The natural occurrence of
2-keto-3-deoxy-D-glycero-D-galacto-nononic
acid (KDN), (
)deaminated neuraminic acid, was first
discovered in 1986 by Nadano et al.(1) . Subsequently,
an increasing number of other KDN-glycoconjugates have been
reported(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) .
Substitution of the aminoacyl group at C-5 on Neu5Ac or Neu5Gc by a
hydroxyl group blocks almost completely the action of bacterial
exosialidases that are commonly used for the identification and
structure-function studies of sialoglycoconjugates (1, 8, 15) .
Recently, we isolated and
identified a Gram-negative soil bacterium, Sphingobacterium
multivorum, that produced a new sialidase that selectively
catalyzed the hydrolysis of different types of KDN-ketosidic
linkages(16) . The partial purification of this enzyme,
designated KDNase SM, and its substrate specificity were also described
previously(16) . The most important feature of KDNase SM was
the complete absence of sialidase activity that could release Neu5Ac
and Neu5Gc residues from a variety of naturally occurring Neu5Ac- and
Neu5Gc-containing glycoconjugates. This unique specificity was in
distinct contrast with other eukaryotic KDN-sialidases that hydrolyzed
both KDN and N-acylneuraminic acid
-ketosidic linkages (17, 18) . Since the first report of KDN residues in
fish egg polysialoglycoproteins(1) , it has been presumed that
KDN residues would not likely be restricted in occurrence to only lower
vertebrates but rather would also be expressed on mammalian cells. With
the recent findings that KDN-glycoconjugates are implicated in
fertilization in fish egg
polysialoglycoproteins(4, 7, 8) , it became
important to determine the extent of the natural occurrence of
KDN-glycoconjugates in other eukaryotic organisms. While anti-KDN
antibodies have been used to show that KDN-glycoconjugates are
expressed in rat pancreas(12) , confirmation of this conclusion
has awaited the availability of a KDNase specific for the hydrolysis of
KDN-ketosidic linkages but not N-acylneuraminyl linkages.
Indeed, we have shown recently that KDNase SM can be used in
combination with the mAb
kdn8kdn-reactive antibodies as a
diagnostic enzyme for the specific detection of KDN-glycotopes in a
variety of different mammalian cells. (
)
The above
observations raise the interesting question as to how KDNase SM can
discriminate between KDN and N-acylneuraminic acid residues.
Attempts to answer this question have been frustrated by the limited
availability of KDNase SM. Because of the importance and interest in
using KDNase in future investigations, we initiated studies to isolate
and purify large quantities of KDNase SM. This report describes that
KDNase SM is an inducible enzyme in S. multivorum when the
cells are grown in the presence of KDN-oligosaccharide alditols
(KDN-OS),
KDN
2-3Gal
1-3GalNAc
1-3[KDN
2-(8KDN
2-)
-6]GalNAcol
as the sole carbon source. We also show that the enzyme is localized in
the periplasm. Based on these observations, we were successful in
purifying the inducible enzyme 700-fold after osmotic shock, to
homogeneity.
EXPERIMENTAL PROCEDURES
Bacteria
Isolation and growth of a strain of Sphingobacterium multivorum that could grow on
KDN-oligosaccharide alditols as the sole carbon source were carried out
as described previously(16) .
Assay for KDNase Activity
4-Methylumbelliferyl KDN
(4-MU-KDN) was used as a substrate to quantitate KDNase activity.
4-MU-KDN was kindly provided by Dr. T. G. Warner (Genentech, Inc.) (17) . Enzyme assay mixtures containing 1.6 µl of 4-MU-KDN
(0.71 µg; 1.6 nmol) were incubated in a total volume of 21.6 µl
with 20 µl of enzyme dissolved in 100 mM Tris acetate
buffer, pH 6.0, containing 100 mM NaCl at 25 °C. After a
30-min incubation, 3.0 ml of 85 mM glycine carbonate buffer,
pH 9.3, were added to a 20-µl aliquot of each incubation mixture.
The 4-methylumbelliferone released was determined fluorimetrically
(
= 365 nm and 
= 450 nm) with a JASCO 821-FP fluorescence
spectrophotometer as described by Warner and
O'Brien(19) . Control incubations were carried out
without enzyme. One unit of enzyme activity is defined as the amount of
enzyme that catalyzed the hydrolysis of 1 nmol of 4-MU-KDN per min at
25 °C.
Assay for Endoglycosidase, Exoglycosidase, and Protease
Activities
The purified KDNase was assayed for the presence of
contaminating protease, exo- and endoglycosidase, and
peptide:N-glycanase activities as described previously (16) .
Preparation of Inducer Oligosaccharides
KDN and
KDN-OS,
KDN
2-3Gal
1-3GalNAc
1-3[KDN
2-(8KDN
2-)
-6]GalNAcol
with n = 5, were prepared as described
previously(16) . A KDN-oligosaccharide alditol-rich fraction,
designated ``enriched'' KDN-OS, was prepared as follows:
rainbow trout ovarian fluid (12.3 liters) was concentrated and
lyophilized, and 110 g of dried powder were obtained. The lyophilized
powder (50 g) was extracted once with 1.0 liter of chloroform/methanol
(2:1 (v/v)) and then with 1.0 liter of chloroform/methanol (1:2 (v/v))
at room temperature for 2 h(11) . The delipidated residue was
air-dried and weighed 39 g. The delipidated ovarian fluid-derived
powder (10 g) was suspended in 100 ml of 1 M NaBH
,
0.1 M NaOH and incubated at 37 °C with agitating. After 24
h, 50 ml of the same alkaline borohydride solution were added and
incubated for another 24 h. The reaction mixture was centrifuged at
9,000
g for 20 min, and the supernatant was desalted
by passage through a Sephadex G-25 column (2.0
150 cm, eluted
with water) after neutralization to
pH 6 with glacial acetic acid
followed by concentration to
50 ml. The desalted fraction was
enriched in KDN-oligosaccharide alditols. The amount of KDN was
quantitated by the TBA method(20) .
Induction of KDNase SM Activity in S.
multivorum
40 ml of M9 mineral medium containing (in
grams/liter) Na
HPO
, 6.0 g;
KH
PO
, 3.0 g; NH
Cl, 1.0 g; NaCl, 0.5
g; MgSO
, 1 mmol; CaCl
, 0.1 mmol; and 1% (w/v)
of casamino acids and glucose were inoculated with 4
10
cells of S. multivorum and incubated at 25 °C. Cells
in late log or early stationary phase of growth (42-46 h) were
harvested and washed twice with M9 medium. Washed cells (6.1
10
) were inoculated into 2.0 ml of M9 medium with or
without either 0.1% (w/v) enriched KDN-OS, KDN-OS, KDN, Neu5Ac, mild
acid hydrolysate of colominic acid (oligoNeu5Ac)(21) , or Glc
and incubated at 25 °C for times varying between 6 and 48 h. At
each time point, viable cell numbers and KDNase activity were
determined. Cell numbers were determined by counting colonies that grew
up from a series of diluted cell aliquots that were plated on LB agar
plates(16) . For measuring total cellular KDNase activity, the
cells were sedimented by centrifugation at 5,000 rpm (1,500
g) for 10 min, resuspended in 0.5 ml of 100 mM Tris
acetate buffer, pH 6.0, containing 100 mM NaCl, and sonically
disrupted (50 watts, 1 min). KDNase activity was assayed in the
supernatant fraction obtained after centrifugation at 10,000 rpm (6,000
g) for 10 min.
Release of KDNase SM by Osmotic Shock from S.
multivorum
Washed cells (1.2
10
), grown at
25 °C in 160 ml of M9 medium supplemented with 1% (w/v) casamino
acids and glucose, as described above, were inoculated into 500 ml of
M9 medium containing 0.1% (w/v) enriched KDN-OS as the sole carbon
source, and incubated at 25 °C for 43 h. The induced cells were
harvested, and the periplasmic fraction was prepared according to the
cold osmotic shock procedure of Nossal and Heppel(22) . In
brief, cells (8 g, wet weight) were harvested and washed in cold 0.03 M NaCl-0.01 M Tris-HCl buffer (pH 7.1) by
centrifugation at 13,000
g for 20 min. The washed cell
pellet was weighed and resuspended in 10 volumes (v/w) of 0.033 M Tris-HCl buffer, pH 7.1, followed by the addition, with rapid
stirring, of 10 vol (v/w) of 40% (w/v) sucrose in the same buffer that
contained 10
vol (v/w) of 0.1 M Na
EDTA, pH 7.1. After incubation with shaking at room
temperature for 10 min, the sucrose-treated cells were collected by
centrifugation (13,000
g/10 min), and resuspended in
20 vol (v/w) of ice-cold 1 mM Mg(CH
COO)
with gentle stirring for 10 min. After the addition of 2 vol
(v/w) of ice cold 1 M NaCl, 1 M Tris-HCl buffer (pH
7.1), the suspension was centrifuged (13,000
g/30
min). Enzymes localized in the periplasm were recovered in the
supernatant(22) .
Purification of KDNase SM from the Periplasm of S.
multivorum
KDNase SM was precipitated from the periplasmic
fraction by the gradual addition of solid ammonium sulfate, with gentle
stirring, to 90% saturation. After sitting overnight at 4 °C, the
precipitate was collected by centrifugation at 17,000
g for 30 min, dissolved in 10 ml of 0.1 M NaCl-100 mM Tris acetate, pH 6.0, and dialyzed against 0.1 M NaCl,
0.25 M sucrose, 20 mM Tris acetate, pH 6.0. The
dialyzed solution was applied to a CM-Toyopearl 650 M column
(2.2
11 cm, 42 ml; equilibrated with the same dialysis buffer)
and eluted first with 60 ml of the same buffer, followed by elution
with 400 ml of a linear NaCl gradient (0.1-0.6 M) in
0.25 M sucrose-20 mM Tris acetate, pH 6.0. The
flow-through fractions were combined, concentrated to 15 ml by limited
filtration (Amicon, YM10), and applied to a DEAE-Toyopearl 650 M column (2.2
11 cm, 42 ml), which had been equilibrated
with 0.1 M NaCl, 0.25 M sucrose, 20 mM Tris-HCl buffer, pH 8.0. The column was eluted initially with 60
ml of the same buffer as used for the equilibration, and subsequently
with 60 ml of 0.5 M NaCl, 0.25 M sucrose, 20 mM Tris-HCl buffer, pH 8.0. The unbound fractions were pooled,
concentrated to 13 ml by limited filtration, and chromatographed on a
CM-Toyopearl 650 M column, as described above. Six
ml-fractions were collected and assayed for KDNase activity and
protein, the latter by measuring absorbance at 280 nm.
Chemical Analysis
Protein was quantitated by the
modified Lowry method (BCA, Pierce Chemical Co.) using bovine serum
albumin as the standard.
Properties of KDNase SM
The apparent molecular
weight of KDNase SM was estimated by gel filtration on Sephacryl S-200
and SDS-polyacrylamide gel electrophoresis, as described previously (16) .The effect of pH on the KDNase SM-catalyzed
hydrolysis of 4-MU-KDN was studied by incubating the enzyme (2 µl;
7.0 milliunits) with 4-MU-KDN (1.6 µl; 1.6 nmol) in 20 µl of
0.1 M NaCl-containing 0.1% (w/v) BSA and 0.1 M Tris
acetate buffer that was adjusted to pH 4.5-8.0. The effects of
sodium cholate and Triton X-100 on the activity were examined at
concentrations up to 0.5% (w/v).
The effect of temperature on KDNase
activity was tested by incubating the enzyme at 4, 15, 25, and 37
°C. Thermal stability of the enzyme was also estimated by measuring
the activity after preincubation of the reaction mixture at 4, 25, and
37 °C for 0, 15, 30, and 60 min.
RESULTS
Growth Properties of Sphingobacterium multivorum and
Induction of KDNase SM
The doubling time of S. multivorum in LB medium at 25 °C was approximately 50 min. A decrease in
KDNase activity in both the periplasmic fraction and the sonically
disrupted cell homogenate was observed when cells grown initially in
the presence of KDN-oligosaccharide alditols were grown without
inducer. For example, after five transfers of an induced culture in the
absence of inducer, the KDNase activity decreased by 90% of that of the
original level and then remained constant at about 1.2 unit/g wet cell
weight. During this attenuation in KDNase activity, the doubling time
and cell volumes appeared unchanged. However, when these cells were
incubated with 0.1% KDN-OS as inducer in M9 minimal medium, KDNase
activity was restored. As shown in Fig. 1and Table 1,
KDNase activity per cell was 15-40-fold higher, compared with
growth in the absence of inducer. On the basis of these results, we
conclude that KDNase is an inducible enzyme in S. multivorum and that KDN-oligosaccharides appear to be required for induction.
To confirm this finding, cells were grown for 24 h in M9 minimal medium
containing the various mono- and oligosaccharides shown in Table 1, as sole carbon sources. KDN-OS and enriched KDN-OS were
shown to be the only inducers of KDNase SM activity (Table 1).
Free KDN had little effect on induction of KDNase SM, indicating that
the ketosidic linkage of bound KDN residues was a structural
requirement for induction. It is also noted in Table 1that the
cell number remained essentially unchanged during the 24 h period when
0.1% KDN, Neu5Ac, or oligoNeu5Ac were the sole carbon sources. In
contrast, KDN-OS, enriched KDN-OS, and glucose were effective carbon
sources, although glucose was not an inducer.
Figure 1:
Induction of
KDNase SM in S. multivorum with enriched KDN-OS. Cells grown
initially in M9 medium supplemented with 1% casamino acids and glucose
were harvested and incubated in M9 minimal medium containing 0.1%
KDN-OS as the sole carbon source. At the times indicated, KDNase
activity and viable cell number were determined, as described under
``Experimental Procedures.''
KDNase SM Is a Periplasmic Enzyme in S.
multivorum
Because a number of bacterial hydrolytic enzymes
including acid and alkaline phosphatases, carboxypeptidases,
polyphosphatases, sugar phosphate phosphohydrolyases, 5`-nucleotidase,
and sugar hydrolylases are localized in the periplasmic
space(23) , we sought to determine if KDNase SM was a
periplasmic enzyme. Cold osmotic shock is a method commonly used to
release periplasmic proteins(22) . As shown in Table 2,
nearly all (99%) of the KDNase activity in the cell homogenate of
induced cells of S. multivorum was released by osmotic shock.
Importantly, the specific activity of the enzyme in the periplasm was
4.4-fold higher than in the cell homogenate (120 versus 27
units/mg of protein). As described below, we were able to exploit this
finding and purify the enzyme after osmotic shock 700-fold to
homogeneity.
Purification of KDNase SM from S.
multivorum
KDNase SM was released by osmotic shock from the
periplasm of induced cells of S. multivorum grown for 43 h in
M9 medium containing 0.1% enriched KDN-OS and purified to homogeneity.
The enzyme was first precipitated by 90% ammonium sulfate before being
applied to a cation-exchange CM-Toyopearl 650 M column. The
KDNase activity obtained in the flow-through fractions (1st
CM-Toyopearl fraction) was applied to an anion-exchange DEAE-Toyopearl
650M column. The KDNase active fractions were not retained by this
column, and the flow-through fractions (DEAE-Toyopearl fraction) were
subjected to a second CM-Toyopearl 650M chromatography. As shown in Fig. 2, the KDNase activity now eluted at an NaCl concentration
of 0.2-0.22 M (2nd CM-Toyopearl fraction). The KDNase
activity was not adsorbed to the first CM-Toyopearl column, because the
enzyme appears to have formed a complex with highly negatively charged
molecules, possibly DNA or RNA, that were removed by the DEAE-Toyopearl
column. The KDNase fractions at each purification step were analyzed by
SDS-polyacrylamide gel electrophoresis (Fig. 3), and the 2nd
CM-Toyopearl fraction was shown to be pure, based on the fact that only
a single band was observed when the gel was stained by the silver
staining method (Fig. 3, lane 4). As summarized in Table 3, KDNase was purified 700-fold in 43% yield from the
periplasmic fraction. The specific activity of the pure enzyme was
82100 unit/mg of protein. The KDNase SM thus obtained was highly
specific for KDN ketosidic linkages of natural and synthetic substrates
and, as reported for the partially purified enzyme, did not hydrolyze
4-MU-Neu5Ac, or other N-acylneuraminyl glycan
chains(16) . No protease, other glycosidases, or
peptide:N-glycanase activities were detected. The purified
enzyme was also free of the low levels of
-fucosidase and
-
and
-hexosaminidase activities that were detected previously in
the affinity-purified KDNase SM(16) .
Figure 2:
Purification of KDNase SM from S.
multivorum by CM-Toyopearl 650M chromatography. The flow-through
fractions from the DEAE-Toyopearl chromatography step were applied to a
column of CM-Toyopearl 650 M and eluted first with 0.25 M sucrose-20 mM Tris-acetate buffer, pH 6.0, containing 0.1 M NaCl, followed by elution with a linear NaCl gradient
(0.1-0.6 M) in the same buffer. 6-ml fractions were
collected and assayed for KDNase activity. The protein concentration in
the KDNase active fractions is not shown because it was too low to be
determined without significant
concentration.
Figure 3:
SDS-polyacrylamide gel electrophoresis of
fractions obtained at each purification step. Molecular weights are
indicated on the side. Lane 1, periplasmic fraction; lane
2, first CM-Toyopearl pooled fractions; lane 3, pooled
DEAE-Toyopearl fractions; lane 4, second CM-Toyopearl
fraction.
Determination of Apparent Molecular Weight
When
the purified KDNase was chromatographed on an analytical Sephacryl
S-200 column, a single peak of KDNase activity was obtained that eluted
with an apparent molecular weight of 40,000. The apparent molecular
weight of the enzyme was also estimated to be 47,500 by
SDS-polyacrylamide gel electrophoresis (Fig. 3, lane
4), both in the presence and absence of 2.5% mercaptoethanol. This
finding indicated that KDNase SM consisted of a single polypeptide
chain with an apparent M
of approximately 47,500.
Instability
The purified KDNase SM was extremely
unstable in the absence of added protein, and lost a considerable
amount of activity. Hence, the enzyme was protected from denaturation
by the addition of bovine serum albumin at 0.1-1.0 mg/ml to the
incubation mixtures. KDNase activity remained unchanged on storage at 4
°C in the presence of 0.1% BSA for at least 6 months. As shown in Fig. 4, KDNase activity decreased by 90% after incubation at 37
°C for 15 min even in the presence of 0.1% BSA. The effect of
detergents often used to increase the susceptibility of glycolipid
substrates to glycosidases had variable effects on KDNase activity.
Triton X-100 at concentrations of 0.1-0.5% (w/v) stimulated the
activity by 10-20%. In contrast, 82% of the enzyme activity was
inhibited by 0.5% (w/v) sodium cholate, while 90% of the activity was
retained at 0.1%.
Figure 4:
Thermal stability of KDNase SM. KDNase
activity was determined after incubation at 4 °C (
), 25 °C
(
), 37 °C (
) for 15, 30, and 60 min in the presence of
0.1% BSA.
Effect of pH, Temperature, and Salt on KDNase SM
Activity
The pH activity profile of KDNase SM was examined in
the presence of 0.1% BSA. Activity was maximal in the pH range between
6.0 and 7.0, and at 25 °C. Activity decreased at 37 °C, because
of the instability of the enzyme at this temperature (Fig. 4).
No requirement for divalent cations could be demonstrated. Exposure of
the enzyme to low concentrations of NaCl (less than 0.1 M) for
30 min reversibly inactivated the enzyme. Exposure for more than 30 min
irreversibly inactivated the enzyme.
DISCUSSION
Our new findings show that KDNase SM in S. multivorum is an inducible enzyme that is localized in the periplasm. The
specific activity in the periplasm was 9- and 4-fold higher than that
in the sonic cell homogenate of the uninduced and induced cells,
respectively: 6.4 versus 0.737 units/mg of protein for
uninduced cells, 120 versus 27 units/mg protein for induced
cells. After induction with KDN-OS or enriched KDN-OS, KDNase activity
was increased 15-40-fold, and the specific activity in the
periplasm was 117 units/mg of protein, which is 20-fold higher than
before induction. These facts allowed us to release the enzyme from
induced cells by osmotic shock, and to purify it 700-fold to
homogeneity. A ketosidic linkage of KDN was necessary for induction,
because neither free KDN, Neu5Ac, or oligoNeu5Ac was an inducer. Thus,
a regulatory protein responsible for KDNase induction would presumably
require a KDN ketosidic linkage, although the molecular mechanism for
induction is not known.
KDNase SM consists of a single polypeptide
chain having an estimated molecular weight of 47,500. It is a weakly
basic protein, based on its retention on a cation-exchange column of
CM-Toyopearl at pH 6.0. KDNase SM has a pH optimum at 6.0-7.0.
The purified enzyme is extremely labile, but it can be stabilized by
the addition of 0.1% BSA. Complete loss of the activity was observed
after a 15-min incubation at 37 °C, but below 25 °C the enzyme
lost only about 10% of its activity after 60 min. KDNase SM is active
in the presence of 0.5% Triton X-100, conditions which facilitate the
hydrolysis of KDN residues in KDN-gangliosides. The enzyme is highly
specific for ketosidic linkages of KDN in oligosaccharides,
glycoproteins, and glycolipids as well as a synthetic substrate,
4-MU-KDN, and devoid of any N-acylneuraminidase activities, as
was previously shown for the partially purified enzyme(16) .
These properties and substrate specificity indicate the usefulness of
KDNase SM for studies that seek to elucidate the structure-function of
KDN-glycoconjugates. In view of the recent success in cloning of a
number of N-acylneuraminidases and in determining their
tertiary structures(24) , it will be of particular interest to
obtain KDNase SM in pure crystalline form for x-ray crystallographic
analysis.