(Received for publication, November 25, 1996, and in revised form, January 7, 1997)
From the Department of Clinical Biochemistry,
Rigshospitalet, University of Copenhagen, Copenhagen Ø, Denmark and
Novo Nordisk A/S, Bagsværd, Denmark
Precursors of the human regulatory peptide
cholecystokinin (CCK) have been expressed in Saccharomyces
cerevisiae, and the post-translational processing of secreted
CCK-related products analyzed. Recombinant plasmids expressing native
human prepro-CCK and a hybrid molecule encompassing the prepro leader
of the yeast -mating pheromone fused to pro-CCK were examined. The
latter construct resulted in considerably higher levels of pro-CCK
secretion and was therefore analyzed in more detail. Two of the protein modifications essential for CCK bioactivity, C-terminal
-amidation and tyrosyl sulfation, were not detected in S. cerevisiae.
Proteolytic cleavage of pro-CCK occurred C-terminally of three basic
sites; (i) Arg105-Arg106 which, upon exposure
to carboxypeptidase activity, leads to the production of
glycine-extended CCK; (ii) Arg95 to produce CCK-8 related
processing intermediates; and (iii) Lys81 resulting in
CCK-22 related products. To elucidate which protease(s) are involved in
these endoproteolytic cleavage events, pro-CCK was expressed in yeast
mutants lacking various combinations of the Mkc7, Yap3, and Kex2
proteases. Only in S. cerevisiae strains deficient in Kex2
function was any of the above mentioned pro-CCK cleavages abolished,
namely processing at the Arg105-Arg106 and
Arg95 sites. This suggests that mammalian Kex2-like serine
proteases may process pro-CCK at single arginine residues. Our data
suggests that an as yet uncharacterized endopeptidase(s) in the
S. cerevisiae secretory pathway is responsible for the
lysine-specific cleavage of pro-CCK.
Cholecystokinin (CCK)1 is a vertebrate
neurohormonal peptide which controls a wide variety of functions,
predominantly in the digestive tract and the brain (for review see;
Crawley and Corwin (1994)). During precursor maturation pro-CCK is
subjected to a number of post-translational modifications, including
C-terminal
-amidation, tyrosyl sulfation, and endoproteolytic
processing (Rehfeld et al., 1988
). Bioactive CCKs possess
the same amidated C terminus which results from endoproteolytic
cleavage C-terminally of the dibasic
Arg105-Arg106 residues followed by removal of
these basic amino acids by carboxypeptidase digestion, exposing
Gly104 to peptidyl-monooxygenase activity (Fig.
1A). In contrast, endoproteolytic processing
of pro-CCK is far more heterogeneous at the N terminus with
examples of CCK-58, -39, -33, -22, -8, and -5 having been identified in
various tissues or plasma samples (Fig. 1A) (Rehfeld and
Hansen, 1986
; Rehfeld et al., 1988
; Eberlein et
al., 1992
). Many of these forms of CCK are produced via cleavage
of pro-CCK C-terminally of single arginine residues (Rehfeld and
Hansen, 1986
; Eberlein et al., 1992
). However, CCK-22, the
predominant hormonal form of CCK, is produced by endoproteolytic
processing at Lys81. Consequently this lysine processing
event is of crucial significance for understanding the biology of CCK
(Liddle et al., 1984
, 1985
; Cantor and Rehfeld, 1987
;
Rehfeld, 1994
; Paloheimo and Rehfeld, 1995
).
Many foreign proteins have been expressed in yeast (Hadfield et
al., 1993). Often these proteins are directed through the yeast
secretory pathway, either by the endogenous N-terminal signal sequence
of the protein or via production of a hybrid protein which includes the
leader region of a secreted yeast preproprotein. Passage through the
secretory pathway exposes proteins to a number of post-translational
capabilities, including disulfide bridge formation, glycosylation and
proteolytic processing (Hadfield et al., 1993
). The best
characterized yeast endopeptidase is the Kex2 serine protease, which
was identified for its ability to cleave precursors of the
-mating
pheromone and killer toxin at dibasic sites (Julius et al.,
1984
). Overexpression studies in kex2 mutants resulted in
the isolation of the yeast aspartyl protease (Yap3), which is able to
rescue the mating deficiency of kex2 deficient
Saccharomyces cerevisiae (Egel-Mitani et al.,
1990
). More recently a third endopeptidase, Mkc7, with considerable
homology to Yap3 has been detected (Komano and Fuller, 1995
). This
protease, which was isolated as a multicopy suppressor of the cold
sensitive growth phenotype of kex2 mutants, has the ability
to cleave an internally quenched flurogenic substrate C-terminally of a
Lys-Arg site (Komano and Fuller, 1995
).
In the present study, we have examined the extent of post-translational processing of human pro-CCK expressed and secreted from S. cerevisiae. Evidence was found for an, as yet, uncharacterized endopeptidase in yeast which is able to cleave CCK at a single lysine residue to produce CCK-22, a predominant form of circulating CCK in mammals.
Yeast
strains used in this study are listed in Table I.
S. cerevisiae grown in YPD medium were transformed with
plasmid DNA using the lithium acetate procedure (Ito et al.,
1983). Transformants were selected and propagated at 30 °C in
synthetic complete medium lacking uracil (Sherman, 1991
).
|
A full-length cDNA clone encoding
human prepro-CCK was generously supplied by Karin Pedersen. The
S. cerevisiae -mating pheromone 1 gene
(MF
1) was kindly provided by Ira Herskowitz (Kurjan and Herskowitz, 1982
). Two pro-CCK expression plasmids were constructed in
the URA3-2µ plasmid pJ399, in which gene transcription is
driven by the yeast glyceraldehyde-3-phosphate dehydrogenase promoter (Bitter and Egan, 1984
) and terminated by the yeast TPI1
termination signal (Alber and Kawasaki, 1982
). Plasmid pJ399 was
constructed from pYES2.0 (Invitrogen).
The coding region of human prepro-CCK was amplified by PCR using the
Pwo DNA polymerase (Boehringer Mannheim). The forward primer
(5-ACGTGAATTCTAAACTAAAAATGAACAGCGGCGTGTGCCTGTGCGTG-3
) resulted in an EcoRI site 10 nucleotides upstream of
the start codon, while the reverse primer
(5
-ACGTTCTAGACTAGGAGGGGTACTCATACTCCTCGGC-3
) introduced an
XbaI site immediately downstream of the TAG stop codon. The
PCR product was purified after agarose gel electrophoresis using the
Sephaglas Band Prep procedure (Pharmacia), cleaved with EcoRI and XbaI (Boehringer Mannheim), and cloned
into pJ399.
To ensure high secretion levels of CCK, we constructed a second
expression plasmid where the sequence encoding the prepropeptide of the
MF1 yeast
-factor (Kurjan and Herskowitz, 1982
) was
fused to the coding region of pro-CCK (amino acids 31-115) (Fig.
1B). This expression plasmid was constructed by a two-step
process. First, the yeast MF
1 gene was amplified by PCR
(forward primer, 5
-ACGTGAATTCTAAACTAAAAATGAGATTTCCTTCAATTTTTACTGCAG-3
; reverse primer, 5
-ACGTTCTAGATTAGTACATTGGTTGGCCGGGTTTTACA-3
) and cloned into pJ399 as described above. The resulting plasmid was cleaved with HindIII and XbaI to remove the coding region
of the four tandem
-factor repeats, but leaving the sequence
encoding the MF
1 prepropeptide. The region encoding
pro-CCK was amplified using PCR (forward primer, 5
-ACGTTCTAGACTAGGAGG
GGTACTCATACTCCTCGGC-3
; reverse primer was the same as used for
amplification of the coding region of prepro-CCK) resulting in a
product with a 5
HindIII site and a 3
XbaI site
that could be ligated into the pJ399 plasmid containing the coding
region of prepro-MF
1. The resulting plasmid encodes a
hybrid protein that consists of the first 89 amino acids of
prepro-MF
1 and the last 85 amino acids of prepro-CCK
starting at Ser31 (Fig. 1B). It was expected
that pro-CCK would be liberated in the yeast secretory pathway by Kex2
cleavage C-terminally of the Lys-Arg site in the
-factor
prepropeptide, followed by two dipeptidyl aminopeptidase digestion
steps to remove the Glu-Ala-Glu-Ala sequence (Fig. 1B)
(Hadfield et al., 1993
). To ensure the coding
nucleotides had not been altered during the PCR steps, plasmid
inserts were exposed to double stranded DNA sequencing with the dideoxy
chain termination method using the Sequenase 2.0 DNA sequencing
procedure (U. S. Biochemical Corp.).
Radioimmunoassay (RIA) analysis was used
to detect and partially characterize pro-CCK products secreted from
yeast transformants. Four monospecific antisera raised in rabbits which
binds various processing intermediates of CCK were utilized; Ab 2609, Ab 3208, Ab 7270, and Ab 89009 (Table II). Measurements
were performed as described previously (Rehfeld, 1978a; Hilsted and
Rehfeld, 1986
; Paloheimo and Rehfeld, 1994
), either directly on the
liquid media used for propagation of yeast transformants or on
fractions collected from gel chromatography. The molecules used as
tracers and standards in RIA experiments are presented in Table II.
The presence of CCK which had not been processed at the Arg105-Arg106 site, referred to as C-terminally extended CCK, was detected by RIA with Ab 3208 after the samples had been treated with trypsin and carboxypeptidase B (T/C). Trypsin cleaves C-terminally of the Arg105-Arg106 site, and carboxypeptidase B removes these residues leaving glycine-extended CCK. This procedure involves incubating the sample with an equal volume of 2 mg/ml trypsin (Boehringer Mannheim) in 0.1 mM sodium phosphate buffer (pH 7.5) at room temperature for 30 min. The reaction was terminated by boiling for 10 min, after which the samples were cooled to room temperature. Carboxypeptidase B (Boehringer Mannheim) was then added to a final concentration of 100 µg/ml, the reaction was incubated at room temperature for 30 min, and terminated by a second boiling step. Processing products of pro-CCK larger than CCK-22 were detected by RIA analysis with Ab 89009 after samples were exposed to trypsin as described above.
Gel ChromatographyYeast transformants grown to late exponential phase were centrifuged at 3,000 × g for 5 min to collect the cells and 100-500 µl of the culture supernatant was loaded directly onto a Sephadex G-50 superfine (Pharmacia) column (1 × 100 cm) at 4 °C. The sample was eluted in VBA buffer (20 mM barbital buffer, 0.6 mM thiomersal, and 0.11% bovine serum albumin) at a flow rate of 2.4 ml/h, with 800-µl fractions being collected every 20 min. The column was calibrated with trace amounts of [125I]albumin and 22NaCl to indicate the void and total volumes, respectively.
Anion exchange chromatography was conducted on a 5 × 50-mm Mono-Q HR/R anion exchange column (Pharmacia) which had been pre-equilibrated with 20 mM sodium phosphate buffer (pH 8). The column was eluted with a gradient from 0 to 20% acetic acid over 70 min, with 1-ml fractions being collected every minute. Sulfated CCK-8-Gly and non-sulfated CCK-8-Gly (Cambridge Research Biochemicals) were used for calibration. Samples were dried under vacuum and resuspended in VBA buffer before RIA analysis.
Purification of CCK Secreted from Yeast TransformantsA five-step procedure was utilized to purify CCK secreted from transformants of S. cerevisiae.
Step 1: the wild type yeast YNG318 transformed with the plasmid
encoding the hybrid prepro-MF1-pro-CCK construct was
grown in 200 ml of selective media to late exponential phase and cells removed by centrifugation at 3,000 × g for 5 min. The
supernatant was adjusted to pH 3 with trifluoroacetic acid and
centrifuged at 10,000 × g for 10 min. The cleared
supernatant was loaded onto five Sep-Pak plus C18 columns (Waters)
connected in series, which had been pre-equilibrated with 10 ml of
0.5% trifluoroacetic acid in acetonitrile followed by 10 ml of 0.5%
trifluoroacetic acid in H2O. Material was passed through
the columns at a flow rate of 2 ml/min. The column was eluted
sequentially with 10-ml solutions of 0.5% trifluoroacetic acid in 0, 10, 30, 50, 70, 90, and 100% acetonitrile. The fractions collected
were diluted in distilled water and analyzed by RIA using Ab 3208.
The same procedure was utilized with the mkc7 yap3 (HKY24)
and kex2 yap3 (ME938) double mutants expressing the
prepro-MF1-pro-CCK construct. However, the starting
quantity was 2 liters of culture medium and CCK-immunoreactive
fractions were detected using RIA analysis with Ab 89009 and/or Ab 3208 after the samples had been treated with trypsin and carboxypeptidase B.
Step 2: immunoreactive fractions from Sep-Pak purification were concentrated under vacuum to a final volume of 1 ml, adjusted to pH 8 with NaOH and loaded onto a Sephadex G-50 superfine column as described previously.
Step 3: the next stage of purification involved anion exchange chromatography on a Mono-Q column and was performed as outlined above.
Step 4: the fractions containing CCK immunoreactivity were further processed by reversed phase high performance liquid chromatography using a C8 column (2.1 × 150 mm) (Vydac) at 50 °C, eluted at 0.2 ml/min with a gradient from 0.1% trifluoroacetic acid in acetonitrile (0.5%/min). Fractions were collected every 0.5 min.
Step 5: selected immunoreactive fractions from Step 4 were subjected to a further reversed phase high performance liquid chromatography step on a Vydac C18 column using the same solvents but employing a gradient of 0.2%/min. Peak fractions were collected manually (monitored at 214 nm).
Mass Spectrometry and Peptide SequencingMolecular masses
of purified peptides were determined by matrix-assisted laser
desorption mass spectrometry performed in a Biflex instrument
(Bruker-Franzen) using -cyano-4-hydroxy cinnamic acid as a matrix.
Both positive and negative ions were analyzed in the linear mode using
external calibration to give an accuracy of 0.1%. Individual peptides
were subjected to amino acid sequence analysis using an automatic
protein sequencer (Procise 494A, Applied Biosystems) equipped with an
on-line high performance liquid chromatography system for detection of
the amino acid phenylthiohydantoins.
Transformants of
the YNG318 strain expressing the prepro-MF1-pro-CCK
fusion peptide were grown overnight at 30 °C in selective media. The
next morning the cells were collected by centrifugation and resuspended
in 500 µl of synthetic complete media lacking uracil and methionine,
and supplemented with 500 µCi of
L-[35S]methionine (Amersham). After the cells
were incubated for a further 30 min at 30 °C they were washed twice
in 50 mM Tris-HCl (pH 7.4) before being resuspended in 50 mM Tris-HCl (pH 7.4) containing 1 mM
phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, 20 µg/ml leupeptin, and 2 µg/ml pepstatin. An approximate equal volume of
pre-chilled acid washed glass beads (Sigma) was added and the sample
vortexed 10 times for 30 s with cooling on ice between each
vortexing. SDS was then added to a final volume of 1% and the cell
lysate boiled for 3 min. An equal volume of 2 × sample buffer
(100 mM Tris-HCl (pH 8.3), 380 mM NaCl, 12 mM EDTA (pH 8), 5% Triton X-100) was included and a
cleared supernatant prepared by centrifugation at 13,000 × g for 10 min at 4 °C.
Immunoprecipitations were performed at 4 °C for 2 h following the addition of 5 µl of Ab 7270 which binds to glycine extended CCK (Table II). Immune complexes were bound to Protein A-Sepharose (Pharmacia) during a second 2-h incubation at 4 °C with gentle agitation. The sample was then centrifuged at 13,000 × g for 10 s and the pellet washed 8 times in 1 × sample buffer, boiled for 5 min, and loaded directly onto a Sephadex G-50 superfine column. Radiolabeled CCK was detected in the column fractions by a combination of liquid scintillation counting and RIA analysis utilizing Ab 3208.
Transformants of
the YNG318 strain expressing prepro-CCK and the
prepro-MF1-pro-CCK fusion protein were grown to late
exponential phase and the culture media was subjected to RIA analysis
for the presence of glycine-extended, C-terminally extended, and
-amidated CCK. In each case two independent transformants were
analyzed. No major differences in CCK immunoreactivity were detected
with transformants harboring the same plasmid constructs (Table
III).
|
Secretion of glycine-extended CCK was determined by immunoreactivity to
Ab 3208 (Table III). Both constructs resulted in the secretion of
glycine-extended CCK from yeast transformants. However, approximately
hundredfold higher levels of secretion were seen when CCK was expressed
as a fusion to the -factor prepropeptide compared with the native
prepro-CCK. Treatment of the samples with trypsin and carboxypeptidase
B increased the quantity of glycine-extended immunoreactivity at least
2-fold indicating that considerable quantities of secreted CCK was
C-terminally extended (Table III).
In contrast to glycine and C-terminally extended CCK, no -amidated
CCK was found in media supernatants of yeast expressing pro-CCK, as
indicated by the absence of immunoreactivity to Ab 2609 in media in
which yeast expressing the prepro-CCK or the prepro-MF
1-pro-CCK constructs had been cultured (Table
III).
To further characterize the extent of endoproteolytic
processing of CCK produced in S. cerevisiae, secreted
material of strain YNG318 expressing the
prepro-MF1-pro-CCK fusion protein were subjected to gel
chromatography. Analysis of the fractions collected for
glycine-extended CCK uncovered two major peaks of CCK immunoreactivity, one at an elution constant (KD) of 0.75, the other
1.15 (Fig. 2A). These KD
values are similar to the known elution constants of CCK-22-Gly and
CCK-8-Gly, respectively, determined by the same procedures (Cantor and
Rehfeld, 1987
). To confirm the identity of the CCK-immunoreactive
products they were purified to homogeneity and subjected to mass
spectrometry and protein sequence analysis. As predicted, the latter
immunoreactive peak was identified as CCK-8-Gly and the earlier as
CCK-22-Gly (Fig. 3, A and B). It
should be noted that the peptides isolated during this study were
oxidized to varying degrees (Fig. 3). All human CCK peptides contain
methionine and tryptophan residues. It is a common observation that
methionine residues are prone to oxidation in the presence of oxygen.
In addition, we often find that tryptophan containing peptides undergo
oxidation in the mass spectrometer.2 Both
processes appear to be more pronounced in short peptides and at low
peptide concentrations.
Since the initial RIA data indicated that a considerable quantity of CCK secreted from yeast was not processed at the C terminus (Table III), the column fractions were treated with trypsin and carboxypeptidase B before RIA analysis with Ab 3208. This resulted in the detection of two additional CCK-immunoreactive peaks not present in untreated fractions, one at a KD of 0.6 and the other at a KD of 1 (Fig. 2B). Thus, it would appear the new peaks represented C-terminally extended CCK-22 and CCK-8, respectively. Fractions collected from Sephadex G-50 gel chromatography were also analyzed with Ab 89009 which binds the N terminus of CCK-22. Two immunoreactive peaks were observed, one corresponding to CCK-22-Gly and the other at KD 0.6 (Fig. 2C), strongly suggesting this elution constant represents C-terminally extended CCK-22. A similar experiment was performed using an antibody which binds the N terminus of non-sulfated CCK-8 (Ab 94179).3 The results showed two immunoreactive peaks at KD values of 1 and 1.15, the latter being CCK-8-Gly, and the former being C-terminally extended CCK-8 (data not shown).
Expression of the pro-CCK fusion in strain MT960 (Table I), which is
the wild type for the PEP4 protease, but defective in the
BAR1 encoded "barrier" protease, an exported pepsin-like
protease which cleaves -factor (Mackay et al., 1988
),
resulted in the formation of CCK-22-Gly and CCK-8-Gly in a manner
similar to that of strain YNG318 (data not shown). Thus, neither
protease A nor the barrier protease seems to be responsible for the
observed proteolytic cleavages of pro-CCK.
The prepro-MF1-pro-CCK plasmid
was transformed into S. cerevisiae strains deficient in the
function of known yeast secretory pathway endopeptidases in an attempt
to determine which enzyme(s) were responsible for the endoproteolytic
processing of pro-CCK. Analysis of the extracellular products from
transformants of the mkc7 yeast mutant HKY21 by gel
chromatography and RIA analysis with Ab 3208 showed that both
CCK-22-Gly and CCK-8-Gly were produced (Fig.
4A). Pro-CCK processing was then analyzed in
yeast deficient in both the Mkc7 and Yap3 aspartyl proteases (HKY24).
However, the three proteolytic cleavage events occurring in wild type
yeast persisted in the mkc7 yap3 double mutant (Fig.
4B). Treatment of the column fractions with trypsin and
carboxypeptidase B uncovered two additional immunoreactive peaks (Fig.
4B) corresponding to C-terminally extended versions of
CCK-22 and CCK-8. Processing at Lys81 to produce
C-terminally extended CCK-22 was confirmed in the HKY24 strain by the
purification of the CCK-immunoreactive material eluting at a
KD of 0.6 followed by protein sequencing and mass
spectrometry (Fig. 3C).
When the prepro-MF1-pro-CCK construct was expressed in a
kex2 mutant, ME598, the pattern of pro-CCK endoproteolytic
processing was altered (Fig. 4C). No glycine extended
products were detected, and a single major CCK-immunoreactive peak was
found eluting at a KD of 0.6 after the Sephadex G-50
column fractions had been exposed to trypsin and carboxypeptidase B
treatment (Fig. 4C). A similar situation was observed when
the fusion protein was expressed in the kex2 yap3 double
mutant ME938 (Fig. 4D). Immunoreactive material in the
medium of this double mutant was purified, exposed to protein
sequencing and mass spectrometry (Fig. 3D), and its identity
shown to be C-terminally extended CCK-22. However, several other CCK
species were recovered during the large scale preparation of pro-CCK
processing products secreted by the ME938 transformant, each
constituting a minor fraction (less than 5%) of total secreted CCK.
Most were N- or C-terminally truncated fragments, indicating that a low
level of degradation by amino- and carboxypeptidases had occurred (data
not shown). Although, a small amount of C-terminally extended CCK-39,
N-terminally truncated by one residue, was also identified (data not
shown).
CCK processing patterns were also examined in the triple protease mutant strain HKY25 (Table I). Analysis of secreted pro-CCK products revealed a large degree of variation between experiments in molecular forms of CCK produced, however, cleavage at Lys81 persisted (data not shown).
Intracellular Processing of Pro-CCK in Yeast TransformantsMetabolic labeling and immunoprecipitation
experiments were performed to determine which, if any, of the
proteolytic cleavage events of pro-CCK occurred within the yeast cell.
Both radiolabeled and CCK-Gly immunoreactive material was found to
elute at KD values of 0.75 and 1.15 (Fig.
5), previously shown to correspond to CCK-22-Gly and
CCK-8-Gly, respectively (Figs. 2 and 3, A and B).
Since no larger forms of CCK were detected it appears that only the
smaller forms of CCK are glycine extended in the yeast cell. In
addition, when CCK-33-NH2 was incubated for 5 h at
30 °C with S. cerevisiae, as well as with media in which
the yeast had been grown, only CCK-33-NH2 was recovered
(data not shown), indicating that the cleavage at Lys81
is occurring entirely within the cell.
Analysis for Tyrosyl Sulfation of CCK Secreted from Yeast Transformants
Anion exchange chromatography was employed to
determine if the Tyr97 residue of CCK was sulfated in the
yeast secretory pathway. Transformants of YNG318 expressing the
prepro-MF1-pro-CCK fusion construct were grown in a 5-ml
culture to late exponential phase. The supernatant was then treated
with trypsin and carboxypeptidase B to convert all CCK-related peptides
into CCK-8-Gly which contains the potential sulfation site. The digest
was loaded on a Sephadex G-50 superfine column from which a single peak
of CCK immunoreactivity was detected with Ab 7270 at the elution
position known for CCK-8-Gly (data not shown). The immunoreactive
material was subjected to anion exchange chromatography where it eluted
at a position similar to the non-sulfated control (Fig.
6), indicating that yeast were unable to sulfate CCK. In
addition, all purified CCK species possessed the same molecular mass
when measured in the positive and negative mode, confirming that these
peptides are not sulfated (Talbo and Roepstorff, 1993
).
Human prepro-CCK has been expressed in S. cerevisiae,
resulting in the secretion of numerous processing intermediates of
pro-CCK. However, many native signal sequences are inefficient at
directing foreign proteins through the yeast secretory pathway (for
review see; Hadfield et al. (1993)). When pro-CCK was
expressed as a fusion protein, N-terminally linked to the prepro region
of MF
1, a hundredfold increase in the secretion of
partially cleaved CCK was observed. Due to the higher expression
levels, yeast expressing the prepro-MF
1-pro-CCK fusion
construct were utilized to further analyze pro-CCK processing in the
yeast secretory pathway.
Peptide -amidation, a widespread protein modification occurring in
invertebrates and vertebrates (Eipper et al., 1993
), is essential for the biological activity of CCK (Martinez et
al., 1986
). The substrate for
-amidation is invariably a
C-terminal glycine, which is processed to an
-amide group by the
bifunctional protein, peptidyl-
-amidating monooxygenase, located in
the late Golgi and/or secretory vesicles (Eipper et al.,
1993
). Amidated CCK was not found in media supernatants of yeast
expressing native prepro-CCK or the prepro-MF
1-pro-CCK
construct. Consequently it appears that S. cerevisiae lack
the ability to
-amidate proteins.
Typically CCK is synthesized in the tyrosyl-sulfated
(Tyr97) form, which is essential for binding to the
CCKA receptor in the gastrointestinal tract and some
regions of the brain (Dourish and Hill, 1987). Analysis of CCK-Gly
secreted from yeast transformants by both anion exchange chromatography
and mass spectrometry revealed only the non-sulfated forms. The
inability of S. cerevisiae to sulfate tyrosine residues was
also observed for the leech anticoagulant, hirudin, which requires
tyrosyl sulfation for full activity (Riehl-Bellon et al.,
1989
).
Analysis of the proteolytic processing products of CCK secreted from
transformants of wild type yeast strains proficient in known secretory
pathway endopeptidases uncovered four pro-CCK products,
glycine-extended CCK-8 and CCK-22, as well as C-terminally extended
CCK-8 and CCK-22. Therefore S. cerevisiae is able to cleave
pro-CCK at three sites: (i) C-terminally of the dibasic Arg105-Arg106 residues, a cleavage which is an
essential step in production of bioactive CCK in vertebrates, (ii)
between the Arg95-Asp96 amino acids producing
CCK-8 related products, the most common form of CCK in the mammalian
brain (Rehfeld, 1978b; Rehfeld and Hansen, 1986
); and (iii) between the
Lys81-Asn82 residues producing CCK-22 related
products, which is a predominant form of CCK in human plasma and plasma
from most other mammals (Liddle et al., 1984
, 1985
; Cantor
and Rehfeld, 1987
; Rehfeld, 1994
; Paloheimo and Rehfeld, 1995
).
Processing at the Lys81 residue is extremely efficient,
abolishing detection of endoproteolytic cleavage events which may have
occurred N-terminally of Lys81 with the antisera utilized
in this study. However, a small quantity of C-terminally extended
CCK-39, lacking one amino acid at the N terminus, was purified from the
kex2 yap3 double mutant ME938 expressing the
pre-proMF
1-pro-CCK construct.
In an attempt to identify which yeast endopeptidase(s) were responsible
for the above mentioned cleavage events, the processing patterns were
analyzed in yeast strains deficient in known secretory pathway
endopeptidases. When pro-CCK processing was examined in strains lacking
the structurally and functionally related Mkc7 and Yap3 proteases, all
forms of CCK secreted from wild type yeast were produced by the
aspartyl protease-deficient strains. Transformants of the mkc7
yap3 double mutant seemed to secrete more C-terminally extended
forms of CCK than glycine extended forms when compared with the wild
type strain. However, since yeast lacking Kex2, but proficient in Mkc7
and Yap3 function, do not produce any CCK-Gly this result suggests that
pro-CCK expressed in yeast is not cleaved at
Arg105-Arg106 by either the Mkc7 or Yap3
proteases within the yeast cell. Incubating CCK-33 with purified Yap3
in vitro resulted in the liberation of both CCK-22 and CCK-8
(Cawley et al., 1996). Since processing to produce these
forms of CCK persists in a mkc7 yap3 double mutant it is
presently difficult to determine if either of the proteases are able to
cleave pro-CCK at Lys81 or Arg95 in
vivo.
Two pro-CCK endoproteolytic processing events were abolished in a yeast
strain lacking the Kex2 serine protease, namely the dibasic
Arg105-Arg106 directed cleavage and processing
between Arg95-Asp96, suggesting that Kex2 is
responsible for both processing events. However, the possibility that
the intracellular trafficking of pro-CCK may be altered in the
kex2 mutant, thus directing the pro-CCK molecule to a
distinct area of the secretory pathway where it cannot be cleaved at
Arg105-Arg106 or Arg95 cannot be
ruled out. Recently the first unequivocal evidence for at least two
distinct vesicles in the yeast secretory pathway has been reported,
where it appears that one of these vesicles may represent proteins
bound for the plasma membrane, with the other vesicle type bound for
secretion from the cell (Harsay and Bretscher, 1995).
The Kex2 enzyme is widely known to be responsible for processing of
both yeast and foreign proteins at dibasic sites (Fuller et
al., 1988; Hadfield et al., 1993
). Thus, it was not
surprising that pro-CCK processing was abolished at the
Arg105-Arg106 residues in a kex2
mutant. As seen in other instances where Kex2 has been responsible for
post-translational processing, not all of the proprotein was processed
at this site (Miyajima et al., 1986
; Thim et al.,
1986
; Zsebo et al., 1986
; Moody et al., 1987
; Driedonks et al., 1995
). Notably, even when low amounts of
pro-CCK were passed through the yeast secretory pathway, as was the
case when prepro-CCK was expressed in yeast, there were still
relatively high quantities of C-terminally extended CCK secreted. Since
large amounts of glycine-extended CCK were secreted from S. cerevisiae transformants expressing the
prepro-MF
1-pro-CCK construct, it seems improbable that
yeast expressing the prepro-CCK construct lack the capacity to cleave
all pro-CCK present in the secretory pathway at the
Arg105-Arg106 site.
The second pro-CCK proteolytic processing event which occurred in wild
type yeast, but not the kex2 mutant, was the single arginine
directed cleavage resulting in CCK-8 related products. There is an
increasing number of instances where Kex2 and related serine proteases
of the mammalian secretory pathway have been shown to preferentially
process proproteins at single arginine residues (Zhu et al.,
1992; Bourbonnais et al., 1994
; Dupuy et al.,
1994
; Galanopoulou et al., 1995
; Vollenweider et
al., 1995
). For instance, prohormone convertase 1, but not
prohormone convertase 2, was shown to cleave rat prodynorphin at a
single arginine residue when co-expressed in mouse AtT-20 cells (Dupuy
et al., 1994
). Our data raises the possibility that some
members of the mammalian Kex2-like proteases may also play a role in
the processing of pro-CCK at the Arg95-Asp96
site to release CCK-8. A non-serine endopeptidase which displayed a
high selectivity for cleavage of CCK-33, resulting in CCK-8, has been
partially purified from rat brain synaptosomes (Viereck and Beinfeld,
1992
), although there is currently no direct evidence to suggest that
this protease cleaves pro-CCK in vivo. Experiments similar
to those performed on prodynorphin by Dupuy and co-workers (1994) would
be interesting to determine if any of the known mammalian Kex2-like
proteases are able to process pro-CCK at the
Arg95-Asp96 site. It is possible that the
tissue specificity observed in the N-terminal processing of pro-CCK in
mammalian tissues is a result of differential expression of a single
arginine-specific endopeptidase and Kex2-like serine proteases, some of
which may have the ability to process pro-CCK to CCK-8.
The ability to cleave pro-CCK at Lys81 to produce CCK-22
related products was maintained in a variety of recombinant yeast
strains deficient in three known putative endopeptidases of the
S. cerevisiae secretory pathway. Hence our data suggests
that another endopeptidase is present in the secretory pathway of
yeast. Previously, two groups have suggested that an uncharacterized
protease was cleaving the human -amyloid precursor after the lysine
residue in the His-Gln-Lys-Leu-Val sequence, when expressed and
secreted in yeast (Hines et al., 1994
; Zhang et
al., 1994
). However, one of these studies did not examine
kex2 or yap3 mutant yeast strains (Hines et
al., 1994
), whereas the other did not express
-amyloid
precursor in yap3 mutants (Zhang et al., 1994
).
The lysine-directed processing of
-amyloid precursor in yeast is
identical to that performed by
-secretase in the mammalian brain, to
produce the
/A4 peptide found in senile deposits of Alzheimer's
disease patients (Hines et al., 1994
; Selkoe, 1994
; Zhang
et al., 1994
). At present it is debatable if the same
protease is responsible for the lysine-directed processing of pro-CCK
and
-amyloid precursor in yeast. This can only be determined when
the gene encoding the endopeptidase responsible for the lysine-directed
cleavage in pro-CCK is identified.
In conclusion, the expression of pro-CCK in S. cerevisiae
has uncovered important features of the yeast secretory pathways ability to post-translationally process secreted peptides/proteins. First, it appears that yeast lack the tyrosyl sulfation and
-amidation protein modifying capabilities. Second, our evidence
suggests that the Kex2 protease has the ability to cleave Arg-Asp
bonds. Finally, we provide evidence that a novel lysine-specific
endopeptidase is present in the yeast secretory pathway. Attempts to
isolate and characterize the gene encoding this putative endopeptidase are currently underway.
We are grateful to Ira Herskowitz for
supplying the MF1 plasmid, as well as Michi Egel-Mitani
and Robert Fuller for providing yeast strains. We thank Robert Eggert
and Allan Kastrup for their skillful technical assistance.