Vol. 64, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL
ENDO-B-GALACTOSIDASE
OF ESCHERICIA
RESEARCH COMMUNICATIONS
FREUNDII
HYDROLYSIS OF PIG COLONIC MUCIN AND MILK OLIGOSACCHARIDES BY ENDOGLYCOSIDIC ACTION Michiko Department University
Received
Fukuda
of Physical Chemistry, of Showa, Shinagawa-ku,
March
and Go Matsumura Faculty Tokyo
of Pharmaceutical 145, Japan
Sciences
25,1975
The purified keratansulfate degrading enzyme from Eschericia freundii could hydrolyze desialyzed pig colonic mucin and milk ollqosacchariaes. Desialyzed pig colonic mucin was digested to produce GlcNAcB(1+3)Gal, GlcNAc-6SB(1+3)Gal and resistant polymer. Lacto-N-tetraose and lactoN-tetraitol were hydrolyzed endoglycosidically to release qlucose and sorbitol, respectively. Therefore, this enzyme was found to be an endoB-galactosidase of rather wide specificity. To study
the
specifically
structure
hydrolyze
sugar
as exoglycosidases. release
the
glycoprotein
structure
carbohydrates
action.
In this
keratansulfate sulfated
are the
found,
are required,
as well that
linked
degrading
In the glycoprotein
potential
and blood
pig colonic
mucin
and lacto-N-tetraitol
group
the freundii
and small
observation
465
substances, it
has yet
that
can hydrolyze oligosaccharides
as an endo-B-galactosidase.
facto-N-tetraose, GalB(1+3)GlcNAcB(1+3)GalB(l+4)Glc lacto-N-tetraitol, GalB(l~)GlcNAcB(1+3)GalB(1-)4)Sorbitol GlcNAc-6S, N-acetylglucosamine-6-O-sulfate
Copyright Q I9 75 by Academic Press, Inc. All rights of reproduction in anv form reserved.
of
them by endoqlycosidic
present
Eschericia
in with
group
to protein,
can hydrolyze we will
utility
associated
case of another
0-glycosidically
enzyme from from
and these
can
linked
of oligosaccharides
(l-4).
enzymes
such as lacto-N-tetraose Abbreviations:
were
communication,
glycoprotein
which
endo-B-N-acetylglucosaminidases
such as sulfated
been shown that
of glycoprotein
and function
has been shown
glycoproteins,
endoglycosidases
from di-N-acetylchitobiosyl-asparagine
and glycopeptide
clarifing
not
portion
Recently,
oligosaccharides
glycoprotein
in which
of glycoproteins,
a
Vol. 64, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS Pig colonic donated
mucin
by Dr.
Desialyzed
S. Inoue
mucin
in 0.1 N HCl,
water.
In this
and Miss
80",
of galactose
tetraitol
(prepared
were
donated
sulfate,
with
by M. Kitamikado galactosidase
was free
aminidase
1 umole
of reducing
following
assay
enzyme solution
is
of whale
by Dr.
freundii
(6)
A.
was
the culture
nasal
according
filtrate
cartilage
keratan-
the method Purified
(8).
reducing
at 37",
and Johnson
defined
as the amount
(as galactose)
described endo+-
exo-@-N-acetylglucos-
The incubation
cartilage
of enzyme required
from
the
produced
to release per min. under
consisted
of 50 ~1 of
buffer,
pH 5.8,
containing
After
incubation
for
keratansulfate.
power
substrate
mixture
and 50 ~1 of 0.1 M Na-acetate
min.
was measured
by the method
30
of Park
(9).
chromatography
was carried
in solvent
1 (butanol:acetic
(butanol:pyridine:water Sugars
Radioactivity scintillation
donated
from
exe+-galactosidase,
condition.
nasal
12:5:4).
and r3H]-lacto-N-
kindly
some modification
sugar
150 pg whale
descending
and a small
and keratosulfatase.
One enzyme unit
Paper
against
77% of fucose
purification
was obtained
from
colonic
dialysis
of Eschericia
in the presence
with
kindly
Kyushu University.
(4400-fold
(7)
of pig
after
were
M. Kitamikado,
45% recovery)
hydrolysis
acid,
were
laboratory.
Lacto-N-tetraose
The strain
cultured
mucin
of this
and lyophylized
removed.
Endo-B-galactosidase freundii
acid
by NaB3H,reduction)
by Dr.
colonic
M. Miyawaki,
95% of sialic
Kobe University.
kindly
pig
by mild
1 hr,
treatment
amount
of r.
and desialyzed
was prepared
mucin
Kobata,
(5)
5:3:2) were
detected
of paper fluid
on Toyo No.51A acid:water
and solvent with
50:15:35),
466
paper
with
solvent
2
(10).
was determined
0.7% 2,5-diphenyloxazole (POPOP),
filter
3 (ethylacetate:pyridine:water
AgN03-NaOH
chromatograms
containing
bis[2-(5-phenyloxazoil)]benzene
out
in a dioxan (PPO),
and 10% naphthalene,
based
0.03% pwith
an
BIOCHEMICAL
Vol. 64, No. 2, 1975
Aloka with
LSC-502
liquid
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
scintillation
spectrometer,
after
cutting
the
paper
1 cm width. Hexose was determined
was by the method
by the anthrone
of Park
reaction
and Johnson
(ll),
reducing
sugar
(8).
RESULTS ANDDISCUSSION Pig Colonic ed with
Mucin
Intact
and without
the enzyme,
As indicated
ed.
and desialyzed
in Fig.1,
enzyme.
In the case of the
galactose
per mg mucin
desialyzed
mucin
The reaction to polymer
pig
and reducing
intact
mucin
desialyzed
was liberated;
colonic power
was not
mucin, thus
mucin liberated
hydrolyzed
56 pgreducing
27.7% of galactose
were
incubat-
was determinby this sugar residue
as of
was hydrolyzed. mixture
and small
was separated oligosaccharides
through
a column
(Fig.2).
1
2
of Sephadex
G-50
The oligosaccharide
3
DAYS
Fig.1 Hydrolysis of pig colonic mucin by endo-6-qalactosidase. The intact (-+-) or desialyted (&) pig colonic mucin (20 mg) were dissolved in 200 ul of 0.1 M Na-acetate buffer, pH 5.8, and 100 pl (0.5 units) of purified enzyme were added, respectively. Incubation was carried out at 37" in the presence of toluene, and at 24 hr intervals a portion (10 ~1) of incubation mixture was removed for the measurement of reducing power. In the control experiment, the enzyme and substrate were incubated separately, and mixed only before assaying for reducing power.
46’7
Vol. 64, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0.6
?
10
20
30
40
FRACTION
50
60
70
NUMBER
Fig.2 Gel filtration of enzymatic digest of desialyzed mucin on Sephadex G-50. Desialized mucin (20 mg) was digested for 3 days in the condition indicated in Fig.1. The digest was applied to a column (1 x 130 cm) equilibrated with 0.2 M NaCl, and eluted with same solution (I). Fractions of 1 ml were collected, and assayed for hexose. The elution profile of an identically treated sample of desialited mucin but without added-enzyme, is shown (II).
fraction
was subjected
to paper
two oligosaccharides GlcNAc6(1+3)Gal mobility
were (Rgal
chromatography
observed.
0.62
digestion
products
One had the
in solvent
as GlcNAc-6%3(1+3)Gal
1) and the
(Rgal
of bovine
in solvent
0.26
cornea1
1 and 2, and
same mobility other
in solvent
keratansulfate
as
had the same 1) that
were
the
by endo-B-galactosid-
ase (8). Uhen the it
polymer
was eluted
and the
in the void
control
These
fraction
without
findings
the nonreducing
disaccharides
volume
with
on the column no difference
of Sephadex
between
the
G-200,
digest
enzyme.
suggest
and GlcNAc-6SB(l+3)Gal near
was applied
that
the pig
structure
similar
terminal
and residual
colonic
468
has GlcNAcB(H3)Gal
to keratansulfate
and was hydrolyzed polymer.
mucin
in the area
by the enzyme to
BIOCHEMICAL
Vol. 64, No. 2, 1975
Lacto-N-Tetraose with
and Lacto-N-Tetraitol
the purified
determined
using
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Lacto-N-tetraose
endo-B-galactosidase, glucose
glucose
liberation
actions
of exo-B-galactosidase
oxidase
and the (12).
was observed.
was incubated
rate
of hydrolysis
As indicated
Thus
it
in
is excluded
Fig.3,
that
was no lag
concerted
and exo-B-N-acetylglucosaminidase
released
glucose.
HOURS
Fig.3 Release of glucose from lacto-N-tetraose by endo-B-galactosidase. The lacto-N-tetraose (170 ug) in 400 ul of 0.1 M Na-acetatebuffer, pH 5.8, was incubated with 100 ~1 (0.02 units) of purified enzyme at 37'. At the times indicated in the inset, 50 ul of aliquots were withdrawn. After heating for 2 min. at 100" to inactivate endo-B-galactosidase, released glucose was determined by the glucose-oxidase method (12).
[3H]-Lacto-N-tetraitol enzyme at 37" for paper
only
When the
indicate of hydrolysis
that
galactosyl
the
liberation
a greater
reaction
was found
or sorbitol
action.
is more rapidly
469
material
alcohol
of the
was subjected
and starting
of glucose
sorbitol.
amount
mixture
oligosaccharide
by endoglycosidase glucose
with
[3H]-sorbitol
and no intermediate
These
found
20 hrs.
chromatography,
observed
result
was incubated
were (Fig.4).
was directly
In addition, hydrolyzed
to
than
it
the was
galactosyl
of
BIOCHEMICAL
Vol. 64, No. 2, 1975
AND BIOPHYSfCAl
RESEARCH COMMUNICATIONS
6
5 III STANCE
15
10 FROM
ORIGIN
( CM 1
Fi
.4 Hydrolysis of [3H]-lacto-N-tetraitol by endo-B-galactosidase. H -lacto-N-teraitol (20 ~1, 5 x f04 cpm) in 0.1 M Na-acetate buffer, pH 5.8, was incubated with 10 ul (0.2 units) of purified enzyme at 37' for 20 hrs. Incubation mixture with (I) and without (II) enzyme were subjected to paper chromatography in solvent 3, and radioactivity was The position of the sorbitol is indicated by the arrow. monitored.
r+-
Therefore,
this
enzyme catalyze
GalB(1B)GlcNAcB(1+3)Galf3(l--)4)Glc
the reaction (or
sorbitol)
~-)
GalB(1-+3)GlcNAcB(1+3)Gal Endo+-galactosidase can act
both
molecular
appear
low molecular
substances
of these
substances
addition,
enzymatic
N-acetylglucosamine
t
to have wide
substance
(in
(or
specificity;
or pig
the common structure
hydrolysis
Glc
sorbitol)
i.e.
the
such as lacto-N-tetraose
such as keratansulfate having
as follows.
occured
keratansulfate)
enzyme
and high
colonic
mucin,
both
of GlcNAcg(lXi)Gal.
in both
linkages
and galactosyl
In
of galactosyl glucose
(in
lacto-
N-tetraose). These for
results
studying
indicate
the structure
that
the endo+-galactosidase
of glycoproteins
will
be useful
and oligosaccharides.
ACKNOWLEDGEMENTS The auther wish to express their gratitude to Dr. M. Kitamikado for the gift of the strain of E. freundii, to Dr. A. Kobata for the gift of milk oligosaccharides,and To Dr.noue and Miss M. Miyawaki for the
470
Vol. 64, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
gift of pig colonic mucin. This research Research Fund of the Ministry of Education
was supported of Japan.
by the
Scientific
REFERENCES Koide, N., and Muramatsu, T. (1974) J. Biol. Chem. 249, 4897 Arakawa, M., and Muramatsu, T. (1974) J. Biochem. mkyo), 76, 307 Tarentino, A. L., and Maley, F. (1974) J. Biol. Chem. 249, 8n Tarentino, A. L., Plummer, T. H. Jr., and Maley, F. (1974) J. Biol. Chem. 249, 818 Inoue, S., and Yosizawa, Z. (1966) Arch. Biochem. Biophys. 117, 257 Z: Kitamikado, M., Ueno, P., and Nakamura, T. (1970) Bull. Jap??oc. Sci. Fish. 36, 592 M., Ueno, R. (1970) Bull. 7. KitamiEdo, Jap. Sot. Sci. Fish. 3&, 1175 Fukuda, M., and Matsumura, G. Manuscript in preparation. i: Park, J. T., and Johnson, M. J. (1949) J. Biol. Chem. 181, 149 10. Trevelyan, W. E., Procter, 0. P., and Harrison, J. S. (1950) Nature, Lond. 166, 444 11. Trevelyan, W. E., and Harrison, J. S. (1952) Biochem. J. 50, 298 12. Huggett, A. St. G., and Nixon, D. A. (1957) Biochem. J. $6-9 12P 1. 2. ? 4:
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