(Received for publication, September 20, 1996, and in revised form, December 6, 1996)
From The Glycosciences Laboratory, Imperial College
School of Medicine, Northwick Park Hospital, Harrow, Middlesex, HA1
3UJ, United Kingdom and § The Department of Pharmacology,
New York University Medical Center,
New York, New York 10016
The monoclonal antibody HNK-1 originally raised
to an antigenic marker of natural killer cells also binds to selected
regions in nervous tissue. The antigen is a carbohydrate that has
attracted much interest as its expression is developmentally regulated
in nervous tissue, and it is found, and proposed to be a ligand, on
several of the adhesive glycoproteins of the nervous system. It is also
expressed on glycolipids and proteoglycans, and is the target of
monoclonal auto-antibodies that give rise to a demyelinating disease.
The epitope, as characterized on glycolipids isolated from the nervous
system, is expressed on 3-sulfated glucuronic acid joined by
1-3-linkage to a neolacto backbone. Here we exploit the
neoglycolipid technology, in conjunction with
immunodetection and in situ liquid secondary ion mass
spectrometry, to characterize HNK-1-positive oligosaccharide chains
derived by reductive alkaline release from total brain glycopeptides.
The immunoreactive oligosaccharides detected are tetra- to
octasaccharides that are very minor components among a heterogeneous
population, each representing less than 0.1% of the starting material.
Their peripheral and backbone sequences resemble those of the
HNK-1-positive glycolipids. An unexpected finding is that they
terminate not with N-acetylgalactosaminitol but with
hexitol (2-substituted and 2,6-disubstituted). In a tetrasaccharide investigated in the greatest detail, the hexitol is identified as
2-substituted mannitol.
Monoclonal antibodies raised with the aim of identifying
developmentally regulated antigens and differentiation antigens that have biological functions are in many cases directed to
oligosaccharides of glycoproteins or glycolipids (1). Prominent among
such antigens are those based on backbone sequences of the
poly-N-acetyllactosamine type that are O- or
N-glycosidically linked to proteins or to a glucose core of
glycolipids (1, 2), several of which are now established ligands for
carbohydrate-binding proteins of animals (3-6); and there is much
interest in the biological events elicited by the carbohydrate-protein
interactions. HNK-1 antigen, recognized by a hybridoma antibody, is a
carbohydrate antigen first identified as a marker of the natural killer
cell population among lymphocytes (7), and later found to be expressed
on glycolipids (8-10) as well as several glycoproteins (11) and
proteoglycans (12) of the nervous system, with a changing distribution
at different stages of development (13-15). HNK-1 antigen, as
characterized biochemically on glycolipids, is expressed on 3-sulfated
glucuronic acid joined by 1-3-linkage to a
(poly)N-acetyllactosamine backbone (8-10). This
carbohydrate sequence has been implicated as a ligand for the
leukocyte-endothelium adhesion molecules, L- and P-selectins (16, 17)
and for a number of cell-interaction systems in nervous tissue (11,
18), notably as a ligand for the major glycoprotein of myelin,
Po, in the peripheral nervous system (19), the
extracellular matrix protein, laminin (20), and an adhesive protein,
amphoterin, of the cerebellum (21). Earlier indications of the presence
of the HNK-1 antigen on N-glycosidically linked oligosaccharides (22-24) have now been followed by structural studies demonstrating the presence of the 3-sulfoglucuronyl
N-acetyllactosamine sequence on a N-glycan
isolated from glycoprotein Po (25). Biosynthetic
experiments suggest that different glucuronyltransferases are involved
in the synthesis of the HNK-1 epitope on glycolipids and glycoproteins
(26).
The present investigation is concerned with HNK-1-immunoreactive oligosaccharides among total brain glycopeptides. In exploratory experiments, oligosaccharides were released by hydrazinolysis to obtain N-glycosidically linked chains, and by reductive and non-reductive alkaline hydrolysis to obtain O-glycosidically linked chains; these were examined for immunoreactivities after conversion to neoglycolipids. Having observed immunoreactive oligosaccharides among all three oligosaccharide populations, a more detailed investigation has been performed of oligosaccharides released by reductive alkaline hydrolysis.
Glycopeptides were prepared from a Pronase digest of a lipid-free protein residue (chloroform-methanol extract) of rabbit brain, after precipitation of glycosaminoglycans with cetylpyridium chloride (27).
OligosaccharidesIn small scale experiments, oligosaccharides were obtained from 10-mg aliquots of brain glycopeptides by three methods. (i) O-Glycosidic oligosaccharides in reducing form were obtained after subjecting the glycopeptides to hydrolysis with ethylamine (70% w/v in water) at 60° C for 6 h as described (28). The oligosaccharide-rich fraction was N-acetylated using acetic anhydride (29) to acetylate free amino groups of any residual peptide materials that might interfere with the reductive amination reaction in the generation of neoglycolipids. (ii) O-Glycosidic oligosaccharides in reduced form were obtained after subjecting the glycopeptides to hydrolysis with 0.05 M NaOH, 1 M NaBH4 at 45 °C for 16 h as described (30), and the oligosaccharide-rich fraction was N-acetylated (29). (iii) N-Glycosidic oligosaccharides in reducing form were obtained after treatment of the glycopeptides with anhydrous hydrazine for 9 h (29). In a larger scale experiment, O-glycosidic oligosaccharides in reduced state were obtained from 600-mg glycopeptides after reductive alkaline hydrolysis (30).
Oligosaccharide StandardsSulfated tri-, tetra-, and
pentasaccharides of the Lea series were synthesized
chemically: HSO3-3Gal1-3(Fuc
1-4)GlcNAc and HSO3-3Gal
1-3(Fuc
1-4)GlcNAc
1-3Gal (31) were
gifts of Dr. K. C. Nicolaou (Scripps Research Institute, La Jolla, CA)
and HSO3-3Gal
1-3 (Fuc
1-4)GlcNAc
1-3Gal
1-4Glc
(32) was a gift of Dr. A. Lubineau (University Paris, Orsay, France).
Man
1-2Man and Fuc
1-2Gal were from BioCarb through Russell Fine
Chemicals (Chester, United Kingdom) and Glc
1-2Glc was from Dextra
Laboratories (Reading, United Kingdom). These disaccharides were
reduced with NaBH4 (29).
Oligosaccharides obtained in each of the small scale experiments were fractionated on DEAE Sephadex A-25 (0.75 ml, acetate form, Pharmacia) into neutral fractions, 1.5 ml (unretained with 10 mM NH4OAc, pH 5) and acidic fractions, 3 ml (eluted with 500 mM NH4OAc, pH 5); these were designated as N and A, respectively. Fractions A were treated with Arthrobacter ureafaciens sialidase (Boehringer Mannheim) and further fractionated as above into neutral (Ns) and sialidase-resistant acidic fractions: As1, and As2 eluted with 100 and 500 mM NH4OAc, respectively. Oligosaccharides recovered are summarized in Table I. An aliquot of oligosaccharides As2 (26 µg of hexose) from the ethylamine hydrolysis experiment was further fractionated by isocratic elution on a normal phase HPLC1 column (TSK amide-80, 4.6 × 250 mm, Anachem, United Kingdom) using 70% acetonitrile/water in 10 mM sodium acetate, pH 5.3, at a flow rate of 1 ml/min. Eight fractions (1.5 ml each), designated As2 a-h, were collected after 2 min of flow, and desalted using AG 50W X-12 H+ (Bio-Rad); whole fractions were converted to neoglycolipids, and 10% aliquots of the neoglycolipid reaction mixtures tested for HNK-1 immunoreactivity.
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Saccharides released in the large scale reductive alkaline hydrolysis
experiment were initially resolved by two chromatographic steps using
Bio-Gel columns (1.5 × 100 cm; Bio-Rad) essentially as described
(33) except that 2 mM trifluoroacetic acid was used as
eluent at ambient temperature. Bio-Gel P-6 was used to separate high
molecular weight materials located at the void volume from the smaller
sized retained fractions, eluting between 61 and 150 ml. The retained
fraction, 80.3 mg of hexose, was concentrated by evaporation and
fractionated on Bio-Gel P-4. Seven pooled fractions were obtained (Fig.
1): 1) 0.5 mg; 2) 3.1 mg; 3) 5.5 mg; 4) 3.6 mg; 5) 13.4 mg; 6) 52.0 mg; and 7) 1.7 mg, determined as hexose. Aliquots of these
pooled fractions (5 µg of hexose) were converted to neoglycolipids
and tested for HNK-1 immunoreactivity (Fig. 2). The
pooled Bio-Gel P-4 fractions 1, 2, and 3 which showed strong HNK-1
immunoreactivities were chromatographed on a weak anion exchange
polymeric HPLC column (Asahipak NH2-P50, 4.6 × 250 mm, Prolabo, United Kingdom) using a start solvent: 2.5 mM NH4OAc, pH 6.8, and eluting with 0-250 mM
NH4OH, pH 11.7 (Fig. 3). A neutral fraction
eluting with the start solvent and two acidic fractions eluting at
about 80 and 150 mM NH4OH were obtained, and
designated N, A1, and A2, respectively (for example 1N, 1A1, and 1A2
for the three subfractions of Bio-Gel P-4 pool 1). Aliquots of these
fractions (5 µg of hexose) were converted to neoglycolipids and
tested for HNK-1 immunoreactivities as neoglycolipids (results not
shown). In each set, fraction A2 showed HNK-1 immunoreactivity and was
further fractionated by gradient elution on a normal phase TSK amide-80
HPLC column as above using acetonitrile/water in 2 mM
trifluoroacetic acid (Fig. 4). Aliquots of these
chromatographic fractions (1 µg of hexose) were monitored for HNK-1
immunoreactivities as neoglycolipids. From each of the A2
oligosaccharide fractions, three HNK-1 immunoreactive subfractions were
obtained (Fig. 4): 1Ab, 1Ac, and 1Ad; 2Ac, 2Ad, and 2Ae; and 3Ad, 3Ae,
and 3Af; the oligosaccharides recovered in terms of micrograms of
hexose are given in Table II.
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The hexose contents of oligosaccharides were estimated by a micro-scale procedure on silica gel plates by applying as spots onto the plates 1 µl of test samples and galactose standards at 0.05-1.0 mg/ml, and staining with orcinol reagent (34).
NeoglycolipidsNeoglycolipids of reducing oligosaccharides and the reduced oligosaccharides were prepared as described previously, incorporating a mild periodate oxidation step with the reduced oligosaccharides (34), and using L-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) from Sigma in the small scale experiments. L-1,2-Dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) from Fluka was used in the large scale experiments to avoid the traces of monoacyl products that are formed when DPPE is used (34). The reaction mixtures were resolved on HPTLC Silica Gel 60 plates (Merck, BDH) using chloroform/methanol/aqueous, 0.2% CaCl2 (v/v): 60/30/6 (solvent A) or 55/45/10 (solvent B) as indicated, and subjected to primulin staining (34) to reveal lipid-containing components.
Liquid Secondary Ion Mass Spectrometry (LSIMS) and TLC/LSIMS AnalysesLSIMS analyses were carried out as described previously (35, 36). Native oligosaccharides (~1 µg) were analyzed after applying onto the target using thioglycerol as liquid matrix and neoglycolipids were analyzed directly on chromatograms after TLC.
Methylation AnalysisFor methylation analysis of oligosaccharides, permethylation (37), hydrolysis, reduction, acetylation, and gas chromatography-mass spectrometry analysis were performed as described previously (38). A modification was introduced into the procedure to include reduction of carboxymethyl groups (39) after permethylation to detect uronic acid residues as their hexitols.
The retention time of -2Manol was established from the partially
methylated alditol acetate standard prepared from the disaccharide alditol Man1-2Manol. This was differentiated from -3Manol, -2Galol, and -2Glcol prepared from Man
1-3Manol, Fuc
1-2Galol, and
Glc
1-2Glcol, respectively; the retention times being 10.03, 9.81, 10.07, and 10.32 min for partially methylated alditol acetate of
-3Manol, -2Glcol, -2Manol, and -2Galol, respectively.
Hybridoma cells secreting the HNK-1 monoclonal antibody (7) were obtained from the American Type Culture Collection (Rockville, MD), and antibody containing ascites fluid was produced by standard procedures following inoculation of BALB/c mice. Antibody binding to neoglycolipids chromatographed on HPTLC plates was assayed as described (40), using a 1:500 dilution of ascites overlaid at room temperature for 2 h. 3-Sulfoglucuronyl neolactotetraosylceramide (HNK-1 glycolipid) isolated from bovine cauda equina (a gift of Dr. F. B. Jungalwala; Eunice Kennedy Shriver Center, Waltham, MA) was used as a positive control.
Small Scale Experiments
In antibody binding experiments using the neoglycolipids obtained
in the small scale experiments, HNK-1 immunoreactivities were observed
predominantly in the most acidic fractions, As2 (Fig.
5), and not in the neutral fractions N and
Ns (not shown). The binding was to minor components which
showed negligible primulin staining (not shown). Among the
neoglycolipids derived from the reducing oligosaccharides released by
hydrolysis with ethylamine, multiple immunoreactive components were
detected, chromatographing in the tri-/tetrasaccharide and higher
oligomer regions. In the same chromatogram binding experiment, using
neoglycolipids derived from acidic oligosaccharides released by
hydrazinolysis, immunoreactivity was observed just above the point of
application (this is the position of migration of neoglycolipids
derived from multi-antennary N-glycosidic
oligosaccharides),2 and at a position
corresponding to tri-/tetrasaccharide neoglycolipids. The
hydrazine-released oligosaccharides were not investigated further in
the present study.
TLC-LSIMS analyses of the HNK-1 immunoreactive neoglycolipids derived
from subfraction g of fraction As2 (Table I) of the ethylamine-released oligosaccharides gave [M H]
ions at m/z 1475 (position a, Fig. 5), and m/z
1840 (position b), consistent with the compositions
HSO3·HexA·Hex2·Hex NAc·DPPE and
HSO3·HexA·Hex3·HexNAc2·DPPE,
respectively (results not shown). These findings, with the evidence in
each case for the presence of an additional hexose, suggested the
presence of the HNK-1 epitope on oligosaccharides having reducing-end
hexose residues that may have been generated by peeling of
O-glycosidic oligosaccharides. Having detected HNK-1
positive components among oligosaccharides in the small scale reductive
experiment (Fig. 5), a larger scale alkaline borohydride reaction was
undertaken to minimize peeling.
Large Scale Experiments
General Features of the Immunoreactive Oligosaccharide AlditolsWhen converted to neoglycolipids after periodate
oxidation, the oligosaccharide alditol fractions 1A2c and d yielded a
triplet of strongly immunoreactive components i, ii, and iii and a
weakly immunoreactive component, iv (Fig. 6). Fractions
2A2c, d, and e and 3A2d, e, and f also yielded immunoreactive
components at positions i and ii, but in addition there were components
with decreasing mobilities v, vi, and vii in fraction 2A2, and vii, viii, and ix in 3A2 (Fig. 6).
The acidic oligosaccharide alditol fractions 1A2c, 2A2d, and 3A2f with
the strongest HNK-1 immunoreactivities as neoglycolipids (Figs. 4 and
6) were selected for negative ion LSIMS analyses as the free
oligosaccharides. They were deduced to contain sulfated tetra- to
octasaccharides with uronic acid in five of seven of them (Table
III) as follows. The mass spectrum of 1A2c with [M H]
at m/z 802 and a fragment ion (loss of
80 Da) at m/z 722 (Fig. 7) was consistent
with that of a reduced tetrasaccharide
HSO3·HexA·Hex2·HexNAc. This composition
is the same as that of one of the components detected as a
neoglycolipid (at position a, Fig. 5) among ethylamine-released oligosaccharides. Oligosaccharide analogs of 1A2c containing an additional HexNAc and additional Hex·HexNAc were deduced to be in
fraction 2A2d; the latter corresponded to the immunoreactive component
detected (at position b, Fig. 5) in the ethylamine experiment. Analogs
of 1A2c with additional Hex2·HexNAc2 and
dHex·Hex2·HexNAc were deduced to be in fraction 3A2f
and the other oligosaccharide alditols to contain sulfate,
dHex·Hex3·HexNAc (fraction 2A2d) and
dHex·Hex4· HexNAc2 (fraction 3A2f). On
account of its relative homogeneity, fraction 1A2c was investigated in
the greatest detail as follows.
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TLC-LSIMS Analyses of the Immunoreactive 1A2c Neoglycolipids
The 1A2c-derived triplet of immunoreactive
neoglycolipid bands i, ii, and iii (Fig. 6) gave [M H]
ions at m/z 1357, 1387, and 1417, respectively (Table IV; see also Fig. 7 for band ii).
Band iv gave too weak a spectrum for interpretation. The 30-Da
differences in the molecular ions derived from bands i to iii are
indicative of increments of CHOH arising from different positions of
periodate cleavage of a terminal hexitol residue at consecutive C-C
bonds (41). This is illustrated for 2-substituted
mannitol3 in Scheme 1, where
R = oligosaccharide and the resulting neoglycolipids contain
remnants of the reduced hexitol with three, four, or five carbons. On
this basis, and the deduced composition of the free, non-oxidized
oligosaccharide alditol 1A2c (Table III), the [M
H]
ions of the triplet of neoglycolipids were
interpreted to arise from neoglycolipids with the composition
HSO3·HexA·Hex·HexNAc-X-DHPE, where
X = the hexitol-derived fragments,
C3H5OH (band i),
C4H6(OH)2 (band ii), and
C5H7(OH)3 (band iii).
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Scheme 1.
A partial sequence, HSO3·HexA·Hex-, was deduced for the neoglycolipid band i from the fragment ions in its spectrum at m/z 1277, 1101, and 939 (Table IV). This, together with the evidence given above for the presence of a terminal hexitol, indicated that band i had the linear sequence HSO3·HexA·Hex·HexNAc·C3H5(OH)-DHPE. Also, the spectrum for band ii (Fig. 7), was consistent with the nonreducing terminal sequence HSO3·HexA·Hex. Therefore, the likely sequences of bands ii and iii were considered to be HSO3· HexA·Hex·HexNAc·C4H6(OH)2·DHPE and HSO3·HexA·Hex· HexNAc·C5H7(OH)3·DHPE, respectively, and the original sequence of the main component in 1A2c before periodate oxidation was assigned as HSO3·HexA·Hex·HexNAc·Hexol.
Linkage and Composition Analyses of the Oligosaccharide Alditol 1A2cMonosaccharide composition and linkage information on the major component in 1A2c was obtained by methylation analysis of the free oligosaccharide alditol preparation. The results showed -3GlcA1-, -3Gal1-, -4GlcNAc1-, and -2Manol, in the form of the partially methylated alditol acetates 2,4-di-O-methylglucitol, 2,4,6-tri-O-methylgalactitol, 3,6-di-O-methyl-2-N-methyl-acetamidoglucitol, and 1,3,4,5,6-penta-O-methylmannitol, respectively. With these and the mass spectrometric data the main oligosaccharide in fraction 1A2c is assigned as: HSO3-3GlcA1-3Gal1-4GlcNAc1-2Manol.
TLC-LSIMS Analyses of the Immunoreactive 2A2d and 3A2f NeoglycolipidsOf the immunoreactive neoglycolipid bands derived
from 2A2d and 3A2f, the bands at i/ii position (Fig. 6) gave the same
[M H]
ion, m/z 1357, as that from
1A2c band i with the three carbon-containing hexitol remnant (Table
IV). A small ion was also present at m/z 1327 corresponding
to an analog with a two carbon-containing remnant. The presence of the
tetrasaccharide-derived neoglycolipids at positions i/ii originating
from alditol fractions 2A2 and 3A2 are unlikely to reflect an
incomplete separation of the tetrasaccharide in the adjacent fractions
1, 2, and 3 during the Bio-Gel P-4 chromatography step. A more likely
explanation is that a proportion of the 2A2 and 3A2 alditols are
branched at the terminal hexitol, and the periodate oxidation cleavage
is limited to two rather than three C-C bonds as shown in Scheme
2 for 2,6-disubstituted
mannitol,4 where R and R
are
oligosaccharides.
Scheme 2.
Thus the mass spectra indicated that the bands at i/ii positions were
derived by a cleavage of a 2,6-branched hexitol (cf. Scheme
2) with the HSO3·HexA·Hex·HexNAc sequence linked at
the 6-position. The neutral neoglycolipids that would be predicted to
arise from the 2-linked branch were not specifically searched for in
the present study by scanning of the di- to tetrasaccharide regions.
However, the compositions of the non-oxidized penta-, hexa-, and
octasaccharide alditols with [M H]
ions 1005, 1167, 1458, and 1532 (Table III) are consistent not only with
unbranched 2-hexitol-terminating sulfated sequences, but also with the
presence of non-sulfated outer arms consisting of HexNAc, Hex·HexNAc,
dHex·Hex·HexNAc, and Hex2·HexNAc2,
respectively, 2-linked to terminal hexitols that also bear 6-linked
sulfated outer arms.
Of the slower migrating triplet of bands derived from 2A2d, band v gave
too weak a spectrum for interpretation, while bands vi and vii gave
[M H]
ions at m/z 1752 and 1782, respectively (Table IV). These correspond to higher molecular mass
analogs of neoglycolipid bands ii and iii derived from 1A2c, but 365 Da
higher, indicating in each case, the presence of an additional
disaccharide unit, Hex·HexNAc. Fragment ions m/z 1672 and
1496 from band vi showed a terminal sulfated HexA residue, and thus
provided partial sequence HSO3-HexA. Bands vi and vii are
deduced to have been derived from a hexitol-terminating polylactosamine-type analog of 1A2c.
Of the slowest migrating duplex of immunoreactive bands viii and ix
from 3A2f, the former gave an [M H]
ion at
m/z 1898, and fragment ions m/z 1818 and 1642 which indicated the presence of a sulfated terminal HexA residue and
provided composition information and the partial sequence
HSO3·HexA- (Table IV). An [M
H]
ion at m/z 1868 was also present in this band and allowed
assignment of composition. Band ix gave two [M
H]
ions at m/z 2117 and 2147; these are 365 Da higher than those from bands vi and vii, respectively, and
consistent in each case, with the presence of an additional
lactosamine-type disaccharide, Hex·HexNAc. Collectively, these data
with 2A2d and 3A2f neoglycolipids, together with the mass spectra from
the parent non-oxidized oligosaccharide alditols (Table III), indicate
that these HNK-1 immunoreactive hexa- and octasaccharides are a
population of polylactosamine-type glycans terminating in 2-substituted
hexitol which upon oxidation is cleaved to produce remnants of the
reduced hexitol with three, four, or five carbons (Scheme 1, Table
IV).
This investigation illustrates the power of the neoglycolipid technology to detect and biochemically characterize bioactive oligosaccharides that are extremely minor components of a heterogeneous population. It has enabled total glycopeptide-derived oligosaccharides from brain, a sample size of 80 mg of hexose, to be monitored for immunoreactivities during successive chromatographies, and provided critical information on sequence and linkage by in situ TLC-LSIMS analyses of discrete immunoreactive components that represent as little as 0.03% of the starting oligosaccharides.
The small scale experiments indicated that both N- and O- glycosidic oligosaccharides contain the HNK-1 determinant. The present study is focused on the immunoreactive oligosaccharides obtained by reductive alkaline hydrolysis, the procedure of choice for releasing O-glycosidic, N-acetylgalactosamine-terminating oligosaccharides with minimal peeling (30). The majority of these were found to be tetra- to octasaccharides. Their peripheral and backbone sequences resembled those of the HNK-1-positive glycosphingolipids isolated from nervous tissue (8, 9) in being of the 3-sulfoglucuronyl lactosamine series. Among the oligosaccharides obtained under non-reductive conditions, using ethylamine or hydrazine, there was immunoreactivity of neoglycolipids chromatographing as trisaccharides. These are likely products of peeling as they were not detected among the oligosaccharides isolated under the reductive conditions (Fig. 4). There was evidence for the presence of a deoxyhexose residue in one of the immunoreactive hexasaccharides released from the brain glycopeptides. With the amounts available, however, the precise location of this presumed fucose residue along the oligosaccharide chain has not been established.
Not all of the sulfated oligosaccharides identified in the non-derivatized fractions 2A2d and 3A2f were uronic acid-containing (Table III): two of the deoxyhexose containing sulfated oligosaccharides, one in 2A2d and another in 3A2f, were lacking in uronic acid, and compositionally they resembled the oligosaccharides identified previously among oligosaccharides from brain chondroitin sulfate proteoglycans (42). These were not detected, however, among the immunoreactive neoglycolipids derived from fractions 2A2d and 3A2f.
In studies which will be described elsewhere,
N-acetylgalactosaminitol-terminating oligosaccharides have
been detected, as expected, by methylation analyses in Bio-Gel P-4
fractions 1 to 4. It is unexpected, however, to find that the
immunoreactive oligosaccharides in fractions 1, 2, and 3 are
hexitol-terminating, some 2-substituted and others 2,6-disubstituted.
In oligosaccharide 1A2c the hexitol was identified as 2-substituted
mannitol. In fraction 3, additional mannitol-terminating
oligosaccharides Gal1-4GlcNAc1-2Manol and
Gal1-4/3(Fuc1-3/4)GlcNAc1-2Manol have been identified after purification and methylation analyses (not shown). Moreover, a recent
report describes an analog of the former oligosaccharide 3-sialylated
at galactose among the oligosaccharides isolated from the Schwann cell
glycoprotein -dystroglycan (43). This raises important questions as
to the origins of this family of hexitol-terminating oligosaccharide
alditols. Are they peeled products of N-glycosidic
oligosaccharides? Or are they members of an unusual family of
O-glycosidic oligosaccharides linked to protein via mannose
residues, of the type previously described to occur among
oligosaccharides released by reductive alkaline degradation from
glycopeptide domains of brain proteoglycans (44)? In the studies which
will be described elsewhere, oligosaccharides of
N-glycosidic type with intact chitobiosyl cores have been
detected among the pooled Bio-Gel P-4 fractions; these are of
high-mannose type in fractions 4 and 5 and complex type in fraction 5;
these findings are consistent with evidence reviewed in Ref. 45 that at
least 10-20% of N-glycosidic oligosaccharides are released from protein after alkaline borohydride degradation. In the present investigation, however, no evidence has been found for the presence of
peeled products of N-glycosidic oligosaccharides with loss of one or two of the N-acetylglucosamines of the chitobiosyl
core, or other intermediate products of sequential degradation among the Bio-Gel P-4 fractions. Moreover, in separate
investigations2 when a purified, asialo-biantennary
N-glycosidic oligosaccharide (100 µg) was subjected to the
reductive alkaline borohydride treatment, and the product mixture was
subjected to HPLC on a TSK amide 80 column, no appreciable degradation
product was detected, and greater than 98% of the starting material
was recovered. Thus, unless extensive peeling of N-glycans
is catalyzed, for unknown reasons, in the course of their release from
the brain glycopeptides under the conditions of reductive alkaline
borohydride degradation, the immunoreactive neoglycolipids detected in
the present study are likely to represent O-glycans.
The 2- and 2,6-disubstitution of the end-hexitol residues is
established here with confidence for the immunoreactive
oligosaccharides from the distinctive spectra of the neoglycolipids
containing the hexitol remnants produced by oxidation of the
end-hexitols. In the tetrasaccharide 1A2c, the end-monosaccharide is
mannitol as shown by methylation analysis. Among the hexa- and
octasaccharides, the presence of 2-substituted and 2,6-disubstituted
end-hexitol is demonstrated, but there is no evidence for the presence
of 3-substituted hexitol. These findings differ from the assignment of
3-substituted mannitol for the oligosaccharide alditols isolated from
the glycopeptide moieties of brain chondroitin sulfate proteoglycans of
the rat (44). In the earlier study, methylation analysis was performed
on a tetrasaccharide alditol, but the partially methylated alditol
acetate of Manol could not be visualized due to interfering peaks in
GC; the inference of 3-substitution of the mannitol was made by
comparing the mass spectra of the trimethylsilyl derivatives (and
permethyl) of a disaccharide GlcNAc-Manol produced after treatments of
the glycopeptides with alkaline NaBH4 or alkaline NaBD4. A fragment at m/z 205 (m/z 89 for the permethylated derivative) containing the carbon atoms 1 and 2 or 5 and 6, and arising by cleavage of different C-C bonds, showed a
10% shift in intensity to m/z 206 in the deuterated
derivatives. Although this is a predicted feature of a 1-3-linked
disaccharide, we have observed a similar shift (13%) in intensity
m/z 205 to 206 in a 1-2-linked disaccharide standard
GlcNAc1-2Manol, generated by NaBH4 and
NaBD4 treatments of the reducing disaccharide2;
this might represent a small degree of rearrangement involving the
deuterium. We find that the spectrum of the deuterated disaccharide is
very similar to that of the deuterated GlcNAc
1-3Manol published earlier (44). It is desirable therefore to re-evaluate the end sequences of oligosaccharide alditols derived from the glycopeptide moieties of brain chondroitin sulfate proteoglycans. The biosynthesis of these mannose-terminating glycans is an important topic for future
investigations, for there is a distinct possibility that they are
products of the O-mannosylation pathway (46) described in
fungi.
We are grateful to J. R. Rosankiewicz, R. A. Carruthers, and Dr. C. G. Herbert for assistance in mass spectrometric analyses and to Drs. A. Lubineau, K. C. Nicolaou, and F. B. Jungalwala for providing synthetic oligosaccharides and glycolipid.