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10
Legends for Figures
Fig.1 Identification of a Kdn-sialidase from Sphingobacterium sp. strain HMA12. (a)
Relative sialidase activities for four candidate genes of the GH33 family from the bacterium.
The recombinant proteins were expressed in E. coli, and measured for the sialidase activity
using 4MU-Neu5Ac and 4MU-Kdn as substrates. (b) SDS-PAGE-CBB staining of the
purified ORF03865 sialidase. The enzyme was affinity-purified as described under Materials
and Methods. (c) The deduced amino acid sequence of the ORF03865 sialidase. Predicted
signal peptide sequence (bold letters), RIP motif (shaded), and Asp-boxes (square). The
numbers represent the amino acid number.
Fig.2 Temperature and pH dependency of the ORF03865 sialidase activity. (a)
Temperature-dependent profiles. The enzyme reaction was performed at pH 7.0 for 30 min at
0-60 ℃, using 4MU-Neu5Ac and 4MU-KDN. Released 4MU was fluorometrically quantified
(excitation at 365 nm, emission at 437 nm). The experiments were performed in triplicate, and
the standard deviations are shown by the bar. (b) pH-dependent profiles. The enzyme reaction
was performed at 37 ℃ for 30 min at pH 4.0-10.0: 0.1 M sodium acetate for pH 4.0-6.0, 0.1 M
HEPES-NaOH for pH 6.0-8.0, and 0.1 M Tris-HCl for pH 8.0-10.0. The experiments were
performed in triplicate, and the standard deviations are shown by the bar.
Fig.3 Substrate specificity of the ORF03865 sialidase. (a) Comparison of the Sia species
specificity with known bacterial sialidases. 4MU-Neu5Ac, 4MU-Neu5Gc, and 4MU-KDN
was digested with sialidases derived from Sphingobacterium sp. (ORF03865), Arthrobacter
ureafaciens (Au), Clostridium perfringens (Cp), Vibrio cholera (Vc), and Streptococcus
pneumoniae (Sp). Values represent relative activities to the 4MU-Neu5Ac hydrolyzing one set
to 1.0. The experiments were performed in triplicate, and the standard deviations are shown
by the bars. (b) Linkage specificity. The sialylglycans, i.e., α2,3Gal, α2,6Gal, α2,8Neu5Ac,
and α2,9Neu5Ac, were digested with the ORF03865. The released Neu5Ac was quantitated
by the DMB-derivatization HPLC method. The experiments were performed in triplicate, and
the standard deviations are shown by the bars. (c) TLC analysis of products from polySia.
α2,8polyNeu5Ac and α2,9polyNeu5Ac were digested at pH 7.0 at 37 ℃ for 15 min. The
digests were analyzed by TLC. +, with enzyme :-, without enzyme. Arrow head, Neu5Ac. (d)
FACS analysis with anti-polySia antibody. CHO cells were treated with and without the
ORF03865 sialidase and applied to FACS analysis.
Fig.4 Inhibition and kinetic analysis of the ORF03865 sialidase. (a) Effect of Neu5Ac2en
on the 4MU-Neu5Ac-hydrolyzing reaction. The reaction was performed in the presence of
0-10 μM Neu5Ac2en at pH 7.0 at 37 ℃ for 30 min. The released 4MU was fluorometrically
11
quantitated (excitation at 365 nm, emission at 437 nm). (b) The Lineweaver-Burk plots for
4MU-Neu5Ac. The substrate (0-80 μM) was incubated with the ORF03865 sialidase in the
presence and absence of 4.6 μM Neu5Ac2en at pH 7.0, at 37 ℃ for 30 min. (c) Effect of
Neu5Ac2en on the 4MU-Kdn-hydrolyzing reaction. See (a) for the detail. (d) The
Lineweaver-Burk plots for 4MU-Kdn. The substrate (0-80 μM) was incubated with the
ORF03865 sialidase in the presence and absence of 1.7 μM Neu5Ac2en at pH 7.0, at 37 ℃ for
30 min. All the experiments were triplicated, and the standard deviations are shown by the
bar.
Footnote
The abbreviations used are: Kdn, deminoneuraminic acid; Neu5Ac, N-acetylneuraminic acid;
Neu5Ac2en,
2,3-didehydro-2-deoxy-N-acetylneuraminic
acid;
Neu5Gc,
N-glycolylneuraminic acid; Sia, Sialic acid;
12
Figure1
Fig. 1 (Iwaki et al.)
(arbitrary fluorescent units)
600000
Relative activity
500000
400000
4MU-Neu5Ac
4MU-Kdn
300000
200000
(kDa)
250
150
100
75
100000
50
37
25
51
101
151
201
251
301
351
MKRIWIMFAFAMLAGICQAQEVNVFVSGEDGYKSYRIPAIVKDKSGQLIA
FAEGRVDHAGDFGNVDIVYKISADNGKTWGSLHIAVDNDNLQVGNPAPVV
DLLDPRYPQGRLLLFYNTGNNHEGEVRKGNGLRECWSISSTDAGKTWSHP
ENITLETHRPNQPLVNTQYNFQEDWRTYANTPGHALQFDSGKYKGRIYIP
ANHSEGNPKANGKDYFAHSYYSDDHGKTFKIGASVKFEGSNETMAAQISN
TGLYMNSRNQQGNVKSRIVSYSNDGGVTWDTTYYDKNLPDPVNQGSVLSW
RRKGRYLLAVCNAATANRRDNLTLRISRDQGKTWFFNQVVSKAPEGVKGD
YAAYSDLVLLDKNRIGVLFEKENYSKIVFVPVNLK
50
100
150
200
250
300
350
385
Fig.1 Identification of a Kdn-sialidase from Sphingobacterium sp. strain HMA12.
(a) Relative sialidase activities for four candidate genes of the GH33 family from the
bacterium. The recombinant proteins were expressed in E. coli, and measured for the
sialidase activity using 4MU-Neu5Ac and 4MU-Kdn as substrates. (b) SDS-PAGECBB staining of the purified ORF03865 sialidase. The enzyme was affinity-purified as
described under Materials and Methods. (c) The deduced amino acid sequence of the
ORF03865 sialidase. Predicted signal peptide sequence (bold letters), RIP motif
(shaded), and Asp-boxes (square). The numbers represent the amino acid number.
Figure2
Fig. 2 (Iwaki et al.)
4MU-Neu5Ac
Released 4MU
(pmol)
Released 4MU
(pmol)
1000
800
600
400
200
4MU-KDN
150
100
50
0 10 20 30 40 50 60
0 10 20 30 40 50 60
Temperature (oC)
Temperature (oC)
2000
4MU-KDN
Released 4MU
(pmol)
Released 4MU
(pmol)
4MU-Neu5Ac
1500
1000
500
pH
9 10
250
200
150
100
50
9 10
pH
Fig.2 Temperature and pH dependency of the ORF03865 sialidase activity.
(a) Temperature-dependent profiles. The enzyme reaction was performed at pH
7.0 for 30 min at 0, 15, 25, 37, 50, and 60 ℃, using 4MU-Neu5Ac and 4MUKDN. Released 4MU was fluorometrically quantified (excitation at 365 nm,
emission at 437 nm). (b) pH-dependent profiles. The enzyme reaction was
performed at 37 ℃ for 30 min at pH 4.0-10.0. 0.1 M sodium acetate for pH 4.06.0, 0.1 M HEPES-NaOH for pH 6.0-8.0, and 0.1 M Tris-HCl for pH 8.0-10.0.
Figure3
Fig. 3 (Iwaki et al.)
4MU-Sia:
Relative activity
1.0
0.5
0.0
α2,9
α2,8
polySia polySia
Hydrolysis rate (%)
Neu5Ac; Neu5Gc; Kdn
1.5
30
20
10
12E3 (anti-polySia)
Count
50
Sialidase＋
Sialidase－
100 101 102 103
Fluorescent intensity
Fig.3 Substrate specificity of the ORF03865 sialidase. (a) Comparison of the Sia species
specificity with known bacterial sialidases. 4MU-Neu5Ac, 4MU-Neu5Gc, and 4MU-KDN
was digested with sialidases derived from Sphingobacterium sp. (ORF03865), Arthrobacter
ureafaciens (Au), Clostridium perfringens (Cp), Vibrio cholera (Vc), and Streptococcus
pneumoniae (Sp) at pH 7.0 at 37 ℃ for 30 min. The released 4MU was fluorometrically
quantitated (excitation at 365 nm, emission at 437 nm). Values represent relative activities
to the 4MU-Neu5Ac hydrolyzing one set to 1.0. (b) Linkage specificity. The sialylglycans,
i.e., α2,3Gal, α2,6Gal, α2,8Neu5Ac, and α2,9Neu5Ac, were digested with the ORF03865
sialidase at pH 7.0 at 37 ℃ for 15 min. The released Neu5Ac was quantitated by the DMBderivatization HPLC method. (c) TLC analysis of products from polySia. α2,8polyNeu5Ac
and α2,9polyNeu5Ac were digested at pH 7.0 at 37 ℃ for 15 min. The digests were
analyzed by TLC. +, with enzyme :-, without enzyme. Arrow head, Neu5Ac. (d) FACS
analysis of polySia on CHO cells after sialidase treatment. CHO cells were treated with the
ORF03865 sialidase at 37℃ for 30 min. The polySia on cell surface were measured with
anti-polySia antibody (12E3) by FACS analysis.
Figure4
Fig. 4 (Iwaki et al.)
35
30
25
20
15
10
0.010
0.01
10 20 30 40
Neu5Ac2en (μM)
4MU-Kdn
00
0.1
0.10
2 4 6 8 10
Neu5Ac2en (μM)
no inhibitor
Neu5Ac2en
0.05
0.05
-0.10
-0.05
-0.1
-0.05
0.10
0.1
1 / [4MU]
0.05
0.05
-0.005
1 / [4MU-Neu5Ac]
no inhibitor
Neu5Ac2en
0.005
0.005
-0.10
-0.05
-0.1
-0.05
Hydrolysis rate (%)
1 / [4MU]
Hydrolysis rate (%)
4MU-Neu5Ac
00
0.05
0.05
0.10
0.1
-0.05
1 / [4MU-KDN]
Fig.4 Inhibition and kinetic analysis of the ORF03865 sialidase. (a) Effect of
Neu5Ac2en on the 4MU-Neu5Ac-hydrolyzing reaction. The reaction was performed in
the presence of 0-10 μM Neu5Ac2en at pH 7.0 at 37 ℃ for 30 min. The released 4MU
was fluorometrically quantitated (excitation at 365 nm, emission at 437 nm). (b) The
Lineweaver-Burk plots for 4MU-Neu5Ac. The substrate (0-80 μM) was incubated with
the ORF03865 sialidase in the presence and absence of 4.6 μM Neu5Ac2en at pH 7.0, at
37 ℃ for 30 min. (c) Effect of Neu5Ac2en on the 4MU-Kdn-hydrolyzing reaction. See
(a) for the detail. (d) The Lineweaver-Burk plots for 4MU-Kdn. The substrate (0-80 μM)
was incubated with the ORF03865 sialidase in the presence and absence of 1.7 μM
Neu5Ac2en at pH 7.0, at 37 ℃ for 30 min. All the experiments were triplicated, and the
standard deviations are shown by the bar.
*Conflict of Interest
Conflict of Interest
On behalf of all the authors for this manuscript, the corresponding author declare：
None declared.
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