Cleavage site Metal bindiing domain Transmembrane domain -1 Transmembrane domain -3 Transmembrane domain -4 Metal binding domain Cleavage site Transmembrane helix domain Exosialidase activity domain 182 to 240 FRIP region Consensus motif Asp motif Asp motif Asp motif Asp motif Asp motif Transmembrane domain Glycine rich motif Glycine rich motif Glycine rich motif Glycine rich motif Endoprotease domain Metal binding Histidine domain Metal binding Histidine domain Metalloprotease consensus domain Metalloprotease consensus domain Glycine rich motif PG0778 PG1724 PG0352 Asp motif Asp motif Inner domain Signal peptide Cleavage site Twin arginine peptide signal 360-370 290-300 410-420 Binding site Binding site Binding site Figure 1. Supplemental data ‡ ▲ ▄ 230-240 ▲ ▄ ◙ ◙ A B C Transmembrane domain -2 Lectin binding CHO domain Lectin binding CHO domain Lectin binding CHO domain ‡
Transcript
Cleavage site
Metal bindiing domain Transmembrane domain -1
Transmembrane domain -3
Transmembrane domain -4 Metal binding domain
Cleavage site
Transmembrane helix domain
Exosialidase activity domain 182 to 240 FRIP region
Figure 1.supplemental data. Amino acid sequences of sialidase/O-sialoglycoprotease genes of P. gingivalis showing various unique domains and motifs.
A. Amino acid sequence of PG0778 showing 4 transmembrane domains, two metal binding domains and one lectin binding carbohydrate domain. There is a twin arginine peptide signal at the 39th position coinciding with the cleavage and specific binding sites.
B. Amino acid sequence of PG0352 showing an inner domain, transmembrane domain and an outer domain. There is a specific exonuclease activity domain consisting of a FRIP region, a non catalytic lectin binding domain and a consensus Asp box domain spanning amino acid positions 182 to 240. The Asp box sequences in the protein are shown in an inset box with positions of their location. Non-specific DxxD (aspartic acid motifs) are noted in 7 positions. The transmembrane helix domain consists of signal peptide sequence and a cleavage site.
C. Amino acid sequence of PG1724 showing N terminal metalloprotease consensus domain bearing a cleavage site. Metal binding histidine domains are indicated at positions 113 and 290. A transmembrane domain spans position 136 to 152. Glycine rich motifs were found distributed throughout the protein. A lectin binding domain is indicated at positions 277 – 282.
867785
482
Porphyromonas gingivalis (PG0352)
Bacteroides fragilis
Actinomyces naeslundii (ANA1493)
Clostridium perfringens
Tannerella forsythia (TF0035)
P.gingivalis(33277)(PGN1608)
Actinomyces naeslundii (ANA2709)
Tannerella forsythia (TN2207)
CONSENSUS
Actinomyces naeslundii (ANA1493)
Clostridium perfringens
Tannerella forsythia (TF0035)
P.gingivalis (33277)(PGN1608)
Actinomyces naeslundii (ANA2709)
Tannerella forsythia (TN2207)
CONSENSUS
Porphyromonas gingivalis (PG0352)
Bacteroides fragilis
▲
Figure 2. Supplemental data.
Figure 2. supplemental data. Consensus amino acid signature sequence of sialidases among oral pathogens. Consensus amino acid signature sequence of sialidase Asp box (SxDxGxTW) found in
PG0352 and the other members of Clad A (Fig. 1).
Identification of Asp box like motifs and a consensus signature motif-DAxG/DAxD in O-Sialoglycoprotease clusture
Figure 3. Supplemental data
Figure 3.supplemental data
Identification of Asp box like motifs(shown in black under score) and consensus sequence motifs of DAxG /DAxD (shown in blue under score) present in the O-sialoglycoprotease clustures.
Figure 4. Supplemental data.
loose confirmation suggestive of weak interactions sites with less hydrophilic
regions
Model of PG1724 O-sialoglyco protease
C
90o
90o
Terminal clef and molecular groove for optimal interaction. The model
shows more hydrophilic regions suggestive
of more active sites.
Model of PG778 O-sialoglycoprotease
A
60o
60o
Compact configuration showing less hydrophilic regions
Model of PG0352 -sialidaseB
90o
90o
1
43
2 1
12
2
3
3
4
4
4
Figure 4.supplemental data. In silico protein modeling
A. Protein model of PG0778 showing terminal clef and molecular groove for optimal interaction. The model shows more hydrophilic regions suggestive of more active sites than the other O-sialoglycoprotease PG1724. The ribbon model showing the sheets forming end propeller and flanking helixes (1). The surface structure of the protein showing an interactive tunnel coinciding with the terminal cleft was shown.The hydrophilic areas in the protein are colored red (2-4).
B. Protein model of PG0352 showing a compact configuration with less hydrophilic
regions. The model shows the sialidase protein is a monomer containing six sheets (1 and 2). The protein surface show less hydrophilic regions (3 and 4).
C. Protein model of PG1724 showing end propeller structure (1 and 2) with a loose
confirmation with less hydrophilic regions on the surface of the protein (3 and 4).
Total protease and Sialidase activity of complemented strains
0
10
20
30
40
50
60
70
80
90
100
FLL401C FLL402C FLL403C W83
Strains of PG
Acti
vit
y %
Sialidase activity
Total protease activity
Gingipain activity of the complemented strains of P.gingivalis
0
20
40
60
80
100
120
FLL401C FLL402C FLL403C W83
Strains
% A
cti
vit
y
Rgp
Kgp
Substrate utilisation of the P.gingivalis complemented mutant strain
0102030405060708090
100
Substrate
Acit
ivit
y(%
)
W83 FLL401C FLL402C FLL403C
Assay of P.gingivalis complemented mutants showing restored defects
Figure 5. Supplemental data
A
B
C
Figure 5.supplemental dataA. Graph showing the total protease and sialidase activity of the P.gingivalis complemented mutants.B. Graph showing the gingipain activity of the P.gingivalis complemented mutantsC. Graph showing the substrate utilisation of the P.gingivalis complemented mutants
