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(Received for publication, September 5, 1995) From the
Dictyostelium discoideum makes multiple developmentally
regulated lysosomal cysteine proteinases. One of these, a lysosomal
enzyme called proteinase I, contains a cluster of GlcNAc-
Dictyostelium discoideum is an eukaryotic amoeba that
grows as single cells, but when the bacterial food source is removed,
the cells initiate a complex multicellular developmental program. Cells
aggregate and differentiate into several different types and, in the
end, 85% of them are converted into spores setting atop a cellular
stalk(1) . We are interested in studying the role of
carbohydrate modifications in this organism(2) . One of these
is the addition of GlcNAc-1-P to serine residues, which has been well
documented to occur on a cysteine proteinase called proteinase I found
in vegetative cells (3, 4, 5) . Although
antibodies against GlcNAc-1-P recognize various proteins in the cells
and in secretions of cells grown in axenic medium ( Previous
studies in Dictyostelium identified two developmentally
regulated members of this gene family, cprA (CP1) and cprB (CP2)(6, 7, 8) , but none have been
identified in vegetative cells. Since cysteine proteinases are highly
conserved in all eukaryotes, we used the active site consensus sequence
of cysteine proteinases and the cprA and cprB cDNAs
to clone two novel vegetative cysteine proteinases, cprD and cprE. They have the predicted conserved regions but also have
an unusual serine-rich domain not previously found in any known
cysteine proteinase that could be the site of GlcNAc-1-P addition. The
cDNA clones were overexpressed and the active enzymes were shown to
have GlcNAc-1-P.
Figure 1:
Nucleotide and deduced amino acid
sequences of cprD and cprE. Panels A and B correspond to cprD and cprE, respectively.
The start of the polyadenylation signal, AATAAA, is underlined. The putative N terminus of the mature proteinase
is boxed. Asterisks signify termination. The amino
acids are indicated by the single letter
code.
Figure 2:
Sequence alignment of CP4 and CP5 to
human, plant, and Dictyostelium cysteine proteinases. Shared
sequences are boxed. Double underlines indicate
putative N-glycosylation sites, and arrows show the
active site cysteine and histidine. * indicates the beginning of the
mature protein. In boldface are the serine-rich domains on CP4
and CP5.
An unusual feature of these deduced amino acid sequences is the
presence of a serine-rich domain near the C terminus of both proteins.
In CP4 it is 115 amino acids long and contains 60 serine residues
(52%), while in CP5 the same region contains 12 serine residues out of
24 amino acids (50%). Another Dictyostelium cysteine
proteinase, CP2, also has an insert in this region (42 amino acids
long), but its serine content is only 11%. Other cysteine proteinases
typically have much shorter sequences (1-12 amino acids) in this
region (Fig. 2). In CP4, the serine residues seem to be
distributed in three distinct repeated motifs: poly-S, SGSQ, and SGSG.
Serines in the insert from CP5 seem to follow the same pattern but in
fewer repeats. The tertiary structures of cysteine proteinases
actinidin, papain, and the human liver cathepsin B are
known(27, 28, 29) . The similarity in the
conserved regions of CP4 and CP5 to these cysteine proteinases suggests
that they may have the same overall structure. Fig. 3shows the
tertiary structure of actinidin and the location of the serine-rich
insert of CP4 and CP5 based on the inferred amino acid sequence
homology and crystal structures. The insertion occurs at Gly-170
(actinidin), and in CP4 it comprises nearly one-third of the predicted
size of the mature protein. As seen in Fig. 3, the insert lies
on the opposite side of the protein away from the active site.
Figure 3:
Location of the serine-rich inserts in
relation to the active site of a cysteine proteinase. The relative size
and location of the serine-rich insertions of CP4 (A) and CP5 (B) are depicted onto the
Figure 4:
Southern blots of cprA, cprB,
cprD, and cprE. Genomic DNA digested with BamHI, BglII, ClaI, and EcoRI/HindIII was
electrophoresed in agarose gels and blotted into nylon. The blot was
probed at high stringency (55 °C) to the entire cDNAs of cprD and cprE. The same filters were reprobed with cprA and cprB. The molecular weight markers are indicated in
kb.
Figure 5:
Analysis
of the mRNA levels corresponding to cprD and cprE during growth and development. A, cells were plated on SM
agar plates with K. aerogenes, and 10
Figure 6:
Glycosylation pattern of cells that
overexpress CP4 and CP5. 40 µg of protein from total cell lysates
of control cells grown in HL-5 or Klebsiella (Ka) and
transformants (CP4-25, CP4-6, and CP5-12) grown in
HL-5 and 1 µg of purified proteinase I were submitted to SDS-PAGE.
The proteins of the gel were then blotted into nitrocellulose filters
and immunologically detected using a monoclonal antibody (ab)
against GlcNAc-1-P (AD7.5) or against fucose (83.5). The primary
antibody binding was detected using a goat anti-mouse antibody
conjugated to alkaline phosphatase.
Figure 7:
Cysteine proteinase activity in cells
overexpressing CP4 and CP5. 10 µg of a total cell lysate was
preincubated for 30 min with or without 10 µM E-64 in 0.1 M phosphate/citrate, pH 5.0, 1 mM DTT, and then 0.3
mM of the substrates N-Cbz-Lys-ON-p and H-D-Val-Leu-Lys-p-NA was added. After 20 min
color development was measured at 405 nM. The graphics
indicate the percent of activity in the transformants (CP4-25,
CP4-6, CP5-6, and CP5-12) in relation to the controls
(AX-4 and AX-2). Values are the average of duplicates. The data are
representative of five different
experiments.
Figure 8:
Cysteine proteinase activity in SDS-PAGE
gels from cells overexpressing CP4 and CP5. 40 µg of protein from a
total cell lysate of transformants (CP4-25, CP4-6,
CP5-6, and CP5-12) and control cells (AX-4 and AX-2) were
submitted to SDS-PAGE without boiling. The gels were then preincubated
or not with 10 µM E-64 in 0.1 M
phosphate/citrate, pH 5.0, 20 mM cysteine for 20 min and then
in the same buffer containing 20 µMN-t-Boc-Val-Leu-Lys-7-MCA. Fluorescence developed
almost immediately and was observed in a UV
transilluminator.
North and Cotter (31) have described cysteine
protease activities in Dictyostelium throughout development
and point out the complex and dynamic activity patterns seen in
vegetative cells(31) . A series of 4-5 different cysteine
proteinase activity bands with apparent M We are interested in studying the function of
GlcNAc-1-P and the signals needed for its addition to proteins. Based
on the previous studies, we screened a vegetative cell cDNA library to
look for typical cysteine proteinase genes that would have serine-rich
region(s). We found two such cDNAs that could code for cysteine
proteinases, cprD and cprE. mRNA for both genes is
detected during vegetative growth and decreases with the start of
development, reappearing in low levels when the fruiting body is formed (Fig. 5). This is in agreement with the observation that general
cysteine proteinase activity slightly increases at the end of
development(35) . A surprising feature is that the amount of
mRNA increases substantially at the end of vegetative growth. This is
typical of the prestarvation responsive genes (36) and occurs
in parallel with a burst of cysteine proteinase activity seen at this
time (35) . The reason for this is unclear, but this may
reflect an increased need for digesting bacteria or for increased
protein turnover known to occur in development. When the cells start
development, the protease may not be necessary and its mRNA levels
decrease. This is consistent with the decrease seen in cysteine
proteinase activity during development(37, 38) .
Southern blot analysis of cprA, cprB, cprD, and cprE shows that they are located in different genomic DNA fragments (Fig. 4). This was confirmed by mapping the genes in the Dictyostelium genome using yeast artificial chromosomes
(YACs). cprD maps to chromosome 3 and cprE maps to
the middle of chromosome 2(39) . CP4 and CP5 have an unusual
domain not present in the other previously studied cysteine
proteinases. CP4 contains a 115-amino acid domain composed of 52%
serine residues divided into three separate contiguous motifs, poly-S,
SGSQ, and SGSG. CP5 contains similar motifs within a 24-amino acid
domain. The serine stretches probably evolved from a series of tandem
duplications. CP5 appears to be the older version of the motifs before
the onset of tandem duplications. The serine-rich inserts in both CP4
and CP5 appear to be located in a non-conserved region of other
cysteine proteinases (Fig. 2). Although they are near the active
site histidine residue, their location in space is expected to be away
from the active site as shown in the three-dimensional structure on Fig. 3. It is possible, though, that the presence of the insert
or of the putative carbohydrate chains may influence the activity of
the enzymes, since the serine-rich domain is connected directly to the
To show that cprD and cprE code for an enzyme that can carry GlcNAc-1-P,
the cDNAs were overexpressed in axenically growing cells. This resulted
in an average 3-fold increase in cysteine proteinase activity (Fig. 7), which corresponded to an increased activity band of 36
kDa (in CP4 transformants) or 29 kDa (in CP5 transformants) on SDS-PAGE (Fig. 8). Significantly, a monoclonal antibody against
GlcNAc-1-P recognizes the same band in the transformants that is found
in very low amounts in non-transformed cells (Fig. 6). CP4
transformants also show some additional bands ranging from 45 to 70
kDa, which are detected in the Western blots but not in the activity
gels. We are currently unable to explain this effect, but they could
possibly represent unprocessed forms of the enzyme due to its
overexpression. The results also show that this antibody recognizes
GlcNAc-1-P in the 38-kDa proteinase I purified from bacterially grown
cells (Fig. 6)(5) . Axenically grown cells also have a
38-kDa protein, but it seems to migrate at a slightly higher molecular
weight, both in control and transfected cells. North and co-workers (32, 33) have shown that 38-kDa cysteine proteinases
are present in cells grown axenically or in the presence of bacteria
but that they have different biochemical properties. Different cysteine
proteinase activity patterns are observed in vegetative cells depending
on the nutrient availability. These interconversions may be due to
differences in post-translational
modifications(31, 32, 33) , and the anomalous
migration of the 38-kDa protein in the transformants may reflect
altered glycosylation of this protein when CP4 is overexpressed.
Resolving these issues will require additional experiments, but it is
clear that CP4 (36 kDa) and proteinase I (38 kDa) are different. This
was confirmed by partial amino acid sequencing of proteinase
I(35) , although both proteins showed similar amino acid
compositions. It is possible that both are members of a closely related
family modified by GlcNAc-1-P, since they at least partially co-purify.
Both proteins also contain fucose, as shown by binding of another
monoclonal antibody (Fig. 6). The location of the fucose
residues is unknown, and they either seem to be absent in CP5 or the
expression levels were not high enough to permit detection with this
antibody even when the blots were overdeveloped. Based on the
processing of other cysteine proteinases, the expected masses of CP4
and CP5 without any modifications would be 32,816 and 24,459 Da,
respectively. Two potential N-linked glycosylation sites occur
in both enzymes, but it is not known if they are actually used. CP4
migrates in SDS-PAGE as an approximate 36-kDa band and CP5 as a 29-kDa
band, but additional experiments will be necessary to determine how
much of this mass is contributed by either N-linked chains or
by GlcNAc-1-P and fucose. GlcNAc-1-P is most probably added to CP4
and CP5 in this newly characterized serine-rich domain. It is
interesting to note that this domain has three distinct motifs,
polyserine, SGSQ, and SGSG. An enzyme activity that transfers
GlcNAc-1-P to serine units in proteins has recently been characterized
in Dictyostelium(40) , and a SGSG peptide can act as
an acceptor in an in vitro GlcNAc-1-P transferase
assay(41) . SGSG repeats are used as sites for the addition of
glycosaminoglycan chains to core proteins such as
serglycins(42, 43) . Polyserine repeats have recently
been described in a secreted acid phosphatase from Leishmania,
which is modified by a new class of phosphoserine-linked glycans,
Man-1-P bound to serines(44, 45) . SP96, a spore coat
protein that is present in prespore vesicles of Dictyostelium(46) , is recognized by the GlcNAc-1-P
antibody. ( The cloning of these two novel cysteine proteinases will allow us to
begin to determine the function of GlcNAc-1-P. By characterizing the
sites of GlcNAc-1-P addition and creating mutations in these sites we
may understand its potential role in targeting these cysteine
proteinases to lysosomes or in affecting enzyme activity. Since these
mutant cDNAs can be expressed in Dictyostelium, we can study
the fate of the protein with altered glycosylation.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s) L36204 [GenBank](CP4) and L36205 [GenBank](CP5).
Volume 270,
Number 48,
Issue of December 1, 1995 pp. 28938-28945
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-1-P-Ser
residues. We call this phosphoglycosylation. To study its function, a
cDNA library from vegetative cells was screened, and two novel cysteine
proteinase clones were characterized (cprD and cprE).
Each of them has highly conserved regions expected for cysteine
proteinases, but unlike any other, each has a serine-rich domain
containing three distinct motifs, poly-S, SGSQ, and SGSG. cprD and cprE cDNAs were overexpressed in Dictyostelium and the active enzymes identified. cprD codes for a
protein of approximately 36 kDa (CP4), which is recognized by
monoclonal antibodies against GlcNAc-1-P and fucose. cprE corresponds to a 29-kDa protein, which is recognized by antibodies
against GlcNAc-1-P. mRNA for both enzymes is present in the vegetative
phase and increases during growth on bacteria but decreases throughout
development. When the formation of the fruiting body is complete the
mRNA for both messages is detected again but in very low levels. Having
cloned cDNAs for proteins that carry GlcNAc-1-P should allow us to
probe the function of the carbohydrate in these putative lysosomal
enzymes.
)the
identity of these proteins is unknown. To study the function of
GlcNAc-1-P on a defined protein, we decided to clone members of the
cysteine proteinase family expressed in vegetative cells.
Materials
Radionucleotides were purchased from
DuPont NEN and ICN Biomedicals, Inc. The random primed labeling kit and
quick spin Sephadex G-25 and G-50 columns were from Boehringer
Mannheim. The messenger RNA isolation kit and the R408
interference-resistant helper phage was from Stratagene Inc., La Jolla,
CA. The Sequenase DNA sequencing kit was from U. S. Biochemical Corp.
The monoclonal antibody 83.5 against fucose was a kind gift from Dr.
Christopher West (University of Florida College of Medicine). Goat
anti-mouse antibody conjugated to alkaline phosphatase was from
Promega. H-D-Val-Leu-Lys-p-NA (
)was from Chromogenix. Geneticin (G418) was from Life
Technologies, Inc. Restriction and modifying enzymes were from New
England Biochemicals and Boehringer Mannheim. Nitrocellulose filters
and prefilters for development were from Millipore. All other chemicals
were from Sigma.Cell Culture and Development Conditions
D.
discoideum strains AX-4 and AX-2 were grown axenically in HL-5 or
with Klebsiella aerogenes(9) . After 50 h of growth,
the bacteria are consumed and the plate appears cleared. This is
referred to as the clearing plate stage and corresponds to late
vegetative growth. Development of 5 
10
cells from
axenic cultures or from clearing plates was done on 47-mm black
nitrocellulose filters resting on prefilters saturated with 20 mM phosphate buffer, pH 6.4, and took 24 h to complete.cDNA Library Construction and
Screening
Poly(A) mRNA was isolated from 5
10
AX-4 cells from clearing plates. The cells were
collected only from plates where no morphological development was
visually observed. Since the first few hours of development occur
without obvious morphological changes, the library could also have very
early development cDNAs represented. Cells were washed twice in cold
water treated with diethyl pyrocarbonate, and poly(A)
mRNA was prepared using the messenger RNA isolation kit. A cDNA custom
phage library was prepared by Stratagene in the EcoRI/XhoI site of Uni-ZAP XR vector. For screening,
the library was plated on SURE cells, blotted into nitrocellulose
filters, and hybridized with the different probes following the
conditions advised by the manufacturer. 1
10
phage
plaques were screened using a 23-base mixed oligonucleotide
corresponding to 8 conserved amino acids (IKNQGQCG) of cysteine
proteinases as follows:
5`-AT(T/C/A)AA(A/G)AA(T/C)CA(A/G)GG(T/C/A/G)CA(A/G)TG(T/C)GG-3` and
3`-TA(A/G/T)TT(T/C)TT(A/G)GT(T/C)CC(A/G/T/C)GT(T/C)AC(A/G)CC-5`. The
oligonucleotide mixture was radiolabeled using T4 polynucleotide kinase
and [-
P]ATP and purified by centrifugation
through columns of Sephadex G-25. Thirty-six of 100 positive clones
were reprobed using a 775-bp BglII/SpeI fragment of cprA (from bases 243 to 1018) and a 405-bp HincII
fragment of cprB (from bases 338 to 743) as
probes(6, 7) . These fragments exclude the 5` end of
the cDNAs, which correspond to unconserved regions of the precursor
protein. The cDNA fragments were radiolabeled by random primed
synthesis with [
-P]dATP and
[
-P]dCTP and purified by centrifugation
through columns of Sephadex G-50. Six positive clones were identified
and sequenced. To sequence, the pBluescript plasmid from the phage
vector was excised using the R408 interference resistant helper phage,
plasmid preparations were performed(10) , and the
double-stranded DNA was sequenced using the Sequenase DNA sequencing
kit. Three clones corresponded to the same cDNA, and one of them was a
full length of 1.1 kb (cprE). The other three clones
corresponded to another cDNA, but none had an initiating methionine.
Three different probes derived from the largest cDNA of this second
group (1.4 kb) were then used to isolate a full-length clone: the
1.4-kb fragment itself, a 373-bp NspI/StuI fragment
corresponding to a serine-rich region not found in cprE, and a
206-bp EcoRI fragment corresponding to the 5` end. 1
10 plaques were rescreened and 1% of these were positives.
A polymerase chain reaction of phage particles from 200 plaques was
then performed (10) using as primers the first 18 bases of the
1.4-kb fragment (5`-AGCGAAAACATTTAAA-3`) and the T3 primer
(5`-AATTAACCCTCACTAAAGGG-3`). Larger polymerase chain reaction products
were rescreened with the 1.4-kb probe and sequenced, and all of them
corresponded to a full sized cDNA (cprD).DNA Preparation and Southern Blot
D.
discoideum DNA minipreparations were performed as
described(11) . 50 µg of DNA were digested with restriction
enzymes according to the manufacturer's instructions,
electrophoresed in 0.8% agarose gels, transferred into nitrocellulose
filters, and prehybridized for 2 h at 55 °C in 5
Denhardt's solution, 4 SSPE, 0.1% SDS, 50 µg/ml
salmon sperm DNA (10) . The filters were hybridized overnight
at 55 °C in the presence of 10 cpm of each probe (the
complete cDNA of cprA, cprB, cprD, and cprE) and washed 3 times for 20 min with 1 SSPE, 1%
SDS and 3 times for 20 min in 0.1 SSPE, 1% SDS at room
temperature (10) . The filters were then dried and exposed to
x-ray films (Kodak X-Omat) overnight.Amino Acid Sequence Alignments
Amino acid sequence
alignments were done using the GENEWORKS program (Intelligenetics
Inc.). Actinidin tertiary structure was displayed using Xfit Program (12) .Subcloning and Transformation Procedures
The
1.4-kb cDNA of cprD or the 1.1-kb cDNA of cprE was
subcloned into the BamHI/XhoI and BamHI/KpnI sites, respectively, of
pDNeo67(13) , which allows expression under control of the
actin 6 promoter. Cloning and DNA preparations followed standard
procedures as described(10) . Stable transformants
overexpressing cprD and cprE were obtained using the
calcium procedure of Nellen et al.(11) . 2
10 cells of AX-4 (for cprD) or AX-2 (for
cprE) were transfected with a calcium phosphate precipitate of 6,
12, or 25 µg of plasmid DNA. After 1 week of selection in 20
µg/ml G418, cells were transferred to flasks and grown under
agitation in HL-5 containing 20 µg/ml G418.RNA Isolation and Northern Transfer
Total RNA was
isolated from 10 cells as described(14) . 20 µg
of RNA were electrophoresed in 1.2% agarose gels containing
formaldehyde and blotted to nitrocellulose membranes(10) . The
filters were prehybridized overnight at 37 °C in 50% formamide, 3
SSC(10) , diethyl pyrocarbonate-treated 5% nonfat dry
milk(15) , 60 mM sodium phosphate, pH 6.4, 10 mM EDTA, and 0.4% SDS and hybridized overnight with 10 cpm of the cprA, cprD, and cprE cDNAs
radiolabeled as above. The mRNA levels on each lane were normalized by
probing the same filters with 1G7, a constitutively expressed
gene(16) .Protein Analysis and Western Blot
Total cell
lysates (40 µg of protein) or 1 µg of purified proteinase I
were analyzed by SDS-PAGE in 10% gels as described(17) . The
proteins were blotted into nitrocellulose filters (18) and
incubated with monoclonal antibodies against GlcNAc-1-P (monoclonal
antibody AD7.5, 1:20 of a culture supernatant) or fucose (monoclonal
antibody 83.5, 1:1000 a culture supernatant). The primary antibody was
detected with conjugated alkaline phosphatase goat anti-mouse secondary
antibody(18) .Cysteine Proteinase Activity Assay
10 µg of
protein from total cell lysates was incubated in the presence of 0.3
mMH-D-Val-Leu-Lys-p-NA or N-Cbz-Lys-ON-p in 0.1 M phosphate/citrate,
pH 5.0, 1 mM DTT for 20 min in a 96-well dish, and color
development was detected at 405 nM using an enzyme-linked
immunosorbent assay reader. For inhibition studies, protein samples
were preincubated for 30 min with 10 µM specific cysteine
proteinase inhibitor E-64 prior to the addition of the substrates.
Cysteine proteinase activity assay in 10% polyacrylamide gels was done
with 40 µg of total cell lysate obtained by freeze-thaw of cells in
10 mM Tris, pH 7.0, 1 mM DTT without boiling. After
electrophoresis, the gel was incubated in 0.1 M phosphate/citrate, pH 5.0, 20 mM cysteine buffer with or
without 10 µM E-64 for 20 min, and then in the same buffer
containing 20 µMN-t-Boc-Val-Leu-Lys-7-MCA. The gel was observed in a
UV transilluminator and photographed using a Polaroid system with a
Kodak yellow A filter.
10 µg of total
cell lysates were assayed in phosphate/citrate buffer (0.05 M in respect to sodium phosphate) using 4-methylumbelliferyl
substrates at concentrations of 0.003 M (pH 4.4) for
-D-Mannosidase and
-D-Glucosidase Activity Assays-D-mannosidase and 0.006 M (pH 4.1) for
-D-glucosidase as described(19) .
Isolation of Cysteine Proteinase cDNAs from Vegetative
D. discoideum
In order to study the role of GlcNAc-1-P
modification on a defined protein, we decided to isolate cDNAs
corresponding to cysteine proteinases of Dictyostelium vegetative cells. A cDNA library was probed with a 23-base mixed
oligonucleotide based on the active site consensus sequence of cysteine
proteinases. Positive clones were then rescreened with cDNA fragments
of Dictyostelium cprA and cprB as described under
``Experimental Procedures,'' and the full-length clones for
two novel cDNAs, cprD (1.4 kb) and cprE (1.1 kb) were
identified and analyzed as follows.Analysis of the cDNAs for cprD and cprE
The
nucleotide sequence of cprD and cprE is depicted in Fig. 1, and from here the enzymes are referred to as cysteine
proteinase 4 (CP4) and cysteine proteinase 5 (CP5), respectively.
Sequence alignment of CP4 and CP5 (Fig. 2) with human cathepsins
(B, H, L, and S)(20, 21, 22, 23) ,
plant cysteine proteinases (actinidin and
papain)(24, 25) , and two cysteine proteinases from Dictyostelium (CP1 and CP2) show that CP4 and CP5 have all the
expected conserved regions and critical active site residues. The first
17 amino acids of both enzymes probably constitute a signal peptide
(pre-region) since it is consistent with other typical signal
sequences(26) . Both CP4 and CP5 also contain the pro-region
(amino acids 18-111) in the N-terminal domain, which is not found
in the mature form of known cysteine proteinases. Two potential N-linked glycosylation sites are found in both CP4 and CP5.
-carbon structure of actinidin.
The active sites Cys-25 and His-162 are indicated. The insertion occurs
at Gly-170 of the actinidin sequence.
Southern Blot Analysis of cprD and cprE
Southern
blots of Dictyostelium DNA samples digested with different
enzymes were probed with the cprA, cprB, cprD, and cprE cDNAs and washed at high stringency (Fig. 4). The results confirm that the cDNA clones described
here are different from cprA and cprB and that they
are from Dictyostelium and not from their bacterial food
source. Some cross-reactivity occurs because there are common sequences
among cprA, cprB, cprD, and cprE.
This is shown by the alignment of faint bands of cprA and cprB when blots are probed with cprD and cprE. A clear example of this is the dark 5.3-kb band in the EcoRI/HindIII digestion of the blot probed with cprB. The blots probed with the other cDNAs show faint 5.3-kb
bands.
Analysis of cprD and cprE mRNA Levels during Growth and
Development
Expression of the mRNA corresponding to cprD and cprE was analyzed in cells grown in axenic cultures
(HL-5) or with bacteria (Ka). AX-2 cells exponentially growing in HL-5
were plated on SM agar plates along with bacteria and collected after
44, 47, and 50 h of growth. These times correspond to log growth,
beginning of clearing, and total clearing plates, respectively. Cells
were then collected at 50 h and plated for synchronous development to
analyze mRNA expression (Fig. 5A). Alternatively, cells
were plated on the filters directly from axenic cultures (Fig. 5C). As seen, mRNA levels corresponding to cprD and cprE increase during growth on bacteria up
to clearing plates and decrease once development starts. Densitometer
scanning of the autoradiograms show that the mRNA levels increase from
2- to 4-fold between 44 and 50 h of growth in bacteria. Cells prepared
from axenic culture also display a decrease in mRNA expression during
development. As seen in Fig. 5C, the mRNAs are not
detected after 4 h of development until the formation of the fruiting
body is complete, when very low levels of mRNA for both messages are
again detected. The same pattern of expression is observed when mRNAs
from AX-4 strain are analyzed (not shown).
cells were
collected after 44 h (growing cells), 47 h (beginning of clearing), or
50 h (clearing plates). Cells were then washed free of bacteria with 20
mM phosphate, pH 6.4, and plated for development on
nitrocellulose filters. Samples of 10 cells were taken
after 0, 2, 4, and 8 h of development. B, 10 cells
were taken from AX-4, AX-2, CP4-25, CP4-6, and CP5-12
axenic cultures. C, exponentially growing cells from axenic
(HL-5) cultures were washed with 20 mM phosphate buffer, pH
6.4, and plated for development over nitrocellulose filters. Samples of
10 cells were taken after 0, 2, 4, 8, 12, 16, 20, and 24 h
of development. Total RNA was isolated, and 20 µg was submitted to
electrophoresis in agarose-formaldehyde gels. The gels were blotted
into nylon membranes and hybridized against radioactive probes
corresponding to cprD, cprE, and cprA (as an
internal control of development) or 1G7 (a constitutively expressed
gene) as indicated.
Overexpression of cprD and cprE in D.
discoideum
We decided to overexpress cprD and cprE in Dictyostelium to investigate whether it codes for
active cysteine proteinases modified by GlcNAc-1-P. The cDNAs were
subcloned into pDNeo67(13) , an expression vector that contains
the G418 resistance marker and where the cDNA is under the control of
the Dictyostelium actin 6 promoter. Transformants were
isolated and analyzed for mRNA expression. As shown in Fig. 5B, transformed clones (CP4-6, CP4-25,
and CP5-12) overexpress the mRNAs 5-10-fold, compared with
control cells. Cells transfected with the pDNeo67 plasmid alone show
the same pattern as non-transfected control cells.Identification of the Overexpressed Proteins in the
Transformed Cells
Cell lysates from control cells grown in HL-5
or on bacteria and from clones overexpressing cprD (CP4-25 and CP4-6) or cprE (CP5-12)
were analyzed by Western blots using monoclonal antibodies against
GlcNAc-1-P or fucose (30) and detected by a secondary
antibody-alkaline phosphatase conjugate. As seen in Fig. 6, a
protein band of approximately 36 kDa is enriched in the transformed
cells (CP4-25 and CP4-6) as detected with antibodies
against GlcNAc-1-P and fucose. A very faint 36-kDa band can also be
seen in the control cells and in a preparation of a 38-kDa cysteine
proteinase purified from cells grown on bacteria that is known to carry
GlcNAc-1-P. It appears that CP4 in control cells is expressed in low
amounts relative to the 38-kDa protein, which co-migrates with
proteinase I(3, 4, 5) . In cells
overexpressing cprE (CP5-12) a 29-kDa band is increased
as detected by the antibody against GlcNAc-1-P, but no increase was
found with the antibody against fucose (not shown).
Cysteine Proteinase Activity Assays on Transformed
Cells
To investigate if cells overexpressing CP4 and CP5 had an
increased cysteine proteinase activity, total cell lysates of control
or transformed cells grown in HL-5 were assayed for activity as
described under ``Experimental Procedures'' using N-Cbz-L-Lys-ON-p or H-D-Val-Leu-Lys-p-NA as substrates. As seen
in Fig. 7, cells that overexpress CP4 and CP5 have 2.5-3.6
times more cysteine proteinase activity with the substrate H-D-Val-Leu-Lys-p-NA and 50% more activity
with the substrate N-Cbz-L-Lys-ON-p. In the
presence of E-64, a specific cysteine proteinase inhibitor, activity is
reduced to 20% of control. As a control, activity levels of two
lysosomal enzymes, -D-mannosidase and
-D-glucosidase, were analyzed using 4-methylumbelliferyl
substrates, and no difference in activity was detected in the
transformed cells when compared with control cells (not shown). To
verify that the increase in cysteine proteinase activity was due to the
overexpressed 36- and 29-kDa proteins, we performed activity assays of
the proteins separated by SDS-PAGE using the fluorogenic substrate N-t-Boc-Val-Leu-Lys-7-MCA. Fig. 8shows that
the 36-kDa band has low activity levels in AX-4 cells, which increases
in the two CP4 transformants analyzed, and that it is inhibited by
E-64. Also, a faint band of 29 kDa is observed in control AX-2 cells
and is increased in the CP5 transformants.
of
30-54 kDa is expressed depending upon whether cells are grown on
bacteria or in axenic media (32, 33) . Gustafson and
co-workers (3, 4) reported a vegetative stage cysteine
proteinase of 38 kDa, proteinase I, that contained up to 20% by weight
GlcNAc-1-P linked to serine residues. Such a serine content is not
typical of cysteine proteinases. Previously, two developmental
stage-specific cysteine proteinase genes, cprA (CP1) and cprB (CP2)(6, 7) , were cloned in Dictyostelium, but their serine content closely resembles that
of other typical eukaryotic cysteine proteinases. A partial sequence
for another developmentally regulated cysteine proteinase (CP3) (34) has also been identified; however, it does not encode a
full-sized enzyme.-strand involved in the active site. This domain may serve special
needs for CP4 and CP5 but is obviously not vital for activity since
most cysteine proteinases are devoid of it.
)SP96 has a 96-amino acid domain with 70% serines
interspersed with alanines and prolines and a 49-amino acid region with
SG and GSQ repeats(47) . SP70, another spore coat protein, also
has repeats of SG and a polyserine region(48) . Both proteins
have been shown to be fucosylated and phosphorylated(49) . It
may be that these prespore vesicle proteins, some putative lysosomal
proteins like CP4 and CP5, and other proteins yet to be identified
share a property influenced or controlled by GlcNAc-1-P and/or fucose.
)))
We thank Dr. A. Kuspa for the physical mapping of cprD and cprE into Dictyostelium chromosomes, Dr. J. Williams for the cprA and cprB clones, Dr. C. West for the antibody against fucose, and Dr. S.
Kudo and Dr. M. Fukuda for technical advice and use of equipment in
early phases of this work. Liying Wang provided excellent technical
assistance. We are also grateful to Dr. R. Doolittle for advice on the
analysis of the cysteine proteinase sequences.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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