Download A Pneumocystis carinii multi-gene family with

Microbiology(1997), 143,2223-2236
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A Pneumocystis carinii multi-gene family with
homology to subtilisin-like serine proteases
Elena B. Lugli, Andrew G. Allent and Ann E. Wakefield
Author for correspondence: Ann E. Wakefield. Tel: +44 1865 222344. Fax: +44 1865 222626.
e-mail: [email protected]
Molecular Infectious
Diseases Group,
Department o f Paediatrics,
lnstitute of Molecular
Medicine, John Radcliffe
Hospita't Oxford OX3 9DUt
UK
Copies of a multi-gene family, named PRTl (protease 3,encoding a subtilisinlike serine protease were cloned from the opportunistic fungal pathogen
Pneumocystis carinii. Comparison of the nucleotide sequence of a genomic
clone and a cDNA clone of PRTl from P. carinii f. sp. carinii revealed the
presence of seven short introns. Several different domains were predicted
from the deduced amino acid sequence: an N-terminal hydrophobic signal
sequence, a pro-domain, a subtilisin-like catalytic domain, a P-domain
(essential for proteolytic activity), a proline-rich domain, a serinerthreoninerich domain and a C-terminal hydrophobic domain. The catalytic domain
showed high homology to other eukaryotic subtilisin-like serine proteases and
possessed the three essential residues of the catalytic active site. Karyotypic
analysis showed that PRTl was a multi-gene family, copies of which were
present on all but one of the P. carinii f. sp. carinii chromosomes. The different
copies of the PRTl genes showed nucleotide sequence heterogeneity, the
highest level of divergence being in the proline-rich domain, which varied in
both length and composition. Some copies of PRTl were contiguous with
genes encoding the P. carinii major surface glycoprotein.
Keywords : Pneumocystis carinii, subtilisin-like serine protease, PRTl , multi-gene
family, major surface glycoprotein
INTRODUCTION
The fungal pathogen Pneumocystis carinii causes potentially fatal pneumonia in the immunocompromised,
including those receiving immunosuppressive therapy
for organ transplantation, those with advanced malignancy and in particular those with HIV infection. The
lack of an effective in vitro culture system still remains
a major obstacle in the understanding of the biology of
P. carinii and its interactions with its host. Molecular
techniques have been employed in the study of the
organism, and a number of genes have now been cloned.
Among these is the multi-gene family encoding the
major surface glycoprotein (MSG or gpA) of the
parasite. In this paper we describe the cloning and
characterization of a second P. carinii multi-gene family,
PRTl (protease I),some copies of which are contiguous
with MSG.
t Presentaddress: Department of Clinical Veterinary Medicine, University
of Cambridge, Cambridge CB3 OES, UK.
The GenBank accession numbers for the PRT7(73j) and PRTl(Paga)
sequences reported in this paper are AF001304 and AF001305 respectively.
0002-1687 0 1997 SGM
The P. carinii major surface glycoprotein is highly
mannosylated and is antigenically distinct in organisms
isolated from different mammalian host species (Lundgren et al., 1991 ; Gigliotti, 1992). The MSG multi-gene
family has been identified in the genome of P. carinii f.
sp. carinii (rat-derived P. carinii) (Kovacs et al., 1993;
Wada et al., 1993; Sunkin et al., 1994), P. carinii f. sp.
mustelae (ferret-derived P. carinii) (Haidaris et al., 1992 ;
Wright et al., 1995), P. carinii f. sp. hominis (humanderived P. carinii) (Stringer et al., 1993; Garbe &
Stringer, 1994) and P. carinii f. sp. muris (mouse-derived
P. carinii) (Wright et al., 1994). The different copies of
the P. carinii f. sp. carinii MSG genes are of similar size
but heterogeneous in sequence. They have been found
on multiple chromosomes and often organized in
tandem arrays. The majority of MSG genes are located
in the subtelomeric regions of the P. carinii f. sp. carinii
chromosomes (Underwood et al., 1996; Sunkin &
Stringer, 1996). The expression of MSG genes has been
shown to be mediated by the upstream conserved
sequence (UCS) which is found on a single chromosome
situated in the subtelomeric region (Wada et al., 199.5).
Different copies of the MSG multi-gene family have been
shown to be linked to the UCS. It has been postulated
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E. B. LUGLI, A. G. A L L E N a n d A. E. W A K E F I E L D
that this differential expression of MSG may represent a
strategy to evade the immune response of the host by
antigenic variation (Sunkin & Stringer, 1996).
The genes comprising the novel P. carinii PRTl multigene family described in this paper show high levels of
homology with subtilisin-like serine proteases. These
are a group of endoproteases which have been characterized from a wide variety of organisms including
bacteria, fungi and higher eukaryotes. Some have been
found to function in the specific endoproteolytic processing of pro-proteins at cleavage sites of paired basic
amino acid residues, to generate regulatory proteins in a
mature and biologically active form. The pro-hormone
processing enzyme kexin, encoded by the KEX2 gene of
Saccharomyces cereuisiae, has been characterized and
found to cleave the precursors of the a-mating-factor
and the killer toxin (Fuller et al., 1989). Genes encoding
a similar processing endoprotease have been identified
in a number of other fungi, the KEXl gene from the
yeast Kluyueromyces lactis (Tanguy-Rougeau et al.,
1988), the gene encoding the KEX2-related protease
(krp) from Schizosaccharomyces pornbe (Davey et al.,
1994) and the XPR6 gene from Yarrowia lipolytica
(Enderlin & Ogrydziak, 1994). Mammalian homologues
have also been identified, including the human fur gene
(fes upstream region) in the region upstream of the fes
proto-oncogene, encoding the enzyme furin (van den
Ouweland et al., 1990). The genes Dfurl and Dfur2
from the insect Drosophila melanogaster encoding furinlike proteins (Roebroek et al., 1992) and the 61i-4 gene
from the nematode Caenorhabditis elegans have also
been studied. Many other members of the subtilisin-like
serine protease family have been identified and the
specific endoproteolytic activity of some of them has
been elucidated. However, for many others, the precise
biological function has not yet been determined.
In this paper we report the identification and characterization of the P. carinii f. sp. carinii PRTl multigene family. We demonstrate high levels of homology of
the PR TI sequences to subtilisin-like serine proteases.
We also show that different copies of the PRTl genes
display D N A sequence heterogeneity and some copies
are contiguous with MSG genes.
METHODS
P. carinii DNA extraction. P. carinii infection was induced in
Sprague-Dawley rats by steroid immunosuppression. The
organisms were isolated and enriched from infected rat lung
tissue by the method described by Peters et al. (1992). Total
DNA was extracted from the enriched parasite preparation by
digestion with proteinase K (1 mg ml-l) in the presence of
0.5% SDS and 10 mM EDTA (pH 8.0) at 50 "C for 16 h,
followed by phenol :chloroform extraction and ethanol precipitation. Samples of DNA for use in PFGE experiments were
prepared in SeaPlaque GTG agarose as described by Banerji
et al. (1993).
Isolation of copies of the PRTl gene from P. carinii f. sp.
carinii genomic and cDNA libraries. A copy of the PRTI gene
was isolated from an unamplified genomic library from
P. carinii f. sp. carinii constructed in IEMBL3 (Banerji et al.,
2224
1993). The library was screened with a cDNA clone containing
a region of a P. carinii f. sp. carinii MSG gene (EMBL
accession number 20870, donated by C. J. Delves and
F. Volpe), as part of a study examining subtelomeric sequences
in P. carinii. A relatively high number of recombinant
plaques gave positive hybridization signals compared to the
number when the library was screened with a probe derived
from the single copy arom locus (Banerji et al., 1993).
Five recombinant phages were isolated from a tertiary screen;
the recombinant DNA was subcloned into the plasmid vector
pBluescript I1 prior to sequencing.
T o isolate a full cDNA clone, a P. carinii f. sp. carinii cDNA
library constructed in AZAPII (donated by C. J. Delves and F.
Volpe, see Dyer et al., 1992) was screened with PCR products
derived from amplification of the 5' end of the gene with
oligonucleotide primer pair pcprot9 and prp4r (9/4r product),
and of the 3' end of the gene with pcprotl3/RI and
pcprotl2/RI (13/12 product) (Fig. 1). A primary screening
was carried out using both probes, and secondary and tertiary
screens were carried out using only the 9/4r product. The
number of positive clones when screening the cDNA library
with the two probes appeared to be relatively high when
compared to the number obtained using a single copy gene.
Four recombinant phage isolated from the cDNA library were
partially characterized. The recombinant DNA was recovered
from the 1 phage by in vivo excision as pBluescript plasmid
DNA. The size of the recombinant DNA ranged from 2-7kb
to 2.9 kb, and sequence analysis revealed that all four clones
contained a poly(A) tail. One recombinant, 73j, was selected
for further analysis and the recombinant DNA was sequenced
in full from both strands.
DNA amplification. Oligonucleotide primers were designed to
hybridize to various regions of the P. carinii PRTI nucleotide
sequences (Fig. 1, Table 1). Some oligonucleotides had an
EcoRI restriction endonuclease site incorporated at the 5' end
to facilitate cloning of the amplification products into EcoRIdigested plasmid vectors pBluescript SK( - ) (Stratagene) or
pUC18 (Pharmacia). The final concentration of the amplification reaction mix was 50 mM KCl, 10 mM Tris (pH 8-0),
0.1 '/o Triton X-100, 3 mM MgCl,, 400 pM (each) deoxynucleoside triphosphate, 1 pM oligonucleotide primer and
0025 U T a q polymerase ml-l (Promega). With primer pair
pcprot9 and pcprotl0, 40 cycles of amplification was performed at 94 "C for 1.5 min, 53 OC for 1.5 min, and 72 "C for
2-0 min. With primer pair pcprot9 and pcprot4r the same
conditions were used, except an annealing temperature of
50 "C was used. With all other primer pairs, ten cycles of
amplification were carried out at 94 OC for 1.5 min, 55 "C for
1.5 min and 72 "C for 2.0 min, followed by 30 cycles of 94 "C
for 1.5 min, 63 "C for 1-5min and 72 "C for 2.0 min. Negative
controls were included in each experiment.
The entire putative gene was amplified as three overlapping
fragments, PrpSe (1626 bp), M14 (1279 bp) and Prp2g (251 bp)
(Fig. 1).Oligonucleotide primer pairs pcprot9 with pcprotlo,
followed by pcprot6/RI with pcprot4/RI were used in a
nested PCR to amplify the 5' fragment, designated PrpSe, of
length 1626 bp. The second portion, called M14, spanning
1279 bp of the central region of PRTI, was amplified using a
nested PCR with primer pairs pcprot2/RI with pcprotl4/RI,
followed by pcprot7/RI with pcprotl2/RI. The third fragment, Prp2g, encompassing the 3' end of the sequence (251 bp),
was amplified using oligonucleotide primers pcprotl3/RI and
pcprotl4/RI (Fig. 1, Table 1).
Five different overlapping regions of the PRTl gene were also
amplified, cloned and the DNA sequences determined. The
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Pneumocystis carinii serine protease
(a)
HR
731
I
I
PRO
-
0
I * O
II
I
I II
*
111
CATALYTIC
I
I
II I
v
IV
o *
PROUNERICH
P-
I
I
VI
sn
HR
VII
I
I
I
I
1
I
I
1
H
H
w
Pcr-l8,Pcr-14,Pcr-5,Pcr-3,Pcr-l ,lam4
t-t
M I
+
I
pcprotlrnl
+
.
t
pcproUrlRI
..................................................................................................................................................................................................................................................,...,.,.,,.,...................,.......................,,......
Fig. 1. Schematic representation of a P. carinii PRTl gene. (a) PRTl domains: HR, hydrophobic region; PRO-, pro-domain;
CATALYTIC, catalytic domain; P-, P-domain; PROLINE-RICH, proline-rich region (the box indicates length and sequence
,,
H,,z and
variation in different copies of PRTI); STR, serine/threonine-rich region; 0, catalytic active site residues D
S423; *, potential glycosylation sites; I , conserved cysteine residues. (b) A genomic copy of PRTl(Paga) showing the
positions of the seven introns (I-Vll), a cDNA copy of PRTI(73j) and the products of amplification of different regions of
PRTl genes. (c) Position of oligonucleotide primers used in the amplification of different regions of PRTl genes.
first region amplified with primer pair pcprotl/RI and
pcprot3/RI spanned approximately half of the subtilisin-like
catalytic domain, the second region amplified with primer pair
pcprot2/RI and pcprot4/RI spanned the end of the subtilisinlike catalytic domain and the start of the P-domain, the third
region amplified with primer pair pcprot7lRI and pcprot8/RI
spanned the P-domain, the fourth region amplified with primer
pair 36ex/RI and Pt3/RI spanned the proline-rich domain and
the fifth region amplified with primer pair pcprotl3/RI and
pcprot 14/RI spanned the C-terminal hydrophobic domain
(Fig. 1, Table 1).The sequences Prpla, Prp3a, Prp7a, Prp2c,
Prp3c, P r p k , Prptaf2, Prpf4, PrpSf, Prpg3 and Prp5g were
amplified from the P. carinii cDNA library, and sequences Pcr19, Pcr-14, Pcr-5, Pcr-3, Pcr-1, Lam-1 and Prpg4 from the
P. carinii genomic DNA (Fig. 2).
DNA sequence analysis. DNA sequence analysis was per-
formed using the dideoxy chain-termination method (Sanger
et al., 1977). Sequence data were obtained in full from both
strands for all sequences. Analysis of the sequence data was
carried out using the University of Wisconsin Genetics
Computing Group (UWGCG) Sequence Analysis Software
Package, version 8 (Genetics Computer Group, Madison, WI,
USA).
PFGE. P. carinii f. sp. carinii was isolated from an infected rat
lung and the chromosomes were separated by PFGE using a
Contour Clamped Homogeneous Electric Field (CHEF) DRII
apparatus (Bio-Rad) operated at 4 "C. Electrophoretic separation was achieved using 0*9°/~Seakem agarose gel with
initial switching time of 10 s, increasing to a final switching
time of 60 s at 180 V for 48 h. A karyotype corresponding to P.
carinii f. sp. carinii form 1was observed (Cushion et al., 1993).
Southern hybridization. Southern blotting and hybridization
were carried out using standard techniques (Sambrook et al.,
1989). PFGE blots were hybridized with three probes derived
from different domains of the PRTl gene. The product 9/4r
was derived from amplification of the 5' end of the PRTI gene
with primer pair pcprot9 and pcprot4r/R1, product 2/4 from
amplification of the central catalytic region with primer pair
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E. B. LUGLI, A. G. A L L E N a n d A.E. W A K E F I E L D
Table 1. Oligonucleotide primer sequences
Primer
PrP4r
pt3/R1
36ex/RI
pctel2
msgterml
pcprot 1/RI
pcprot2/RI
pcprot3/RI
pcprot4/RI
pcprotS/RI
pcprotb/RI
pcprot7lRI
pcprot8/RI
pcprot9
pcprotlO
pcprotl2/RI
pcprot 13/RI
pcprot 14/RI
Sequence
S’-GCTTGTCACTATTAAACC-3’
5’-GGGAATTCTGAAGCTTTTCGAGTGGTTG-3’
S’-GCGAATTCTAACCTTCAGCCAGATTCA-3’
S’-AAGTCACGTGCTCTCTTGGTCA-3’
5’-AATGTTGTTGGGAGTGATTG-3’
S’-GGGAATTCTTATTCTTGTAGCTGGGGAC-3’
S’-GGGAATTCTTCTACACCTCTTGCTGCG-3’
S‘-GGGAATTCCACGCCATGTAAGATTAGGA-3‘
S’-GGGAATTCTAATGCTTAGGATATCCGCG-3’
S’-GGGAATTCATGTGAAATGGTGCCAGTAG-3’
S’-GGGAATTCGTTTTTTTTTAACATTTCATCATG-3’
S’-GGGAATTCTCGGTTATGGAAAACTAGATG-3’
S’-GGGAATTCAAGGTTAGCATCCAGATCTG-3’
5’-AGAATTTCTAATTAAAAAGTTAAG-3’
S’-AAACACCAACATACTCTAAAC-3’
S’-GGGAATTCTTATAGTACATGAAGCTTTTCG-3‘
S’-GGGAATTCTTCATCTACATCTACGACTTC-3’
S’-GGGAATTCTATAGGTTAAAAAGAGTAACCC-3
pcprot2/RI and pcprot4/R17 and product 13/12 from amplification of the 3’ end of the gene with primer pair pcprotl3/RI
and pcprotl2/RI (Fig. 1). The amplification products were
gel-purified (Geneclean 11, BiolOl) and labelled with [a32P]dCTP by random priming (Megaprime, Amersham).
Hybridization was carried out at 45 “C and stringency washing
~
0.1 ‘/o SDS.
at 60 “C in 0 . 2 SSC,
Southern blots of genomic P. carinii DNA digested with
restriction endonuclease PstI or BamHI were probed with
oligonucleotide probes pcprotS/RI, pcprot5/R17 pctel2 and
msgterm (Table 1) labelled with [p3’P]dATP using polynucleotide kinase. Hybridization was carried out at 46 “C and
stringency washing at 52 “C in 5 x SSC, 0.5 o/‘ SDS.
copy from the cDNA library, PRTl(73j), confirmed the
presence of seven short introns in the genomic DNA
sequence. The introns ranged in length from 38 bp to
45 bp, with a base composition ranging from 71 to 84
mol YO A T. In all seven introns, the dinucleotide G T
was present at the 5’ splice donor site and AG at the 3’
splice acceptor site. The sequence YTRAT, which has
been identified as the putative lariat-forming motif in
other P. carinii f. sp. carinii introns (Zhang & Stringer,
1993), was present in the first, second, fourth, fifth and
seventh intron. The eukaryotic lariat consensus sequence, YYRAY, was identified in the third and sixth
intron.
RESULTS
The sequence of the cDNA clone, PRTl(73j), contained
an ORF of 2370 bp, which on translation resulted in a
peptide of 790 amino acids (Fig. 2). The deduced amino
acid sequence was compared to sequences in the
GenBank and EMBL databases and showed homology
to fungal and other eukaryotic subtilisin-like serine
proteases. The A T content of the ORF was 64 mol YO,
with a high A + T content at the third base position of
the codons. The base composition of the 5’ upstream
sequence was 74 mol YO A T and of the 3’ downstream
sequence 75 mol YO A T. A consensus polyadenylation
signal, AATAAA, was observed 68 bp downstream of
the stop codon.
Analysis of DNA and derived amino acid sequence of
copies of the PRTl gene
We have identified a family of genes in the P. carinii f. sp.
carinii genome which shows homology to the subtilisinlike serine proteases. We have named this gene family
PRTl (gotease 1).
A copy of the PRTl gene (Paga) was
isolated from a P. carinii genomic library, the ORF
(3069 bp) containing seven short putative intervening
sequences. A copy of the PRTl gene (73j) of length
2370 bp was also isolated from a cDNA library. Portions
of the gene were amplified by PCR from the cDNA
library as three overlapping fragments at the 5’ end
(PrpSe), the central region (M14) and the 3’ end (Prp2g).
Five other regions of the gene were also amplified, from
either the P. carinii cDNA or genomic libraries, to
determine the extent of diversity among different copies
of the multi-gene family at different domains within the
gene sequence.
Analysis of the DNA sequence of the copy of the PRTl
gene from the genomic library, PRTl(Paga), and of the
2226
+
+
+
+
The deduced amino acid sequence of the genomic clone
PRTl(Paga), the cDNA clone PRTl(73j), the three
fragments obtained by PCR amplification of the cDNA
library and the other recombinant clones generated by
DNA amplification were compared (Fig. 2). Several
regions of homology were found and also a number of
regions in which significant divergence was observed.
These data suggested that the sequences were derived
from different copies of the PRTl multi-gene family.
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Pneumocystis carinii serine protease
Comparison of P. carinii PRTl with other subtilisinlike serine proteases
The deduced amino acid sequence of the cDNA clone
PRTl(73j) was aligned with nine other subtilisin-like
serine proteases including fungal, mammalian, insect
and nematode sequences (Fig. 3). The P R T l sequences
showed homology with all the other sequences, with a
high level of homology in the subtilisin-like catalytic
domain. The three essential residues of the catalytic
active site, aspartic acid (Asp,,,), histidine (His,,,) and
serine (Ser,,,) [residues are numbered with reference to
PRTl(73j)l were conserved in all the PRTl sequences.
The highest levels of homology between all the sequences were around these residues.
The structural organization of the fungal sequences
showed domains characteristic of this class of endoproteases, a hydrophobic signal sequence, a pro-domain
that may be cleaved by autoproteolysis, a subtilisin-like
catalytic domain, a P-domain (known as such because it
is essential for proteolytic activity), a serine/threoninerich domain which may potentially be modified by 0linked glycosylation, a C-terminal hydrophobic transmembrane domain and a C-terminal tail with acidic
residues (Van de Ven & Roebroek, 1993) The P. carinii
PRTl sequences showed a similar putative structural
organization but unlike the nine other subtilisin-like
serine proteases, they also had a proline-rich domain
preceding the serine/threonine-rich domain and the Cterminal hydrophobic domain (Fig. 1). The P. carinii
PRTl(73j) sequence had a hydrophobic signal sequence
at the N-terminus, followed by a putative pro-domain, a
subtilisin-like catalytic domain from Ser,,, to His,,,, a
P-domain from residue Tyr,,, to Ser,,,, a proline-rich
domain from residue Pro,,, to
a serine/threonine-rich domain from residues Thr,,, to Ser,,,, and
a C-terminal hydrophobic domain from residues His,,,
to Phe,,,.
Analysis of subtilisin-like catalytic domain
The three-dimensional structures of four subtilisin-like
serine proteases have been determined, subtilisin
BPN’/Novo from Bacillus amyloliquefaciens (Hirono et
al., 1984; Bott et al., 1988), subtilisin Carlsberg from
Bacillus licheniformis (McPhalen & James, 1988), thermitase from Thermoactinomyces vulgaris (Gros et al.,
1989; Teplyakov et al., 1990) and proteinase K from
Titirachium album (Betzel et al., 1988). The amino acid
sequence of these four proteases has been compared to
that of 31 other subtilisin-like serine proteases isolated
from bacteria, fungi and higher eukaryotes and the
essential core structure of the catalytic domain of this
group of molecules has been identified (Siezen et al.,
1991).
We have compared the deduced amino acid sequence of
the P. carinii PRTl(73j) gene with the multiple sequence
alignment of the other subtilisin-like serine proteases
and have identified, by homology, the three essential
residues of the catalytic active site, aspartic acid, histidine and serine, in the PR T l sequence
His,,,
and Ser,,,). On the basis of the sequence alignment,
the P. carinii PR T l sequence could be assigned to the
class I subtilases, within the subgroup I-E which contains
the pro-hormone processing proteases from yeasts and
higher eukaryotes (Siezen et al., 1991).
Eight a-helical domains and nine /?-sheet regions have
been defined as the structurally conserved regions within
the essential core structure. The variable regions which
connect the core segments have been found to differ both
in length and in amino acid sequence (Siezen et al.,
1991). High levels of homology were observed between
the PR T l sequences and the other sequences in the
regions of the two conserved internal helices, helix C
(residues 252-262) and helix F (residues 422-438) [Fig.
3; residues are numbered with reference to the
PRTl(73j) sequence]. Eleven amino acid residues have
previously been found to be totally conserved in all the
characterized subtilisin-like serine proteases, and most
but not all are conserved in the PR T l sequences. These
amino acid residues are at the active site Asp,,,, His,,,
and Ser,,, [found in all the PR T l sequences except
PRTl(Prp7a)I and in the internal helices at residues
GlY253, Gly,,, and Pro,,,. The residues Ser,,,, Gly,,,,
Gly,,,, Gly,,, and Thr,,,, involved in substrate binding,
were conserved in all the PR T l sequences except Thr,,,,
which was found only in two sequences generated by
PCR, PRTl(Prp1a) and PRTl(Prp7a).
In addition to the totally conserved residues, seven other
amino acid residues have been identified which are
highly conserved. Of these, six were conserved in the P.
carinii PR T l sequences and included the oxyanion hole
residue (Asn,,,), residues GlyZl6 and Thr,,, near the
active site, and also residues Gly205, Gly,,, and Gly,,,.
Seven conserved cysteine residues were found in all the
P. carinii PR T l sequences : c y S 2 5 6 , cys268, Cys,,,, Cys,,,,
Cys389, Cys,,, and Cys,,,. Nineteen variable regions,
generally located in loops on the surface of the molecule,
have been identified in the subtilase family, of which 14
were found in the P . carinii PR T l sequences. Three
positions have been identified at which charge is totally
conserved in all the subtilisin-like proteases examined,
and these were also conserved in the P. carinii PRTl
sequences :the positive charge on Arg,,, and the negative
charges on residue Asp,,, (active site) and Asp,,,.
~~
Fig. 2. Alignment of the f . carinii PRTl deduced amino acid sequences from the genomic clone Paga, the cDNA clone 73j
and the three overlapping PCR products amplified from a cDNA library correspondingt o the 5’ region (PrpSe), the central
region (M14) and the 3’ region (Prp2g). The deduced amino acid sequences of PCR products amplified from five different
regions of the fRTl gene family were also aligned: the catalytic domain (Prpla, Prp3a, Prp7a); the boundary of the
catalytic domain and the P-domain (PrpZc, Prp3c, Prp4c); the P-domain (Prptaf2, Prpf4, Prp5f); the proline-rich region
(Pcr-19, Pcr-14, Pcr-5, Pcr-3, Pcr-1, Lam-1); and the C-terminal region (Prpg4, Prpg3, Prp5g). Gaps were introduced t o
maximize homology; identical amino acids are boxed.
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E. B. LUGLI, A. G. ALLEN a n d A . E . WAKEFIELD
Prpla
prp3a
prp7a
- - - -- - -- - - - - - ----- - --- - - - - - - - - --- --
~ J ~D GI YY A N
sPYT
ITIA A I
411
420
413
0
DOE 0 KnFVi S ifit
107
108
107
411
480
473
0
prp2c
Prp3c
PrpQc
----
--
- - -
132
132
131
-- --- - -
50
50
50
Paga Y G K L D A Y R
73j F G K L D A
PrpSe Y G K L D A Y R
Ml4'-[Y
R
prpla . . . . . . . .
~rp7a
Prp2c Y G K L D A
Prp3c Y G K L D A
Prp4c Y G K L D A
Prptaf2 - - - - _ A
Prpf4 - - - - PrpS f
- A
n V E K A R T F K
Y R ~ ~ E ~ A
M V E K A R T F K
M VT-E
F K
T
~
T
T
L N P
T F
L N P
LNm
Q
K
Q
Q
T n F S
T L N
T M F S
TM FS
T Q L I P L N K K F S E N G G H I T S S F Y I H R G Y P
~ Q T ~ F ~ T Q L I P L N K ~ F S E N G G H I T S ~ F Y I ~ G ~ P
T Q L I P L N K K F S E N G G H I T S S F Y I H R G Y P
T PQ -L 1
S R RLI T S S F Y I H m G Y P
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
131
105
-- - - - - - 105
- - - - -105
Y K F K 55
55
55
Y R M V E K A R T F K T L N P Q T H F S T Q L I P L N K K F S E N G G H I T S S F Y I H R G Y P K H Y K F K
Y
Y
Y
Y
R
R
R
R
M
M
M
M
~
V
V
V
E
E
E
E
K
K
K
K
A
A
A
A
R
R
R
R
T
T
T
T
F
F
F
F
K
K
K
K
T
T
T
T
L
L
L
L
N
N
N
N
P
P
P
P
Q
Q
Q
Q
T
T
T
T
M
M
M
M
F
F
F
F
S
S
S
S
T
T
T
T
Q
Q
Q
Q
L
L
L
L
I
I
I
I
P
P
P
P
L
L
L
L
N
N
N
N
K
K
K
K
K
K
K
K
F
F
F
F
S
S
S
S
E
E
E
E
N
N
N
N
G
G
G
G
G
G
G
G
H
H
H
H
I
I
I
I
T
T
T
T
S
S
S
S
S
S
S
S
F
F
F
F
Y
Y
Y
Y
I
I
I
I
H
H
H
H
R
R
R
R
G
G
G
G
Y
Y
Y
Y
P
P
P
P
K
K
K
K
~
H
---_ AYR~VEKAROFKTLNPQTMF~TQLIPLNKKFSENGGHITSS
_ _ _ _ _ - - - - - _ _ _ - _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - - - - - - - - - - - - - - - - - - - - 105
S L E Y V G V S F H Y Q H Q R R G H L E F N I T S P S G V T S V L A H R R N R D K H G G S I L W T F M T V K H W G E S I 115
S L E Y V G V S O H Y P H Q R R G H L E F N I T S P S G V T S V L A H R R N R D K H G G S I L W T F M T V ~ H W G E S I 115
S L E Y V G V S F H Y Q H Q R R G H L E F N I T S P S G V T S V L A H R R N R D K H G G S I L W T F M T V K H W G E S I 115
P ~ ( C - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Fig. 2. For legend see p. 2227.
2228
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Pneumocystis carinii serine protease
-
- - - - - - - -
177
38
..
28
19
29
35
19
734
693
529
271
177
179
177
98
69
59
66
71
59
Paga
73j
PrpSe
ni 4
Per-19
Pcr-14
Pcr-5
Pcr-3
Pcr-1
Lam- 1
Paga
73j
PrpSe
ni4
Pcr-19
Pcr-14
Pcr-5
Pcr-3
Pcr-1
Lam-1
Pm2u
P E
---
--
- E P
E T
Q S
_______--_-__-_
----
858
724
529
394
162
192
165
169
94
138
0
924
790
529
406
171
201
174
178
103
147
63
61
69
62
..................................,........................................................................................,.....................................................................................................................................................................................
Fig. 2 (cont.) For legend see p. 2227.
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2229
E. B. LUGLI, A. G. A L L E N a n d A.E. W A K E F I E L D
It has been proposed that the high specificity of the class
I-E subtilisin-like serine proteases for paired basic
residues Lys-Arg or Arg-Arg may be facilitated by a high
density of negative charge at the substrate-binding face,
provided by nine highly conserved Asp residues and one
Glu residue (Siezen et al., 1991). T w o of the Asp
residues, Asp,,, and Asp,,,, were found in all the P.
carinii PRTl sequences and also the Glu,,, residue. In
addition, four other Asp residues were found in some
but not all of the copies of PRT1.
Analysis of the domains flanking the subtilisin-like
catalytic domain
The putative domains of the PRTl(73j) polypeptide are
summarized in Fig. 1. A hydrophobicity plot of the
PRTl(73j) sequence revealed a hydrophobic region at
the N-terminus, suggesting that this may be a signal
sequence. Residues 1-23 of the N-terminus of the
sequence showed a high level of homology to the Nterminus of the P. carinii f. sp. carinii multifunctional
folic acid synthesis fas gene which encodes dihydroneopterin aldolase, hydroxymethyldihydropterin
pyrophosphokinase and dihydropteroate synthase
(Volpe et al., 1992, 1993). This region was followed by
the presumptive pro-domain, which may be cleaved by
autocatalysis. Potential autocatalytic sites of paired
basic residues were identified in the P R T l (Paga)
and PRTl (Prp5e) sequences at Ly~,,~-Arg,,, and
Arg13,-Arg13,, but were absent in the PRTl(73j) sequence. Five other semi-conserved autocatalytic sites
were found in some, but not all, copies of the P. carinii
PRTl sequences, two in the catalytic domain
(Lys,,,-Arg,,,
,Arg4,,-Arg,,,) and three in the P-domain
(Arg521-Argjr22, Arg555 Or Lys555-Arg556, Arg576-Arg577) *
One potential autocatalytic site at the start of the Cterminal hydrophobic region (Lys,,,-Arg,,,)
was found
in all the sequences. The PRTl(73j) sequence contained
two of the potential autocatalytic sites, Arg5,,-Arg5,,
and Lys769-Arg770s
The PRTl sequences showed homology with the other
subtilisin-like serine proteases in the region of the Pdomain, the highest homology being with the derived
amino acid sequence of the Schis. pombe krp gene. Four
potential sites for N-linked glycosylation were observed
in all the PRTl sequences, three in the subtilisin-like
catalytic domain (Asn,,,, Asn,,,, Asn,,,), and one in the
P-domain (Asn,,,).
A serine/threonine-rich region was also identified in the
PRTl(73j) sequence from residue Thr,,, to Ser765,and
the hydrophobicity plot of the PRTl(73j) sequence
revealed a hydrophobic region at the C-terminal end,
residues His,,,-Phe,,,,
suggesting a membrane-associated domain. Unlike most other serine protease
sequences, however, all the copies of the P R T l polypeptide contained a proline-rich region downstream of
the P-domain.
2230
Genetic organization of the PRTl multi-gene family
Analysis of the alignments of the DNA and the deduced
amino acid sequences of copies of the PRTI gene from
genomic DNA, the cDNA sequence and the three
fragments obtained by PCR of the cDNA library
revealed domains in the PRTI gene which were highly
conserved and also regions where significant divergence
was observed, again suggesting that PRTI comprises a
multi-gene family (Fig. 2). The subtilisin-like catalytic
domain and the P-domain appeared to be conserved
whereas high levels of heterogeneity were observed in
the proline-rich domain and the C-terminal domain.
The variation in this region was both in length and in
sequence. A number of repeated DNA sequence motifs
were found in the proline-rich region. Nucleotide
sequences encoding polyproline were found in all the
sequences, and also the dipeptides Pro-Glu and Pro-Gln
and the tetrapeptides Pro-Glu-Pro-Gln and Pro-GluThr-Gln. The order and number of tandem repeats
varied in each sequence. The overall length of this region
varied from 67 amino acid residues in the shortest
sequence, PRT1(73j), to 233 residues in the longest
sequence, PRTl(M14).
T o further substantiate the presence within the P. carinii
genome of multiple copies of the PRTI gene, P. carinii f.
sp. carinii chromosomes were separated by PFGE and a
karyotype corresponding to P. carinii f. sp. carinii form
1 was observed (Cushion et al., 1993). Under the
conditions used, the rat chromosomes were too large to
be resolved and remained at the band of limiting
mobility. The separated chromosomes were analysed by
hybridization with three probes derived from different
domains of PRTI. All three probes showed similar
patterns of hybridization, annealing at high stringency
to all the chromosome bands except for one, the third
smallest in size, approximately 350 kb (Fig. 4). This
provided further evidence that the P. carinii f. sp. carinii
genome contained many copies of the PRTI gene, which
were present on most of the P. carinii f. sp. carinii
chromosomes.
The sequences of the PRTZ gene family showed high
levels of homology with ORF3, an ORF which was
reported to encode a P. carinii protein of unknown
function and was demonstrated to be contiguous with a
copy of the gene encoding the major surface glycoprotein
MSGIOO (Wada & Nakamura, 1994). This gene arrangement was reported in 15 other Iz clones, in which a
gene showing high homology to ORF3 was located
downstream of a copy of MSG (Wada & Nakamura,
1994). Most copies of the MSG genes have been
demonstrated to be located in the P. carinii f. sp. carinii
subtelomeric regions (Underwood et al., 1996 ; Sunkin
& Stringer, 1996). The copy of the PRTZ gene encoded
by the PRTl(Paga) sequence was cloned from a IzEMBL3
genomic library as a single 14 kb fragment and was
approximately 1150 bp downstream of a copy of MSG.
Four other Iz clones isolated from the same library
contained a copy of PRTI contiguous with a copy of
MSG.
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Pneumocystis carinii serine protease
P. carinii f. sp. carinii genomic DNA was digested with
either restriction endonuclease PstI or BamHI and
probed sequentially with four oligonucleotide probes,
derived from the 5’ end of PRTl gene (pcprotS/RI),
from the catalytic domain of the gene (pcprot3/RI), an
M S G probe (msgterm) and a subtelomeric probe
(Pctel2). All probes hybridized to multiple bands. The
hybridization patterns of some of the bands, ranging in
size from 7 kb to greater than 12 kb, were the same for
all four probes. However, hybridization to other fragments was not coincident, with the PRTl probes alone
hybridizing to some high-molecular-mass fragments and
also low-molecular-mass fragments of less than 7 kb
(Fig. 5).
DISCUSSION
We describe the cloning and characterization of copies
of the PRTI multi-gene family from P. carinii f. sp.
carinii. A copy of the PRTl gene was isolated from a P.
carinii f. sp. carinii genomic library. A different copy
was isolated from a cDNA library, indicating that this
copy of the gene was transcribed, and also identifying
the presence of seven short introns in the genomic
sequence. Consistent with many other P. carinii genes,
the coding region and the flanking sequences of the
PRTl sequences showed a strong bias for adenine or
thymine, and in particular at the third base position of
the codons. Similarly, the presence of short A+T-rich
introns has been reported in other P. carinii genes. In the
PRTl sequences, the introns were not distributed
throughout the gene, but six of the seven introns were
found in the subtilisin-like catalytic domain and the
seventh in the P-domain. It is possible that the introns
may play a role in restricting the variation in this region
of the gene, whereas no introns were observed in the
highly heterogeneous proline-rich region.
The high level of homology of the P. carinii PRTl
sequences to subtilisin-like serine proteases, and in
particular in the region of the catalytic domain, strongly
suggested that this gene encoded a protease of this type.
The predicted P. carinii PRTl polypeptide sequences
possessed the three essential residues of the catalytic
active site as well as many other highly conserved
motifs. The domain organization of the PRTl gene
strongly resembled that of the fungal prohormoneprocessing proteases, with the exception of the prolinerich domain. This proline-rich region is very uncommon
in the subtilisin-like serine protease superfamily, although the KRP6 gene from Y . lipolytica is reported to
contain a short region of a tetrapeptide repeat, the
consensus sequence of the four amino acids being Glu-
(Asp/Glu)-Lys-Pro (Enderlin & Ogrydziak, 1994). A
proline-rich region has also been found in the C-terminal
tail domain of the mammalian serine protease acrosin, a
proteolytic enzyme of sperm cells, located in the
acrosome at the apical end of the spermatozoan (Klemm
et al., 1991).
In the African trypanosome, Trypanosoma brucei, a
proline-rich domain has been identified in the procyclic
acidic repetitive proteins (PARPs). These proteins are
found on the cell surface of the insect form of the
parasite and are encoded by a family of polymorphic
genes which contain a variable region with heterogeneity
in both length and sequence. The variable region
contains the proline-rich domain and is primarily
composed of the dipeptide Glu-Pro (Roditi et al., 1989).
Unlike any of the other fungal prohormone-processing
proteases, which appear to be single-copy genes, the
data reported in this study suggested that the PRTl
sequence is present in many copies, which are similar
but not identical, in the genome of P. carinii f. sp. carinii.
The relatively large number of recombinants present in
both the genomic and the cDNA libraries suggested a
multi-copy gene and this was substantiated by PFGE
data revealing that at least one copy of a PRTl gene was
present on all but one of the P. carinii chromosomes.
Southern hybridization of restriction endonucleolytic
digests of P. carinii f. sp. carinii DNA probed with PRTl
sequences also confirmed the presence of many copies of
the gene. Analysis of sequence data generated by the
amplification of the locus showed heterogeneity, suggesting that a variety of different copies of the gene were
present in the P. carinii genome. Some domains,
including the subtilisin-like catalytic domain and the Pdomain, were highly conserved between gene copies,
whereas the highest levels of divergence were observed
in the proline-rich domain, which varied both in length
and in sequence.
Of five genomic clones analysed in this study, all
possessed a copy of PRTl contiguous with an M S G gene.
It has been reported that 15 independent genomic clones
which encoded M S G were contiguous with the ORF3
sequence, which from our analysis appears to encode the
proline-rich domain of PRT2 (Wada & Nakamura,
1994). It has been demonstrated that most copies of
MSG are subtelomeric (Underwood et al., 1996; Sunkin
& Stringer, 1996). It is therefore highly likely that many
copies of the PRTl multi-gene family are located in the
subtelomeric regions of the P. carinii f. sp. carinii
genome. However, PFGE analysis has shown that not
every P. carinii f. sp. carinii chromosome contains a
copy of PRTl ,and the preliminary characterization of a
Fig. 3. Alignment of the P. carinii PRTl(73j) deduced amino acid sequence with subtilisin-like serine proteases from Schiz.
pornbe (spkrp), Y, lipolytica (ylipo), Kluveromyces lactis (klkexl), Sacch. cerevisiae (sckex2), C. elegans (celeg), 0.
melanogaster (furs-d), rat (ratkex) and human (furin and nec2-h). GenBank accession numbers are X82435, 540697,
X07038, M24201, L29438, P26016, M83745, X17094 and P16519, respectively. Residues are numbered with reference to
the P. carinii PRTl(73j) sequence. Gaps were introduced to maximize homology. -, catalytic domain; ---, P-domain;
> > > and ---,predicted conserved secondary structure elements, a-helix (hA-hH) and P-sheet Wl-pS), respectively;
conserved catalytic active site residues, Asp,,, (D), His,,, (H) and Ser,,, ( 5 ) ; *, conserved residues in the catalytic domain.
+,
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2231
E. B. LUGLI, A. G. A L L E N a n d A.E. W A K E F I E L D
73 j
SPkrp
YliPO
klkexl
sckex2
celeg
furs-d
ratkex
furin
nec2-h
................................................................................
..................................................................... C IERPST#HPA
..........
.MLRXPILOL LLASQAVAQL PHlCEMYDSR VYVALSLRDQ LDPREWASV SOLDHQQWTP EHPVGTIPNT
YSLIFRTIVM ILSSQLMLU IAVSQYGKAM QvPKKDHENa QYPAIESYDD V-L.....
..AEHSDWSF EHDMGLANH
. m Y I T L CRIWAF.... .STSALVSSQ Q I P W m S R QYPAVESNET LSRLE.....
. . m y EHDVRQLPNH
RV
..............................................................................
................................................................................
.....................................................................
S NPEPGAVCRV
............................................................ .AGTWBPRPY
................................................................................
DgAlcETQAw
..........................
54
ILLTPPLYWI YLVRVRCEMV PVDFENM)YY YYPHLSEDVD IEEPSBAVOF
VEPE VDPWAAEAI GAKYVRPLLN L . K m I K L
ElELnarmEKLQ XKY-EEDV
EKRLVALERL DYDWSERGLG SLEVLSERRI
El.........
DAI DTOYSENIID pHDL.PPVQL
RS........
SLEEL QGDNNDHILS VHDLPPILNOL
TIEHDSICDE SI.....OAC GEPIBTVIRL AKRD.DE..L ARRIAADHwd
73j
OPkrp
YliPO
klkexl
sckex2
celeg
furs-d
ratkex
furin
nec2-h
YIFK
LLCGPILAIP LQPLVSSCSP LENDDLPLVQ
YVFSAPKEYA PIBNIRDQDR L E V A Q G W
YVFSKPLQ..
SLOK
WPSKELL..
KLGK
bfRISIGRIAW QILAV.....
LIAVAP
73j
.Pkrp
YliPO
klkexl
sckex2
celeg
furs-d
ratkex
furin
nec2-h
KygMltvogLD
73 j
SPkrp
YliPO
klkexl
sckex2
celeg
furs-d
ratkex
furin
nec2-h
LYNDEEIVNN
KRGIDAGILE
DASPAMPVQW
LPIQ.
.LPVPA
RRAQRQQPQS
73j
SPkrp
YliPO
klkexl
eckex2
celeg
furs-d
ratkex
furin
nec2-h
TVAIADNQLD YTNXDLAPNY NSQGSYDFVS KTDDPNPK..
TVAFVDWID PKEPDLQAAY TSLGSWDFND NIADPLPK..
VTAVVDDGLD - D I M
PAEaswDFNF NKSDPKPS..
VAALVDWLD YENEDLKDNF CVEWWDFND "PLPKPR..
V M I M W L D Y E N E D W N F CAEOSWDFND "LPKPR..
SVSILDWIQ RDHPDLAANY DPLASTDIND HDDDPTPQNN
W T I L D W L E SDEPDIQDNY DPKhSYDVNS HDDDPNPEYD
VITVLDWLE WNHTDIYANY DPEASYDFND NDHDPPPRYD
W S I L D W I E lWHPDLAONY DPQASFDVND QDPDPQPRYT
TIGIHDDOID YLEPDLASNY NAEASYDPSS NDPYPYPRYT
PI
@2
73j
'Pkrp
ylipo
klkexl
sckex2
celeg
furs-d
ratkex
furin
nec2-h
PSO.LSYELE SLALSYKPNV NYIYSCSWOP PGDGYMIPM YPTTYSAIIK GImORNGLG SIyvpoT13Na
S A P . ITDAVE SEALNYGPQT NHIYSCSWGP ADDORAMDAP NTATRRALBW GVLNQLLNQLO SIPWASSKE.IAEDIE ALA-K
NDIYSCSWGP PDNUQTluBP GKVVKDAMVN AITNURQOKG NVPWASQNG
SQQ.ITAEDE AASLIYGLDV NDIYSCSWGP SDWKTMQAP DTLVKKAIIK GVTEGRDAKG A L Y W A S m
SGD.ITTEDE AASLIYGLDV NDIYSCSWGP ADW-P
SDLVKXALVK GVTBQRDSKO AIYWASDGA.VSDSVE MSLSLNQDH IDIYSASWOP E D W K T F W P O
P
G I m R Q G K O NIFVWASGNG
DGD.VTDAVE ARSLSXNPQH IDIYSASWGP DDDOKTVWP GELASRAFIE GTTKGRWKG SIPIKASWI.VTDAIE ASSIOPNWH VDIYSASWOP NDWKTVEGP GRLAQKAFEY UVKQGRQGKG SIFVWASWE-VTDAVE ARSLGLNPNB IHIYSASWGP EDDQKTVWP ARLAEEAFPR GVSQORWLG S I F V W A S m
DQPPMTDIIE ASSIS-L
IDIYSASWGP TDNQKTVDQP RDVTLQAMAD GVNKORGGKG SIYVWASODG
-83
> > > > > > > > > > > > > >----h D P4
>>>>>>>>>>>>hE
P5
................
................
.................
................
...............
....
................................................................................
ERSSLAGVFL ILLLPSQPPL L m Q R m TLQCTAPTLP CWOCALNSVK AKRQFVNEWA AEIH.QGPBA ASAIAEELGY
SQPPVP-L
RPWLL..... . . . . w W M . ...TGTLVLL M . ....DAQ QQKVFTNTWA VRIP.WPAV A N S V m G F
......................... mQGCVSQWKMAOP LFCVHVFASA ERPVFTNKFL VELHllOaEDK ARQVAAEHGF
132
NHHIPPIEKQ VLEDEIK..E KIENYFSLEK GELNAIWPNS DKLPYYEKQK LVKPVNROAI RDDIYFDNQD
0 SDDSVQSSIR
HlcILApVNWTE E-YLICEIK
RUEEAQMQDDKGDKKBDQ KDDKKBQQBA QKBQDKEDNK G D D W W E E D DDDDEDEDDD
...........................................................
HKR.......
......................................................................
FKR.......
......................................................................
EVXGDP..PL DTHYPLYHSE TTRTRRXKRA IVERLDSHPA VEWVEEQRPK KRVKRDYILL DND-NPP
RRSVLNRDOT
................................................................................
DLLQQIOSLE NEYLFlcHlwa PRRSRRSALH ITKRLSDDDR VIWAEQQYEK ERRXRS.... ....................
LNLoQI..FG DY-OV
TKRSLSPHRP RHsRLQREPQ VQWLEQQVAX RRTKRD.... ....................
GVR.KLPPAE GLYHFYBNOL AKNCRRRSLH EnrQQLERDPR m E G P DRKKRGYRDI ....................
HII........
*208
VVlCDPTVDQA KKSTEDLKRE
LERQTPRWRY XRDASESDEL
KPVDESMYQG HPDDSLYDVY
DSSI(EQ1 QNARILP...
PPWDSSLLPV KEABDKLPAEIPSL...
VKEIKKELOI
LNEPSNEFOI
RKYYPDEVGI
........NI
.SI
PF
SF
LF
EP
.....
...
....
.........
..................
.......... . . . . . A M . . . ..................
.........V PRDSALN.. ...................
..............WQ... ..................
............NEIDI... .......... ........N#
SDPCFDKQWY
SDPLPYQQWE
KDPSLWKQWY
SDPLPDQQWE
NDPLFERQWE
PDPLYKVQWY
NDSKWPQMWY
LPNTEKPQ..
IPNSNNPGH.
H
.
LINPNY-.
LVNPSFPQS.
LHWAVQG.. ..YDlQWRQA WLQGYAGRNV
LNR..WQ.. ..LDWIPA WlR(a1TGKGV
NDPMWNQQWY LQDTRMTASL PKLDLEVIPV WQKOITQKGV
TDPKFPQQWY LSQ..VTQ.. ..RDLNVKAA WAQGYTQHOI
NDPLPTKQWY LINTQQAWT POLDLNVAEA WELGYTGKOV
>>>>>>>>>hA
>>>>>hB - - - -
* *
+**
_--------*
------
..VDINVTQV WLQOITOKGV
...DLHLIIEV WDAGYFOENV
...DLNVTGL m O V
...DvNvT(;IL WKENITQYGV
... D m L WYNNITGAQV
.SSSDTEOTR
.LSDDQHOTR
SHDDYHGTR
LXDDYHGTR
.LSDDYHGTR
.QDNAHoTR
* *
* a 4
CAOEVAMR. NDPCGLGVAY ESNISQLRFL
CAGEVAAAW. NDVCGVGIAP RAKVAGLIIIL
CAOEIAAVR. NNVCQVOVAY DSKVAGIRIL
CAOEIAAFR NDICOVOVAY NSKVSGIRIL
CAQEIAAKKG "FCGVQVGY NAKISQIRIL
CAOEVAALAO W C G V G V A P ICMIQQVRML
MTDSNREGTR CAGEVMTAN NSFCAVGIAY OAsV~VEudL
CAOEIAMQAN -CQVGVAY
NSKVQGIRML
P-GTR
m N R E G T R CAGEVAAVAN NGVCGVGVAY NARIQGVRML
DDWFNSHGTR CAGEVSAAAN NNICGVOVAY NSKVAOIRHL
>>>>>>>>>>>>hC
.
.
.
--------
**
**
------
08L.D-
GHYHD"FD
OSRGDNCNPD
OldWDSCNPD
GTRGDNCNYI)
OSSQDSCSAD
OREQDNCNCD
GRQGDNCDCD
QRBHDSCNCD
OSYDD.CNCD
................................................................................................................................................................................................................................................................1
Fig. 3. For legend see p. 2231.
2232
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On: Fri, 05 May 2017 06:37:59
*362
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...
2233
E. B. L U G L I , A. G. A L L E N a n d A.E. W A K E F I E L D
Fig. 4. Southern hybridization analysis of P. carinii
chromosomes separated by PFGE and probed with three
different regions of the PRTl(Paga) gene. Lanes: 1, 3, 5 and 7,
Sacch. cerevisiae DNA; 2, 4, 6 and 8, P. carinii DNA; 1 and 2:
ethidium-bromide-stained gel; 3 and 4, probed with the 5‘ end
of the PRTl(Paga) gene (9/4r product); 5 and 6, probed with
part of the catalytic domain (U4product); 7 and 8, probed with
the 3’ end of the gene (13112 product). The arrow indicates the
P. carinii chromosome which did not hybridize to the PRTl
probes.
clone of one of the subtelomeric regions of P. carinii f.
sp. carinii has not revealed a copy of PRTl (Underwood
& Wakefield, unpublished results). Hybridization of
MSG and subtelomeric probes to endonuclease-digested
P. carinii f. sp. carinii DNA resulted in positive
hybridization to fragments greater than approximately
7 kb in size. Probes derived from the PRTl sequence
hybridized to these bands but also to low-molecularmass fragments, again suggesting that not all copies of
PRTl are subtelomeric.
The P. carinii PRTl gene family shows some striking
similarities to that of MSG. Both are composed of many
genes, copies of which are found on most P. carinii
chromosomes and show sequence heterogeneity. Some
copies of PRTl are contiguous with MSG and are
located in the subtelomeric regions of the P. carinii
chromosomes.
It is interesting to note that one of the major components
of the cell surface of Leishmania has proteolytic activity.
The Leishmania major surface protease (msp or g p 6 3 ) , a
zinc endoprotease, is found in all species of Leishmania
and is encoded by a family of genes, some of which are
tandemly arrayed (Bouvier et al., 1989; Webb et al.,
1991). Expression of different copies of the gene is
regulated during the development of the parasite and
different isoforms of the protein are found in the
2234
Fig. 5. Southern hybridization analysis of P. carinii genomic
DNA digested with restriction endonucleases and probed with
oligonucleotides designed to hybridize to the PRTl sequences.
Lanes: 1 and 3, P. carinii DNA digested with Pstl; 2 and 4,
P. carinii DNA digested with BamHI; 1 and 2, probed with
an oligonucleotide designed to hybridize to the 5’ end
(pcprotYRI); 3 and 4, probed with an oligonucleotide designed
to hybridize to the catalytic domain (pcprotYRI).
promastigote stage in the gut of the sand fly and in the
amastigote stage in the phagolysosomes of the macrophages (Frommel et al., 1990; Roberts et al., 1995;
Ramamoorthy et al., 1995).The major surface protease
is thought to play an important role in the virulence of
Leishmania by involvement in the degradation of
components of the extracellular matrix and by facilitating promastigote attachment to host macrophages
(McMaster et al., 1994). Immunization with MSP
protein confers partial protection of mice against
Leishmania infection (Abdelhak et al., 1995).
The proteins encoded by the P. carinii PRTl gene family
show highest homology to subtilisin-like serine proteases. A wide diversity of different types of precursor
proteins are processed by this family of proteases to
mature and active regulatory proteins, but the precise
function of many of these proteases has not yet been
determined. Some of the fungal homologues have been
shown to function in the processing of several proteins,
such as the Sacch. cerevisiae K E X 2 gene product which
processes both the pheromone a-factor and the killer
toxin (Fuller et al., 1989). The krp gene product from
Schiz. pombe, which cleaves the pheromone precursor
pro-P-factor to its active form, is thought to also
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Pneumocystis carinii serine protease
function in the processing of other regulatory proteins
since its activity is essential for cell viability (Davey et
al., 1994). The XPR6 gene product from Y . lipolytica,
although not essential for cell viability, when disrupted
was found to cause aberrant growth and morphology
(Enderlin & Ogrydziak, 1994). The function of the
products of the P. carinii PRTl gene family is not yet
understood but they are likely to play an important role
in the life cycle and possibly also the pathogenicity of the
organism.
life stages of Leishmania. Mol Biochem Parasitol 38, 25-32.
Fuller, R. S., Brake, A. & Thorner, J. (1989). Yeast prohormone
processing enzyme (KEXZ gene product) is a Ca2+-dependent
serine protease. Proc Natl Acad Sci USA 86, 1434-1438.
Garbe, T. R. & Stringer, J. R. (1994). Molecular characterization of
clustered variants of genes encoding major surface antigens of
human Pneumocystis carinii. lnfect lmmun 62,3092-3 101.
Gigliotti, F. (1992). Host species-specific antigenic variation of
a mannosylated surface glycoprotein of Pneumocystis carinii.
] lnfect Dis 165,329-336.
Gros,
ACKNOWLEDGEMENTS
This research was supported by the Medical Research Council
(E. B. L.) and the Royal Society (A. E. W.), and formed a part of
the European Concerted Action, Biomed I, PL941118. We
should like to thank D r C. J. Delves and D r F. Volpe, Glaxo
Wellcome Research, for the gift of the P. carinii cDNA library
and the MSG clone, and the Oxford Pneumocystis Research
Group for helpful discussion.
REFERENCES
Abdelhak,
Louzir, H., Timm, J., Blel, L., Banlasfar,
Lagranderie, M., Gheorghiu, M., Dellagi, K. & Gicquel, B. (1995).
5.8
Frommel, T. O., Button, L. L.8 Fujikura, Y. & McMaster, W. R.
(1990). The major surface glycoprotein (GP63) is present in both
z.8
Recombinant BCG expressing the leishmania surface antigen
Gp63 induces protective immunity against Leishmania major
infection in BALB/c mice. Microbiology 141, 1585-1592.
Banerji, S., Wakefield, A. E., Allen, A. G., Maskell, D. J., Peters,
S. E. & Hopkin, J. M. (1993). The cloning and characterization of
the mom gene of Pneumocystis carinii. J Gen Microbiol 139,
2901-29 14.
Betzel, C., Pal, G. P. & Saenger, W. (1988). Three-dimensional
structure of proteinase K at 0.15 nm resolution. Eur J Biochem
178, 155-171.
Bott, R., Ultsch, M., Kossiakoff, A,, Graycar, T., Katz, B. & Power,
5. (1988). The three-dimensipnal structure of Bacillus amyl-
oliquefaciens subtilisin at 1-8 A and an analysis of the structural
consequences of peroxide inactivation. J Biol Chem 263,
7895-7906.
Bouvier, J., Bordier, C., Vogel, H., Reichelt, R. & Etges, R. (1989).
Characterization of the promastigote surface protease of Leishmania as a membrane-bound zinc endopeptidase. Mol Biochem
Parasitol37, 235-246.
Cushion, M. T., Kaselis, M., Stringer, 5. L. & Stringer, J. R. (1993).
Genetic stability and diversity of Pneumocystis carinii infecting
rat colonies. lnfect Zmmun 61, 48014813.
Davey, 1. Davis, K., Imai, Y., Yamamoto, M. & Matthews, G.
(1994). Isolation and characterization of krp, a dibasic endo-
peptidase required for cell viability in the fission yeast Schizosaccharomyces pombe. EMBO ] 13,5910-5921.
Dyer, M., Volpe, F., Delves, C. J., Somia, N., Burns, S. & Scaife,
1. G. (1992). Cloning and sequence of a B-tubulin cDNA from
Pneumocystis carinii : possible implications for drug therapy. Mol
Microbiol6, 991-1001.
Enderlin, C. S. & Ogrydziak, M. (1994). Cloning, nucleotide
sequence and functions of XPR6, which codes for a dibasic
processing endoprotease from the yeast Yarrowia lipolytica.
Yeast 10, 67-79.
P.8
Betzel, C., Dauter,
z.8
Wilson, K. 5. & Hol, W. G. J. (1989).
Molecul$r dynamics refinement of a thermitase-eglin-c-complex
at 1.98 A resolution and comparison of two crystal forms that
differ in calcium content. ] Mol Biol210, 347-367.
HaidariS, P. J., Wright, T.
w
.
8
Gigliotti, F. & Haidaris, C. G. (1992).
Expression and characterization of a cDNA clone encoding an
immunodominant surface glycoprotein of Pneumocystis carinii.
J lnfect Dis 166, 1113-1123.
Hirono, 5.8 AkagFwa, H., litaka, Y. & Mitsui, Y. (1984). Crystal
structure at 2.6 A resolution of the complex of subtilisin BPN
with Streptomyces subtilisin inhibitor. J Mol Biol 178, 389414.
Klemm, U., MUller-Esterl, W. 81 Engel, W. (1991). Acrosin, the
peculiar sperm-specific serine protease. Hum Genet 87,635-641.
Kovacs, J. A., Powell, F., Edman, 1. C., Lundgren, B., Martinez, A.,
Drew, B. & Angus, C. W. (1993). Multiple genes encode the major
surface glycoprotein of Pneumocystis carinii. J Biol Chem 268,
6034-6040.
Lundgren, B., Lipschik, G.Y. & Kovacs, 1. A. (1991). Purification
and characterization of a major human Pneumocystis carinii
surface antigen. J Clin Znvest 87, 163-170.
McMaster, R. R., Morrison, C. J., MacDonald, M. H. & JOShi, P. B.
(1994). Mutational and functional analysis of the Leishmania
surface metalloproteinase GP63 : similarities to matrix metal-
loproteinases. Parasitology 108, S29-S36.
McPhalen, C. A. 81James, M. N. G. (1988). Structural comparison
of two serine proteinase-protein inhibitor complexes : eglin-csubtilisin Carlsberg and CI-Zsubtilisin Novo. Biochemistry 27,
65824598.
van den Ouweland, A. M. W., van Duijnhove, H. L. P.8 Keizer,
G. D., Dorssers, L. C. 1. & Van de Ven, W. 1. M. (1990). Structural
homology between the human fur gene product and the subtilisinlike protease encoded by yeast KEX2. Nucleic Acids Res 18,664.
Peters, 5. E., Wakefield, A. E., Banerji, 5. & Hopkin, J. M. (1992).
Quantification of the detection of Pneumocystis carinii by DNA
amplification, Mol Cell Probes 6, 115-117.
Ramamoorthy, R., Swihart, K. G., McCoy, 1. J., Wilson, M. E. &
Donelson, J. E. (1995). Intergenic regions between tandem gp63
genes influence the differential expression of gp63 RNAs in
Leishmania chagasi promastigotes. ] Biol Chem 270,
12133-12139.
Roberts, 5. C., Wilson, M. E. & Donelson, J. E. (1995). Developmentally regulated expression of a novel 59-kDa product
of the major surface protease (Msp or gp63) gene family of
Leishmania chagasi. J Biol Chem 270, 8884-8892.
Roditi, I., Schwan, H., Peanon, T. W., Beecroft, R. P., Liu, M. K.,
Richardson, J. T., Buhring, H. J., Pleiss, J., Bulow, R., Williams,
R. 0. & Overath, P. (1989). Procyclin gene expression and loss of
the variant surface glycoprotein during differentiation of Trypanosoma brucei. J Cell Biol 108,737-746.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 05 May 2017 06:37:59
2235
E. B. LUGLI, A. G. A L L E N a n d A . E . W A K E F I E L D
Roebroek, A. 1. M., Creemers, J. W. M., Pauli, 1. G. L., KurzikDumke, U., Rentrop, M., Gateff, E. A. F., Leunissen, 1. A. M. &Van
de Ven, W. 1. M. (1992). Cloning and functional expression of
Dfurin2, a subtilisin-like proprotein processing enzyme of Drosophila melanogaster with multiple repeats of a cysteine motif.
J Biol Chem 267, 17208-17215.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular
Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor,
NY: Cold Spring Harbor Laboratory.
Sanger, F., Nicklen, 5. & Coulson, A. R. (1977). DNA sequencing
with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74,
5463-5467.
Siezen, R. J., de Vos, W. M., Leunissen, 1. A. M. & Dijkstra, B. W.
(1991). Homology modelling and protein engineering strategy of
subtilases, the family of subtilisin-like serine proteinases. Protein
Eng 4,719-737.
Stringer, 5. L., Garbe, T., Sunkin, 5. M. & Stringer, J. R. (1993).
Genes encoding antigenic surface glycoproteins in Pneumocystis
from humans. J Eukaryot Microbiol40, 821-826.
Sunkin, 5. M. & Stringer, J. R. (1996). Translocation of surface
antigen genes to a unique telomeric expression site in Pneumocystis carinii. Mol Microbiol 19, 283-295.
Sunkin, 5. M., Stringer, 5. L. & Stringer, J. R. (1994). A tandem
repeat of rat-derived Pneumocystis carinii genes encoding the
major surface glycoprotein. J Eukaryot Microbiol41, 292-300.
Tanguy-Rougeau, C., Wesolowski-Louvel, M. & Fukuhara, H.
(1988). The Kluyveromyces lactis K E X l gene encodes a subtilisin-
type serine proteinase. FEBS Lett 234, 464470.
Teplyakov, A. V., Kuranova, 1. P., Harutyunyan, E. H. & Vain:
shtein, B. K. (1990). Crystal structure of thermitase at 1-4 A
resolution. J M o l Biol214, 261-279.
Underwood, A. P., Louis, E. J., Borts, R. H., Stringer, J. R. &
Wakefield, A. E. (1996). Pneumocystis carinii telomere repeats are
composed of TTAGGG and the subtelomeric sequence contains
a gene encoding the major surface glycoprotein. Mol Microbiol
19,273-281.
Van de Ven, W. J. M. & Roebroek, A. J. M. (1993). Structure and
function of eukaryotic proprotein processing enzymes of the
subtilisin family of serine proteases. Crit Rev Oncog 4, 115-336.
2236
Volpe, F., Dyer, M., Scaife, J. G., Derby, G., Stammers, D. K. &
Delves, C. 1. (1992). The multifunctional folic acid synthesis fas
gene of Pneumocystis carinii appears to encode dihydropteroate
synthase and hydroxymethyldihydropterin pyrophosphokinase.
Gene 112,213-218.
Volpe, F., Ballantine, 5. P. & Delves, C. J. (1993). The multifunctional folic acid synthesis fas gene of Pneumocystis carinii
encodes dihydroneopterin aldolase, hydroxymethyldihydropterin
pyrophosphokinase and dihydropteroate synthase. Eur J Biochern
216,449458.
Wada, M. & Nakamura, Y. (1994). M S G gene cluster encoding
major cell surface glycoproteins of rat Pneurnocystis carinii. D N A
Res 1, 163-168.
Wada, M., Kitada, K., Saito, M.,Egawa, K. & Nakamura, Y. (1993).
cDNA sequence diversity and genomic clusters of major surface
glycoprotein genes of Pneumocystis carinii. J Infect Dis 168,
979-985.
Wada, M., Sunkin, 5. M., Stringer, J. R. & Nakamura, Y. (1995).
Antigenic variation by positional control of major surface
glycoprotein gene expression in Pneumocystis carinii. J Infect Dis
171, 1563-1568.
Webb, J. R., Button, L. L. & McMaster, W. R. (1991). Heterogeneity
of the genes encoding the major surface glycoprotein of Leishmania donovani. Mol Biochem Parasitol48, 173-184.
Wright, T. W., Simpson-Haidaris, P. J., Gigliotti, F., Harmsen,
A. G. & Haidaris, C. G. (1994). Conserved sequence homology of
cysteine-rich regions in genes encoding glycoprotein A in Pneumocystis carinii derived from different host species. Infect Zmmun
62, 1513-1519.
Wright, T. W., Bissoondial, T. Y., Haidaris, C. G., Gigliotti, F. &
Simpson-Haidaris, P. J. (1995). Isoform diversity and tandem
duplication of the glycoprotein A gene in ferret Pneumocystis
carinii. D N A Res 2, 77-88.
Zhang, J. & Stringer, J. R. (1993). Cloning and characterization of
an alpha-tubulin-encoding gene from rat-derived Pneumocystis
carinii. Gene 123, 137-141.
Received 21 January 1997; revised 7 March 1997; accepted 20 March
1997.
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