BlOCHEMlCAl
Vol. 81, No. 2, 1978 March 30,1978
PARTIAL CHARACTERIZATION
OF THE ACIDIC OLIGOSACCHARIDES FROM RAT SUBLINGUAL GLYCOPROTEIN
Bronislaw Gastroenterology Biochemistry, Received
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 391-396
L. Slomiany
and Amalia
Slomiany
Research Laboratory, Departments New York Medical College, New York,
February
of Medicine 10029 N.Y.
and
2,1978
- Five sialic acid-containing oligosaccharides composed of nine, ten, thirteen and fifteen sugar residues, respectively, have been isolated twelve, Each oligosaccharide contained sialic acid, from rat sublingual glycoprotein. N-acetylglucosamine, N-acetylgalactosaminitol and galactose. The partial structures as determined by sequ ntial degr dation with of desialyzed oligosaccharides, are proposed to be: GlcNA&al G cNA Gal+G cNA Galg ;;ziF&af&cosidges, 1cNAc ,alNAc-ol (oligosaccharide I), GlcN!&!Gal@~cN$Gal@~cNA$ Gal-&l NA&GalNAc-ol (oligosaccharides II and III) and GlcNAc+Gal+GlcNAc+Gal+ GlcNAc 4 GalNAc-ol (oligosaccharides IV and V). SUMMARY
INTRODUCTION - The mucous consist
of a high
properties
molecular
and biological
carbohydrate
content,
glycoprotein
(l-5).
mainly
derived
contain
very
chemical rat
little
the
isolation
saccharides
of mucins
in most
acid
called
exceeds
(1,6,7), (6).
mucins.
the
the
immunological
in great elucidated.
of rat
total
submaxillary
to protein
of the
glycoprotein
by their
high weight
of
mucus glycoproteins
of carbohydrate
characterization
animals
The physical
6% of the
while
Although
was never
of higher
are determined
have been studied groups
glands
acid-containing
glands
and the nature
the major
species
the sialic
of sialic
and partial from
functions
sublingual
prostetic
salivary
glycoproteins
which
glycoprotein
of carbohydrate
of the
weight
In rat
from
composition
sublingual
secretions
detail In this
sialic
sublingual
are glands
properties, attachment
(1,8), report
acid-containing
the
in structure
we describe oligo-
gland.
MATERIALS AND METHODS - Rat sublingual glands (50), dissected from the submaxillary glands, were provided by Dr. J. Moschera of our school. 8-Galactosidase and B-N-acetylhexosaminidase were kindly supplied by Drs. Y.T. Li and S.C. Li (Tulane University, La.). The V. cholerae neuraminidase was obtained from Worthington Biochemical Corporation (Freehold, N.J.). The major rat sublingual glycoprotein, which was not absorbed onto hydroxywas isolated according to the procedure of Tettamanti and Pigman (9) as apatite, modified by Moschera and Pigman (1). The major glycoprotein, isolated by this procedure, was obtained in a yield of 40.2 mg per 2.1 g of wet weight of the tissue. 0006~291X/78~0812~0391$01,00/0 391
Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
Vol. 8 1, No. 2, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Alkaline borohydride degradative hydrolysis of the isolated glycoprotein was performed according to the procedure of Carlson (10). The glycoprotein (30 mg) was incubated in 0.05 M KOH and 1 M NaBH4 for 15 h at 4!i°C in a final volume of 30 ml. After destruction of the excess of borohydride by tritration to pH 5 with dilute acetic acid, the solution was dialyzed against distilled water, dialysable material was concentrated and boric acid was removed as methyl borate. Separation of neutral oligosaccharides from the acidic was accomplished on a column (1.2 x 10 cm) of Dowex l-X2 (Cl-, SO-100 mesh). The neutral oligosaccharides were eluted with water, and the sialic acid-containing oligosaccharides were eluted from the column with 0.5 M NaCl. The latter fraction was desalted by passing through BioGel P-2, lyophilized and acetylated (11). Acetylated oligosaccharides, recovered from the acetylation mixture by evaporation with toluene (ll), were dissolved in a small volume of chloroform/methanol (4/l, v/v) and purified to homogeneity by thin-layer chromatography in chloroform/methanol (9/l, v/v), and chloroform/ methanol (85/15, v/v). The purified individual oligosaccharides were deacylated with sodium methoxide (11) and desalted on a P-2 column. Removal of sialic acid from the isolated deacylated oligosaccharides was accomplished with neuraminidase (1). Enzymatic degradation of the desialyzed oligosaccharides was performed by sequential treatment of the oligosaccharides with 8-galactosidase and &hexosaminidase (12). The degraded oligosaccharides were isolated by gel filtration of the boiled digest on a P-Z column (13). The liberated monosaccharides were monitored by paper chromatography and quantitated by gas-liquid chromatography (14) . The protein content of the isolated glycoprotein was measured by the method of Lowry et al (15). The carbohydrate components in glycoprotein and in the isolated oligosaccharides were determined by gas-liquid chromatography following methanolysis and derivatization with silylating reagent (16). RESULTS - The chemical given
in Table
glucosamine,
were
glycoprotein
The principal
also
after
indicated
destruction
alkaline loss
production
were
of the
in 0-glycosyl
also
components
of the
(98.7%)
were
reductive
sialic
cleavage, which
and other
in the dialyzable
prior
to protein,
and that
to dialysis, by a
No significant
sugars
was observed.
galactose
in this
mannose
of this
was accompanied
fraction.
residues
N-acetyl
of mannose
components
of N-acetylgalactosaminitol. (1.2%)
is acid,
quantities
carbohydrate
N-acetylgalactosaminitol, found
glycoprotein
Lesser
of N-acetylgalactosamine,
N-acetylgalactosamine
linkage
alkali-lablile
and fucose,
These
results
glycoprotein
is not
Sialic
suggest are
a constituent
but
involved of the
fraction.
The sialic components
Analyses
of N-acetylglucosamine
not mannose,
sublingual
and galactose.
borohydride
N-acetylglucosamine,
all
carbohydrate
present.
a complete
corresponding
that
of the major
N-acetylgalactosamine
and fucose
acid,
I.
composition
acid-containing
on a Dowex l-Cl-
oligosaccharides, Column,
represented
392
separated about
two-third
from
the neutral of the dialyzable
BIOCHEMICAL
Vol. 81, No. 2, 1978
Table
Chemical
I.
composition
AND BIOf’HYSICAL
of the major
RESEARCH COMMUNKATIONS
glycoprotein
of rat
sublingual
gland.
Component Sialic
Relative
acid
a
GlcNAc
18.5
GalNAc
10.8
Gal
15.2
Man
2.0
Fuc
0.7 17.8
a Sialic
acid
fraction
and contained
is
and galactose. fraction.
(%)
25.2
Protein
were
weight
expressed
sialic
Fucose
Following
purified five
togram
of these
111,IV
and V were
was found
per
hydrates
in the
Complete
is
isolated
given
after
saccharides
resistant
were
24 h treatment
is
were
treated
with
0.37,
given
in Table
from
with
neuraminidase.
was lost &galactosidase.
393
when treated from
rigorous
purifichroma-
oligosaccharides 0.27
and 0.51 ratios
I,II, uEl, resof carbo-
II. oligosaccharides
The desialyzed but with
each compound Stepwise
fraction
The thin-layer
the deacylated
of S-galactosidase, residue
After
and molar
residues
to the action
of galactose
1. The purified 0.26,
of the neutral
of the acidic
isolated.
The composition
one N-acetylglucosamine
One residue
were
of 0.15,
oligosaccharides acid
components
chromatography.
in Fig.
in a yield
of sialic
N-acetylgalactosaminitol
oligosaccharides
(I-V)
30 mg of glycoprotein.
was accomplished
oligosaccharides
the major by thin-layer
obtained
removal
lost
N-acetylglucosamine,
oligosaccharides
compounds
acid.
among the carbohydrate
acetylation,
individual
pectively,
saminidase.
acid,
to homogeneity
cation
saccharide
as N-acetylneuraminic
oligo-
each oligoB-N-acetylhexowhen the recovered
treatment
of the
Vol. 8 1, No. 2, 1978
Fig.
1.
from rat
BIOCHEMICAL
Thin-layer
chromatogram
sublingual
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the
oligosaccharides
with
B-galactosidase,
resulted
samine enzymatic rat
residues
glycosidases,
disaccharide.
degradation
in conversion This
linked
presented
glycoprotein
II; V. v/v).
in sequence of all
disaccharide
and N-acetylgalactosaminitol
sublingual
oligosaccharides
purified
glycoprotein.
1, Oligosaccharide I; 2, oligosaccharide 4, oligosaccharide IV; 5, oligosaccharide developed in chloroform/methanol (85/15,
GalNAc-ol
acetylated
3, oligosaccharide III; Conditions: Silica gel Visualization: orcinol
g-N-acetylhexosaminidase, five
compounds
was in turn
converted
to a G1cNAc-t to N-acetylgluco-
by S-N-acetylhexosaminidase. above
consist B to the GlcNAc+GalNAc
indicate
that
the
of a variable
number
disaccharide
adjacent
394
HR 250 nm reagent.
Theresults
carbohydrate
of
chains
of BGlcNAc-tBGal to the protein
of
disaccharide core.
Vol. 81, No. 2, 1978
Table
II.
BIOCHEMICAL
The composition
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and molar
ratios
of carbohydrates
in the
isolated
oligosaccharides.
Oligosaccharide
Gal 3.85
4.93
1.0
4.91
II
2.97
3.86
1.0
4.90
III
3.03
3.90
1.0
3.87
IV
2.05
3.10
1.0
3.92
V
2.01
2.91
1.0
2.84
as N-acetylgalactosaminitol.
DISCUSSION - Studies
of Moschera
have established
to the protein
core
and in the presence obtained
(10,17)
Using we have
optimal
through
of alkaline
I II
are
This reduced
of rat linked
bond is
sublingual
0-glycosidically alkali-labile,
oligosaccharide
chains
can be
for
were
of intact
to homogeneity
fron
the rat
found
to be composed
respectively,
were
the release
five
sublingual of nine,
and contained and galactose.
elucidated
with
the
aid
oligosaccharides
(lo),
sialic
acid-containing
glycoprotein.
These
ten,
twelve,
sialic
acid,
oligo-
thirteen
and fifteen
N-acetylglucosamine,
The partial
structures
of specific
glycosidases
of these
oligo-
as:
f-3 GlcNAc @Gal~GlcNAc~Gal$GlcNAc$Gal$GlcNAc~al$G1GalNAc-01. and III
IV and V Although number pect
chains
borohydride
(I-V)
N-acetylgalactosaminitol saccharides
on glycoprotein
N-acetylgalactosamine.
and purified
residues,
(1)
the oligosaccharide
conditions
isolated
saccharides
that
and Pigman
.
oligosaccharides
sugar
NeuAc
I
a Determined
gland
Molar ratios GlcNAc GalNAca
GlcNAc @Gal,GlcNA&Gal~GlcNA~Gal~GlcNAc~GalNac-01. B GlcNA&Gal~GlcNA&Gal~GlcNA&GalNAc-01. the oligosaccharides
of hexose to the number
II
and hexosamine of sialic
and III,
residues, acid
residues
395
they
and IV and V contained
identical
differ
with
(Table
II).
from It
each other is not
known,
resas
Vol. 81, No. 2, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
yet, which sugar residues in the oligosaccharide sialic
acid. In the salivary
chains are substituted
mucins of other animals, sialic
linked to galactose, N-acetylgalactosamine
with
acid was found
and N-acetylglucosamine
(10,17,18).
ACKNOWLEDGEMENTS - This research was supported by Grant # AM-00068-26 from NIAMDD, NIH, PHS. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Moschera, J. and Pigman, W. (1975) Carbohyd. Res. 40, 53-67. Downs, F. and Herp, A. (1977) Int. J. Pept. Prot. Res.10, 229-234. Roukema,P.A., Oderkerk, C.H. and Salkinoja-Salonen, M.S. (1976) Biochim. Biophys. Acta 428, 432-440. Keryer, G., Herman, G. and Rossignol, B. (1973) Biochim. Biophys. Acta 297, 186-191. Lombart, C. and Winzler, R.J. (1972) Biochem. J. 128, 975-977. Spicer, S.S. and Warren, L. (1959) J. Histochem. Cytochem. 8, 135-137. Quintarelli, G., Tsuiki, S., Hashimoto, Y. and Pigman, W. (1961) J. Histothem. Cytochem. 9, 176-183. Kent, S.P. and Sanders, E.M. (1969) Proc. Sot. Exp. Biol. Med. 132, 645-650. Tettamanti, G. and Pigman, W. (1968) Arch. Biochem. Biophys. 124, 41-50. Carlson, D.M. (1968) J. Biol. Chem. 243, 616-626. Slomiany, B.L. and Slomiany, A. (1977) Biochim. Biophys. Acta 486, 531-540. Li, S.C., Mazzota, M.Y., Chien, S.F. and Li, Y.T. (1975) J. Biol. Chem. 250, 6786-6791. Tsay, G.C., Dawson, G. and Sung, S.J. (1976) J. Biol. Chem. 251, 5852-5859. Slomiany, B.L. and Meyer, K. (1973) J. Biol. Chem. 248, 2290-2295. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Hughes, K.W. and Clamp, J.R. (1972) Biochim. Biophys. Acta 264, 418-425. Baig, M.M. and Aminoff (1972) J. Biol. Chem. 247, 6111-6118. Bertolini, M. and Pigman, W. (1970) Carbohyd. Res. 14, 53-63.
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