A bio-informatics study of the c25 cysteine protease family

doi:10.4236/ojgen.2012.24B004

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Open Journal of Genetics, 2012, 2, 18-22 OJGen

Published Online December 2012 (h ttp://www.SciRP.org/journal/ojgen/)

Published Online March 2012 in SciRes. http://www.scirp.org/journal/ojgen

A bio-informatics study of the c25 cysteine protease family

K.J. Cross, N.L. Huq, E.C. Reynolds

Bio21 and Oral Health CRC, University of Melbourne, Melbourne 3010, Australia

Email: [email protected]

Received 2012

ABSTRACT

The oral pathogen Porphyromonas gingivalis is recog-

nized as one of the major aetiological agents of

chronic periodontitis. The gingipains, which are the

principal virulence factors of P. gingivalis, are mul-

ti-domain proteins containing an N-terminal C25

cysteine protease domain. We have conducted a

bio-informatics study of the C25 cysteine protease

domains and have identified related domains in over

two thousand proteins from 739 organisms in 35 dis-

tinct phyla. Proteins having significant similarity to

the gingipain C25 cysteine protease domain are also

found in Gram +ve bacteria, Archaea, algae, higher

fungi, and a wide variety of Eukaryotic species.

Keywords: C25 Cysteine Proteases; Evolution;

Substrate Preference

1. INTRODUCTION

Gingivitis is an inflammatory disease of the gum tissue.

If not checked, the disease can progress to periodontitis

leading to inflammation of the soft tissues surrounding

the teeth, resorption of bone, and eventual loss of teeth.

Porphyromonas gingivalis, is a major pathogen

associated with chronic periodontitis in adults.

Gingipains were identified as the outer membrane,

multi-domain virulence factors of the oral pathogen

Porphyromonas gingivalis [1]. The N-terminal domain

of the gingipains is a C25 cysteine protease domain. The

evolution of the C25 cysteine protease family has been

difficult to elucidate due to both the limited number of

family members identified and their narrow distribution

by species. The aim of this study was to undertake a

detailed search for C25 protease-like sequences in public

genome databases.

The MEROPS database [2] defines clan CD as

containing families of proteases with either a protein fold

or a sequence motif similar to those found in the caspase

family (C14) and a histidine-cysteine catalytic dyad with

the histidine located N-terminal to the cysteine. The

catalytic histidine is usually in a histidine-glycine motif

and is preceded by a block of hydrophobic residues. The

catalytic cysteine is found predominantly in an

alanine-cysteine motif and is preceded by a second block

of hydrophobic residues.

Enzyme specificity in clan CD is determined primarily

by the P1 residue of the substrate, which is normally an

asparagine in family C13 (legumains), an aspartate in

family C14 (caspases), and either an arginine or lysine in

C11 (clostripains), C25 (gingipains), and C50 (separases),

and a leucine in C80 (RTX toxin) [2]. The C25

(gingipain) specificity preference is based on the limited

experimental data available for three proteins (RgpA,

RgpB, and Kgp) from Porphyromonas gingivalis: RgpA

and RgpB share greater than 97% sequence identity

through their respective catalytic domains and display

specificity toward arginine residues, while the more

divergent Kgp displays specificity toward lysine

resid ues.

Tertiary structures are only available for members of

families C14 [3], C25 [4] and C80 [5]. These show

α/β-proteins with a fold consisting of an α/β/α sandwich.

The β-sheet contains six strands (in the order 213456)

with strand 6 anti-parallel to the rest. The fold is believed

to be unique to members of clan CD [2]. Other protein

families are included in clan CD because of the

conservation of motifs around the catalytic residues [6].

2. MATERIALS AND METHODOLOGY

2.1. Fugue Runs

We used a “pattern initiated hit and search” strategy to

identify possible C25 domain sequences in the ‘nr’

database [7]. In brief, a preliminary alignment of C25

cysteine protease domain sequences was used to develop

a regular expression that described the key features of the

alignment. The regular expression was then used to pare

down the number of sequences to be considered in the

second step of the process.

Sequences that passed the regular expression were

aligned against the RgpB sequence using Fugue [8]

taking into account the structural preferences of residues.

Sequences with a Z-score greater than 5.94 were

accepted for further study. Fugue alignments were

K.J. Cross et al. / Open Journal of Genetics 2 (2012) 18-22

performed using the structurally annotated sequence

(hs1cvra) for RgpB available from the HOMSTRAD

database [9] and the Fugue program [8]. The sequence

and structure of RgpB used throughout this paper is that

of Eichinger et al. [4].

2.2. Sequence Alignments and Analysis

T-Coffee [10] was used in the ‘espresso’ mode to

generate an initial (master) alignment of the C25 domain

sequences identified using Fugue. MAFFT [11] was used

to generate sequence alignments of the cysteine protease

domains identified using PSI-BLAST [12]. To ensure

consistency, all alignments were performed using the fft

option and ‘re-treeing’ twice. The quality of the hits

identified as possible C25 cysteine protease domains was

assessed using sequence alignments and the Shannon

information content of those alignments [13]. A Tcl/Tk

script was developed that populated the EPS template

used by WebLogo and allowed selected columns from

the sequnce alignments to be plotted.

2.3. PSI-BLAST Runs

Preliminary runs of PSI-BLAST were performed using a

single C25 domain sequence (RgpB) and varying the

‘inclusion threshold’ and maximum ‘E value’. Two runs

were performed with a maximum E-value of 1e-3 and

inclusion threshold values of 5e-4 and 5e-3. A further

two runs were performed with an inclusion threshold of

5e-4 and maximum E-values of 1e-2 and 1e-3.

Two production runs were performed. The first using a

master sequence alignment of 103 protein sequences

(MSA) identified by Fugue as being C25 domains. The

inclusion threshold was set at 5e-4, the maximum

E-value of 1e-4, and 6 iterations of the PSI-BLAST algo-

rithm were performed. The master sequence index was

incremented in consecutive runs so that a PSI-BLAST was

performed for each of the C25 sequences identified by

Fugue. The PSI-BLAST data was aggregated into an sqlite

database for further analysis. The second run used a cu-

rated subset of the sequences identified in the third itera-

tion of the first run with 336 sequences (MSA) and three

PSI-BLAST iterations were performed and analysed as for

the first run.

3. RESULTS AND DISCUSSION

Identification of Seed Sequences

A total of 103 sequences were identified as possible C25

cysteine protease domains using the Fugue-filter

criterion. They constitute a subset of the 185 sequences

identified in the Pfam database [14]. However, at least

six of the sequences identified by Pfam as C25 cysteine

protease sequences appear to lack the catalytic cysteines.

These non-cysteine protease sequences are

A9B8P8_HERA2,A4BYP5_9FLAO,E1K598_9EURY,

P96966_PORGI, E1K599_9EURY, and A7BN31_9GAMM.

PSI-BLAST uses two parameters to control the search for

related sequences. The ‘expectation value’ (E-value)

threshold determines whether a hit is accepted or not,

smaller values are associated with increased significance.

The ‘inclusion threshold’ is the maximum expectation

value for a hit to be used to calculate the PSSM (Position

Sensitive Substitution Matrix), the matrix describing the

probability of particular mutations occurring at specific

locations in the protein sequence.

In preliminary experiments, we demonstrated that

changing the E-value threshold from 1.0e-3 to 1.0e-2,

while keeping the inclusion threshold fixed at 5.0e-4,

increased the number of hits from 327 to 332 after five

iterations starting from the RgpB C25-domain sequence.

Changing the ‘inclusion threshold’ had no effect on the

number of hits located. Having demonstrated that the

PSI-BLAST algorithm was fairly insensitive to these

parameters at these levels (results not shown), we used a

significance level of 5.0e-4 for the inclusion threshold

and 1.0e-4 for the E-value threshold to minimize the risk

of incorporating misassigned sequences.Figure 1 shows

Figure 1: Schematic representation of the ‘BLAST connectivity’

of sequences in the ‘seed’ set of C25 sequences. Heavier lines

represent smaller, more significant expectation values in the

range 1e-10 to 1e-420. Note the location of the archetypical RgpA,

RgpB, and Kgp C25 sequences on the periphery of the cluster,

and the tight cluster of sequences primarily associated with

bacteria from the Prevotella phylum.

the ‘BLAST connectivity’ network for the C25 domains

K.J. Cross et al. / Open Journal of Genetics 2 (2012) 18-22

identified by the Fugue-filter process: the plot was

prepared using Cytoscape [15]. Heavier lines indicate

smaller expectation values for the blast connectivity

between the C25 domains (i.e. higher significance). All

the connectivities shown in Figure 1 have expectation

values of 1e-10 or less and can be considered as highly

significant. Figure 1 emphasizes the fact that the C25

domains of RgpA, RgpB, and Kgp (the archetypical

C25-proteins) are in fact on the edge of a cluster of

proteins that should be thought of as having typical C25

domain sequences.

As shown in Figure 2 there is a rapid increase in the

number of sequences identified after the third iteration of

PSI-BLAST accompanied by a rapid decline in the infor-

mation content of the alignments whether considered as a

sum over all positions in the alignment, or as information

per position as shown. This behaviour could be consis-

tent with ‘contamination’ of the PSSM used by PSI-BLAST.

The observation that the rapid increase in the number of

hits occurred even after the removal of suspected

non-gingipain sequences in the second, production

PSI-BLAST run suggests that many of these additional hits

are to significantly related sequences.

Figure 2. Number of unique sequences identified and the in-

formation content per position in the sequence alignments as a

function of PSI-BLAST iteration using the 103 C25-sequences

identified by Fugue as the ‘seed’ set.

The putative C25 cysteine protease domains were

curated by inspection of the alignments. For example,

inspection of the alignment of 362 sequences identified

at iteration 3 of the first PSI-BLAST run identified 44

sequences that either lacked a conserved cysteine at the

catalytic site, or had residues such as valine or proline

aligned with the catalytic histidine, or had a cluster of

three bulky residues aligned with the ‘GHG’-motif.

These sequences were removed from the alignment and

their GI numbers added to a ‘black list’ of sequences to

be excluded from future PSI-BLAST runs. The information

per position increased from 0.45954 ± 0.00029 to

0.49367 ± 0.00034 after removal of the ‘bad’ sequences

(compared to 0.50987 ± 0.00038 at the first iteration).

Figure 3 shows a ‘WebLogo’-style representation [16]

of those columns that have fewer than 40% gaps in the

alignment of the 2,333 proteins identified as being re-

lated to C25 domains in this work. The gaps between

conserved portions (note the indices are discontinuous)

are consistent with a family of proteins having a con-

served core connected by more variable regions.

Table 1 summarizes the progress of the search for C25

cysteine protease domains. The number of Archaeal and

Bacterial proteins identified increases at each stage of the

search process, as does the number of Eukaryotic species.

This suggests the families of proteins within Clan CD are

significantly overlapped in sequence space.

Figure 3. A ‘WebLogo’-style representation of the mafft

alignment of the 2,333 proteins identified in the final, curated

psi-blast data set. To compress the graphic, only those columns

with at most 40% gaps are represented as a consequence the

column indices labelling the x-axis are discontinuous.

Figure 4 emphasizes the structural similarities between

RgpB (C25), Yca1 a meta-caspase from yeast (C14B),

the RTX toxin from V. cholerae (C80), and human cas-

pase-7 (C14). Not only is there a strong topological

K.J. Cross et al. / Open Journal of Genetics 2 (2012) 18-22

Table 1. Summary of the number of different species and

proteins organized by phyla at various stages of the search for

C25 cysteine protease domains. The ‘Seed Set’ consists of the

103 proteins confirmed by Fugue alignment to be ‘C25 cysteine

protease domains’. The ‘1st Round’ proteins are those identified

in three iterations of PSI-BLAST starting from the ‘Seed Set’, but

excluding sequences as described in the text. The ‘Final’ set

represents the proteins identified after a further three iterations

from the “1st Round” set. The figure in brackets represents the

number of proteins.

Groups

Phylum

Seed Set

1st Round

Fina l

Archaea

Crenarchaeota 2 (4) 1 (1)

Eurya r chae ota 2 (2) 7 (19) 17 (26)

Korarchaeota

1 (1)

Thaumarchaeota 1 (2)

Bacteria

Acidobacteria

1 (1)

6 (18)

Actinobacteria 4 (4) 89 (208)

Aquificae

1 (1)

Bacteroidetes

56 (65)

131 (210)

149 (333)

Caldiserica 1 (1) 1 (1)

Chl amyd iae

1 (13)

Chlorobi 1 (1) 1 (2) 6 (11)

Chloroflexi 6 (7) 8 (19) 10 (73)

Cyanobacteria

3 (3)

53 (677)

Deinococcus-

The rmus

1 (1)

Firmicutes 1 (1) 12 (13)

Fusobacteria 1 (1)

Gemmatimonadetes

1 (1)

Ignavibacteria 2 (3) 2 (4)

Nitrospirae

2 (7)

Planctomycetes

3 (3)

5 (5)

6 (14)

Proteobacteria 5 (8) 31 (42) 176 (317)

Spirochaetes 13 (13) 18 (19)

Verrucomicrobia

1 (1)

1 (2)

2 (2)

Fungi

Ascomycota

2 (4)

76 (184)

Basidiomycot a

21 (248)

Eukaryota

Arthropoda

3 (3)

Bacillar iop hyt a 2 (3)

Chordata 29 (33)

Cni daria

1 (24)

Ec hinodermata 1 (1)

Platyhelminthes 1 (1)

Porifera

1 (2)

Viridi pl antae

Chlorophyta 2 (2)

Streptophyta

13 (30)

Brown algae

Phaeophyceae 1 (3)

similarity as shown by the order and orientation of the

β-strands forming the core of the proteins, but the cata-

lytic histidine and cysteine are consistently two residues

Figure 4. Cartoon representations of the β-strand core of RgpB

from Porphyromonas gingivalis (PDB:1cvr) a C25 cysteine

protease (A), caspase-7 from Homo sapiens (PD B: 1 k88) a C14

cysteine protease (B), the RTX-toxin from Vibrio cholerae

(PDB:3gcd) a C80 cysteine protease (C), and Yca1 a metacas-

pase from Saccharomyces cerevisiae (PDB:4f6o) a C14B

cysteine protease (D). The location of the catalytic histidine and

cysteine shown in stick form, are always located two residues

C-terminal of the fin al residues in the third and fourth β-strands

respectively.

C-terminal to the last residues of the 3rd and 4th

β-strands respectively. Given this strong structural simi-

larity, it is not surprising that C25 domain proteins dis-

play a marked sequential similarity with other Clan CD

proteins.

The sequence specificity of the C25 proteases is de-

termined by the residues that line the ‘S1’ specificity

pocket. As shown in Figure 5 there is very little se-

quence conservation in this region apart from the

‘GHG’-motif and the catalytic cysteine. For example, in

RgpB the side-chain of Asp163 hydrogen bonds to the

guanidine group of the arginine substrate whereas in Kgp

the analogue of Asp163 is a threonine and it is probably

Asp516 that is the predominant hydrogen-bonding part-

ner to the lysine substrate. Inspection of the alignment of

the putative gingipain sequences identifies three se-

quences that had previously been annotated as either

“propeptide peptidase C25” (GI: 373458037) or “pepti-

dase C25” (GI:326279641 and 307565663) from Cal-

dithrix abyssi, Odoribacter splanchnicus, and Prevotella

amnii [7] respectively, where Thr209 of RgpB is re-

placed by an arginine residue. Thr209 is located near the

bottom of the ‘S1’ specificity pocket, and an arginine

residue would have its side-chain extend to near the top

of the specificity pocket suggesting that these three bac-

terial gingipains have a caspase-like specificity toward

aspartate (or possibly glutamate) residues N-terminal to

the cleavage point. In, for example, RgpB a short-chain

residue near the bottom of the specificity pocket is used

to ‘recognize’ the long-chain of the arginine substrate.

The three proteins identified here, appear to use a

long -chain arginine at a neighbouring location to ‘recog-

nize’ a short-chain substrate. These specific examples

underscore the wide range of substrate specificities sug-

K.J. Cross et al. / Open Journal of Genetics 2 (2012) 18-22

gested by Figure 5.

Figure 5. A plot of the frequency of occurrence of various re-

sidues that align with the ‘S1’-pocket residues of RgpB in the

‘seed’ set of 103 C25 cysteine protease sequences. As shown in

Table 1, these are Bacterial or Archaeal proteins. These resi-

dues determine the substrate specificity of the various C25

proteases. The low overall conservation suggests a broad range

of C25 substrate preferences.

4. CONCLUSION

The bacterial C25 cysteine proteases share significant

sequential and structural similarity with other Clan CD

cysteine proteases. The number of identified bacterial

and archaeal sequences increases at each stage of the

search procedure as seen in Table 1, while the number of

sequences associated with other phyla increases dramat-

ically in the final round of the search.

The lack of sequence conservation in the ‘S1’-binding

site argues for a wide-range of substrate specificities

among the C25 cysteine proteases further blurring the

distinctions between the various protease families within

the Clan CD proteases.

5. ACKNOWLEDGEMENTS

We acknowledge funding from the Oral Health CRC and NH&MRC.

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