Photoaffinity labeling of sialidase with a biotin-conjugated phenylaminodiazirine derivative of 2,3-didehydro-2-deoxy-N-acetylneuraminic acid

Article abstract of DOI:10.1248/bpb.31.352

A biotin-conjugated photoactivatable phenylaminodiazirine derivative of 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA) was synthesized to identify sialidase. The free carboxylic group and N-acetyl substituent of sialic acid, which are important for recognition and enzymatic activity of sialidase, were conserved by the photolabeling compound as confirmed using analytical methods. The synthesized compound and DANA competitively inhibited starfish sialidase with a K_i value of 7.6 μM and 4.6 μM, respectively. Photo incorporation of the labeling compound to sialidase increased with irradiation time; 90% photo incorporation was achieved with more than 10-min irradiation, and labeling was completely inhibited by the addition of a competitive inhibitor. Starfish sialidase purified using high-performance gel filtration chromatography was subjected to photoaffinity labeling. A 50-kDa band was revealed to contain the sialidase active site by the photolabeling compound, and labeling was completely hindered in presence of the competitive inhibitor. Labeling specificity was ensured by the addition of the heat-deactivated standard protein chymotrypsinogen A to the reaction mixture.

Full text of DOI:10.1248/bpb.31.352

352
Biol. Pharm. Bull. 31(3) 352—356 (2008)
Vol. 31, No. 3
Photoaffinity Labeling of Sialidase with a Biotin-Conjugated
Phenylaminodiazirine Derivative of 2,3-Didehydro-2-deoxy-N-
acetylneuraminic Acid
a
a
b
,a
*
Ramaswamy KANNAPPAN, Masayuki ANDO, Kimio FURUHATA, and Yutaka UDA
a Department of Health Chemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life
Sciences; Niigata 956–8603, Japan: and b School of Pharmaceutical Sciences, Kitasato University; Minato-ku, Tokyo 108–
8641, Japan. Received November 14, 2007; accepted November 21, 2007; published online December 25, 2007
A biotin-conjugated photoactivatable phenylaminodiazirine derivative of 2,3-didehydro-2-deoxy-N-acetyl-
neuraminic acid (DANA) was synthesized to identify sialidase. The free carboxylic group and N-acetyl sub-
stituent of sialic acid, which are important for recognition and enzymatic activity of sialidase, were conserved by
the photolabeling compound as confirmed using analytical methods. The synthesized compound and DANA com-
petitively inhibited starfish sialidase with a Ki value of 7.6 mM and 4.6 mM, respectively. Photo incorporation of the
labeling compound to sialidase increased with irradiation time; 90% photo incorporation was achieved with
more than 10-min irradiation, and labeling was completely inhibited by the addition of a competitive inhibitor.
Starfish sialidase purified using high-performance gel filtration chromatography was subjected to photoaffinity
labeling. A 50-kDa band was revealed to contain the sialidase active site by the photolabeling compound, and la-
beling was completely hindered in presence of the competitive inhibitor. Labeling specificity was ensured by the
addition of the heat-deactivated standard protein chymotrypsinogen A to the reaction mixture.
Key words photoaffinity labeling; Asterina pectinifera; sialidase; inhibitory kinetics; protein identification
Sialidase [EC 3.2.1.18], which catalyzes the hydrolysis of protease, and its activity was also present in all the purifica-
terminal sialic acid residues of oligosaccharides, glycopro- tion steps. We also demonstrated the separation of cathepsin
teins, and glycolipids, is widely distributed in species from D activity from sialidase activity.15) The 47-kDa band was
prokaryotes to eukaryotes.1,2) The physiological significance believed to be the decomposed product of the 50-kDa pro-
of sialidase was revealed from studies of the pathology of tein. Hence the protein band derived from sialidase could not
lysosomal sialidase deficiency diseases such as isolated siali- be identified.
dase deficiency and galactosialidosis (sialidase deficiency
Photoaffinity labeling of sialidase using the radioiodinat-
combined with partial b-galactosidase deficiency).3,4) How- able arylazide derivative of 2,3-didehydro-2-deoxy-N-acetyl-
ever, the isolation and characterization of mammalian siali- neuraminic acid (DANA) has been reported.16,17) Because of
dase have been difficult because of its extremely poor stabil- its radioactive compound and low affinity toward human pla-
ity and membrane-bound character. So its enzymatic features cental sialidase, however, it is not convenient to use it for
of the enzyme isolated from mammalian tissues remains un- photoaffinity labeling of sialidase. In this study, we describe
clear. Recent studies have shown that lysosomal sialidase ex- the photoaffinity labeling and identification of the sialidase
ists as a multienzyme complex with b-galactosidase, protec- from starfish using a nonradioactive arylaldehyde derivative
tive protein/cathepsin A, and other proteins including a-N- of DANA.
acetylgalactosaminidase.5—9) Previously, we have highly-
purified a sialidase from human placenta.10) The final prepa- MATERIALS AND METHODS
ration of sialidase still gave five protein bands on sodium do-
decylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
Materials DANA was purchased from Boehringer
with molecular masses of 78 kDa, 64 kDa, 46 kDa, 32 kDa, Mannheim GmbH (Germany). Affilight CHO (biotionyl-N-
and 20 kDa. Of the five protein components, the 64-kDa pro- boc-phenylaminodiazirine) was from Seikagaku (Tokyo,
tein was thought to be b-galactosidase and the 32-kDa and Japan). Streptavidin-horseradishperoxidase (HRP) was pur-
20-kDa proteins were thought to be the components of pro- chased from Invitrogen (Carlsbad, CA, U.S.A.). Chemilumi-
tective protein, which has recently been identified as cathep- nescence Western blotting detection reagent (ECL Plus) was
sin A.7,11) We have since confirmed that the 46-kDa protein is purchased from GE Healthcare Bio-sciences (Piscataway, NJ,
a-N-acetylgalactosaminidase using cDNA cloning,12) while U.S.A.). Bicinchoninic acid was from Sigma-Aldrich (St.
the 78-kDa protein was identified as the heavy chain of im- Louis, MO, U.S.A.). 4-Methylumbelliferyl-a-D-N-acetylneu-
munoglobulin M by van der Horst et al.13) Thus the protein raminic acid (4MU-NeuAc) was synthesized in our labora-
band on SDS-PAGE derived from human placental sialidase tory. All other chemicals used in the experiments were of
remains unknown.
analytical grade.
We previously purified a sialidase from the starfish Aster-
General Analysis Melting points were measured on a
ina pectinifera and compared the enzymatic properties with Yamato melting point apparatus without correction. Elemen-
those of human placental sialidase.14) On SDS-PAGE, the tal analyses were performed on an MT-5 elemental analyzer
purified preparation generously gave two protein bands: an (Yanaco, Japan). Optical rotations were measured with a
upper (50-kDa) and a lower (47-kDa) band. We confirmed JASCO JIP-4 digital polarimeter (at 23 °C).
that the 50-kDa protein contains cathepsin D, a lysosomal
Chromatography Column chromatography was con-
∗ To whom correspondence should be addressed. e-mail: uda@nupals.ac.jp
© 2008 Pharmaceutical Society of Japan

March 2008
353
ducted on silica gel 60 (70—230 mesh, Merck). Thin-layer 160 °C]. The 9-(p-toluenesulfonyl) derivative was dissolved
chromatography (TLC) was performed on silica gel 60 F254 in acetone and 2,2-dimethoxypropane (5 ml) and anhydrous
(Merck) plates, and spots were detected under ultraviolet ir- camphorsulfonic acid (20 mg) were added with stirring
radiation or by spraying with 5% sulfuric acid.
Infrared Spectroscopy Infrared (IR) spectra were Dowex-1 (OHꢁ) anion-exchange resin to remove the acid,
recorded using FT/IR-460 Plus (JASCO, U.S.A.) with KBr. and the resin was filtered off and washed with acetone. The
overnight at room temperature. The mixture was treated with
Nuclear Magnetic Resonance Spectroscopy The nuclear filtrate and washings were combined and dried by evapora-
magnetic resonance (NMR) spectra were recorded at 300 tion to yield the 7,8-O-isopropylidene-9-O-(p-toluenesul-
MHz (Varian VXR-300) in solution in CDCl3 (internal fonyl) derivative of 1 as an amorphous powder, which was
Me4Si, d 0 ppm), or D2O (internal 3-trimethylsilyl-2,2,3,3-d4- subjected to the next reaction step without purification.
propionic acid sodium salt, d 0 ppm).
Acetic anhydride (5 ml) was added to a solution of the crude
Mass Spectrometry Mass spectra were measured on a 7,8-O-isopropylidene-9-O-(p-toluenesulfonyl) derivative in
JEOL JMS-AX-505HA or a JMS700MA Station mass spec- pyridine (10 ml). The mixture was stirred for 3 h at room
trometer.
temperature and evaporated to give a syrup, which was puri-
SDS-PAGE Fifty nanograms of protein were separated fied on silica-gel column chromatography with chloroform–
on 10—20% SDS-PAGE following the procedure described methanol (50 : 1) to give 2 (0.81 g, 50%) as a colorless
by Laemmli.18) The proteins separated on the gels were powder. 2: [a]D ꢂ10.4 ° (cꢃ1, MeOH). Anal. Calcd for
visualized using Daiichi silver staining kit (Daiichi Pure C24H31NO11S: C, 53.91; H, 5.77; N, 2.59. Found: C, 53.90;
Chemicals, Tokyo, Japan).
H, 5.81; N, 2.56. MS (EI) m/z: 541 (M+). IR (KBr) cmꢁ1:
1
Electroblotting/Chemiluminescence Detection The pho- 1740, 1660, 1600, 1540. H-NMR (CDCl3): d 1.30 and 1.36
tolabeled high-performance gel filtration chromatography-pu- (each 3H, s, isopropylidene methyl groups), 1.97 (3H, s,
rified fraction (50 ng protein) or preparative PAGE-purified NAc), 2.06 (3H, s, OAc), 2.30 (3H, s, Ph-methyl group), 3.77
(100 ng protein) starfish sialidase with 50 ng of heat-deacti- (3H, s, ester methyl group), 3.95 (1H, br q, Jꢃ8.5 Hz, 5-H),
vated chymotrypsinogen A (CTG-A) were separated on a 4.28—4.49 (5H, m, 6-H, 7-H, 8-H, 9-H2), 5.69 (1H, dd,
10—20% SDS-PAGE using the above method. Then the pro- Jꢃ3.0, 8.0 Hz, 4-H), 5.73 (1H, d, Jꢃ8.0 Hz, 5-NH), 5.88 (H,
teins were electroblotted on a polyvinylidene difluoride d, Jꢃ3.0 Hz, 3-H), 7.33, and 7.82 (each 2H, dd, Jꢃ8.0 Hz,
(PVDF) membrane using an electroblot apparatus (ATTO, phenyl group).
Model AE-6677). The membrane was exposed to strepta-
Methyl 5-Acetamido-4-O-acetyl-2,6-anhydro-9-azido-
vidin–HRP (Invitrogen) (1 : 10000) in 0.1% Tween-20 Tris 3,5,9-trideoxy-7,8-O-isopropylidene-D-glycero-D-galacto-
buffered saline (20 mM Tris–HCl, 140 mM sodium chloride, non-2-enonate (3) Sodium azide (0.20 g, 3.1 mmol) was
pH 7.5) after blocking with 5% nonfat milk. Chemilumines- added to a solution of 2 (0.76 g, 1.4 mmol) in dry dimethyl
cence was performed with an ECL Plus Western blotting de- sulfoxide (20 ml). The mixture was stirred for 6 h at 80 °C,
tection reagent (Amersham Biosciences, U.S.A.).
poured into water (100 ml), and extracted twice with chloro-
Starfish Sialidase Sialidase from starfish ovary was iso- form (50 ml). The extract was washed with brine, dried over
lated and purified using the previously described proce- sodium sulfate, and evaporated to dryness. The residue was
dures.15)
purified on silica-gel column chromatography with chloro-
Synthesis of 9-N-(4-Formylbenzoyl)Neu5Ac2en (6) The form–methanol (100 : 1) to give 3 (0.46 g, 80%) as a color-
starting material, methyl 5-acetamido-2,6-anhydro-3,5- less powder. 3: [a]D 3.5 ° (cꢃ1, MeOH). Anal. Calcd for
dideoxy-9-O-(p-toluenesulfonyl)-D-glycero-D-galacto-non-2- C17H24N4O8: C, 49.51; H, 5.87; N, 13.59. Found: C, 49.48;
enonate (2), was prepared following the previously described H, 5.90; N, 13.55. IR (KBr) cmꢁ1 : 2100, 1740, 1660, 1550.
1
procedure.19,20) We prepared methyl 5-acetamido-4-O-acetyl- MS (EI) m/z: 412 (M+). H-NMR (CDC13): d 1.34 and 1.48
2,6-anhydro-9-azido-3,5,9-trideoxy-7,8-O-isopropylidene- (each 3H, s, isopropylidene methyl groups), 1.97 (3H, s,
D-glycero-D-galacto-non-2-enonate (3) utilizing the condi- NAc), 2.08 (3H, s, OAc), 3.52 (1H, dd, Jꢃ13.0, 4.5 Hz, 9-H),
tions reported by Warner.17) 5-Acetamido-2,6-anhydro-3,5,9- 3.76 (1H, dd, Jꢃ13.0, 8.0 Hz, 9-H), 3.79 (3H, s, ester methyl
trideoxy-9-(4-formylbenzamido)-D-glycero-D-galacto-non-2- group), 4.02 (1H, br q, Jꢃ7.5 Hz, 5-H), 4.27—4.38 (2H, m, 7-
enoic acid (6) was synthesized as described in Chart 1a. This and 8-H), 4.50 (1H, dd, Jꢃ3.0, 8.5 Hz, 6-H), 5.63 (1H, dd,
1
structure was ascertained by analysis of H-NMR spectra, Jꢃ3.5, 7.0 Hz, 4-H), 5.98 (1H, d, Jꢃ3.5 Hz, 3-H), 6.18 (1H,
since chemical shifts at d 3.79 for 9-Hꢀ are strongly indica- d, Jꢃ8.0 Hz, 5-NH).
tive of the position of the 4-formylbenzamide group.
Methyl 5-Acetamido-4-O-acetyl-2,6-anhydro-3,5,9-
Methyl 5-Acetamido-4-O-acetyl-2,6-anhydro-3,5dideoxy- trideoxy-9-(4-formylbenzamido)-7,8-O-isopropylidene-D-
7,8-O-isopropylidene-9-O-(p-toluenesulfonyl)-D-glycero-D- glycero-D-galacto-non-2-enonate (5) solution of 3
A
galacto-non-2-enonate (2) p-Toluenesulfonyl chloride (0.42 g, 1 mmol) in methanol (30 ml) was hydrogenated with
(0.90 g, 4.6 mmol) was added to a solution of methyl 5-ac- hydrogen over Lindlar catalyst (0.1 g) for 3 h at room temper-
etamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non- ature. The solution was filtered through celite and evaporated
2-enonate19) (1, Neu5Ac2en1Me) (0.91 g, 3 mmol) in pyri- to dryness at 20 °C to the yield the 9-amino derivative (4) as
dine (20 ml) at 0 °C. The mixture was stirred for 3 h at room a brownish residue, which was subjected to the next reaction
1
temperature, poured into water (50 ml), and extracted twice step without purification. 4: H-NMR (CDCl3): d 1.34 and
with ethyl acetate (100 ml). The extract was washed with 1.48 (each 3H, s, isopropylidene methyl groups), 1.99 (3H, s,
brine, dried over sodium sulfate, and evaporated to dryness. NAc), 2.08 (3H, s, OAc), 2.93 (1H, ddd, Jꢃ13.0, 4.5 Hz, 9-
The residue was crystallized from ethyl acetate to give the 9- H), 3.09 (1H, ddd, Jꢃ13.0, 8.0 Hz, 9-H), 3.40 (2H, br s, 9-
(p-toluenesulfonyl) derivative of 1 as colorless needles20) [mp NH2), 3.80 (3H, s, ester methyl group), 4.12 (1H, dt, Jꢃ6.0,

354
Vol. 31, No. 3
7.0 Hz, 5-H), 4.27—4.38 (1H, ddd, Jꢃ5.0, 4.5, 8.0 Hz, 8-H), yield (0.9 mg, 1.2 mmol) was determined from the UV spec-
4.37 (1H, br t, Jꢃ6.0 Hz, 6-H), 4.53 (1H, dd, Jꢃ5.0, 6.0 Hz, tra of methanol solution using the e value of compound a
7-H), 5.53 (1H, dd, Jꢃ3.5, 6.5 Hz, 4-H), 6.02 (1H, d, (e360ꢃ400) as a standard. TLC was carried out following the
Jꢃ3.5 Hz, 3-H), 6.18 (1H, d, Jꢃ7.0 Hz, 5-NH).
4-Formylbenzoyl chloride (0.25 g, 1.5 mmol) was added to as the mobile phase to confirm the purity of 7 (Rfꢃ0.37).
a solution of 4 (0.40 g, 1.04 mmol) in pyridine (5 ml) at 0 °C. Photoaffinity Labeling Photolabeling was carried out in
above procedure using chloroform : methanol : water (8 : 5 : 1)
The mixture was stirred for 3 h at room temperature, poured 100 mM of acetate buffer, pH 4.3, in the presence of 15 mM of
into ice-water, and extracted twice with chloroform (20 ml). probe 7, 200 ng of protein, and with or without 1 mM of
The extract was washed with brine, dried over sodium sul- DANA. The mixture was allowed to equilibrate by incubating
fate, and evaporated to dryness. The residue was purified on at 4 °C on ice in the dark for 30 min. The mixture was kept
silica-gel column chromatography with chloroform–methanol on ice, 7 cm away from the light source in a UV light box
(100 : 1) to give 5 (0.38 g, 73%) as a colorless powder. 5: (Model LS-D, Super Light Hayashi Rikagaku, Japan). Irradi-
[a]D ꢂ23.0 ° (cꢃ0.27, MeOH). Anal. Calcd for C25H30N2- ation was carried out for 5 min at 365 nm.
O10S: C, 57.91; H, 5.83; N, 5.40. Found: C, 57.95; H, 5.83;
Enzyme Assay and Inhibitory Kinetics Sialidase activ-
N, 5.42. MS (FAB) m/z: 519 (M+ꢂ1). IR (KBr) cmꢁ1: 1740, ity toward the synthetic substrate 4MU-NeuAc was deter-
1
1700, 1660, 1605, 1540. H-NMR (CDCl3): d 1.33 and 1.46 mined according to the method reported in the literature.21)
(each 3H, s, isopropylidene methyl groups), 1.99 (3H, s, One unit of enzyme is defined as the amount of enzyme
NAc), 2.08 (3H, s, OAc), 3.61 (1H, ddd, Jꢃ14.0, 5.0, 6.0 Hz, which catalyzed the release of 1 nmol of sialic acid per
9-H), 3.89 (3H, s, ester methyl group), 3.99 (1H, ddd, Jꢃ minute. For inhibitory studies, stock solutions of DANA and
14.0, 4.5, 7.0 Hz, 9-H), 4.11 (1H, br q, Jꢃ9.0 Hz, 5-H), 4.37 7 were added to the enzyme preparation to yield final con-
(1H, dd, Jꢃ2.5, 6.0 Hz, 7-H), 4.41 (1H, br q, Jꢃ6.0 Hz, 8- centrations of 20 mM and 10 mM, respectively. The inhibitors
H), 4.63 (1H, dd, Jꢃ2.5, 9.0 Hz, 6-H), 5.74 (1H, dd, Jꢃ3.5, were allowed to bind to the enzyme for 5 min at 4 °C prior to
7.5 Hz, 4-H), 5.89 (1H, d, Jꢃ8.5 Hz, 5-NH), 5.98 (H, d, Jꢃ adding the substrates. The residual sialidase activity was
3.5 Hz, 3-H), 7.53 (1H, dd, Jꢃ4.5, 6.0 Hz, 9-NH), 7.92, and determined using the above procedure.
7.95 (each 2H, d, Jꢃ8.0 Hz, phenyl group), 10.05 (1H, s,
formyl group).
RESULTS AND DISCUSSION
5-Acetamido-2,6-anhydro-3,5,9-trideoxy-9-(4-formyl-
benzamido)-D-glycero-D-galacto-non-2-enoic Acid (6)
A
Synthesis and Characterization of Photoaffinity Label-
solution of the fully protected 9-(4-formylbenzoylamido) de- ing Compound Previously, we demonstrated the inhibitory
rivative (5) (0.3 g, 0.57 mmol) in 90% acetic acid (50 ml) was effects of sialic acid derivatives against starfish sialidase and
stirred for 1.5 h at 60 °C. The mixture was evaporated to dry- the importance of the free carboxylic group and N-acetyl
ness. The residue was dissolved in 4% aqueous sodium hy- substituent of sialic acid for recognition and enzymatic activ-
droxide (5 ml), kept at room temperature for 3 h, diluted with ity of sialidase.14) Therefore throughout the synthesis of the
water (20 ml), and acidified to pH 3.0 with Dowex-50 (Hꢂ) photoaffinity labeling compound, we preserved these two
resin. The resin was filtered off and washed with water. The functional groups of the inhibitor DANA and introduced the
filtrate and washings were combined and lyophilized. The photoreactive group at the C-9 position which is a primary
residue was purified by recrystallization with water–methanol hydroxyl group and open for selective derivatization. The
(1 : 2) to give 6 (0.14 g, 58%) as a colorless needles. 6: mp starting material, compound 2, was prepared following the
(dec.) 155 °C. [a]D ꢂ20.3 ° (cꢃ0.36, MeOH). Anal. Calcd previously described procedure.19,20) Compound 6, was syn-
for C19H22N2O9: C, 54.03; H, 5.25; N, 6.63. Found: C, 54.09; thesized as shown in Chart 1a. Each compound was con-
H, 5.27; N, 6.55. IR (KBr) cmꢁ1: 3300, 1720, 1700, 1640, firmed by mass, IR, and NMR spectroscopy. Compound a
1
1600, 1550, 1520. MS (FAB) m/z: 423 (M+ꢂ1). H-NMR was deprotected by TFA to give b and coupled to 6. The out-
(D2O): d 2.00 (3H, s, NAc), 3.57 (1H, dd, Jꢃ14.0, 7.5 Hz, 9- line of the coupling reaction is shown in Chart 1b. After cou-
H), 3.60 (1H, dd, Jꢃ1.0, 9.5 Hz, 6-H), 3.79 (1H, dd, Jꢃ14.0, pling, the mixture was purified on silica gel 60 to give the
3.5 Hz, 9-H), 4.07 (1H, ddd, Jꢃ10.5, 7.5, 3.5 Hz, 8-H), 4.08 pure glycoconjugates 7. The completion of the reaction,
(1H, t, Jꢃ9.5 Hz, 5-H), 4.28 (1H, dd, Jꢃ1.0, 10.5 Hz, 7-H), product formation, and purity of the synthesized compound
4.48 (1H, dd, Jꢃ2.5, 9.0 Hz, 4-H), 5.94 (H, d, Jꢃ2.5 Hz, 3- were confirmed on TLC.
H), 7.90, and 8.02 (each 2H, d, Jꢃ8.5 Hz, phenyl group),
Inhibitory Kinetics We first investigated whether 7 has
9.98 (1H, s, formyl group).
the same high affinity for sialidases as DANA by comparing
Synthesis of Photoreactive Neu5Ac2en Derivative The the inhibitory effects of these compounds on the activity of
photoreactive Neu5Ac2en derivative was synthesized as shown CM-Sephadex purified A. pectinifera sialidase. Figure 1
in Chart 1b. Deprotection of compound a (1.6 mg, 2.3 mmol), shows the double-reciprocal plot of the enzyme activity as a
dissolved in dichloromethane (20 ml), was performed with function of the substrate concentration. The Lineweaver–
trifluroacetic acid (20 ml) at 0 °C for 60 min and after evapo- Burk analysis data obtained at four concentrations of 4MU-
ration the residue was dissolved in 50 ml of 80% acetonitrile NeuAc (Kmꢃ58.7 mM) indicated that 7 and DANA were com-
containing compound 6 (0.5 mg). The pH was adjusted to petitive inhibitors with Ki values of 7.5 mM and 4.6 mM,
5—6 with diisopropylethylamine and the mixture was incu- respectively. This shows that the incorporation of the pho-
bated at 37 °C for 40 h in the dark. After evaporation to dry- toreactive carbene-generating group, biotinyl-phenylamino-
ness and dissolving in 80% acetonitrile (10 ml), it was puri- diazirine, in to DANA did not influence the affinity of this
fied on a silica gel 60 column by gradient elution with chlo- compound for the enzyme.
roform : methanol : water (10 : 2 : 0.1 to 8 : 5 : 1) to give 7. The
Next we investigated the time-dependent photo incorpora-

March 2008
355
Chart 1a
Chart 1b
Fig. 2. Time Course of Label Incorporation
Residual starfish sialidase activity against 4MU-NeuAc was measured after photo-
labeling with 7 (ꢂ) in the presence of DANA (ꢃ) or nonirradiated dark control (ꢄ).
Fig. 1. Effects of the Synthesized Photolabeling Compound on the Kinetic
Parameters of the Hydrolysis of 4MU-NeuAc by Starfish Sialidase
CM-Sephadex-purified starfish sialidase was assayed in the absence (ꢀ) or presence
of 7 (ꢁ) or DANA (ꢂ). The results were calculated with the Lineweaver–Burk recipro-
cal plot.
detectable photolabeling (inhibition of sialidase activity) was
observed. From the graph, it is clearly seen that photolabel-
ing is effective when exposed to UV light for 10 min and it is
tion of 7 in to the CM-Sephadex-purified starfish sialidase completely inhibited by the excess addition of DANA. How-
fraction. For this study, photolabeling of sialidase with 7 was ever, 10-min exposure also increased nonspecific photolabel-
performed as described in Materials and Methods at various ing. When the 10-min photolabeled protein sample was
time intervals in the presence or absence of DANA. Then the examined for biotin on a PVDF membrane, even the DANA-
reaction mixture was dialyzed to wash out the unbound pho- inhibited sample showed biotin incorporation (data not
tolabeling compound, and the residual enzyme activity was shown). This means that nonspecific labeling also occurred
measured to plot the graph of photoirradiation time vs. per- with 10-min exposure. For this reason, 5-min irradiation,
centage of inhibition (Fig. 2). The enzyme inhibition by pho- where more than 60% of sialidase activity was inhibited, was
tolabeling reached the maximum after 10 min and did not in- set as the standard procedure.
crease further with time. This photolabeling was completely
Photoaffinity Labeling High-performance gel filtration
inhibited by the addition of 1 mM DANA. In the dark, 7 is chromatography-purified sialidase (Fig. 3a) was photoaffinity
sufficiently stable under our experimental conditions, and no labeled with 7 in presence or absence of the competitive in-

356
Vol. 31, No. 3
The active site of sialidase from starfish ovary was labeled
effectively and we clarified that the 50-kDa band in SDS-
PAGE has the sialidase catalytic site. The arylaldehyde deriv-
ative of 2,3-didehydro-2-deoxy-N-acetylneuraminic acid con-
jugated with biotinyl phenylaminodiazirine will be a valuable
tool to identify the human protein that contain sialidase ac-
tive site.
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Fig. 3. Photoaffinity Labeling of Starfish Sialidase
(a) Silver-stained high-performance gel filtration chromatography (HPGFC) fraction
of starfish sialidase. (b) Photoaffinity labeling of HPGFC-purified starfish sialidase with
7 in the presence (lane 1) or absence (lane 2) of DANA. (c) CTG-A (25-kDa band) was
added to the preparative PAGE-purified starfish sialidase as a standard to confirm
equimolar loading, and photoaffinity labeling was performed with 7 in the presence
(lane 1) or absence (lane 2) of DANA.
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hibitor DANA (Fig. 3b). The 50-kDa protein (lane 2) was la-
beled effectively by 7 and its labeling was inhibited in pres-
ence of DANA (lane 1). This reveals that 7 has high affinity
toward the catalytic site of starfish sialidase, and this affinity
is competitively inhibited by DANA. The preparative gel
electrophoresis-purified starfish sialidase after removing
cathepsin D activity using high-performance gel filtration
chromatography was subjected to photolabeling and the
specificity of photoaffinity labeling was confirmed by the
addition of a standard protein CTG-A (25-kDa) (Fig. 3c).
The photolabeling compound, which has no affinity for CTG-
A, bound to CTG-A nonspecifically and this binding was not
influenced by DANA. Whereas, the 50-kDa protein, which
has the sialidase catalytic site influenced the photolabeling
process of 7 and was subsequently inhibited by DANA.
In our earlier studies, we showed the purification of a siali-
dase from the starfish A. pectinifera,14) and the copurification
of cathepsin D. On SDS-PAGE, the purified preparation gen-
erously gave two protein bands: an upper (50-kDa) and a
lower (47-kDa) band. We confirmed that the 50-kDa band
has cathepsin D domains and also demonstrated the separa-
tion of cathepsin D activity from sialidase activity.15) The
current study clearly showed that after removal of cathepsin
D activity, the 50-kDa protein band remained in the SDS-
PAGE and contained the sialidase catalytic site, which con-
cludes that starfish cathepsin D and sialidase have similar
molecular weights.
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In this study, we established a reliable procedure for pho-
toaffinity labeling of sialidase using a nonradioactive probe.