2377, raised against a synthetic peptide corresponding to amino acids Tyr47-Glu61 within the yapsin 1 propeptide. Endoglycosidase H (Sigma) treatment of the enzymes was performed prior to the Western blots in order that a clearer size comparison could be made between the two forms of the enzyme. The media (1.1 µg of protein) was also assayed for yapsin 1 enzymatic activity by the standard ACTH1-39 assay (15).

Expression and Analysis of Proyapsin 1 from Baculovirus

Proyapsin 1 and yapsin 1(Delta proyapsin 1) were engineered for expression in the baculovirus expression system. Recombinant YAP3 gene constructs, encoding amino acids 22-532 (proyapsin 1) and 68-532 (Delta proyapsin 1), were engineered by PCR using the primer pairs 1/3 and 2/3, respectively (see Table I for primer sequences), and the previously characterized expression vector, pYAP3LC (8) as template. Both PCR fragments containing a BamHI and a PstI restriction site at their 5' and 3' ends, respectively, were subcloned into the pAcGP67 baculovirus transfer vector downstream of the strong signal peptide of the acidic glycoprotein, gp67. The pAcGP67 plasmids containing the YAP3 inserts were cotransfected with the BaculoGold viral DNA (Pharmingen, San Diego, CA) into Spodoptera frugiperda (Sf9) cells. Recombinant viral particles were identified by plaque assay and harvested for high titer stock generation. For the expression of the recombinant proteins, Sf9 cells were grown in an 80-cm2 canted neck tissue flask at 27 °C with 18 ml of serum-free medium (Sf-900 II SFM, Life Technologies, Inc.) supplemented with penicillin and streptomycin. After reaching 80% confluency, the cells were infected with 18 ml of fresh media containing 2 ml of purified recombinant baculovirus (1.1×108 pfu/ml). Two days postinfection, the cells and media were collected for analysis by Western blot and by the yapsin 1 activity assay. The media were concentrated and partially purified by concanavalin A (ConA) affinity chromatography (22) prior to analysis, and the soluble cell extracts were analyzed without manipulation except for an aliquot of the Delta proyapsin 1 sample which was treated with and without N-glycanase, according to the company's guidelines (Genzyme, Cambridge, MA) and analyzed by Western blot.

Other experiments described below utilized the culture supernatant, from multiple flasks of cells expressing proyapsin 1, that was concentrated by centrifugation through a Macrosep 10-kDa omega membrane and frozen at -20 °C until analysis. A fresh preparation of a protease inhibitor mixture (CompleteTM, Boeringer Mannheim, Germany) containing EDTA but not pepstatin A was added prior to the experiments to minimize nonspecific degradation during the procedures.

pH-dependent Activation of Proyapsin 1-- The effect of pH on the activation of proyapsin 1 was investigated. Five µl of the proyapsin 1 sample were incubated in 20 µl of 0.1 M sodium citrate or sodium citrate/sodium phosphate buffers with pH values ranging from pH 2.6 to 7.2 for 20 h at 30 °C. Five µl from each incubate were adjusted to pH 4.3 and assayed for yapsin 1 activity by the ACTH1-39 assay which was carried out at 30 °C for 30 min.

Time Course Studies on the Activation of Proyapsin 1-- The time course of ACTH1-39 cleavage was investigated to determine the profile of proyapsin 1 activation. An aliquot (5-45 µl) of the proyapsin 1 was incubated in 900 µl of 0.1 M sodium citrate/sodium phosphate buffer, pH 4.3, at 30 °C, in the presence of ACTH1-39 (22 µM). At regular intervals, 100-µl aliquots were removed, and the presence of ACTH1-15, as a specific indicator of yapsin 1 activity, was quantitated by HPLC. Note that the lower amounts of proyapsin 1 were used so that sufficient substrate remained when the longer incubations were performed. It was calculated that >= 70% of the substrate remained at the end of the time courses. The amount of ACTH1-15 that was generated was then normalized to 5 µl of the proyapsin 1/100-µl reaction.

The rate at which yapsin 1 activity was generated from proyapsin 1 was investigated by a pre-activation experiment. Forty five µl of the proyapsin 1 were incubated in 900 µl of 0.1 M sodium citrate/sodium phosphate buffer, pH 4.3, at 30 °C, in the absence of ACTH1-39. At regular times after the start of the incubation, 90 µl were removed, added to 10 µl of ACTH1-39 (220 µM), and incubated for a further 30 min at 30 °C. ACTH1-15 was then quantitated by HPLC. To investigate if active yapsin 1 itself could activate proyapsin 1, 5 µl of proyapsin 1 were incubated in the presence and absence of 2.2 units of purified yapsin 1 from yeast for 30 min at 30 °C in 0.1 M sodium citrate/sodium phosphate buffer, pH 4.3. ACTH1-39 (220 µM) was then added, and the tubes were incubated for a further 30 min at 30 °C. The presence of ACTH1-15 was then quantitated by HPLC.

To study the molecular changes that occurred after activation, a pre-activation experiment was performed and analyzed by Western blot and assayed for enzymatic activity. Ten µl of the proyapsin 1 was incubated for 22 h in 40 µl of either 0.1 M sodium citrate/sodium phosphate buffer, pH 4.3, or 0.1 M sodium phosphate buffer, pH 7.2, after which 5 µl of the incubates were assayed for the presence of yapsin 1 activity by the ACTH1-39 assay for 30 min at 30 °C. 1.25 µl of the incubates were also analyzed by Western blot using antisera MW283 and VO2377 after SDS-PAGE under reducing conditions.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Purification and Analysis of Yapsin 1 from Yeast

Yapsin 1 was purified from the culture supernatant of the yeast expression system yielding ~1 µg of yapsin 1/g of wet yeast. The purification procedure resulted in an overall recovery of 29.7% of yapsin 1 from the starting media with an overall fold purification of 43.6 (Table II). Anion exchange chromatography of the concentrated culture supernatant on the MonoQ1 column resulted in the step with the highest fold purification of 8.7 and a recovery of ~64%. Yapsin 1 eluted from the column in 100-150 mM NaCl (Fig. 1A). Application of the MonoQ1 pool of activity on the Superdex G-200 column resulted in a bimodal distribution of yapsin 1 activity (Fig. 1B) which was attributed to the two hyperglycosylated forms of secreted yapsin 1, a ~150-180-kDa and a ~90-kDa form that have previously been characterized (8). Fractions 54-63, corresponding to the ~150-180-kDa molecular mass form of yapsin 1, were pooled for the next purification step resulting in a ~57% recovery of activity in the G-200 step. The inclusion of a second MonoQ column at this time was necessary to remove a contaminating protein, identified by amino acid sequencing as glucan-1,3-beta -glucosidase (Fig. 2, lanes 2 and 3), that appeared to be co-purifying with yapsin 1. The shallow NaCl gradient used to elute the protein from the column generated a broad peak of yapsin 1 activity (data not shown); however, only the first half of the peak (fractions 52-57) was pooled, to avoid the glucosidase, and referred to as purified yapsin 1. 

                              
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Table II
Summary of the purification procedure of yapsin 1 from yeast


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Fig. 1.   Purification profiles of secreted yapsin 1. The concentrated culture supernatant from yeast overexpressing yapsin 1 was applied to a MonoQ column (A) under the conditions described under "Experimental Procedures." The pool of yapsin 1 from this column was further concentrated and applied to the Superdex G-200 column (B). Activity of yapsin 1 was measured by the ACTH1-39 assay (15), and the results are represented in relative units by the bar chart in both panels.


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Fig. 2.   Analysis of yapsin 1 from yeast by SDS-PAGE. 280 ng of the purified yapsin 1 was analyzed by silver stain under reducing conditions. One protein was stained, demonstrating the apparent homogeneity of the enzyme (lane 1). 1.6 µg of a partially purified preparation of yapsin 1 was deglycosylated by endoglycosidase H, described previously (8), and analyzed by Coomassie Blue under nonreducing (lane 2) and reducing conditions (lane 3). Note the shift in mobility of yapsin 1 (upper band) and the appearance of a diffuse staining band at ~34 kDa when the protein was reduced. Amino-terminal amino acid analysis confirmed the identity of the upper band as the beta -subunit of yapsin 1, whereas the diffuse band was identified as the alpha -subunit. The focused band present in both lanes was identified as glucan-1,3-beta -glucosidase.

Silver stain analysis demonstrated the apparent homogeneity of the yapsin 1 preparation by staining only one protein (Fig. 2, lane 1), and amino-terminal amino acid sequence analysis resulted in two yapsin 1 sequences as follows: sequence 1, A68DGYEEIIITNQQSF; sequence 2, D145INPFGWL(T)GTGSAI. The internal sequence of yapsin 1, starting at Asp145, has been obtained on four different occasions, each with a different enzyme preparation, rendering this cleavage consistent and specific. When the picomole recovery of amino acids from each cycle of the sequencer was plotted, a least squared linear regression fit allowed an initial concentration of each sequence to be calculated by extrapolation. The average relative ratio of the two sequences was 1.22:1 for sequence 1 relative to sequence 2, demonstrating that ~90% of the yapsin 1 molecules has been processed into two subunits, alpha  and beta . To determine if the two yapsin 1 subunits were associated by a disulfide bond, a sample of a partially purified preparation of yapsin 1 was analyzed by SDS-PAGE under nonreducing and reducing conditions followed by Coomassie Blue staining and amino-terminal amino acid sequencing. Coomassie Blue staining of 1.6 µg of unreduced protein from the Superdex G-200 pool of yapsin 1 activity that had been deglycosylated by endoglycosidase H showed a major protein between the 66- and 97-kDa molecular mass standards (Mark12, Novex, San Diego, CA) and a minor protein at ~31 kDa (Fig. 2, lane 2). Amino-terminal amino acid sequencing of the upper band gave a similar relative picomole ratio of the alpha - and beta -subunit sequences of yapsin 1 as the purified preparation described above, whereas the lower band contained only yeast glucan-1,3-beta -glucosidase. When a similar aliquot of the protein was run in the presence of beta -mercaptoethanol, a reduction in the molecular mass of the yapsin 1 band to ~65 kDa was observed concomitant with the appearance of a diffuse protein at ~34 kDa (Fig. 2, lane 3). Amino-terminal amino acid sequencing of the upper band resulted in the beta -subunit sequence of yapsin 1, whereas the lower band resulted in the alpha -subunit sequence of yapsin 1 in addition to the glucosidase sequence.

Analysis of an Active Site Mutant of Yapsin 1 Expressed in Delta Yapsin 1 Yeast

It was demonstrated by Western blot using antiserum MW283 that yapsin 1 and yapsin 1(Asp101 right-arrow Glu101) were successfully expressed and secreted at similar levels in the Delta yapsin 1 yeast strain (Fig. 3A, lanes 3 and 4). Yapsin 1 was ~5 kDa smaller than the active site mutant. No enzymatic activity was present from the culture supernatant of the mutant yapsin 1, although abundant activity was observed with the normal yapsin 1 (Fig. 3B). Western blot analysis using antiserum VO2377, which is specific for the propeptide of yapsin 1, showed strong immunostaining of yapsin 1(Asp101 right-arrow Glu101) (Fig. 3A, lane 1), whereas only trace amounts were evident in the normal yapsin 1 sample (Fig. 3A, lane 2).


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Fig. 3.   A, Western blot analysis under nonreducing conditions of mutant and normal yapsin 1. The yapsin 1 propeptide-specific antiserum, VO2377 (lanes 1 and 2), and anti-yapsin 1 antiserum, MW283 (lanes and 4), were used. The samples were treated with endoglycosidase H prior to the Western blot to reduce the hyperglycosylated forms of secreted yapsin 1 to ~65 kDa. Lanes 3 and 4 show equivalent intensity of staining of the mutant and normal yapsin 1, respectively, by antiserum MW283, whereas an antiserum specific for the propeptide shows strong staining of the mutant yapsin 1 (lane 1) and only minor staining of the normal yapsin 1 (lane 2). The mutant yapsin 1 is ~5 kDa bigger than the normal yapsin 1. Western blots were performed on 8-16% Tris/glycine gradient polyacrylamide gels. M represents the Mark 12 molecular mass markers from Novex, San Diego, CA. B, enzymatic activity analysis of the active site mutant of yapsin 1 compared with normal yapsin 1. Yapsin 1 activity was measured by the ACTH1-39 assay and expressed as units/µg protein. See Table II legend for definition of yapsin 1 units. Abundant activity was obtained from the normal yapsin 1, and no activity was observed from the mutant yapsin 1.

Analysis of Baculovirus-expressed Yapsin 1 Recombinants

Western blot analysis of ConA-purified media (1.6 µg of protein) from Sf9 cells expressing proyapsin 1 and Delta proyapsin 1 demonstrated that proyapsin 1 was secreted into the growth media as a ~60-kDa protein (Fig. 4A, lane 1), whereas the Delta proyapsin 1 was not secreted (Fig. 4A, lane 2). Western blot analysis of the corresponding cell extracts (3.8 and 4.3 µg of protein) showed that proyapsin 1 was present as a ~75-kDa protein and Delta proyapsin 1 was present as a ~70-kDa protein (Fig. 4A, lanes 3 and 4). Furthermore, Delta proyapsin 1 shifted to a lower molecular mass upon treatment by N-glycanase indicating the presence of N-linked sugars on Delta proyapsin 1 (data not shown).


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Fig. 4.   Western blot analysis of the ConA-purified media (lane 1 and 2) and soluble cell extract (lane 3 and 4) from Sf9 cells expressing proyapsin 1 or Delta proyapsin 1. Both proyapsin 1 and Delta proyapsin 1 were expressed; however, only proyapsin 1 was secreted (lane 1), and Delta proyapsin 1 was retained intracellularly (lane 4). M represents SeeBlue molecular mass markers from Novex, San Diego, CA. Western blots were performed on 8-16% Tris/glycine gradient polyacrylamide gels.

The ConA-purified media and the soluble cell extracts (1.3 and ~12 µg of protein, respectively) were further analyzed by the ACTH1-39 activity assay with and without a pre-activation step at pH 4.3 and 37 °C. The ConA-purified media were pre-activated for 12 h, and the soluble cell extracts were pre-activated for 2 h. Yapsin 1 enzymatic activity was found only in the proyapsin 1 samples yielding a ~31-fold increase in yapsin 1 activity from the ConA-purified media and a 5.6-fold increase in yapsin 1 activity from the soluble cell extract as a result of the pre-activations (Table III).

                              
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Table III
Summary of the enzymatic activity of proyapsin 1 and Delta proyapsin 1 expressed in the baculovirus/Sf9 expression system
12 µg of protein from the soluble cell extracts and 1.3 µg of protein from the ConA-purified culture supernatant of proyapsin 1 and Delta proyapsin 1 expressing Sf9 cells were assayed by the ACTH1-39 assay. In a duplicate experiment, the cell extracts were pre-activated at pH 4.3 and 37 °C for 2 h while the ConA purified media was pre-activated for 12 h before the ACTH assay. Activity is expressed as ng of ACTH1-15 generated per reaction. ND, not detectable.

pH-dependent Activation of Proyapsin 1-- After incubation of 5 µl of proyapsin 1 for 20 h at various pH values, an aliquot of each incubate was assayed for yapsin 1 activity. A plot of nanogram of ACTH1-15 generated versus preincubation pH demonstrated a pH-dependent generation of yapsin 1 activity (Fig. 5). Activity was generated at acidic pH values between 3.0 and 5.0 with an optimum at pH 4.0. The samples that were incubated below pH 3.0 were unable to be activated by a subsequent incubation at pH 4.3 indicating that the enzyme had been irreversibly denatured. However, the samples that were incubated above pH 5.0 were able to be activated by a subsequent incubation at pH 4.3 indicating that the enzyme had not been denatured (data not shown).


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Fig. 5.   pH activation profile of proyapsin 1 expressed in Sf9 cells. Thirteen aliquots of proyapsin 1 (5 µl) were incubated for 20 h at 30 °C with a range of pH values between 2.6 and 7.2. An aliquot of each sample was then adjusted to pH 4.3 and analyzed by the ACTH1-39 assay at 30 °C. Activity is plotted as nanograms of ACTH1-15 generated/5 µl of proyapsin 1/100-µl reaction as a function of the preincubation pH. Activity was generated in a pH-dependent manner with an optimum at pH 4.0.

Time Course Studies on the Activation of Proyapsin 1-- When proyapsin 1 was incubated at 30 °C and pH 4.3 in the presence of ACTH1-39 for various time points, the activity profile obtained demonstrated an initial lag in the generation of ACTH1-15 after which the product was generated in an apparent linear fashion (Fig. 6A). In reality, however, the mechanism governing the generation of product is a function of two processes, i.e. the activation of the enzyme and the activity of the enzyme, and should be described by nonlinear second-order kinetics. The observed apparent linearity is due to the fact that the rate of activation of the proyapsin 1 is significantly lower than the rate of ACTH1-39 hydrolysis. In such a case, the second-order process would appear as a single-order process.


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Fig. 6.   Kinetic studies on the activation of proyapsin 1. A, proyapsin 1 was incubated for 7 h with 22 µM ACTH1-39, pH 4.3, 30 °C, and at regular time points, the presence of ACTH1-15 was quantitated by HPLC. The data presented are a combination of four experiments, and the activity is expressed as nanograms of ACTH1-15 generated/5 µl of proyapsin 1/100-µl reaction. Note the lag phase in the profile indicative of the formation of an intermediate. A nonlinear regression fit of the data, calculated by SigmaPlotTM, version 4.0, resulted in the following equation, y = -1782.6 + 1162.5x + 67.9x2, r2 = 0.96. B, proyapsin 1 was preincubated without ACTH1-39, pH 4.3, and 30 °C. At different time points, aliquots were removed, added to ACTH1-39, and allowed to incubate for a further 30 min at 30 °C. The amount of ACTH1-15 generated was then quantitated by HPLC. The data presented are a combination of three experiments, and the activity is expressed as nanograms of ACTH1-15 generated/5 µl of proyapsin 1/100-µl reaction. Note the linearity in the generation of yapsin 1 activity demonstrated by its equation, y = 123.6x + 81.5, r2 = 0.99. Inset, 5 µl of proyapsin 1 were preincubated with and without 2.2 units of active yapsin 1 from yeast for 30 min, pH 4.3, 30 °C, and then total yapsin 1 activity was determined by the ACTH1-39 assay at 30 °C. The results presented are the average of two experiments. Yapsin 1 alone (1) generated 406.6 ng of ACTH1-15 and proyapsin 1 alone (2) generated 219.6 ng of ACTH1-15. When combined (3) the resulting total activity of 594 ng of ACTH1-15 demonstrated that active yapsin 1 did not activate the proyapsin 1.

When proyapsin 1 was incubated at 30 °C and pH 4.3 in the absence of ACTH1-39 for various time points and then assayed for yapsin 1 activity, it was found that yapsin 1 activity was generated with apparent first-order kinetics (Fig. 6B). When 2.2 units of purified yapsin 1 were incubated with proyapsin 1 at 30 °C and pH 4.3 for 30 min prior to the ACTH1-39 assay, the resulting total activity (594 ng of ACTH1-15) was approximately equal to the combined activity of the pure yapsin 1 from yeast (406.6 ng ACTH1-15) and the activated yapsin 1 from the proyapsin 1 (219.6 ng of ACTH1-15) (Fig. 6B, inset). This experiment was performed in duplicate with similar results indicating that active yapsin 1 did not activate proyapsin 1 intermolecularly.

The molecular changes of proyapsin 1 that occurred after a 22-h activation were analyzed by Western blot using antisera V02377 and MW283. In addition to the observed increase in yapsin 1 activity (Fig. 7A), proyapsin 1 was converted from ~65 kDa (Fig. 7B, lanes 1 and 3) to a slightly smaller protein that was immunoreactive with both MW283 and VO2377 (Fig. 7B, lanes 2 and 4). The intensity of the immunostained band of proyapsin 1 (unactivated and activated) under nonreducing and reducing conditions remained the same (data not shown) and indicated that baculovirus-expressed proyapsin 1 was not processed into subunits.


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Fig. 7.   Proyapsin 1 was incubated for 22 h at pH 7.2 and pH 4.3. A, an aliquot of each was assayed for yapsin 1 activity, and the results were expressed as nanogram of ACTH1-15 generated/5 µl of proyapsin 1/100-µl reaction. B, additional aliquots were analyzed by Western blot under reducing conditions on a 12% Tris/glycine SDS-PAGE gel. Lanes 1 and 2 were probed with antiserum MW283, and lanes 3 and 4 were probed with antiserum VO2377. Note the size difference between the immunoreactive bands in the unactivated (lanes 1 and 3) and the activated samples (lanes 2 and 4) stained with both antisera.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Our previous work demonstrated that recombinant yapsin 1, devoid of its GPI membrane-anchoring domain, was efficiently overexpressed and secreted from a yeast expression system. These forms of yapsin 1 were found to be hyperglycosylated and stable to lyophilization and long term storage under a variety of conditions. In contrast to the yapsin 1 obtained from the cellular extract of the same expression system (14), the stability of the secreted enzyme rendered it useful for kinetic studies since the specific activity remained constant for long periods. In addition, the abundance of yapsin 1 activity in the culture supernatant as compared with the cell extract (8) represented a source of enzyme in quantities potentially sufficient for structural analysis. Yapsin 1 was purified to apparent homogeneity from the culture supernatant yielding ~1 µg of yapsin 1/g of wet yeast. The procedure utilized anion exchange chromatography (Fig. 1A) based on the finding that the isoelectric point (pI) of yapsin 1 was ~4.5 (8), thus rendering this enzyme negatively charged at pH 7.0. Gel filtration chromatography was also used (Fig. 1B), since secreted yapsin 1 was previously determined to be hyperglycosylated, primarily giving a molecular mass of ~150-180 kDa (8, 9). A combination of these procedures was used with an overall recovery of 29.7% and a fold purification of 43.6 (Table II). The unexpected increase in total activity observed in the concentration step (168%, Table II) was initially thought to be from the removal of a low molecular mass inhibitor; however, upon addition of the filtrate back to the retentate, no inhibition of the activity was observed (data not shown). The presence of a small amount of proyapsin 1 in the media (Fig. 3A, lane 2) that became activated during the initial stages of the concentration step may in part account for the increase. The major increase, however, is most likely due to compounded small errors in the pipetting introduced during the many dilutions of up to 1 × 104 which were required to obtain quantifiable yapsin 1 activity in the highly sensitive ACTH1-39 assay for yapsin 1 (picogram range). This was confirmed when the procedure was repeated on a smaller scale where such large dilutions were not necessary. In that instance the recovery of total yapsin 1 activity in the retentate was ~95% of the starting material.

Purity of the final preparation of enzyme was confirmed by silver stain (Fig. 2, lane 1) and amino-terminal amino acid analysis. As expected, the mature enzyme was shown to start at Ala68 indicating that the proregion had been removed, but surprisingly, an additional internal sequence was obtained. Processing in this region resulted in the generation of two subunits designated here as alpha - and beta -subunits each with one of the active site triads (Fig. 8D). The amino-terminal amino acid of the beta -subunit, identified as Asp145, is situated within a unique loop domain of yapsin 1 arising from a large insertion of ~76 residues in comparison with pepsin, rendering this site susceptible to cleavage by proteases. Whether the loop gets cleaved at this residue only or amino-terminally to this residue followed by aminopeptidase trimming is still unknown. This loop domain is also found in yapsin 2 and may represent an important domain of the yapsins in general. Similar processing of cathepsin D has also been observed in a species- and tissue-specific manner where both active site triads are separated into two subunits by cleavage at a site within a 9-residue insertion (23). Processing into subunits appears to be a rare event for mammalian aspartic proteases, with cathepsin D as one exception, but it has been documented for Aspergillus niger proteinase A (24) and a barley aspartic protease (25). However, until now, association of these subunits was found to be noncovalent. We have shown here that the yapsin 1 subunits are associated by a disulfide bond (Fig. 2, lanes 2 and 3), and by comparison with the known structure of pepsin (26), the disulfide bond is most likely between Cys117 of the alpha -subunit and Cys186 of the beta -subunit. The relevance of such a processing is unknown; however, preliminary pulse-chase experiments3 have indicated that it occurs early in the yeast secretory pathway and may represent a key event in the structure/function relationship of yapsin 1 in vivo.


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Fig. 8.   Schematic diagram of the structural components of yapsin 1. A, the molecule contains a typical signal sequence (ss, amino acids 1-21), a proregion (pro, amino acids 22-67), and the mature enzyme (amino acids 68-569). The GPI membrane anchor is situated in the extreme carboxyl-terminal domain of the molecule. B, from the studies of the baculovirus-expressed proyapsin 1, an intermediate or pseudo-yapsin 1 has been identified presumably as a result of a self-cleavage at Lys37 (i.e. Lys16 of the propeptide). C, the remainder of the proregion is then likely to be removed at the Lys66-Arg67 cleavage site by an intra-molecular mechanism. D, from the studies on yapsin 1 expressed in yeast, it was verified that the proregion had been completely removed at Lys66-Arg67. The mature enzyme is also processed into an alpha - and beta -subunit resulting in Asp145 as the amino-terminal amino acid of the beta -subunit. The subunits are associated by a disulfide bond predicted to be between Cys117 and Cys186. The carboxyl terminus is removed during the GPI-anchoring process which is predicted to occur at Asn548. The two active site aspartic acid residues are indicated by an asterisk.

The amino-terminal amino acid of the alpha -subunit, identified as Ala68, indicated that the proregion was removed at the paired basic residue site, Lys66-Arg67, a site similar to that in the activation of pro-renin to renin (27, 28). Since this propeptide processing site represents an efficient motif for yapsin 1 cleavage, RXXKR (15), it suggested that processing of the propeptide may occur by autoactivation in contrast to renin which relies on a trypsin-like activity, although active renin has been reported to contain some intrinsic activity capable of activating prorenin (29). We investigated this hypothesis by expressing an active site mutant of yapsin 1 and demonstrated that the inactive enzyme was ~5 kDa bigger than the normal active yapsin 1 (Fig. 3, lanes 3 and 4). This expected difference in size corresponded to the theoretical molecular mass of the propeptide, the presence of which was confirmed by Western blot (Fig. 3, lane 1) and is consistent with the inability of the active site mutated yapsin 1 to remove its own propeptide. Also, since both constructs were expressed in yapsin 1-deficient yeast strains, we can also conclude that the two other known endogenous processing enzymes with specificity for this type of cleavage site, Kex2p and Mkc7p (yapsin 2), were not able to process the mutant proyapsin 1 either. It appears, therefore, that removal of the propeptide is dependent upon active yapsin 1 molecules.

To study further the activation of yapsin 1 in vitro, yapsin 1 was expressed in the baculovirus/Sf9 system, with and without its proregion (proyapsin 1 and Delta proyapsin 1, respectively), and analyzed with respect to its properties. Both recombinant proteins were expressed in this system; however, only proyapsin 1 was secreted (Fig. 4, lane 1). Absence of the proregion resulted in the expression of an intracellularly localized protein (Fig. 4, lane 4) that contained no apparent activity even when pre-activated (Table III). These observations are consistent with a misfolded protein that was transport-incompetent. However, that it had entered the secretory pathway is supported by the result that it was N-glycanase-sensitive (data not shown). In contrast to Delta proyapsin 1, proyapsin 1 was secreted from the cells and possessed significant levels of activable yapsin 1 activity (Table III), showing that the proregion was required for the correct folding and secretion of yapsin 1, and only as a correctly folded enzyme was it able to be activated. This activation process was characterized further and shown to occur optimally at pH 4.0 with <10% activated in the pH range of 5.5-6.0 and no apparent activation at the more neutral pH values (Fig. 5). In addition, the enzyme was irreversibly denatured below pH 3.0 (data not shown), a result that had been observed previously (30). The observed progressions of product formation in both the activity profile experiment (Fig. 6A) and the pre-activation experiment (Fig. 6B) indicated that the mechanism of activation was intra-molecular, a result that was further supported by the result that purified yapsin 1 from yeast was unable to activate the proyapsin 1 (Fig. 6B, inset). Previous reports by us (8) and others (9) have deduced that yapsin 1 is located to the extracellular side of the plasma membrane via its GPI membrane anchor and is presumed to be its site of action. Since the pH of yeast growth media is acidic (pH 5.0-6.0) and becomes more acidic with growth, the periplasmic space may represent the primary compartment where autoactivation occurs. However, yapsin 1 was first cloned based on its ability to process the yeast prohormone, pro-alpha -mating factor (6), in the secretory pathway of KEX2-deficient yeast. Since the synthesis of biologically active alpha -mating factor requires the subsequent action of two other Golgi resident enzymes, Kex1p (31), a carboxypeptidase B-like enzyme and a dipeptidyl-aminopeptidase (32), it is assumed that a sufficient amount of proyapsin 1 must become activated in the late Golgi in order for mature alpha -factor to be generated. This is supported by the fact that proyapsin 1 was expressed and functionally active in mammalian PC12 cells (17) where the pH ranges from pH 6.2 in the trans-Golgi network (33) to pH 5.0-5.5 in mature secretory vesicles (34).

Investigation of the biosynthesis and maturation of aspartic proteases such as endothiapepsin (35), cathepsin D (36), pepsin (37), Rhizopus niveus aspartic proteinase-I (38), and yeast proteinase A (39, 40) have all demonstrated the importance of their proregion in the folding of the enzyme and the regulation of their activity. For pepsin, the model aspartic protease, its proregion, which contains 11 basic residues, is partially stabilized by ionic interactions with the negatively charged aspartic acid residues of the active site (41). Upon acidification, the aspartic acid residues become protonated and less charged (pKa = 4.3) causing destabilization and an intramolecular cleavage at Leu16-Ile17 within the propeptide resulting in the generation of an intermediate form of the enzyme or pseudo-pepsin. The intermediate form undergoes a further intermolecular or intramolecular cleavage of the remainder of the proregion to generate the mature active enzyme. It is interesting to note the similarity to yapsin 1 in this respect in that a potential cleavage site exists at Lys16-Phe17 within the propeptide. Because of this structural similarity and that cleavage of a mono-lysine residue by yapsin 1 has previously been documented (15), it is predicted that a yapsin 1 intermediate, formed from an intramolecular cleavage presumably at this site, exists. Our activation studies support this prediction, since during the time course experiment, an initial lag phase (within 3 h) in the generation of yapsin 1 activity was observed (Fig. 6A). This observation is consistent with a molecular event that involves the generation of an intermediate, a process that has been documented for other aspartic proteases (36, 37, 42). Further support came from the pre-activation experiment where the immunoreactive proyapsin 1 was converted to a slightly smaller protein upon activation (Fig. 7A). Since this band still contained the propeptide antigenic site for antiserum VO2377, i.e. Tyr47-Glu61 (Fig. 7A, lane 4), the size shift is consistent with removal of an amino-terminal portion of the proregion and the assignment of this band as an intermediate, i.e. pseudo-yapsin 1. Although trimming at the carboxyl terminus cannot be completely ruled out, the use of serum-free medium, a 2-day infection to limit cell lysis and the addition of protease inhibitors, renders the presence of potential proteases specific for the carboxyl terminus of proyapsin 1 highly unlikely. Furthermore, the inability of 6 µM pepstatin A to inhibit formation of pseudo-yapsin 1 (data not shown) indicates that another aspartic protease was not responsible for the processing of proyapsin 1 and provides further evidence that the process was intra-molecular since the active site, already tightly bound to the proregion, was inaccessible to the inhibitor.

Although the generation of pseudo-yapsin 1 appears to be fairly rapid, within 3 h as evidenced by the lag in Fig. 6A and a time course study (data not shown), the generation of yapsin 1 from pseudo-yapsin 1 appeared to be extremely slow. The concentration of proyapsin 1 in the starting material was estimated by Western blot from a standard curve made with purified yapsin 1 from yeast (data not shown) and based on 100% conversion of this amount, the amount of enzymatic activity obtained was found to be <1% of that expected. Such inefficient activation explains why no mature processed yapsin 1 was detectable in the Western blot in the activated sample (Fig. 7). Attempts at increasing the efficiency with either calcium or dithiothreitol had no effect on the rate (data not shown) making it apparent that either some other co-factor is necessary or that some structural requirement has not been met by the baculovirus-expressed proyapsin 1 for activation to occur efficiently. The most obvious structural difference of proyapsin 1 expressed in Sf9 cells is its lack of processing into alpha - and beta -subunits. We speculate that the lack of this processing may result in conformational restraints on pseudo-yapsin 1 to allow efficient removal of the remainder of the proregion at Lys66-Arg67 and may be the reason why pseudo-yapsin 1 appears to get stuck in that form. Other pseudo-aspartic proteases, such as pseudo-chymosin and pseudo-cathepsin D (42, 43) under certain conditions, have also been found to be poorly converted.

In conclusion, our studies with yeast have demonstrated that mature yapsin 1 is a heterodimer stabilized by an intramolecular disulfide bond, and the final proregion cleavage site, which was yapsin 1-dependent, was confirmed to occur at the Lys66-Arg67 (Fig. 8D). From our baculovirus expression studies, the proregion of yapsin 1 was required for the formation of intact, single-chain proyapsin 1 which was then capable of being self-activated by incubation at acidic pH. The in vitro activation process resulted in the generation of pseudo-yapsin 1 (Fig. 8B) which appeared to be converted to mature yapsin 1 at an extremely slow rate by an apparent intramolecular mechanism (Fig. 8C). It is hypothesized that the slow rate of in vitro activation may be a result of the difference between single-chain proyapsin 1 and two-chain proyapsin 1 found in yeast. The enzyme(s) responsible for the processing of yapsin 1 into the subunits may ultimately play an important role in the regulation of yapsin 1 activity in vivo and is currently under investigation.

    ACKNOWLEDGEMENT

We thank Dr. Ying Zhang for expert help in the baculovirus expression of the yapsin 1 constructs.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Bldg. 49, Rm. 5A38, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-8222; Fax: 301-496-9938; E-mail: cawley{at}codon.nih.gov.

1 The abbreviations used are: GPI, glycophosphatidylinositol; YAP3, yeast aspartic protease 3; ACTH, adrenocorticotropin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; ConA, concanavalin A; HPLC, high pressure liquid chromatography.

2 N. X. Cawley, V. Olsen, and Y. P. Loh, unpublished observations.

3 V. Olsen, N. X. Cawley, and Y. P. Loh, manuscript in preparation.

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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